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Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Keratin: Structure, Properties and Applications : Structure, Properties and Applications, edited by Renke Dullaart, and João

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Keratin: Structure, Properties and Applications : Structure, Properties and Applications, edited by Renke Dullaart, and João

PROTEIN BIOCHEMISTRY, SYNTHESIS, STRUCTURE AND CELLULAR FUNCTIONS

KERATIN

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

STRUCTURE, PROPERTIES AND APPLICATIONS

No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

Keratin: Structure, Properties and Applications : Structure, Properties and Applications, edited by Renke Dullaart, and João

PROTEIN BIOCHEMISTRY, SYNTHESIS, STRUCTURE AND CELLULAR FUNCTIONS Additional books in this series can be found on Nova‟s website under the Series tab.

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Keratin: Structure, Properties and Applications : Structure, Properties and Applications, edited by Renke Dullaart, and João

PROTEIN BIOCHEMISTRY, SYNTHESIS, STRUCTURE AND CELLULAR FUNCTIONS

KERATIN

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

STRUCTURE, PROPERTIES AND APPLICATIONS

RENKE DULLAART AND

JOÃO MOUSQUÈS EDITORS

Nova Science Publishers, Inc. New York

Keratin: Structure, Properties and Applications : Structure, Properties and Applications, edited by Renke Dullaart, and João

Copyright © 2012 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‟ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book. Library of Congress Cataloging-in-Publication Data Keratin : structure, properties, and applications / editors, Renke Dullaart and Joco Mousquhs. p. ; cm. Includes bibliographical references and index.

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I. Dullaart, Renke. II. Mousquhs, Joco. [DNLM: 1. Keratins. QU 55.3] LC classification not assigned 572'.67--dc23 2011033101

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Keratin: Structure, Properties and Applications : Structure, Properties and Applications, edited by Renke Dullaart, and João

CONTENTS Preface Chapter 1

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Chapter 2

Chapter 3

Chapter 4

Chapter 5

Chapter 6

vii Simple Epithelial Keratins K8 and K18: from Structural to Regulatory Protein Anne-Marie Fortier and Monique Cadrin

1

Extraction, Processing and Applications of Wool Keratin M. Zoccola, A. Aluigi, A. Patrucco and C. Tonin

37

Appearance and Distribution of Cytokeratins 13, 14 and 18 in the Lingual Epithelium during Morphogenesis of the Rat Tongue Shin-ichi Iwasaki, Tomoichiro Asami and Hidekazu Aoyagi

63

Water Sorption of Human Keratinized Fibers: Effect of Wool Keratin Proteins and Peptides Clara Barba, Meritxell Martí, Alisa RoddickLanzilotta, Albert M. Manich, Josep Carilla, Jose L. Parra and Luisa Coderch

89

Lipid Structures of Various Stratum Corneum Investigated by Electron Paramagnetic Resonance Kouichi Nakagawa

113

Keratin Expression in the Human Pituitary Gland and its Application to Neoplastic Pituitary Cells Hidetoshi Ikeda

133

Keratin: Structure, Properties and Applications : Structure, Properties and Applications, edited by Renke Dullaart, and João

vi Chapter 7

Contents Keratin Fibers from Chicken Feathers: Structure and Advances in Polymer Composites Ana Laura Martínez-Hernández and Carlos Velasco-Santos

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Index

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149

213

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PREFACE Keratins represent a group of fibrous proteins produced in some epithelial cells of vertebrates such as reptiles, birds and mammals. These proteins are abundantly present in nature and they constitute the major part of hair, wool, horns, nails, feathers and stratum corneum of the skin. In this book, the authors present current research in the study of the structure, properties and applications of keratin. Topics discussed include simple epithelial keratins K8 and K18; extraction, processing and applications of wool keratin; water sorption of human keratinized fibers and keratin expression in the human pituitary gland and its application to neoplastic pituitary cells. Chapter 1 - K8 and K18 (K8/18) are the major heteropolymeric intermediate filaments (IFs) present in simple layer epithelia. In hepatocytes, keratin filaments form an extensive cytoplasmic network that is denser at the cell periphery and around bile canaliculi. Keratin filaments are attached to the plasma membrane via desmosomes. K8/18 IFs have long been linked to human chronic liver diseases. In fact, modifications in keratin IFs network organization and the formation, in hepatocytes, of K8/18 containing aggregates, named Mallory-Denk bodies (MDBs), are characteristic of alcoholic and non-alcoholic steatohepatitis, copper metabolism diseases such as Wilson disease and Indian childhood cirrhosis and hepatocellular carcinoma. The formation of MDBs is the consequence of an increase of K8/18 mRNA and proteins, alterations in K8/18 post-translational modifications such as phosphorylation on multiple sites, transglutaminase mediated keratin crosslinking and a defect in K8/18 degradation by ubiquitinproteasome pathway. The use of transgenic mouse models has allowed unravelling the significance of these changes in K8/18 dynamic in hepatocytes and revealed a function for keratins in protecting hepatocytes against

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viii

Renke Dullaart and João Mousquès

mechanical and non-mechanical stresses. For instance, mice expressing an ectopic human K14, a mutated human K18 (Arg89→Cys) or K18 (Ser52→Ala) or K18-Gly(-), K8 (Ser73→Ala) or K8 deficient mice are more susceptible to various mechanical and toxic injuries. The recent observation of the existence of K8 and K18 mutations in cases of cryptogenic and noncryptogenic forms or human liver disease is in total agreement with a role for keratin in maintaining cellular integrity under various threatening conditions. In a pursuit for understanding the molecular mechanism by which keratins could accomplish their protective role in cells, researchers have investigated the relationship between K8/18 and different regulatory pathways. There is now evidence that keratins are involved in signalling pathways regulating apoptosis, cell growth, and motility of various simple epithelial cells. It is noteworthy that expression of keratins is maintained during malignant transformation of simple epithelial cells. The PI3K/Akt pathway plays a pivotal role in apoptosis, cell growth, and motility and over-expression of the active form of Akt1 and Akt2 increase K8/18 protein levels suggesting that IFs are involved in PI3K/Akt pathway. The direct binding of K8 to Akt1 suggests that K8/18 IFs might provide a scaffold for Akt1 in cells. K8/18 interacts directly with other molecules involved in the apoptotic signalling pathway such as tumor necrosis factor receptor (TNFR), TNFR1-associated death domain protein (TRADD), Mrj co-chaperon, with Hsp/c70 and caspase 3. K8/18 also binds to key regulatory proteins of mitosis and cell proliferation such as 14-3-3, Cdc25 and Raf-1 kinase. Therefore keratins which were considered as only structural in the early 90s are now considered as key regulatory elements in modulating multiple signalling pathways. Chapter 2 - Keratins are proteins characterised by a high sulphur amount and by the presence of strong disulphide bonds which make keratins water insoluble and resistant to different chemical agents. Keratins with molecular weight ranging from less than 10 to 60 kDa are obtained by cleavage of the cystine bonds with reducing or oxidising agents or by sulphitolysis. Keratin oligopeptides can be obtained by cleavage of peptide bonds using strong acids or strong bases. Recently, green hydrolysis with superheated water and steam-explosion has been proposed with the aim of avoiding the use of harmful agents. Proteins extracted from wool can be processed alone, or in blend with other polymers, with crosslinking agents, or in the presence of additives (e.g. plasticisers). Keratins are biocompatible, biodegradable, hygroscopic, adsorb heavy metal ions, formaldehyde and other volatile organic compounds. So, keratin can be used in different fields in the form of films, sponges, nanofibres,

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ix

microcapsules, hydrogels and powders especially for biomedical applications and filtration. In this review details on the extraction and processing of keratins from wool are reported, with some example of utilization for different practical applications. Chapter 3 - Appearance and distribution of different kinds of cytokeratins are closely related to morphogenesis of the various organs. On the morphogenesis of the rat tongue during prenatal and postnatal development, we could recognize the specific appearance and distribution of some kinds of cytokeratins, such as cytokeratins 13 (K13), 14 (K14) and 18 (K18). No immunoreactivity specific for K13 and K14 was detected on the dorsal epithelium of the anterior part of the tongue in fetuses on embryonic day 15 after conception (E15), at which time the lingual epithelium was composed of a few layers of cuboidal cells. No lingual papillae are recognizable in this area. K14-specific immunoreactivity was first detected on the lingual epithelium of fetuses on E17 and K13-specific immunoreactivity on E19. The number of layers of cuboidal cells in the lingual epithelium also increased from E17 to E19. At all postnatal stages, K13-specific immunoreactivity became to be gradually evident in the suprabasal cells of the interpapillary cell columns and K14-specific immunoreactivity did in the basal and suprabasal cells of the papillary and interpapillary cell columns according to the development of filiform papillae. On the other hand, no immunoreactivity specific for K13 and K14 was detected in the lingual epithelium on E15, at which time the primitive rudiment of the circumvallate papillae was detectable by the thickening of several layers of cuboidal epithelial cells. On E17 and E19, the developing circumvallate papillae were clearly recognizable. No K13 and K14-specific immunoreactivity was evident in the lingual epithelium around these structures. K14-specific immunoreactivity was first detected in the basal layer of the epithelium of the circumvallate papillae on postnatal day 0 (P0) and K13-specific immunoreactivity was detected on P7. Morphogenesis of the circumvallate papillae progressed significantly from P0 to P14, and K13 and K14-specific immunoreactivity was clearly recognizable after P7. K13specific immunoreactivity was generally evident in cells of the intermediate layer of the epithelium, while K14-specific immunoreactivity was detected in cells of the basal and suprabasal layers. K18-specific immunoreactivity was detectable in the single layer of periderm cells that covered the dorsal epithelium of the tongue of fetuses on E13. The immunoreactivity was sparsely distributed throughout the cytoplasm

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in some periderm cells. On E15, K18-specific immunoreactivity was also detectable in the periderm cells. The distribution of the K18-specific immunoreactivity was sparsely distributed in the cytoplasm in some periderm cells on E13 and E15. On E17, the K18-specific immunoreactivity was very distinct in the periderm cells, which had become swollen and elliptical and covered the dorsal epithelium of the fetal tongues. The pattern of distribution of the immunoreactivity was different from that on E13 and E15, and K18specific immunoreactivity was compactly distributed over almost the entire cytoplasm in most periderm cells. No K18-specific immunoreactivity was detectable on the dorsal epithelium of the tongue of fetuses on E19. Periderm cells had disappeared completely by E19. Chapter 4 - Water produces changes in the properties of human keratinized fibers, such as hair and nails, and is therefore of fundamental interest. Water diffusivity in wool, horn, and the corneocyte phase of stratum corneum considerably increases with increased water content in the tissue. However, water sorption of wool is well documented whereas there are few data on human hair and nails. Human hair is a keratinized fiber which is divided into three structural zones: medulla, cortex, and cuticle. Reactive cosmetic treatment of hair often impairs the fiber structure. The resulting damage has an adverse effect on hair water absorption at ambient humidities and leads to an increase in swelling or to liquid retention on wetting. Like hair, the nail plate consists of hard keratin and lipids. The nail plate is an indicator of overall health. The degree of hydration is thought to be the most important factor influencing the physical properties of the nail. The use of nail care products and procedures to beautify and groom the nails is extremely common. Unfortunately, when improperly used, nail cosmetics can lead to nail diseases. Brittleness in the nail may be caused by trauma, such as repeated wetting and drying, repeated exposure to detergents and water, and excessive exposure to harsh solvents, such as those found in nail polish remover. There is a growing consumer trend toward natural actives that have the potential to maintain hair and nails with a healthy, youthful appearance. Wool proteins are mild, natural, biodegradable and sustainable with multiple functionalities and have potential for use in the personal care and detergent markets. In this work the effect on hair and nails of two keratin proteins isolated from wool has been investigated, an intact keratin intermediate filament protein extract (K-protein) and a low molecular weight keratin peptide from intermediate filament proteins (K-peptide). Both keratins have cystine present in the active S-sulphonated form. This unique chemistry might enable the keratin peptide and protein to reform disulphide bonds in damaged

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hair and nails and to replenish the natural disulfides bonds of the hair fibres, directly affecting their properties. The determination of water sorption isotherm by isothermally applying discrete, cumulative humidity changes involves dynamic and static aspects from which diffusion coefficients and equilibrium water contents have been deduced. Time/absorption isotherms provide a complete description of the absorption phenomenon under particular conditions such as initial regain of the sample, temperature and relative humidity. A number of equations have been proposed for modeling sorption isotherms, being in our case the GAB (Guggenheim-Anderson-deBoer) equation successfully applied. The main aim of this work is to apply two different S-sulfonated wool keratins, K-protein and K-peptide to untreated hair and nail and to hair and nail subjected to different chemical cosmetic treatments. The moisture absorption/desorption isotherms curves for untreated and treated hair and nail and the kinetics of these processes are studied in this work. The effectiveness of these keratin ingredients at restoring the water sorption characteristics of the keratin tissues is determined. Chapter 5 - Stratum corneum (SC) is the outermost layer of skin and the skin barrier against chemicals, surfactants, UV irradiation, and environmental stresses. The SC has a heterogeneous structure composed of corneocytes embedded in the intercellar lipid lamellae as illustrated in Figure 1. The morphology of the SC lipids is closely associated with the main epidermal barrier. Knowledge of the lipid structure is important in understanding the mechanism of irritant dermatitis and other SC diseases. The structural information of the SC lipid is obtained by the analysis of aliphatic spin probes incorporated into intercellar lamella lipids using EPR (Electron Paramagnetic Resonance). EPR in conjunction with spin probe method non-destructively measures the ordering of the lipid bilayer of SC. EPR (or ESR: Electron Spin Resonance) utilizes spectroscopy, which measures the freedom of an unpaired electron in an atom or molecule. The principles behind magnetic resonance are common to both EPR and nuclear magnetic resonance (NMR), but there are differences in the magnitudes and signs of the magnetic interactions involved. EPR probes an unpaired electron spin, while NMR probes a nuclear spin. EPR can measure 10-9 M (moles per liter) concentration of the probe and one of the most sensitive spectroscopic tools. Therefore, EPR is able to elucidate skin lipid structures as well as dynamics.

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Chapter 6 - The detection of specific keratins may be of diagnostic value in terms of dividing epithelial tissues into various classes depending upon their cellular origin and morphologic and/or growth features. Therefore, this review will focus on the normal patterns of keratin expression in the developing human pituitary gland, in non-neoplastic pituitary cells, and in several pathological conditions such as Rathke‟s cleft cyst and pituitary adenomas and its associated pathology (i.e. fibrous bodies and Crook‟s hyaline change). Chapter 7 - Natural fibers have been involved in an emerging kind of polymer composites taking advantage of the outstanding properties that nature confers them. Thus, several types of biofibers have recently attracted increasing interest of engineers and scientists since mimic their structures or make use of all their potential is a motivating challenge in polymeric materials field. Biofibers can be obtained from different renewable natural sources, those from vegetal origin have been the most exploited, however alternative raw materials, such as keratin biofibers are coming into view. Keratin, as fiber, can be found in hair and feathers. Human hair keratin and wool have been studied since many years ago, due to textile and medical implications, whereas keratin from feathers has not been highlighted enough. Keratin fiber has a hierarchical structure, with a highly ordered conformation, is by itself a biocomposite, product of a large evolution of animal species. Keratin fibers from feathers are non-abrasive, eco-friendly, biodegradable, insoluble in organic solvents, and also have good mechanical properties, low density, hydrophobic behavior and finally low cost. These characteristics make keratin fibers from chicken feather a suitable material to be used as a high structural reinforcement in polymer composites. However the development of keratin fibers as a new reinforcement must be based on a complete characterization in order to know their features, advantages and restrictions. The analysis of keratin fiber from chicken feather include spectroscopic analysis using both Fourier transform infrared and Raman, thermal studies including differential scanning calorimetry and thermogravimetric analysis, also contact angle determinations reflecting the hydrophobic behavior are shown. Morphological details are observed with optical, transmission and scanning electron microscopy. During the last decade these new fibers have been studied by different research groups as reinforcement for several synthetic polymers; their results are reviewed together by first time in concerned literature. Thus, keratin fibers from chicken feathers are shown as a novel eco-friendly material that must be adequately applied in the development of green composites. Finally, the studies reviewed in this chapter can be the scaffolding to increase the use of this protein taking as base the knowledge of its properties and scope,

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and therefore the current and future research in keratin could be related to high importance areas as nanotechnology or environmental decontamination, opening interesting gates in multidisciplinary areas that could take advantage of the high performance that nature confers to keratin fibril protein.

Keratin: Structure, Properties and Applications : Structure, Properties and Applications, edited by Renke Dullaart, and João

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Keratin: Structure, Properties and Applications : Structure, Properties and Applications, edited by Renke Dullaart, and João

In: Keratin: Structure, Properties and Applications ISBN 978-1-62100-336-6 Editors: Renke Dullaart et al. pp. 1-35 ©2012 Nova Science Publishers, Inc.

Chapter 1

SIMPLE EPITHELIAL KERATINS K8 AND K18: FROM STRUCTURAL TO REGULATORY PROTEIN Anne-Marie Fortier and Monique Cadrin*

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Molecular Oncology and Endocrinology Research Group, Department of Chemistry-Biology, University of Québec at Trois-Rivières, Trois-Rivières, Québec G9A 5H7, Canada

ABSTRACT K8 and K18 (K8/18) are the major heteropolymeric intermediate filaments (IFs) present in simple layer epithelia. In hepatocytes, keratin filaments form an extensive cytoplasmic network that is denser at the cell periphery and around bile canaliculi. Keratin filaments are attached to the plasma membrane via desmosomes. K8/18 IFs have long been linked to human chronic liver diseases. In fact, modifications in keratin IFs network organization and the formation, in hepatocytes, of K8/18 containing aggregates, named Mallory-Denk bodies (MDBs), are characteristic of alcoholic and non-alcoholic steatohepatitis, copper metabolism diseases such as Wilson disease and Indian childhood cirrhosis and hepatocellular carcinoma. The formation of MDBs is the consequence of an increase of K8/18 mRNA and proteins, alterations in K8/18 post-translational modifications such as phosphorylation on multiple sites, transglutaminase mediated keratin crosslinking and a *

Corresponding author, e-mail: [email protected]

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2

Anne-Marie Fortier and Monique Cadrin defect in K8/18 degradation by ubiquitin-proteasome pathway. The use of transgenic mouse models has allowed unravelling the significance of these changes in K8/18 dynamic in hepatocytes and revealed a function for keratins in protecting hepatocytes against mechanical and nonmechanical stresses. For instance, mice expressing an ectopic human K14, a mutated human K18 (Arg89→Cys) or K18 (Ser52→Ala) or K18Gly(-), K8 (Ser73→Ala) or K8 deficient mice are more susceptible to various mechanical and toxic injuries. The recent observation of the existence of K8 and K18 mutations in cases of cryptogenic and noncryptogenic forms or human liver disease is in total agreement with a role for keratin in maintaining cellular integrity under various threatening conditions. In a pursuit for understanding the molecular mechanism by which keratins could accomplish their protective role in cells, researchers have investigated the relationship between K8/18 and different regulatory pathways. There is now evidence that keratins are involved in signalling pathways regulating apoptosis, cell growth, and motility of various simple epithelial cells. It is noteworthy that expression of keratins is maintained during malignant transformation of simple epithelial cells. The PI3K/Akt pathway plays a pivotal role in apoptosis, cell growth, and motility and over-expression of the active form of Akt1 and Akt2 increase K8/18 protein levels suggesting that IFs are involved in PI3K/Akt pathway. The direct binding of K8 to Akt1 suggests that K8/18 IFs might provide a scaffold for Akt1 in cells. K8/18 interacts directly with other molecules involved in the apoptotic signalling pathway such as tumor necrosis factor receptor (TNFR), TNFR1-associated death domain protein (TRADD), Mrj co-chaperon, with Hsp/c70 and caspase 3. K8/18 also binds to key regulatory proteins of mitosis and cell proliferation such as 14-3-3, Cdc25 and Raf-1 kinase. Therefore keratins which were considered as only structural in the early 90s are now considered as key regulatory elements in modulating multiple signalling pathways.

INTRODUCTION Intermediate filaments (IFs) are with microtubules and actin microfilaments the major cytoskeletal components of most mammalian cells. They also constitute an important part of the nucleoskeleton through the nuclear lamina. However, contrary to microtubules and actin microfilaments, which have known specific functions, IFs functions as a whole are still not fully understood. Even though more needs to be known about microtubules and microfilaments some of their functions are well documented in comparison to what we understand of IFs functions. For instance, microtubules are directly involved in intracellular transport of vesicles, granules, organelles

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Simple Epithelial Keratins K8 and K18

3

like mitochondria, and chromosomes, while actin microfilaments are known to play a role, in establishment and maintenance of cell membrane organization, cell junctions and cell shape, in cell motility and muscular contractions [1]. IF proteins, encoded by 70 genes in the human genome [2-4], all present similar structures composed of a central α-helical core domain flanked by variable extensible head and tail regions [5]. This large family of proteins has been divided into six groups according to their sequences similarities, gene structure and tissue distribution [6-8]. For instance, type I and II are respectively the acidic and neutral basic keratins expressed in epithelial cells; type III is composed of vimentin, desmin, and GFAP respectively expressed in mesenchymal, muscular and glial cells; type IV includes the nervous system associated nestin and synemin and the neurofilaments (NF-L, -M, and –H); type V, the lamins (A, B and C) constitute an important part of the nuclear skeleton; finally, type VI comprises filensin and phakinin expressed in the eye lens, tanabin expressed in the growth cones of embryonic vertebrate neurons, transitin and nestin are present in myogenic and neurogenic cells, and synemin is found in undifferentiated and mature muscle cells of mammals [9]. As Professor Traub wrote in 1995: „The expression of a large number of cell typespecific and developmentally regulated subunit proteins is believed to provide multicellular organisms with different IF systems capable of differential interactions with the various substructures and components of their multiple, differentiated cells‟[10]. This statement has served to justify a large number of proposals for having grant money to pursue studies on IFs. Present in epithelial cells, keratins of type I and type II are expressed by 54 different genes and represent the largest family of IF proteins [11]. The two keratin types are coordinately expressed in pairs in various epithelia [12-14] and this differential gene expression is linked to epithelial cell lineage and differentiation (e.g. K18 and 14 of type I interact respectively with type II, K8 and K5 in liver and epidermal basal keratinocytes) [15]. More than two keratins can associate to form a filament but the ratio of type I and type II is obligatory 1:1. Type I and type II keratins form obligate heterodimers (i.e. at least one type I and one type II keratins) that assemble through a coiled coil interaction of the central α-helical rod domain of the proteins which are flanked by the largely variable flexible N- and C-terminus. Because of their variability from one protein to the other, the variable N- and C-terminus which are subjected to post-translational modifications such as phosphorylation and glycosylation are assumed to play a central role in regulating the functions of specific keratin combinations. The 10-12 nm filaments form by the association

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of the rod domains and gather to form a complex network in cells (Figure 1) [7, 16-18].

Figure 1. Schematic representation of K8/18 filaments formation. A) K8/18 heterodimers showing the non-covalent interaction of the alpha helical rod domain interrupted by linker 1 and linker 2. The head and the tail are non-alpha helical most variable and flexible domain of keratins. B) Lateral association of heterodimer form the 10-12 nm filaments. C) Elongation occurs by the association of the 1A domain of a tetramer with the coil 2 domain of a second tetramer. The head and the tail of keratins are subjected to post-translational modifications such as phosphorylation and glycosylation [23-25].

It is important to note that IFs and keratin IFs were for a long time considered to be relatively stable structures with little turnover activity [19]. This interpretation was due to the apparent stability of the IFs when isolated. This myth slowed down the beginning of the study on keratin assembly and disassembly which was initiated by Professors R.D. Goldman and E. Fuchs [20-22]. The aim of the present chapter is to review the progress that has been made over the recent years in our understanding of simple epithelial keratins. The chapter will be divided into three major sections: 1) study of liver K8/18 in health and disease, 2) K8/18 mutation and liver disease 3) involvement of K8/18 in major cellular signalling pathways.

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Simple Epithelial Keratins K8 and K18

5

1. LIVER K8/18 IN HEALTH AND DISEASE In single layer epithelia (simple epithelia) present in digestive organs, K8 is expressed with different levels of K7, K18, K19 and K20 depending on the cell type and the organ (Table 1). Table 1. Keratin expression in digestive organs Organ Liver

Epithelial cell type Hepatocytes Hepatobiliary ductal cells Oval cells

Pancreas

Acinar cells

Intestine

Ductal cells Enterocytes small intestine Enterocytes colon Goblet cells

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Gallblader

Keratins K8/K18 K7/K8/K18/K19/K20 [26, 27] K7/K8/K18/K19 [26, 27] K7/K8/K18/K19/K20 [27] (cancer K23 ) [28] K7/K8/K18/K19/K20 [27] K8/K18/K19/K20 [27] K8/K18/K19/K20 [27] K8/K18/K19/K20* [27, 29] K7/K8/K18/K19 [27]

K20* : presence of multiple phosphorylation. K20 : Low level of expression.

As depicted in Table 1, hepatocytes are unique among simple epithelia since they express solely K8 /18 in a 1:1 stoichiometric ratio. They have been known to be linked to liver diseases for a long time [30-32]. For instance, alcoholic hyaline bodies described for the first time by Professor F.B. Mallory in 1911 [33] and currently named Mallory Denk bodies (MDBs) [34] have been shown by Professor H. Denk and colleague in 1979 to be associated with modifications in keratin organization [32]. During the same period, Professor S.W. French‟s team performed biochemical analysis of MDBs to investigate the possibility that MDBs could be composed of IFs [35]. Professor H. Denk and Professor S.W. French are the pioneers in the field of intermediate filaments and MDBs, they have shaped the field and, with their team, they brought major contributions to our current understanding of the composition and mechanism of formation of MDBs.

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1.1. Composition of MDBs

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MDBs are now recognized to be common to many chronic human liver diseases such as alcoholic and non-alcoholic steatohepatitis, copper metabolism diseases such as Wilson disease and Indian childhood cirrhosis, and hepatocellular carcinoma (reviewed in [34]). The existence of animal models for MDB formation has facilitated the study of their composition and mechanism of formation. Indeed three models that are chronic feeding of mice with a diet containing griseofulvin (GF) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or dieldrin are well recognized for the induction of MDBs in hepatocytes. The formed MDBs are morphologically and biochemically very similar to human MDBs [34, 36-39]. Different studies have shown that MDBs are composed of native K8/18, post-translationally modified K8 and K18 as well as bile ductular epithelial keratins. They also contain stress proteins and chaperone, kinases and protein related to proteasome system of protein degradation. In non-diseased hepatocytes, K8/18 form in hepatocytes a complex cytoplasmic network that is denser at the cell membrane (Figure 2A).

Figure 2. Immunofluorescence staining of keratin IFs in mice and human livers. A and B are respectively liver sections from control and 4 months GF-treated mice. C and D are sections from human cirrhotic liver. Arrows show MDBs. The liver has a heterogeneous response. Note that in D there is no MDB.

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Chronic treatment of mice (4 months) with a diet containing 2.5% GF induces the formation in hepatocytes of keratin containing aggregates MDBs (Figure 2B) [40]. These MDBs are morphologically similar to human MDBs (Figure 2C). It is important to note that in diseased livers, all hepatocytes do not contain MDBs and that keratin IFs appear normal in hepatocytes in parts of the liver (Figure 2D). The major constituents of MDBs are native K8/18 and post-translationally modified K8/18 by cross-linking and phosphorylation. Early biochemical analysis showed that an important portion of MDBs is insoluble which suggested that MDBs contained cross-linked proteins [41-43]. Further analysis demonstrated that in MDBs some keratins are ubiquitinated or transaminated by tissue transglutaminase giving rise to high molecular weight keratins [4448]. Related to ubiquitinated keratins and the ubiquitin-proteasome system, valosin-containing protein (VCP), NEDD8 and p62 molecular chaperone, are also present in variable amounts in MDBs [49-51]. Pressure tuning infrared spectroscopy showed the molecular structure of IFs in MDB containing liver to be largely modified [52]. Phosphorylation is one of the most studied keratin post-translational modification and is believed to modulate their function. By using antibodies against specific phosphorylation sites on K8 and K18 (K8 Ser79; K8 Ser436; K18 Ser33), it was shown that hyperphosphorylation of keratins is also an important post-translational modification that affects keratins in MDBs [53-57]. The presence on MDBs of phospho-p38 MAPK and phosphothreonine as detected by immunostaining suggests that these kinases are playing a role in the phosphorylation of keratins present in MDBs [58, 59]. The significance of keratin hyperphosphorylation is not totally understood. K8 Ser79 has been proposed to function as a phosphate sponge for stress activated kinases in the liver undergoing chronic stress [60]. However, this interpretation is difficult to generalize since, while all cells in the livers are subjected to chronic stress, only a small number of cells and MDBs contain K8 pSer79 [57]. Moreover the cells containing K8 pSer79 are in close association with apoptotic cells suggesting that in chronic liver disease K8 pSer79 is related to apoptosis [57]. While the major constituents of MDBs are K8/18, other keratins, proteins such as chaperones stress proteins, proteins related to protein turnover in cells and protein kinases are also present in the aggregates [34]. By immunostaining, K7, K19 and K20, which are not detectable in hepatocytes from normal liver, are sometimes present in MDBs [61-64]. This indicates that chronic diseases related to MDB formation are associated with

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changes in keratin gene expression toward a pattern of expression similar to biliary tract epithelia. Non-keratin components of MDBs are proteins from the Heat stress protein family (Hsp). The presence of Hsp70 was first described by Omar et al. in 1990 [65]. Further immunocytochemical analyses confirmed the colocalization of Hsp70, Hsp90 and Hsp25 with MDBs [55, 59, 66]. Moreover, an increase in Hsps expression is observed in livers treated with GF [55]. Since Hsp70 has been shown to directly interact with K8 in normal and stressed cellular conditions [67, 68], one can conclude that Hsp70 tentatively attempts to rescue the conformational change of K8 in diseased conditions. The same interpretation can be proposed for the other chaperone Hsp proteins. Conversely, other studies have shown a decrease in Hsp70 in MDB formation in DDC livers [69]. There is accumulating data concerning the composition of MDBs. However, since the composition varies depending on the hepatotoxic agent used, more need to be done to determine the relative importance of each component. The aim of the next section is to put some light into the mechanism of MDB formation in hepatocytes.

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1.2. Molecular Mechanism of MDB Formation The formation of MDBs is without a doubt the consequence of chronicliver stress. In humans, the stress is caused by long term alcohol abuse or copper metabolism dysfunction. In the animal model used for the study of MDBs, chronic feeding of mice with diets containing hepatotoxic agents like GF, DDC or dieldrin induces their formation [32, 34, 36-38]. The existence of these animal models has made possible the study of the changes in cellular and molecular events that precede MDB formation and that could on the long term lead to the development of MDBs. A second approach to studying the molecular mechanism of MDB formation is the analysis of livers during the re-induction of MDB development after a recovery period [34, 70]. Feeding mice with a diet containing GF or DDC for 3 to 4 months (induction of MDB formation) followed by 1 month drug withdrawal (drug-primed mice) and 7 days of drug refeeding induce an increase, in the liver, of MDB formation. The number of MDBs is more numerous than after the 3 to 4 months pre-treatment [70]. Even though researchers are using the same model for their studies, important discrepancies exist in the results and interpretations about the mechanism of MDB formation. One important factor that was neglected for a

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long time, but is now considered as being crucial for the different analyses, is the genetic background of the animal used [55, 71]. The genetic background component can also explain why some humans can abuse of alcohol for their whole life and develop no liver disease while others after a short period of alcohol abuse develop hepatitis and cirrhosis. In humans, MDBs are an important cellular sign of a chronic progressive liver injury. This is related in mice to a 3 to 4 month period with drugs such as GF and DDC (Figure 2B) [36]. Changes in K8/18 protein and mRNA expression are observed as early as one day after the initiation of the treatment [40, 72]. Both K8/18 protein and mRNA levels are higher than control value from day one up to the end of treatment i.e. after MDBs have formed in hepatocytes [40, 55, 72]. As shown in figure 3, double labelling for the detection of keratin IFs by immunofluorescence and K8 mRNA by in situ hybridisation, shows that mostly all hepatocytes present an increased in K8 mRNA content in 2 weeks and 4 months of GF treatment (Figure 3D). The increase in K8/18 mRNA is also observed in MDB containing cells (Figure 3E, F). By studying the effect of GF treatment of C3H mice liver, our laboratory showed that both keratins K8 and K18 as well as their respective mRNAs increased in a parallel manner suggesting that the two genes were regulated in a coordinated manner and that the regulation was post-transcriptional [40]. Other authors have suggested that MDB formation depends on an increase in K8/18 protein levels associated with an imbalance between K8/K18 ratio K8 > K18 (reviewed in [34, 38]). The general interpretation is that K8/18 increase expression is necessary for the response of cells to the oxidative stress that is generated by the GF treatment [50, 52]. This interpretation is supported by the results obtained using genetically modified mice that either expressed no K8 (K8-/-) no K18 (K18-/-) or heterozygous mice K8+/- or K18+/- [73-75]. For instance, FVB/n mice that do not express K8 are extremely sensitive to GF or DDC treatment [76-78] while heterozygous mice are more sensitive than the wild type animal (Table 2). Morphological analysis of the livers from these animals is consistent with the result presented in Table 2. Haematoxylin/eosin staining of the liver following different period of GF treatment shows that the liver of K8 deficient mice are more susceptible to the treatment than K8 heterogeneous and wild type mice (Figure 4). Immunofluorescence staining of IFs and actin shows that livers containing less keratins are more prone to be affected by GF treatment than wild type animals (Figure 5).

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Figure 3. Double staining of keratin IFs (A, C, E) and K8 mRNA (in situ hybridization) (B, D, F) in normal (A, B), 2 weeks GF (C, D) and 4 months GF (E, F). These results show that K8 mRNA level is very low in hepatocytes of control liver and that an increase is observed before the formation of MDBs in hepatocytes (D, F).

The extreme sensitivity of K8-null mice livers to GF indicates that the early modifications in keratin organization and dynamics are important phenomena in the response of hepatocytes to GF intoxication and that increases in K8/K18 levels represent a survival reaction. Thus, keratins play an active rather than a passive role in protecting hepatocytes from the GF- and DDC-induced toxic stress [74, 76, 77]. Moreover, the two alleles of the K8 gene are necessary for proper protection of hepatocytes against the stress induced by GF intoxication.

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Simple Epithelial Keratins K8 and K18 Table 2. Rate of mortality - weeks of GF treatment Time with GF

FVB/n +/+

FVB/n +/-

FVB/n -/-

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1 week 0 0.04 0.4 2 weeks 0 0.05 1 3 weeks 0 0.076 4 weeks 0 0.4 5 weeks 0 0.25 6 weeks 0 0.33 Mice from each group were given GF for different periods of time. FVB/n K8-/- are significantly more susceptible to GF treatment than FVB/n K8+/-, which are significantly more susceptible to GF treatment than control mice.

Figure 4. Histology analysis of GF-treated wild-type mice (WT), K8 heterozygous mice (HTZ) and K8-null mice (HMZ) livers. Hematoxylin/eosin staining of WT livers (a,d,g), HTZ livers (b,e,h) and HMZ livers (c,f). Mice were on the control diet (a,b,c) or treated with GF for 7 days (d,e,f) and 28 days (g,h). Large arrows in e and h highlight necrotic cells. Small arrow in g shows bile steatosis. Arrowheads in e, g and h show vacuolation of hepatocytes.

Phosphorylation is the major post-translational modification that affects keratins and is increased during the exposition of hepatocyte to oxidative stress. It is noteworthy that keratin phosphorylation sites are located in the head and the tail of the protein (Figure 1). As mentioned above, analysis of K8/18 phosphorylation with specific phospho-epitope antibodies showed that K8 and K18 phosphorylation increased during stress.

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Figure 5. K8 and actin filaments organization in hepatocytes of GF-treated wild-type (WT) and K8-heterozygous (HTZ) mice. Immunofluorescence staining of K8 in WT livers (a, f, k, o) and HTZ livers (c, h, m, q). Mice were on the control diet (a-e) or treated with GF for 1 week (f-j), 1 month (k-n) and 4 months (o-r). Arrows point to bile canaliculi.

In a recent study we have shown that the known K8/18 phosphorylation sites (K8 Ser23, K8 Ser73, K8 Ser431, K18 Ser33 and K18 Ser52) could not explain the number of isoforms observed by performing 2D gels on GF-treated livers. For instance, an increase in phosphorylation is observed on K8 Ser73, K8 Ser431 and K18 Ser33. However, K8 showed 2 phospho-isoforms in control and 7 and 6 phospho-epitopes after respectively two weeks and five months of GF treatment [57]. K18 has 2 phospho-isoforms in control liver and 3 in treated livers. The number of isoforms that we detect by 2D gels is compatible with the number predicted (http://www.cbs.dtu.dk/services/ NetPhos/). This increase in keratin phosphorylation is an indication that K8/18 phosphorylation correlates with disease progression. In recent studies, using gene-targeted mice mutated on keratin phosphorylation sites, Professor M.B. Omary‟s laboratory demonstrated that these mice presented remarkably fragile hepatocytes (reviewed in [23]). They proposed that K8 Ser73 serves as a

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phosphate sponge and in that way protects cells from the numerous stress protein kinases that are activated in diseased cells [60]. This proposed function for phosphorylation is mostly based on K8 Ser73 phosphorylation, which is increased upon stress, and does not take into account the significance of the increased phosphorylation of other sites. We believe that further analysis of keratin phosphorylation sites is necessary to fully understand the significance of these post-translational modifications during the cellular response to stress. As an example, it is possible that phophosite specific combinations be modulated during stress response and creates a cellular environment that protects cells from stress. This interpretation is in agreement with the fact that post-translational modifications occur on the head and the tail of the protein, affect the conformational reorganization of these portions of the filaments and modify their interaction with cellular components involved in cell protection against stress. The failure of the ubiquitin-proteasome pathway to properly degrade proteins is an important phenomenon in the development of MDBs [79]. Ubiquitination of keratins in a phosphorylation modulated fashion is the normal mechanism for regulating keratin turnover in cells [80]. The presence of UBB+1, a frame shift mutant of ubiquitin that cannot target protein to the proteasome, in GF- or DDC-treated hepatocytes would inhibit the degradation of keratins by the ubiquitin-proteasome pathway and induce their aggregation [79, 81, 82]. Valosin-containing protein (VCP), a protein related to proteasome inhibition, has been observed to directly bind polyubiquitinated proteins [83] and is present in MDBs [50]. The inhibition of proteasome activity by VCP is most likely playing a role in keratin aggregation and MDB formation in hepatocytes [50]. The defect in Hsps chaperoning capacity could also play a key role in the process of MDBs. The presence of Hsps on MDB suggests that the oxidative stress caused by the toxic agent alters keratin structure [52, 59]. Since Hsps levels increase during the long process of MDB formation, its chaperone capacity might help to maintain keratin structure for a certain period of treatment [69]. However, on the long term the chaperoning capacity may be reduced, leading to misfolded proteins aggregation. During the last five years, Professor S.W. French and collegues focused their effort to determine the signalling pathways activated in hepatocytes which lead to the development of MDB in hepatocytes (ref 85-95). For this purpose they utilised the primed mice model in which MDB form in 7 days [70]. They postulate that rapid MDB formation in these mice is an epigenetic phenomenon [84]. Analysis of proteasomes in these animals show that the 26S proteasome formation shifts to an immunoproteasome formation, which

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cannot degrade ubiquitinated keratins. This change would be the consequence of an IFNγ- and TNFα-mediated pro-inflammatory response. Drug toxicity induces these mechanisms and the activation of the signalling pathway can be prevented by methyl donors SAMe and betaine [85-88]. More needs to be done to understand the mechanism of MDB formation and especially to characterize the signalling pathways, which induce the different phenomenon that are observed during the development of MDBs. The understanding of the signalling pathways could serve to develop molecules that could maintain the positive aspect of the hepatocyte response to oxidative stress while ending the processes of liver degeneration.

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2. FROM K8/18 GENETICALLY MODIFIED MICE TO HUMAN LIVER DISEASE The first breakthrough in the search for keratin IFs functions come from studies on transgenic mice lacking or expressing mutated epidermal keratins by Coulombe et al. [89-91]. These mice display blistering skin disease and led to the identification of keratins as the cause of human skin diseases. From these initial studies, it was clear that IFs functions take part of their senses in the context of whole tissues and that their alteration is deleterious. As for epidermal keratins, the use of gene-targeting has been useful to unravel roles for IFs in hepatocytes. For instance, mice expressing in hepatocytes an ectopic human K14 [92] presented an increased susceptibility to hepatotoxic agent. In an effort to understand the role of site specific function, mice carrying mutated keratin were generated. Mutation on IFs highly conserved site (human K18 Arg89→Cys) [93-95] or phosphorylation sites (K18 Ser52→Ala, K8 Ser73→Ala) [60, 96] all presented increased sensitivity of hepatocytes to mechanical and toxic stress. Increased sensitivity to hepatotoxic agent was also observed in K8 [76, 78, 97] and K18 deficient mice [98]. Importantly, glyscosylation which is another post-translational modification that affect keratin 8 and 18 protect also epithelial tissue from injuries [99]. Analysis of humans for the presence of mutations on K8/K18 has shown that K8 and K18 may present mutations. However, keratin mutations in humans are not found at the beginning or end of the rod domain where epidermal keratin mutations concentrate. The mutated keratins found in humans are not necessarily deleterious for the liver but are overrepresented in human suffering from cryptogenic and non-cryptogenic forms of human liver

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disease [23, 100-103]. These findings are in agreement with the results deducted from studying K8/18 mice livers and indicate that K8/18 play a significant role in maintaining hepatocytes integrity upon stress in humans. Since mutation at the beginning and end of the rod domain which are hot spot mutation sites in epidermal keratins that such mutations must be lethal. Moreover, absence of K8 or 18 is most likely lethal too. Mutations that would disrupt formation of filamentous K8/18 filaments would also be lethal or cause serious liver dysfunction [104]. These results all converge to the general interpretation that as observed in mice K8/18 are proteins that play essentials survival functions in human hepatocytes.

3. K8/18 IN CELLULAR SIGNALLING

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Multiple signalling pathways regulate cell homeostasis, survival and death. During the last decade, several studies reveal that the protective role of K8/18 against stress and injuries may be related to their numerous interactions with cell signalling mediators. The next section summarizes the involvement of K8/18 in major cell signalling events accountable for cell fate.

3.1. K8/18 in Cell Division and Cell Death Homeostasis Cell division and cell death are fundamental processes that together maintain tissue/organ homeostasis in both physiological and pathological conditions. Disturbances in this equilibrium underlie uncontrolled proliferation or death of cell population. Multiple signalling pathways have been identified to initiate and regulate these processes and during the last years, increasing evidences suggest a role for keratins IFs in the regulation of cell cycle and apoptosis. Cell cycle is an ordered sequence of events largely controlled by cyclindependent kinases that leads to cell duplication and division. Thus, proper progression through the cell cycle is governed by activation of the appropriate cyclin-dependent kinase at the right time. In this regard, K8 has been shown to moderate cell cycle progression since K8-null hepatocytes enter more efficiently into S phase [105]. K8 loss promotes EGF- and insulin-stimulated cycle progression through a specific increase of cyclin A level [105]. Moreover, K18 can associate with and sequester the ERα-target/coactivator

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gene LRP16, thus attenuating the oestrogen-stimulated cell cycle progression of MCF-7 breast cancer cells [106]. On the opposite, K18 promotes cell cycle by interacting with 14-3-3 family proteins in a phosphorylation-dependent manner [107, 108]. In resting cells, 14-3-3 sequesters the cell cycle phosphatase Cdc25 in the cytoplasm and attenuates its nuclear import [109]. During hepatocytes mitosis, phosphorylation of K18 on Ser33 increases significantly with consequent sequestration of cytoplasmic and nuclear 14-3-3 by keratins [110]. These results suggest that keratins may sequester 14-3-3 during cell cycle progression and disrupt 14-3-3-Cdc25 complexes, thus promoting the cell cycle-dependent Cdc25 nuclear translocation and its function [109]. Thereby, these studies revealed that K8/18 could modulate different steps of cell cycle mainly by sequestering key regulators. Cells undergo apoptosis through two major pathways, namely the extrinsic pathway (death receptor pathway) or the intrinsic pathway (the mitochondrial pathway). The extrinsic pathway is activated by the tumor necrosis factor (TNF) family and their receptors (TNFR). For example, FasL binds to FasR, TNF binds to TNFRI and TRAIL binds to DR4 and DR5 [111]. The intrinsic pathway is mediated by diverse apoptotic stimuli (DNA damage, high level of ROS, UV or ionizing radiation, growth factor withdraw and anoikis), which converge at the mitochondria where the release of cytochrome c initiates the apoptotic signalling [112]. Both pathways involve the activation of a cascade of “caspase” proteases that cleave regulatory and structural molecules leading to the death of the cell. At an early stage of apoptosis, preceding caspase activation and nuclear changes, the death effector domain containing DNA binding protein (DEDD) directs procaspase 3 to keratin filaments in epithelial cells under apoptosis induced by either the death-receptor or mitochondrial dependent pathway [113]. Furthermore, the active caspase 9 is similarly concentrated on keratin fibrils [114]. These results support a model in which keratin K8/18 filaments provide a scaffold for accumulation and autoactivation of procaspase 9, which in turn cleaves procaspase 3 that is within close proximity. The active caspase 3 can in turn activate more procaspase 9, thereby facilitating a caspase amplification loop [114]. In line with this model, knockdown of DEDD or expression of its keratin-targeting-defective mutant inhibits activation of caspase 3 [113]. Furthermore, the ability of DEDD to associate with K8/18 filamentous network strongly correlates with an increased sensitivity to apoptosis [115]. Thus, the keratin network regulates apoptotic machinery and confers a caspase- activation/amplification platform. Amongst the primary caspase-targets in epithelial cells are found keratins type I family (Figure 6) [116].

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Simple Epithelial Keratins K8 and K18

Asp238

Asp397 48 kD

st

1 cleavage fragments

45 kD; 3kD 26 kD; 22 kD

both cleavage fragments

AGGREGATES

26 kD; 19 kD; 3 kD

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Figure 6. Shematic representation of K18 caspase-cleavage sites during apoptosis.

K18 is first cleaved at Asp397 site in the COOH terminal (tail) domain by caspase 3, 7 and 9 [116-118]. This initial K18 cleavage does not affect filamentous organization to a great extent, but liberates a carboxy-terminal fragment that is rapidly translocated to the nucleus to interfere with topoisomerase I-mediated chromatin condensation during apoptosis. This leads to preservation of transcriptional activity required during early stages of programmed cell death [119]. Moreover, cleavage at Asp397 generates a neoepitope reactive with the commercially available antibody M30 that can be used to monitor caspase 3 activation [118]. A second caspase cleavage site was identified in the conserved L1-2 linker region at Asp238 site [116, 120]. This event is responsible for the collapse of the keratin cytoskeleton into punctuate aggregates and coincides with loss of intracellular contacts and detachment of cells from their substrates [117]. These K8/18 cytoplasmic inclusions, which exhibited the same immunohistochemical and morphological features as MDBs, also contain several pro-apoptotic factors, such as caspase 3 and 9, DEDD, tumor necrosis factor receptor type-1-associated death domain protein (TRADD) and heat-shock proteins [114, 115, 121]. Many of their constituents, including active caspases, remain sequestered within these inclusions, even after detergent treatment and isolation [114]. As the apoptosis program proceeds, it is currently unclear whether active caspases are released from these inclusions to accomplish the cleavage of other cellular substrates. However, a recent study shows that the smallest subunit of eukaryotic translation initiation factor 3 complexes, eIF3k, also localizes to keratins network where it promotes the release of active caspase 3 from the K8/18 inclusions during apoptosis [122]. A sequential process regulated by K8/18

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emerges from all these observations. At early stage of apoptosis, effective recruitment of procaspase 3 and 9 at the keratin‟s filamentous scaffold through DEDD leads to an increased local availability of caspases, which renders cells more apoptosis-prone [115]. Later in the process, the collapse of keratin‟s filaments induced by the caspase cleavage of K18 sequesters all these proapoptotic proteins that could injure neighboring cells [114]. Thus, keratin filaments may both control key stages of the caspase cascade to facilitate an ordered cell death process and minimize the effects of this process on adjacent cells. The fact that keratins of type I are a target of caspases suggest an antiapoptotic role for these IFs proteins. Indeed, several studies demonstrate that K8/18 provide resistance to extrinsic and intrinsic pathways. Normal and malignant epithelial cells deficient in K8 and/or K18 are more sensitive to FasL and TNF-induced death [123, 124]. First, K8 moderates FasR targeting to the hepatocytes membrane in a microtubule-dependent manner [124]. Moreover, K8 and K18 both bind the cytoplasmic domain of TNFR2 [123] and K18 sequesters the adaptor protein TRADD [125]. These interaction moderate Jun NH(2)-terminal kinase (JNK) intracellular signalling and NFkappaB activation in simple epithelial cells [123]. In vivo study also shows that disrupted K8/18 filament network by mutant overexpression predispose mice hepatocytes to Fas- but not TNF-mediated apoptotic injury [60, 94]. K8 mutation on a phosphorylation site (Ser73) increases nonkeratin pro-apoptotic substrate phosphorylation by stress-activated kinases, thus predisposes to Fasinduced liver injury in transgenic mice [60]. The authors suggest that K8 can protect tissue from injury by serving as a phosphate sponge for stress-activated kinases [60]. All these studies provide evidence that moderation of death receptor downstream signalling may be a fundamental function of K8 and K18, particularly in liver regeneration. K8/18 also provide resistance to intrinsic apoptosis pathway by a direct or indirect effect on mitochondria. Knockdown of K18 as well as its disassociation from Pirh2, a novel interacting protein which maintains keratin filaments organization, affected mitochondrial localization through a microtubule-mediated mechanism and led to nuclear clustering of mitochondria aggregates, thus to enhance UV-induced apoptosis in human lung cancer cell lines [126]. Absence of K8 also induced an irregular mitochondrial distribution pattern in hepatocytes from K8-null mice [127], that correlates with a significant reduction of mitochondrial size. These morphological modifications result in a functional decrease in cytochrome c content, an increased mitochondrial permeability and a higher sensitivity to

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oxidative injury [127]. K18 point mutation (Arg89→Cys) in transgenic mice modulates several oxidative stress-related genes and protein oxidation byproducts to prime hepatocytes to oxidative injury [95]. Conversely, K8knockout mouse hepatocytes and K8-knockdown H4-II-E-C3 rat hepatoma cells are more resistant to ROS-mediated cell death by altering PKCδ activity at mitochondria [128]. The authors propose that keratin loss in hepatic cells displaces PKCδ away from its death-related mitochondrial target, whereas mutated K8/K18 promote PKCδ mediation of mitochondria-dependent cell death in response to excess ROS [128]. Nonetheless, these findings establish a link between K8/18 filament network and mitochondrial functional integrity in intrinsic apoptotic signalling.

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3.2. K8/18 in Cell Motility First evidences linking keratin IFs expression with cell motility, invasion and metastasis come from observations that metastatic cancer cells presenting a dedifferentiated phenotype co-expressed keratins and vimentin, a mesenchymal-specific IF, whereas the poorly metastatic counterpart expressed only one type of the IFs proteins [129-131]. At this time, several studies indicate a role for keratin IFs in modulating cell migration. Mouse L fibroblasts and melanoma cells overexpressing human K8/18 filamentous network showed higher invasiveness through matrigel matrix than cells transfected with K8 or K18 alone [132, 133]. This migratory activity was directly correlated with the spreading ability of the cells on the same substrate, in which the K8/18 transfectants maintain a round morphology for a longer duration [132]. This study suggested that keratins may play a role in migration, by influencing cell shape. On the contrary, overexpression of K18 in MDAMB-231 metastatic breast cancer cell line causes a dramatic reduction of the invasive and metastatic potential [134]. These differential effects of ectopic K8/18 expression raise the hypothesis that the role of IFs in these processes could depending on the epithelium type and differentiation state. It is well known now that cell motility, invasion and metastasis are closely related to the cell differentiation state in regard to epithelial-mesenchymal phenotype transition (EMT), that constitute a hallmark of malignant transformation [135]. In this context, keratins should not be considered merely as markers but also as regulators of differentiation in that inappropriate IFs expression strongly correlates with altered differentiation, invasiveness and metastatic potential.

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However, the molecular mechanisms regulated by K8/18 in cell motility and invasion remains elusive. Given the extensive K8/18 filamentous network into the cell, IFs can act as signal transducers from the extracellular matrix to the nucleus. For instance, keratin IFs are connected to cell-cell and cell-matrix junctions by linking to desmosomes and hemidesmosomes respectively [136, 137]. By this interaction, K8/18 maintain epithelial cell junction integrity, which prevents desmosomes remodelling required for cell motility. Indeed, K8 loss leads to alterations in desmosomes distribution at the surface membrane of mouse hepatocytes [138, 139], mouse embryonic epithelia [140] and human epithelial cancer cell lines MCF-7, HeLa and Panc-1 [141]. Interestingly, vimentinpositive HeLa and Panc-1 cells fail to target desmoplakin, a desmosome component, at cell borders [141]. A previous study has already shown that desmoplakin associates more strongly with keratins than vimentin network [137]. As a consequence of reduced cell-cell contacts, knockdown of K8 with siRNA results in accelerated wound closure in HeLa and Panc-1 cell lines and in appearance of cells with a mesenchymal, irregularly spread morphology [141]. Thus, K8 is required for the maintenance of epithelial integrity during migration. Moreover, K8/18 complete filamentous network seems to be essential to exert the moderating role in cell motility since perinuclear reorganization of K8/18 network by sphingosylphosphorylcholine increases cellular elasticity and augments migration through limited-sized pores [142]. We also demonstrated that perinuclear reorganization of K8/18 network occurs in HeLa and HepG2 epithelial cancer cells overexpressing constitutively active Akt isoforms [143], and that is associated with increased K8/18 protein levels [143] and invasion through matrigel (Fortier et al., unpublished results). Disruption of K8/18 filamentous network in hepatocytes by transfection of mutant K18 or by proteasome inhibition also affects localization of desmoplakin, zonula occludens-1, beta-catenin and 14-3-3-zeta, which are relocated to keratin inclusions [144]. On the other hand, K8 has been identifed as a binding protein for plasminogen and urokinase-type plasminogen activator expressed on the external surfaces of hepatocytes and breast carcinoma cells [145, 146]. The authors propose that the small fraction of total cellular K8, which is expressed on the outer cell surface, may promote cellular invasiveness by enhancing proteinase activation in the pericellular spaces [147]. Keratins are regarded as relatively stable cytoskeleton components that are important for epithelial flexibility to mechanical stress [148]. However, there is increasing evidence that they are highly dynamic.

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Indeed, keratins may also be important in migration by influencing cell shape, intercellular junctions and epithelial sheet integrity during collective cell migration. A key step during metastatic process is the invasion of cancer cells through the basal membrane and the endothelial layer, which requires both migration and cellular elasticity. Thus, keratins loss or network reorganization in perinuclear compartment could indeed facilitate cell migration and resilience.

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CONCLUSION Keratin IFs constitute a major cytoskeletal component of simple epithelial cells. Previously considered merely as a structural scaffold, keratin network is now known to notably regulate cell survival and motility. Since the hepatocytes only express K8/18, first evidence for their cell protective role came from liver diseases, which display characteristic keratin aggregates called Mallory Denk bodies. Over the years, the accurate analyses of these inclusions revealed that keratin network reorganization involves their post-translational modifications and their association with cell signalling proteins. Indeed, K8/K18 mutations or deficiencies in transgenic mice show that keratins play a pivotal role in hepatocytes resistance to mechanical and toxic stress. Today, the K8/18 function in cell survival is extended to other epithelial tissue and carcinomas. Increasing findings of keratin associated proteins define more precisely the role of K8/18 in cell homeostasis as a signalling platform.

ACKNOWLEDGEMENTS The authors are grateful to researchers in the field for their contribution to the work cited in this chapter and apologize to those who were not cited because of lack of space. We also want to thank Dr. M. Bishr Omary for providing the anti-K8 antibody, Dr. Hélène Baribault and Dr. Normand Marceau for providing FVB/n knockout mice. We are thankful to Hervy Brisson Jr. (MSc), Hélène Hovington and Dr. Mutsumi Satoh for their work on in situ hybridization and K8 knockout mice. This project was supported by a grant to Monique Cadrin from Natural Sciences and Engineering Research Council of Canada.

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[100] Strnad, P., Lienau, T. C., Tao, G. Z., Ku, N. O., Magin, T. M., and Omary, M. B. (2006) Denaturing temperature selection may underestimate keratin mutation detection by DHPLC, Hum Mutat 27, 444-452. [101] Strnad, P., Lienau, T. C., Tao, G. Z., Lazzeroni, L. C., Stickel, F., Schuppan, D., and Omary, M. B. (2006) Keratin variants associate with progression of fibrosis during chronic hepatitis C infection, Hepatology 43, 1354-1363. [102] Ku, N. O., Gish, R., Wright, T. L., and Omary, M. B. (2001) Keratin 8 mutations in patients with cryptogenic liver disease, N Engl J Med 344, 1580-1587. [103] Ku, N. O., Darling, J. M., Krams, S. M., Esquivel, C. O., Keeffe, E. B., Sibley, R. K., Lee, Y. M., Wright, T. L., and Omary, M. B. (2003) Keratin 8 and 18 mutations are risk factors for developing liver disease of multiple etiologies, Proc Natl Acad Sci U S A 100, 6063-6068. [104] Omary, M. B., Ku, N. O., Strnad, P., and Hanada, S. (2009) Toward unraveling the complexity of simple epithelial keratins in human disease, The Journal of clinical investigation 119, 1794-1805. [105] Galarneau, L., Loranger, A., Gilbert, S., and Marceau, N. (2007) Keratins modulate hepatic cell adhesion, size and G1/S transition, Exp Cell Res 313, 179-194. [106] Meng, Y., Wu, Z., Yin, X., Zhao, Y., Chen, M., Si, Y., Yang, J., Fu, X., and Han, W. (2009) Keratin 18 attenuates estrogen receptor alphamediated signaling by sequestering LRP16 in cytoplasm, BMC Cell Biol 10, 96. [107] Liao, J., and Omary, M. B. (1996) 14-3-3 proteins associate with phosphorylated simple epithelial keratins during cell cycle progression and act as a solubility cofactor, J Cell Biol 133, 345-357. [108] Ku, N. O., Liao, J., and Omary, M. B. (1998) Phosphorylation of human keratin 18 serine 33 regulates binding to 14-3-3 proteins, Embo J 17, 1892-1906. [109] Tzivion, G., Shen, Y. H., and Zhu, J. (2001) 14-3-3 proteins; bringing new definitions to scaffolding, Oncogene 20, 6331-6338. [110] Ku, N. O., Michie, S., Resurreccion, E. Z., Broome, R. L., and Omary, M. B. (2002) Keratin binding to 14-3-3 proteins modulates keratin filaments and hepatocyte mitotic progression, Proc Natl Acad Sci U S A 99, 4373-4378.

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[111] Locksley, R. M., Killeen, N., and Lenardo, M. J. (2001) The TNF and TNF receptor superfamilies: integrating mammalian biology, Cell 104, 487-501. [112] Jin, Z., and El-Deiry, W. S. (2005) Overview of cell death signaling pathways, Cancer Biol Ther 4, 139-163. [113] Lee, J. C., Schickling, O., Stegh, A. H., Oshima, R. G., Dinsdale, D., Cohen, G. M., and Peter, M. E. (2002) DEDD regulates degradation of intermediate filaments during apoptosis, J Cell Biol 158, 1051-1066. [114] Dinsdale, D., Lee, J. C., Dewson, G., Cohen, G. M., and Peter, M. E. (2004) Intermediate filaments control the intracellular distribution of caspases during apoptosis, The American journal of pathology 164, 395-407. [115] Schutte, B., Henfling, M., and Ramaekers, F. C. (2006) DEDD association with cytokeratin filaments correlates with sensitivity to apoptosis, Apoptosis 11, 1561-1572. [116] Caulin, C., Salvesen, G. S., and Oshima, R. G. (1997) Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis, J Cell Biol 138, 1379-1394. [117] Schutte, B., Henfling, M., Kolgen, W., Bouman, M., Meex, S., Leers, M. P., Nap, M., Bjorklund, V., Bjorklund, P., Bjorklund, B., Lane, E. B., Omary, M. B., Jornvall, H., and Ramaekers, F. C. (2004) Keratin 8/18 breakdown and reorganization during apoptosis, Exp Cell Res 297, 1126. [118] Leers, M. P., Kolgen, W., Bjorklund, V., Bergman, T., Tribbick, G., Persson, B., Bjorklund, P., Ramaekers, F. C., Bjorklund, B., Nap, M., Jornvall, H., and Schutte, B. (1999) Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis, J Pathol 187, 567-572. [119] Schutte, B., Henfling, M. E., Verheyen, F. K., Li, G., Tolstonog, G. V., and Ramaekers, F. C. (2009) The caspase-9 derived C-terminal fragment of cytokeratin 18 modulates topoisomerase action, Int J Oncol 35, 625630. [120] Omary, M. B., and Ku, N. O. (1997) Intermediate filament proteins of the liver: emerging disease association and functions, Hepatology 25, 1043-1048. [121] Nakamichi, I., Hatakeyama, S., and Nakayama, K. I. (2002) Formation of Mallory body-like inclusions and cell death induced by deregulated expression of keratin 18, Molecular biology of the cell 13, 3441-3451.

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[122] Lin, Y. M., Chen, Y. R., Lin, J. R., Wang, W. J., Inoko, A., Inagaki, M., Wu, Y. C., and Chen, R. H. (2008) eIF3k regulates apoptosis in epithelial cells by releasing caspase 3 from keratin-containing inclusions, J Cell Sci 121, 2382-2393. [123] Caulin, C., Ware, C. F., Magin, T. M., and Oshima, R. G. (2000) Keratin-dependent, epithelial resistance to tumor necrosis factorinduced apoptosis, J Cell Biol 149, 17-22. [124] Gilbert, S., Loranger, A., Daigle, N., and Marceau, N. (2001) Simple epithelium keratins 8 and 18 provide resistance to Fas-mediated apoptosis. The protection occurs through a receptor-targeting modulation, J Cell Biol 154, 763-773. [125] Inada, H., Izawa, I., Nishizawa, M., Fujita, E., Kiyono, T., Takahashi, T., Momoi, T., and Inagaki, M. (2001) Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD, J Cell Biol 155, 415-426. [126] Duan, S., Yao, Z., Zhu, Y., Wang, G., Hou, D., Wen, L., and Wu, M. (2009) The Pirh2-keratin 8/18 interaction modulates the cellular distribution of mitochondria and UV-induced apoptosis, Cell Death Differ 16, 826-837. [127] Tao, G. Z., Looi, K. S., Toivola, D. M., Strnad, P., Zhou, Q., Liao, J., Wei, Y., Habtezion, A., and Omary, M. B. (2009) Keratins modulate the shape and function of hepatocyte mitochondria: a mechanism for protection from apoptosis, J Cell Sci 122, 3851-3855. [128] Mathew, J., Galarneau, L., Loranger, A., Gilbert, S., and Marceau, N. (2008) Keratin-protein kinase C interaction in reactive oxygen speciesinduced hepatic cell death through mitochondrial signaling, Free Radic Biol Med 45, 413-424. [129] Hendrix, M. J., Seftor, E. A., Chu, Y. W., Seftor, R. E., Nagle, R. B., McDaniel, K. M., Leong, S. P., Yohem, K. H., Leibovitz, A. M., Meyskens, F. L., Jr., and et al. (1992) Coexpression of vimentin and keratins by human melanoma tumor cells: correlation with invasive and metastatic potential, J Natl Cancer Inst 84, 165-174. [130] Thompson, E. W., Paik, S., Brunner, N., Sommers, C. L., Zugmaier, G., Clarke, R., Shima, T. B., Torri, J., Donahue, S., Lippman, M. E., and et al. (1992) Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines, Journal of cellular physiology 150, 534-544. [131] Ramaekers, F. C., Haag, D., Kant, A., Moesker, O., Jap, P. H., and Vooijs, G. P. (1983) Coexpression of keratin- and vimentin-type

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intermediate filaments in human metastatic carcinoma cells, Proc Natl Acad Sci U S A 80, 2618-2622. [132] Chu, Y. W., Runyan, R. B., Oshima, R. G., and Hendrix, M. J. (1993) Expression of complete keratin filaments in mouse L cells augments cell migration and invasion, Proc Natl Acad Sci U S A 90, 4261-4265. [133] Chu, Y. W., Seftor, E. A., Romer, L. H., and Hendrix, M. J. (1996) Experimental coexpression of vimentin and keratin intermediate filaments in human melanoma cells augments motility, The American journal of pathology 148, 63-69. [134] Buhler, H., and Schaller, G. (2005) Transfection of keratin 18 gene in human breast cancer cells causes induction of adhesion proteins and dramatic regression of malignancy in vitro and in vivo, Mol Cancer Res 3, 365-371. [135] Hanahan, D., and Weinberg, R. A. (2011) Hallmarks of cancer: the next generation, Cell 144, 646-674. [136] Green, K. J., and Simpson, C. L. (2007) Desmosomes: new perspectives on a classic, J Invest Dermatol 127, 2499-2515. [137] Kouklis, P. D., Hutton, E., and Fuchs, E. (1994) Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins, J Cell Biol 127, 1049-1060. [138] Loranger, A., Gilbert, S., Brouard, J. S., Magin, T. M., and Marceau, N. (2006) Keratin 8 modulation of desmoplakin deposition at desmosomes in hepatocytes, Exp Cell Res 312, 4108-4119. [139] Toivola, D. M., Nieminen, M. I., Hesse, M., He, T., Baribault, H., Magin, T. M., Omary, M. B., and Eriksson, J. E. (2001) Disturbances in hepatic cell-cycle regulation in mice with assembly-deficient keratins 8/18, Hepatology 34, 1174-1183. [140] Vijayaraj, P., Kroger, C., Reuter, U., Windoffer, R., Leube, R. E., and Magin, T. M. (2009) Keratins regulate protein biosynthesis through localization of GLUT1 and -3 upstream of AMP kinase and Raptor, J Cell Biol 187, 175-184. [141] Long, H. A., Boczonadi, V., McInroy, L., Goldberg, M., and Maatta, A. (2006) Periplakin-dependent re-organisation of keratin cytoskeleton and loss of collective migration in keratin-8-downregulated epithelial sheets, J Cell Sci 119, 5147-5159. [142] Beil, M., Micoulet, A., von Wichert, G., Paschke, S., Walther, P., Omary, M. B., Van Veldhoven, P. P., Gern, U., Wolff-Hieber, E., Eggermann, J., Waltenberger, J., Adler, G., Spatz, J., and Seufferlein, T. (2003) Sphingosylphosphorylcholine regulates keratin network

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architecture and visco-elastic properties of human cancer cells, Nat Cell Biol 5, 803-811. [143] Fortier, A. M., Van Themsche, C., Asselin, E., and Cadrin, M. (2010) Akt isoforms regulate intermediate filament protein levels in epithelial carcinoma cells, FEBS Lett 584, 984-988. [144] Hanada, S., Harada, M., Kawaguchi, T., Kumemura, H., Taniguchi, E., Koga, H., Yanagimoto, C., Maeyama, M., Ueno, T., and Sata, M. (2007) Keratin inclusions alter cytosolic protein localization in hepatocytes, Hepatol Res 37, 828-835. [145] Hembrough, T. A., Vasudevan, J., Allietta, M. M., Glass, W. F., 2nd, and Gonias, S. L. (1995) A cytokeratin 8-like protein with plasminogenbinding activity is present on the external surfaces of hepatocytes, HepG2 cells and breast carcinoma cell lines, J Cell Sci 108 ( Pt 3), 1071-1082. [146] Obermajer, N., Doljak, B., and Kos, J. (2009) Cytokeratin 8 ectoplasmic domain binds urokinase-type plasminogen activator to breast tumor cells and modulates their adhesion, growth and invasiveness, Mol Cancer 8, 88. [147] Hembrough, T. A., Kralovich, K. R., Li, L., and Gonias, S. L. (1996) Cytokeratin 8 released by breast carcinoma cells in vitro binds plasminogen and tissue-type plasminogen activator and promotes plasminogen activation, Biochem J 317 ( Pt 3), 763-769. [148] Fuchs, E., and Cleveland, D. W. (1998) A structural scaffolding of intermediate filaments in health and disease, Science 279, 514-519.

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Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Keratin: Structure, Properties and Applications : Structure, Properties and Applications, edited by Renke Dullaart, and João

In: Keratin: Structure, Properties and Applications ISBN 978-1-62100-336-6 Editors: Renke Dullaart et al. pp. 36-62 ©2012 Nova Science Publishers, Inc.

Chapter 2

EXTRACTION, PROCESSING AND APPLICATIONS OF WOOL KERATIN M. Zoccola, A. Aluigi, A. Patrucco and C. Tonin

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Italian National Research Council, Institute for Macromolecular Studies, C.so G. Pella 16, Biella, Italy

ABSTRACT Keratins are proteins characterised by a high sulphur amount and by the presence of strong disulphide bonds which make keratins water insoluble and resistant to different chemical agents. Keratins with molecular weight ranging from less than 10 to 60 kDa are obtained by cleavage of the cystine bonds with reducing or oxidising agents or by sulphitolysis. Keratin oligopeptides can be obtained by cleavage of peptide bonds using strong acids or strong bases. Recently, green hydrolysis with superheated water and steam-explosion has been proposed with the aim of avoiding the use of harmful agents. Proteins extracted from wool can be processed alone, or in blend with other polymers, with crosslinking agents, or in the presence of additives (e.g. plasticisers). Keratins are biocompatible, biodegradable, hygroscopic, adsorb heavy metal ions, formaldehyde and other volatile organic compounds. So, keratin can be used in different fields in the form of films, sponges, nanofibres, microcapsules, hydrogels and powders especially for biomedical applications and filtration.

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M. Zoccola, A. Aluigi, A. Patrucco et.al. In this review details on the extraction and processing of keratins from wool are reported, with some example of utilization for different practical applications.

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1. INTRODUCTION Keratins represent a group of fibrous proteins produced in some epithelial cells of vertebrate such as reptiles, birds and mammals. These proteins are abundantly present in nature; in fact they constitute the major part of hair, wool, horns, nails, feathers and stratum corneum of the skin [Bradbury, 1973; Fraser, 1972]. Keratins are distinguished from other structural proteins such as silk fibroin, collagen and fibrin by the number of cysteine residues in the protein molecules (7-20% of the total amino acid residues). Particularly, the cysteine amino acid residues form inter- and intra-molecular disulphide bonds (cystine residues), giving rise to a compact three-dimensional structure that confers to the keratin proteins a high resistance to chemical and enzymatic attacks [Dowling, 1986]. There are two kinds of keratins: the “hard-keratins” and the “soft-keratins” according to the physical and chemical properties, particularly to the sulphur amount. The soft-keratins, with a low sulphur content (< 3% wt), are found in the stratum corneum of the skin, whereas the hard keratins are found in hair, wool, feathers, nails and horns and have a high sulphur content (> 3% wt) [Fraser, 1972]. A further classification is based on X-ray diffraction pattern obtained from different keratin proteins. The -helix appears to be the basic fibrillar element in the hard keratins from mammals (e.g. wool), while keratin from birds and reptiles shows a -conformation [MacLaren, 1981; Fraser, 1996].

2. KERATIN FROM WOOL Wool is characterized by a complex histological structure, which has origin from the follicle bulb. Wool is composed of three main morphological components, namely the cuticle, that consist of a thin layer of flat overlapping cells surrounding the cortical cells, the cortex, which is made of spindle shaped cells arranged in the direction of the fiber axis, and the cell membrane

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Extraction, Processing and Applications of Wool Keratin

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complex which performs the function of cementing together cortical and cuticular cells [Aluigi, 2008]. Raw wool, when first shorn from the sheep, often contains less than 50% of clean fibre due to contamination by wool wax, suint (i.e. the secretion of subiferous glands), sand, dirt and vegetable matter. After cleaning, wool is essentially pure protein and on the hydrolysis yields the eighteen amino acids commonly present in the hydrolysates of most proteins. In wool samples, significant variation in amino acid composition can exist, both between fibres from different individual and also along the length of a single fibre from the same animal [Marshall, 1988]. These differences are influenced by genetic origin [Le Roux, 1957; Gillespie, 1971] physiological state of sheep [Marshall, 1988] and nutrition [Ross, 1961; Gillespie, 1969]. Wool fibres aren‟t chemically homogeneous; they consist of a complex mixture of widely different polypeptides. It has been estimated that wool contains about 170 different types of protein molecules, termed keratinous and non-keratinous proteins according to their cystine content. The highly crosslinked keratinous components of the fibre are resistant to chemical attacks and constitute the main part of the total fibre mass [Zahn, 1980].

Figure 1. Picture of wool fibre (SEM, 2000x).

The wool cortex constitutes the major part of the weight of the fibre and it is the stress-bearing element. It is composed of spindle shaped cells of intermediate filament proteins with a predominant α-helical structure

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M. Zoccola, A. Aluigi, A. Patrucco et.al.

embedded in a matrix rich in sulphur and glycine/tyrosine. Finally wool cuticle, which constitutes the outermost surface of the wool fibres, has high cystine content and contains cuticle-specific proteins, arranged in β-sheets and amorphous structure.

3. EXTRACTION AND HYDROLYSIS OF WOOL KERATIN 3.1. Extraction via Reduction, Oxidation and Sulphitolysis

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Keratin extraction may take place after cleavage of disulphide bonds and this can be achieved by oxidation, reduction or sulphitolysis. Indeed, almost all the accumulated data on extracted wool refer to the reduced keratins [Crewther, 1965; MacLaren, 1987].

3.1.1. Reductive Extraction Reductive extraction with an aqueous mixture of urea and thiols followed by reaction with methyl iodide or -iodoacetic acid to produce the corresponding S-alkylated keratins has been proposed by many authors [O‟Donnel, 1964; Miyamoto, 1987]. Wool is washed with water, dried and defatted by Soxhlet extraction using a 1:1 v/v mixture of hexane and dichloromethane. The cleaned wool (10 g) is mixed with urea (7M), SDS (sodium-dodecyl-sulphate) surfactant (0.11M) and 2-mercaptoethanol (1.1M) and then shaken at 50 °C for 12 h. The resulting mixture is filtered through a stainless-steel mesh and the filtrate is dialyzed against distilled water containing 0.08% of 2-mercaptoethanol to afford clear solution of the reduced keratin. The extraction yield is 48% and the keratin aqueous solution is stable at room temperature for at least 1 year without precipitation and rotting. Urea and 2-mercaptoethanol in the method previously described cleave hydrogen bonds and covalent S-S bonds between the protein chains, respectively. The reaction mechanism consists of two reactions of nucleophilic displacement:

WSSW + RS WSSR + RS

WSSR + WSRSSR + WS-

W=Wool

The thiol groups of wool (cysteine residues) are rapidly oxidized by air.

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Besides thiols, there is another efficient class of reducing agents: the phosphines. The wool reductive reaction with phosphine is irreversible [Jenkins, 1963]:

WSSW

R 3P H 2O

2WSH R 3 PO

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3.1.2. Oxidative Extraction An alternative method for preparing soluble proteins from wool involves treatment either with peracetic and performic acid, which oxidize cystine to cysteic acid residues [Alexander, 1950]. Performic acid is preferable because it oxidizes quantitatively the cystine residues without breaking peptidic bonds. Successively, the oxidized wool can be dissolved in alkali (usually ammonia). Ammonia dissolves about 85-90% of the wool. Part of this soluble protein precipitates by acidification (keratoses). The keratin extraction from wool trough oxidative method produces three groups of proteins: and keratoses that derive from the microfibrils, cellular membrane complex and matrix respectively. The use of an excess of oxidant produces cysteic acid, but when the oxidant is not in excess, several intermediate products are formed [Crewther, 1965; MacLaren, 1987]: Cys-S-S-Cys disulphide

Cys-SO-S-Cys monoxide

Cys-SO2-S-Cys dioxide

Cys-SO-3 cysteic acid

3.1.3. Sulphitolysis and Oxidative Sulphitolisis Sulphitolysis describes the cleavage of a disulphide by sulphite to give a thiol and a S-sulphonate anion (or Bunte salt):

RSSR + SO32-

RS- + RSSO3-

The sulphitolysis of cystine has been extensively studied by Cecil and his co-workers. Sulphite ions react with cystine more rapidly than bisulphite ions [Cecil, 1959; Cecil, 1963]. Both ions exist together in equilibrium in aqueous solution and the sulphite ion concentration increases with increasing the pH. Consequently there is a marked increase in the rate of sulphitolysis of cystine. However, the rate decreases above pH 9 due to repulsion of sulphite ions by the carboxylate

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anions of cystine. The net charge influences the rate and extent of this reaction, thus there is an optimum pH at which both the reaction rate and equilibrium constant are maximal. The main species present in the pH range 3-10 are the bisulphite and the sulphite ions. However spectral measurements show that disulphite (metabisulphite) solutions (< 0.06M) at pH < 7 also contain significant amounts of the disulphite ions (S2O52-) in equilibrium with bisulphite and sulphite. Johnson and Thompson have invoked a third reaction to account for the sulphitolysis of wool at low pH values. Under these conditions sulphitolysis involves reaction with disulphide ion that is either S2O52- and HS2O5- or both [Johnson, 1975]:

WSSW + HS2O5-

WSS2O5- + WSH

In table 1 the keratin extraction yield obtained with the sulphitolysis are compared with those obtained by reduction.

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Table 1. Reduction procedures for protein extraction from wool Reducing Agents

Urea

pH

Treatment

Yield %

Mercaptoethanol (1.1M) [1]

7.4M

7

shaking at 50°C, 6 h

48%

Thioglycolic Acid (0.2M) [13]

8M

11

shaking at 50°C, 3 h

70%

Dithiothreitol (0.2M) [14]

8M

11

shaking at 20°C, 16 h

60%

m-Bisulphite (0.5M)

8M

6.5

shaking at 65°C, 5h

33-38%

Oxidative sulphitolysis converts a disulphide to two S-sulphonate anions. The equilibrium reaction of sulphite with a disulphide is displaced by converting the resultant thiol anion to disulphide with suitable mild oxidants:

SO32- + RSSR

RS- + RSSO3-

[o] This oxidative sulphitolysis leads eventually to the complete conversion of each cystine residue to two S-sulphocysteine residues:

RSSR + 2SO32- + H2O

[o]

2RSSO3- + 2OH-

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Oxidants that also react with sulphite ions are obviously unsuitable. Any thiol group present also gives the same product [MacLaren, 1981]:

SO32- + RSH + [o]

RSSO3- + OH-

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3.2. Keratin Hydrolysis While using the previous protein extraction methods the molecular weights of extracted proteins remain unchanged, wool protein hydrolysis leads to the breaking of peptide bonds with the formation of low molecular weight peptides. The hydrolysis of keratin represents one of the most promising ways to extend practical application of keratin materials. Hydrolysis through scission of the protein chains yields oligopeptides and, finally, amino acids: both can be eventually refined and used as building blocks for the synthesis of a range of novel polymers via polycondensation. Hydrolysis can be carried out in different process conditions with different chemical agents. Acid hydrolysis was used in the past to study the biochemical composition of wool fibres [MacLaren, 1981]. The complete hydrolysis of wool fibre for the quantitative amino acid analytical determination is usually carried out in strong acidic conditions (HCL 6N at 110°C for 24 h). Under these conditions most of the amino acids are released in a quantitative way, although tryptophan is completely destroyed and a partial destruction of serine, threonine and tyrosine and a quantitative conversion of asparagine to aspartic acid and glutamine to glutamic acid also occur. Under milder acidic conditions partial protein hydrolysis occurs, but, conversely to other proteins, studies on the peptides obtained by partial acid hydrolysis of wool have yielded relatively little information on sequence of the wool proteins due to the heterogeneous nature of the proteins in wool and their relatively high molecular mass. Alkali treatment attacks wool peptide, disulphide and side-chain amide bonds. Under mild alkali conditions wool keratin is modified through the degradation of cystine, leading to the formation of strong linkages, principally found in lanthionine and lysinoalanine, with a decrease of solubility of wool in alkali and urea-bisulphite solutions. Boiling wool in strong alkali media

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represents the most common way to carry out a strong hydrolysis of keratin by total cleavage of the peptide bonds. Partial hydrolysis under controlled alkaline process conditions, with the aim of tailoring the extent of the peptide length, seems to focus the attention of many researchers, [Cardamone, 2007; Kawahara, 2004], although the processes can give rise to a considerable amount of unhydrolysed solid residue. Recently green processes, solvent-free, have been proposed for the hydrolysis of keratin. These treatments include hydrolysis in superheated water and enzymes. The water in the superheated state, i.e. above the ambient boiling temperature and below the supercritical temperature, is highly effective in the hydrolysis of keratin because of the increased mobility of molecules. Tests on the hydrolysis of keratin were carried out on feathers keratin by Yin et al. [Yin, 2007]. They hydrolysed almost completely feather keratin in superheated water using a temperature of 220°C for 2 h and demonstrated that oligopeptides obtained from the hydrolysis are able to self-assemble into hierarchical crystalline structures. Similarly, keratin from wool was hydrolysed by steam-explosion, i.e. a short time steam treatment at high temperature (220°C for 10 min) followed by an explosive decompression, or in microwave-assisted superheated water [Tonin, 2006; Zoccola, 2011]. Proteins extracted from keratin with superheated water show low molecular weights and low amount of cystine, so they are difficult to process when suitable mechanical properties are required in the final product (i.e. films, sponges, fibres, nanofibres). However in this process there are many advantages, for example the use of a green solvent (water) in the hydrolysis, the speed of operations where time consuming steps like dialysis are avoided, and the purification via crystal formation of proteins which permit its further use for example in the synthesis of novel man-made proteins or polyamides. These advantages make the hydrolysis of keratin in superheated water an interesting alternative process, suitable for new investigations and applications.

4. CHARACTERISATION AND PROPERTIES OF KERATIN FROM WOOL Keratin extracted from wool by reduction [Yamauki, 2002] or sulphitolysis [Aluigi, 2007] shows an amino acid composition very similar to the original wool, namely consistent in a wide variety of amino acids with a

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prevalence of the polar ones. Noticeably, it is the presence of cysteine residues that can crosslink forming disulphur bonds. By the oxidative extraction of wool keratin, keratoses are formed which possess residues of cysteic acid in place of cystine residues. In wool peptides obtained by the alkaline hydrolysis, disulphide and side-chain amide bonds are involved with the partial destruction of cystine, lysine, arginine, treonine and serine and the formation of strong linkages confirmed by the detection of lanthionine, lysinoalanine and ornithine. Whereas, in proteins extracted in superheated water most of the cystine is destroyed by the high temperature of the process and a prevalence of polar amino acids is detected in proteins, oligopeptides or free amino acids in solution [Tonin, 2006]. In general, the molecular weights of proteins extracted from wool keratin are lower when compared with the molecular weights of other fibrous proteins such as silk fibroin or collagen and they are distributed in a quite wide range, each molecular weight corresponding to proteins present in a particular histological part of the fibre. In the electrophoretical separation patterns of proteins extracted from wool by sulphitolysis, are clearly visible (figure 2, line 2) two high molecular mass bands (60-40 kDa) corresponding to low sulphur proteins from the intermediate filaments of cortical cells, different bands in the range 28-11 kDa corresponding to high sulphur proteins in the cuticle, and different bands at molecular weights lower than 10 kDa corresponding to the high glycine, tyrosine proteins of the matrix between cuticular and cortical cells. In proteins extracted from wool by reduction, sulphytolysis or oxidation, because of the breaking of cystine bonds alone, the molecular weights of extracted proteins are unchanged when compared with the weights in original wool, while, when the hydrolysis is carried out using alkaline agents, the molecular weights of the extracted proteins depends on the treatment conditions. In this last case, the molecular weights of extracted proteins range from higher than in original wool (figure 2) due to the formation of non reducible bonds in lanthionine and lysinoalanine, after mild alkali treatments [Patrucco, 2011] to lower than in original wool, after a strong keratin hydrolysis, with the cleavage of peptide bonds [Cardamone, 2008]. Finally, when the proteins are extracted using superheated water, low molecular weight proteins are obtained from wool hydrolysis [Tonin, 2006] (figure 2). Keratin extracted from wool shows many interesting and peculiar properties, often depending on the extraction method used, that makes keratin an interesting material mainly in the biomedical and filtering sectors.

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Figure 2. SDS-Page patterns on 4-12% polyacrilamide gel, from the left: 1) molecular weight marker, 2) wool proteins extracted with DTT-Urea buffer, 3) keratin hydrolysed by superheated water, 4) keratin hydrolysed in mild alkali.

As a biomaterial, keratin is biocompatible, biodegradable and able to support cell attachment and spreading. Keratin proteins extracted by reduction have an intrinsic ability to self-assemble and polymerise into porous, fibrous scaffolds. As concerning the biodegradability, reduced keratins, after the recrosslinking through oxidative coupling of cysteine groups, are quite stable to acidic pH, and they can persist in vivo for weeks or months, unlike keratins extracted by oxidation, which are non-disulfide crosslinkable and degrade relatively quickly in vivo [Hill, 2010]. In addition, keratins contain several peptide binding motifs, such as leucine-aspartic acid-valine (LDV) and glutamic acid-aspartic acid-serine (EDS), that support attachment of a wide variety of cell types, similarly to other proteins of the extracellular matrix, including fibronectin and collagen [Tachibana, 2002]. Like other intermediate filaments, keratins are believed to participate in some regulatory functions that mediate cellular behaviour.

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Keratin extracted from wool is rich in amino acids having polar and ionisable side chains able to bind charged species such as heavy metal ions (copper, chromium, lead, mercury, etc.). In particular, having a pKa value of about 4.5, free carboxyl groups of aspartyl and glutamyl residues are considered the most likely binding sites over a wide pH range [MacLaren, 1981]. Also formaldehyde and other volatile organic compounds are irreversibly bound by wool keratin. The active filtration capacity of wool keratin can be enhanced by the production of keratin-based nanofilaments to form mats with high surface area to volume ratio. In these cases, to improve the spinnability and the mechanical properties of obtained filaments, keratin is blended with higher molecular weight fibrous polymers [Tonin, 2010].

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5. PROCESSING OF WOOL KERATIN Proteins extracted from wool can be processed alone or blended with other polymers, with crosslinking agents, or in the presence of additives (e.g. plasticisers). Much work has been done to fabricate and characterise new keratin-based products such as films, sponges, capsules, gels and fibres. In addition, many researchers have discovered methods for modulating the physical and mechanical properties of keratins in order to create materials that have appropriate characteristics for their application of interest.

5.1. Films and Coatings Films are usually formed by evaporation of the solvent, usually water, from protein solutions. Bio-composite films made of keratin and cortical cells were produced by Patrucco et al. by fibrillation of wool fibres after ultrasonic disruption of fibres treated before with mild alkali (figure 3) [Patrucco, 2011]. A lot of work has been done on the structural characterisation of keratin and keratin-blend cast films in order to investigate and modulate their chemical and mechanical behaviour. Structural characteristics of keratin cast films regenerated from water and from formic acid were compared by Aluigi et al. [Aluigi, 2007]. Keratin extracted by sulphytolysis and regenerated in water was re-dissolved in formic acid and cast for films fabrication. It was shown that formic acid promotes the formation of β-sheet structure in cast films and destabilises the α-helix conformation found in cast films regenerated from water.

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Figure 3. Picture of bio-composite film from wool fibrillation.

Moreover, formic acid because of its strong solvatation properties can be used as a co-solvent to prepare keratin-based polymer materials, such as keratin-fibroin and keratin-polyamide blends; the second polymer is used to improve the mechanical properties. In the past years it has been demonstrated that keratin is a good substrate for cell culture [Yamauchi, 1998]. The attachment and growth of fibroblast cells were studied on keratins extracted from wool in the absence and presence of sodium dodecyl sulphate and cast to form films. The protein extracted in the presence of sodium dodecyl sulphate showed some toxic effect on the cell growth, but upon washing with a phosphate buffer, the protein behaved better. A comparative culture assay on keratin, type I collagen and glass revealed that keratins were more adhesive to the cells and more supportive for cell proliferation than the other substrates. Keratin films obtained after reductive sulphytolisis and regenerated from water are too fragile for practical use, so different approaches have been tried to enhance their mechanical properties. Aqueous solutions of keratin were mixed with glycerol, then cast and dried [Yamauki, 2002]. Tensile properties of glycerol-plasticized cast films vary according to the film thickness and ambient humidity, but plasticized films aren‟t as brittle as those made of keratin alone and they were degraded in vitro by trypsin and in vivo by subcutaneous embedding in mice [Yamauchi, 1996]. A different approach to improve mechanical properties of keratin was tried by mixing reduced keratin solutions before casting with diepoxy crosslinkers (ethylene glycol diglycidyl ether and glycerol diglycidyl ether) [Tanabe, 2004]. Chemically crosslinked keratin films showed good waterproof

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characteristics, didn‟t swell under acidic and neutral aqueous conditions and maintained their mechanical properties upon re-drying after swelling under basic conditions. Moreover, it was shown that the attachment of fibroblast cells on a keratin crosslinked coated surface is delayed but, once attached, cells proliferated well suggesting that the crosslinked keratin was not toxic. For the same purpose keratin was blended with chitosan using acetic acid as co-solvent [Tanabe, 2002]. The addition of chitosan in keratin gave strong and flexible films, improved waterproof characteristics such as swelling behaviour, and mechanical properties after swelling were much ameliorated in the composite films compared with keratin and chitosan alone films. Also silk fibroin is a polymer widely used for biomedical application. Vasconcelos et al. [Vasconcelos, 2008] studied the chemical structure and the degradation behaviour (by in vitro enzymatic incubation with trypsin) of fibroin-keratin films regenerated from aqueous and formic acid solutions. They stated that when fibroin and keratin are blended, they do not follow additive rules but are able to establish inter-molecular interactions. The degradation of this blend is a function of the amount of keratin in the blend and of the co-solvent used and encourages new studies for the proposed application of keratin-fibroin blends in the biomedical field whether in scaffolds for tissue engineering or as controlled release drug delivery vehicles. Always with the aim to improve the mechanical behaviour of keratin films, a different fabrication method was tested. Katoh et al. [Katoh, 2004] investigated the compression moulding process as an alternative method to fabricate keratin films. Keratin powder was obtained by sulphitolysis of wool and was spray-dried and then subjected to compression moulding. A water addition and a proper moulding were necessary to obtain homogeneous and transparent plastic-like films. Such films have been demonstrated to scarcely swell in acidic and neutral aqueous solutions, probably because of the abundance of disulfide crosslinking, and to support fibroblast cells attachment and proliferation. A similar process for the production of transparent keratin film was developed by Reichl et al. [Reichl, 2011]. The multi-step procedure includes keratin extraction from wool or human hair using a reducing agent, dialysis against distilled water and against NaOH solution, mixing of the two dialysates, addition of a softening agent (glycerol), drying and subsequent curing procedure with dry heat. Different steps (percentage of alkaline dialysate and plasticiser, curing temperature) were optimised in order to obtain film characteristics (swelling behaviour, biomechanical strength and light transmission) suitable for applications in ocular surface reconstruction as an alternative to human amniotic membrane. Moreover attachment and

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proliferation of corneal epithelial cells on keratin films has been shown to be comparable to those observed on amniotic membranes.

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5.2. Keratin Sponges The properties of extracted keratin to self-assemble and polymerise into complex three dimensional structures has led to their development as scaffold for tissue engineering. Natural or synthetic polymer scaffolds are used for supporting cells for repairing and for regenerating organs and tissues. Polymers scaffolds should be completely biodegradable and should be eliminated from the body when their functions are finished, they should be able to act as carriers of bioactive substance that may be released in a controlled way to influence the behaviour of in-growing cells. Moreover scaffolds for cell cultivation must have a stable structure in order to not lose their structure in an aqueous environment and to sustain treatments and manipulations such as washing for the removal of chemicals used for their production. Fabrication of keratin sponges for cell cultivation has been first reported by Tachibana [Tachibana, 2002]. Wool keratin sponge scaffolds were fabricated by lyophilisation after controlled freezing of wool proteins extracted by sulphytolysis and dialysed against distilled water. A freezing temperature of -20 °C for 3 days was necessary for the production of stable sponges with a homogeneous structure and with pore size of about 100 μm. Then the procedure for the sponges fabrication established a treatment with iodoacetic acid to protect SH groups of keratin, an accurate washing with phosphate buffer to remove any remaining iodoacetic acid, a sterilisation with an aqueous ethanol solution and finally a washing with the cultivation medium. Keratin sponges fabricated in this way have been demonstrated to be good scaffolds for mouse fibroblast cells adhesion, spreading, and grow also for long term aims, being keratin digested by proteases more slowly in comparisons with other structural proteins (e.g. collagen). An alternative method to fabricate keratin sponges was developed by Katoch and it was based on a compression-moulding/particulate-leaching process [Katoh, 2004]. The keratin solution obtained by sulphytolysis, after dialysis and concentration, was spray dried to obtain keratin powder which was mixed with urea and then compression moulded with sieved NaCl particles. Salt and urea were removed by solubilisation in water and the porosity was obtained by leaking-out salt. Sponges obtained were water insoluble and strong enough to be handled in an aqueous environment. The

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pore size ranges from less than 100 to 300-400 μm and the porosity was well controlled by the size and the amount of NaCl particulate. The pore size is in a range suitable to permit the in-growth of cells, the vascularisation and the regeneration of tissues. Although flexible and strong enough to be handled in a wet state, compression-molded keratin sponges are hard and brittle in the dry state, so the particulate-leaching and the freeze-drying methods were combined utilizing calcium alginate beads as a porogen [Hamasaki, 2008]. Reduced keratin aqueous solution was mixed with dried calcium alginate beads and was lyophilised to produce keratin-calcium alginate complex, which was subsequently treated with ETDA solution to leach out calcium alginate beads and to produce a keratin porous structure. Some experiments were also carried out in order to immobilise bioactive substances on keratin sponges. During the fabrication of keratin sponges, some cysteine residues in keratin contribute to the mechanical characteristics of the sponge via oxidative disulphide formation, whereas other residues may remain in a free form and can be utilised as reaction site for molecules immobilisation. Lysozime, a model of a biologically active substance, was immobilized on a keratin sponge via disulphite and thioether bonds and the stability of lysozime on the sponge was evaluated [Kurimoto, 2003]. It was shown that disulphidelinked lysozyme was gradually released over a 21 day periods, whereas lysozyme linked via thioether bonds were stably maintained for up to two months. So new challenges are open for the applications of keratin sponges in biomedical field, not only as cell scaffolds, but also as a support of bioactive substances or a controlled release carrier for drugs. The hybrid of the keratin sponges with calcium phosphate materials were also fabricated to obtain in keratin sponges some additional functions in the differentiation patterns of osteoblast cells. Chemically modified keratin sponges have been hybridised with calcium phosphate [Tachibana, 2005]. Two kinds of calcium phosphate composite sponges were fabricated by either chemically binding calcium and phosphate ions or trapping hydroxyapatite particles inside the keratin carboxy-sponges. Both hybrid sponges are suitable for the cultivation of osteoblasts and for their differentiation. In a later work [Takibana, 2006], keratin carboxyl-sponges were shown to bind significant amounts of bone morphogenetic protein-2, which regulates the differentiation of various cells involved in bone formation during fracture repair. Osteoblast cells grow and differentiate inside the sponges and don‟t outside, opening perspective for in vivo application because it is expected that these scaffolds

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will promote internal osteogenesis without inducing external heterotopic ossification.

5.3. Nanofibres

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Electrospinning is the simplest way of producing fibres with a nanoscale diameter. In this process, a polymer solution in a spinneret is subjected to an electric field generated by a high voltage power supply. A polymer jet is ejected from the spinneret and when the electric field overcomes the surface tension, travels toward a grounded collector and is stretched and deposited as a non-woven mats on the collector. By adjusting solution properties and operating parameters, fibres with very small diameters and with high surface area-to-volume ratio forming high porosity non-woven mats can be obtained (figure 4). Electrospinning is a promising technique for the fabrication of tissueengineered scaffolds [Huang, 2003], and high efficiency filters [Tsai, 2002; Qin, 2006; Li, 2006; Barhate, 2007]. An important patent on electrospinning was issued in 1934 [Formhals, 1934], but only in recent years electrospinning has been widely studied and applied for many natural and synthetic polymer processing [Huang, 2003; Ahn, 2006; Zoccola, 2007].

Figure 4. Picture of keratin/fibroin (90:10) nanofibres (SEM, 5000x).

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Recently, the electrospinning process has also been extended to include regenerated keratin. However, due to the poor mechanical characteristics of pure keratin, many researchers have resorted to the addition of synthetic or natural polymers in order to increase the processability of keratin for fibre formation. Aluigi et al. extracted keratin from wool by sulphitolysis and added poly(ethylene oxide) to keratin aqueous solutions to enhance electrospinnability [Aluigi, 2008]. The increase of poly(ethylene oxide)/keratin ratio promotes the formation of continuous nanofibres without defects due to the increase in the viscosity of the solution. The authors showed that electrospinning process induces structural modifications in the natural self-assembling of keratin chains, moreover chemical compatibility from poly(ethylene oxide) and keratin was evidenced by viscosity behaviour of melting solutions [Varesano, 2008] and thermal and structural analysis of electrospun nanofibres. Keratin/poly(ethylene oxide) nanofibres mats are soluble in water and blends rich in keratin show poor mechanical properties. They can be proposed as filters for air cleaning. In a recent study, protein material resulting from steam explosion of wool in water was mixed with polyamide 6 using formic acid as common solvent and electrospun to obtain nanofibres [Zoccola, 2007]. Since the molecular weight of keratin was lower than that of keratin extracted by sulphytolysis, blend solutions were electrospun successfully until a 50/50 wt/wt polyamide6/keratin ratio. Authors stated that keratin and polyamide show a better miscibility and a higher thermal stability in electrospun nanofibres compared to cast films. A lot of work has been done for electrospinning wool keratin/silk fibroin blends for potential application in both biomedical and filtering sectors. Keratin extracted from wool by sulphytolysis and fibroin extracted from silk were blended and successfully electrospun in different ratios ranging from pure fibroin until keratin/fibroin 90/10 wt/wt % [Zoccola, 2008] using formic acid as a common solvent. Authors showed that the nanofibres become thinner and more homogeneous by increasing the keratin amount in blends and that the electrospinning process promotes the formation of -helix/random coil structures instead of the -sheet supramolecular structure present in cast films. Moreover the 50/50 keratin/fibroin blend shows higher solution viscosity, different thermal and structural properties and produces by electrospinning thinner filaments compared with the other blend ratio because of the enhanced interactions between keratin and fibroin. Nanofibres obtained have been proposed for potential application in the biomedical field due to the

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biocompatibility of keratin and fibroin and the peculiar properties of antithrombogenicity of this blend [Lee, 1998]. A different application of keratin/fibroin nanofibres was proposed in the filtering sector. Ki et al. [Ki, 2007] blended and electrospun wool keratin, obtained by oxidation, with silk fibroin. Silk fibroin enhances the electrospinnability of keratose, whereas keratin is an excellent material for heavy metal ion absorption due to the abundance of polar groups. Electrospun nanofibers (50/50 wool keratose/silk fibroin) were treated with formaldehyde vapour for stabilising in aqueous environment. Authors showed that nanofibres from blends exhibited higher Cu2+adsorption capacity than fibroin nanofibres, wool sliver or filter paper. Furthermore, the absorption capacity of nanofibres from blends was maintained after several recycling process of adsorption and desorption.

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5.4. Keratin Microcapsules Microencapsulation is defined as enclosing a solid, liquid or gas inside of a polymeric coating. Microcapsules can be used as carriers of fine chemicals such as dyes, flavours, fragrances and drugs and also for biologically active compounds. Encapsulation may sustain the duration of activity of the trapped substances and influence the releasing mode into aimed sites. There are many methods, both chemical and physical, of microencapsulation. The method proposed for keratin was the sonication of an aqueous solution of wool keratin in the presence of an organic solvent [Yamauchi, 1997; Yamauchi, 2002]. Spherical microcapsules with few micrometres in diameter were obtained and they are able to trap oil, fat and dyes dissolved in the solvent, in a yield of more 90%. The keratin microcapsules fabricated in this way were stable in boiling water and in most of organic solvents. Keratin microcapsules show high barrier properties probably imputable to the intermolecular -S-S- bonds between the cysteine residues of the proteins.

5.5. Keratin Hydrogels Hydrogels are crosslinked polymeric networks containing hydrophilic groups that promote swelling due to interaction with water and they are used in the field of regenerative medicine, wound healing, cell encapsulation and controlled drug release. Hydrogels used for regenerative applications are based

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on natural or synthetic polymers. By most definitions, native tissues, particularly the extra-cellular matrix, are hydrogels and derivatives of these and other naturally based systems are in widespread use. Hydrogels are easy to fabricate from keratin. Abuoshwareb et al. [Aboushwareb, 2009] extracted keratin from human hair following a long-established protocol [Goddard, 1934] using thioglycolic acid to reduce cystine bonds. After dialysis, keratin solution was adjusted to a final pH of 7.4, concentrated to 20 wt% using a rotary evaporation system and loaded into syringes. Upon exposure to air overnight, the viscous keratin solution forms a crosslinked hydrogel, that was unable to be re-dissolved in water and was sterilized in the syringe using gamma irradiation prior to use. In the in vivo trial, keratin hydrogel was evaluated as hemostatic biomaterial in rabbit having lethal liver injury. Results obtained show that the keratin treated rabbits (with 2 ml of keratin hydrogel on the bleeding liver surface) performed statistically better than the negative control group and as well as, or better than rabbit treated with commercial hemostats. Moreover an interesting healing response at the keratin liver interface was found at the microscopic observation of tissue. Keratin hydrogel was also experimented in vivo in the management of trauma-associated nerve defects [Sierpinski, 2008]. Keratin hydrogel was fabricated starting from proteins extracted from human hair with Tris-base after oxidation with peracetic acid, filtration to remove remaining solids and dialysis. Keratin hydrogel was fabricated by the re-hydratation of lyophilized material with a phosphate saline buffer and sterilised with γ-irradiation. It was shown that keratin hydrogel can mediate a strong nerve regeneration response, in part by enhancing the activity of Schwann cells, by increasing their attachment and proliferation and by up-regulating expression of important genes. A case control study was carried out on mice for the regeneration of peripheral nerve [Apel, 2008]. Fifty-four mice were randomized into three treatment groups: empty conduit, sural nerve autograft, and keratin hydrogelfilled conduit. Results obtained by the trial show that keratin hydrogels significantly improve electrophysiological recovery at an early time point of regeneration and they also produce long-term electrical and histological results superior to empty conduits and equivalent to sensory nerve autografts. In these experiments keratin hydrogels in vivo behave as a matrix that is permissive of regenerative cell infiltration and may be capable of mediating nerve repair if it can recruit beneficial cells and direct their behaviour toward restoration of functional tissue.

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5.6. Keratin Powders and Solutions Aqueous solutions of keratin powders were proposed in the cosmetic field for human hair, skin, nails care. In a recent paper Barba et al. [Barba, 2010] showed the effectiveness of protein extracted from wool on human hair water sorption kinetics. A 1% aqueous solution of commercial keratin peptides (Keratec Prosina™ with molecular weight lower than 1 kDa) and proteins (Keratec intermediated filaments protein™ with molecular weight of 55 kDa) were used for treatments on untreated and bleached hair samples. The application of the keratin peptide and protein was demonstrated to improve the moisture absorption/desorption capacity of bleached hair by restoring their integrity, and by decreasing, namely improving, their water permeability. Moreover the effectiveness of these keratin products to restore the mechanical properties, hair surface hydrophobicity and moisture content of treated human hair was demonstrated [Barba, Scott, 2010]. Similarly the same keratin commercial products were used on damaged nails and it was demonstrated that they improved their water sorption properties, reducing permeability, especially in the case of treatments with wool proteins [Barba, 2011]. It was postulated that the cystine in the S-sulphonated form present in keratin products used enable the keratin present in damaged hair or nails to reform disulphide bonds by directly affecting hair or nails properties. The cosmetic effectiveness of keratin hydrolysed peptide material was also tested on skin [Barba 2008] in two different formulations: an aqueous solution and an internal wool lipids liposome suspension. Both the formulations were applied topically on skin of healthy volunteers, improving, in both cases, hydration, elasticity and moisture sorption/desorption profiles of the skin area treated. Wool hydrolysates were also proposed for applications [Cardamone, 2007] in the finishing process of wool fabrics. Wool fibres were hydrolyzed in 0.5 N NaOH at 60 °C for 3 h. Soluble proteins were obtained after filtration of solid fibres, dialysis and freeze-drying. Keratin hydrolysates and their lyophylized powders were applied on wool fabrics previously bleached, in combination with transglutaminase enzyme which are able to catalyze crosslinking involving keratin solution and wool fabric. The coating of wool fibres with keratin powder could be proposed as finishing process to control the felting shrinkage of wool fabrics without evident fabric strength loss. The chemistry of tranglutaminase-mediated reaction was investigated in deep by the author [Cardamone, 2008]. Chemical treatments with commercial hydrolyzed keratin from wool have been shown to be effective at enhancing the tensile properties of bast fibres.

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Moreover the treatment with hydrolyzed down keratin from feathers is also useful for suppressing shrinkage during the drying of waterlogged archaeological wood excavated from sites [Kawahara, 2004]. Finally, hydrolysed keratin (0.15 M KOH , 0.05 M NaOH at 120 °C for 20 min) containing 75-80% water-soluble materials including potassium ions from alkali used in the hydrolysis and 20-25% partially degraded highly dispersed keratin, was evaluated as a fertilizer in agriculture [Nustorova, 2006]. It was shown that this organic material positively influenced microbial soil population and ryegrass growth and, when the remaining partially degraded keratin is highly dispersed, it will act additionally as a slow release nitrogen fertilizer.

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CONCLUSION In recent years there is a growing interest in innovative ways of valorisation of natural waste materials. Waste wool from the textile industries and coarse wool not suitable for spinning, form a biomass that can be converted into new added-value materials. In addition to convectional uses as fertilizer or protein source for fodders, keratin has been processed into films, sponges, capsules, powders, and nanofibres, hydrogels for applications in many different fields. Biocompatibility and biodegradability of keratin can be exploited for biomedical applications. Moreover, the reactivity with heavy metals and VOCs makes keratin an interesting candidate for active filtration of air and water.

ACKNOWLEDGEMENTS The authors greatly acknowledge the CARIPLO Foundation (Milan, Italy) for the financial support within the research project “Kebab, keratin-based composite bio-plastics”.

REFERENCES Aboushwareb, T; Eberli, D; Ward, C; Broda, C; Holcomb, J; Atala, A; Van Dyke, M. A keratin biomaterial gel hemostat derived from human hair:

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evaluation in a rabbit model of lethal liver injury. J Biomed Mater Res-A, 2009 90B, 45-54. Ahn, YC; Park, SK; Kim, GT; Hwang, YJ; Lee, CG; Shin, HS; Lee, JK. Development of high efficiency nanofilters made of nanofibers. Alexander, P; Earland, C. Structure of Wool Fibres: Isolation of an α- and βProtein in Wool. Nature,1950 166, 396. Aluigi, A; Vineis, C; Ceria, A; Tonin, C. Composite biomaterials from fibre wastes: characterization of wool-cellulose acetate blends. Composites Part A: Applied Science and Manufacturing, 2008 39, 126-132. Aluigi, A; Vineis, C; Varesano, A; Mazzuchetti, G; Ferrero, F; Tonin, C. Structure and properties of keratin/PEO blend nanofibres. Eur Polym J, 2008 44, 2465-2475. Aluigi, A; Zoccola, M; Vineis, C; Tonin,C, Ferrero, F; Canetti, M. Study on the structure and properties of wool keratin regenerated from formic acid. Int J Biol Macromol, 2007 41, 266-273. Apel, PJ; Garrett, JP; Sierpinski, P; Ma, J; Atala, A; Smith, TL; Koman, L.; Van Dyke, ME. Peripheral nerve regeneration using a keratin-based scaffold: long-term functional and histological outcomes in a mouse model. The Journal of Hand Surgery, 2008 33, 1541-1547. Barba, C; Martì, M; Roddick-Lanzilotta, A; Manich, A; Carilla, J;. Parra, J.; Coderch, L. Effect of wool keratin proteins and peptides on hair water sorption kinetics. J Therm Anal Calorim, 2010 102, 43-48. Barba, C; Martì, M; Roddick-Lanzilotta, A; Maniche, A; Carilla, J;. Parra, JL; Coderch, L. Water sorption of nails treated with wool keratin proteins and peptides. J Therm Anal Calorim, 2011 104, 323-329. Barba, C; Mendez, S; Roddick-Lanzilotta, A; Kelly, R; Parra, JL, Coderch, L. Cosmetic effectiveness of topically applied hydrolysed keratin peptides and lipids derived from wool. Skin Res Technol, 2008 14, 243–248. Barba, C; Scott, S; Roddick-Lanzilotta, A; Kelly, R; Manich, AM; Parra, JL; Coderch, L. Restoring important hair properties with wool keratin proteins and peptides. Fibers and Polymers, 2010 11, 1055-1061. Barhate, RS; Ramakrishna, S. Nanofibrous filtering media: filtration problems and solutions from tiny materials. J Membrane Sci, 2007 296, 1-8. Bradbury, JH. The Structure and chemistry of keratin fibres. Adv. Protein Chem., 1973 27, 111-211. Cardamone, JM; Phillips, JG. Enzyme-mediated crosslinking of wool. Part II: Keratin and transglutaminase. Textile Res. J., 2007 77, 277-283. Cardamone, JM. Keratin transamidation. Int J Biol Macromol, 2008 42, 413419.

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Cecil, R; McPhee, JR. The Sulfur Chemistry of Proteins. Adv. Protein Chem.,1959 14, 299-302. Cecil, R. The proteins. New York: Neurath EH; Academic Press; 1963; 438. Crewther, WG; Fraser, RDB; Lennox, FG; Lindley H. The chemistry of keratins. Adv. Protein Chem., 1965 20, 191. Dowling, LM; Crewther, WG; Parry, DAD. Secondary Structure of Component 8c-1 of α-Keratin. Biochem. J., 1986 236, 705 – 712. Formhals, A. US Patent 1, 975, 504, 1934. Fraser, RDB; MacRae, TP et Rogers, G E, Keratins: their composition, structure and biosynthesis, Springfield: Charles C. Thomas; 1972. Fraser, RDB; Parry, DAD. The molecular structure of reptilian keratin. International Journal of Biological Macromolecules, 1996 19, 207-211. Gillespie, JM; Broad A; Reis, PJ. A further study on the dietary-regulated biosynthesis of high-sulphur wool proteins. Biochem. J., 1969 112, 41-49. Gillespie, M; Darskus, RL. Relation between the tyrosine content of various wools and their content of a class of proteins rich in tyrosine and glycine. Aust. J. Biol. Sci., 1971 24, 1189-1197. Goddard, DR; Michaelis, L. A study on keratin. J Biol Chem, 1934,106, 605– 614. Hamasaki, S; Tachibana,A; Tada, D; Yamauchi, K; Tanabe, T. Fabrication of highly porous keratin sponges by freeze-drying in the presence of calcium alginate beads. Mater Sci Eng C, 2008 28, 1250-1254. Hill, P; Brantley, H; Van Dyke, M. Some properties of keratin biomaterials: kerateines. Biomaterials, 2010 31, 585-593. Huang, ZM.; Zhang, YZ.; Kotaki M.; Ramakrishna, S. A Review on Polymer Nanofibers by Electrospinning and Their Applications in Nanocomposites. Compos. Sci. Technol. 2003, 63, 2223-2253. Jenkins, AD; Wolfram, LJ. The chemistry of the reaction between tetrakis (hydroxymethyl) phosphonium chloride and keratin. J. Soc. Dyers Col., 1963 79, 55. Johnson, A; Thompson, DE. The Sulphitolysis of wool. Proc. Int. Wool Textile Res. Conf., Aachen, 1975 3, 144-153. Katoch, K; Tanabe, T; Yamauchi, K. Novel approach to fabricate keratin sponge scaffolds with controlled pore size and porosity. Biomaterials, 2004 25, 4255-4262. Katoh, K; Shibayama, M; Tanabe, T; Yamauchi, K. Preparation and physicochemical properties of compression-molded keratin films. Biomaterials, 2004 25, 2265-2272.

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Kawahara, Y; Endo, R; Rimura, T. Chemical finishing of bast fibers and woods using hydrolyzed keratin from waste wool or down. Textile Res. J., 2004 74, 93-96. Ki, CS; Gang, EH; Um, IC; Park, YH. Nanofibrous membrane of wool keratose/silk fibroin blend for heavy metal ion adsorption. J Membrane Sci, 2007 302, 20-26. Kurimoto, A; Tanabe, T; Tachibana, A; Yamauchi, K. Keratin sponge: immobilization of lysozyme. J Biosci Bioeng, 2003 96, 307-309. Le Roux, PL; Speakman, JB. The plasticity of wool: Part III: The physical and chemical causes of variation. Text. Res. J., 1957 27, 1-7. Lee, K; Kong, S; Park, W; Ha W; Kwon, I. Effect on surface properties on the antithrombogenicity of silk fibroin/S –carboxymethyl kerateine blend films J. Biomater. Sci- Polym Ed, 1998 9, 905-914. Li, L; Frey MW; Green TB. Modification of air filter media with nylon-6 nanofibers. J Eng Fibers Fabr, 2006 1, 1-24. MacLaren, JA. Maximum Extraction of Wool Proteins by Thiol-Urea Solutions. Textile Res. J., 1987 57, 87. MacLaren, JA; Milligan B. Wool Science. The chemical reactivity of the wool fibre. Marrickville NSW: Science Press; 1981 Marshall R.C; J.M. Gillespie in The biology of wool and hair. G E Rogers, London and New York: Chapman and Hall,1988. Miyamoto, T; Sakabe, H; Inagaki, H. Ordered structure of high-glycine proteins from reduced merino wool. Bull. Inst. Chem. Res. Kyoto Univ. 1987 65, 109-119. Nustorova, M; Braikova, D; Gousterova, A; Vasileva-Tonkova, Eand Nedkov, P. Chemical, microbiological and plant analysis of soil fertilized with alkaline hydrolysate of sheep’s wool waste. World Journal of Microbiology & Biotecnology, 2006 22, 383-390. O‟Donnel, IJ; Thompson, EOP. Austr. J. Biol. Sci., 1964 17, 973-979. Patrucco, A; Aluigi, A; Zoccola, M; Vineis, C and Tonin, C. Keratin BioComposites from Wool Fibrillation, 3rd International Conference on Biodegradable and Biobased Polymers, Strasbourg, 2011. Patrucco, A; Aluigi, A; Vineis C; Tonin, C. Bio-composite keratin films from wool fibrillation. Journal of Biobased Materials and Bioenergy, 2011 5, 1 124-131. Qin, XH; Wang, SY. Filtration properties of electrospinning nanofibers. J Appl Polym Sci, 2006 102, 1285-1290. Reichl, S, Borrelli, M, Geerling, G. Keratin films for ocular surface reconstruction. Biomaterials, 2011 32, 3375-3386.

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Ross, DA. Biological aspects of the sulphur content of Romney wool. Proc. NZ Anim. Prod., 1961 21, 153-165. Sierpinski, P; Garrett, J; Ma, J; Apel, P; Klorig, D; Smith, T; Koman, LA; Atala, A; Van Dyke, M. The use of keratin biomaterials derived from human hair for the promotion of rapid regeneration of peripheral nerves. Biomaterials, 2008 29, 118-128. Tachibana, A; Furuta, Y; Takeshima, H; Tanabe, T; Yamauchi, K. Fabrication of wool keratin sponge scaffolds for long-term cell cultivation. J Biotechnol, 2002 93, 165-170. Tachibana, A; Kaneko, S; Tanabe,T; Yamauchi, K. Rapid fabrication of keratin–hydroxyapatite hybrid sponges toward osteoblast cultivation and differentiation. Biomaterials, 2005 26, 297-302. Tachibana, A; Nishikawa,Y; Nishino,M; Kaneko,S; Tanabe, T and Yamauchi, K. Modified Keratin Sponge: Binding of Bone Morphogenetic Protein-2 and Osteoblast Differentiation. J. of Bioscence and Bioengineering,2006 102,425-429. Tanabe, T; Okitsu, N; Tachibana, A; Yamauchi, K. Preparation and characterisation of keratin-chitosan composite films. Biomaterials, 2002 23, 817-825. Tanabe, T; Okitsu, N; Yamauchi, K. Fabrication and characterization of chemically crosslinked keratin films. Mater Sci Eng C, 2004 24, 441-446. Thompson, EOP; O‟Donnel IJ. Studies on oxidized wool I: a comparison of the completeness of oxidation with peracetic and performic acids. Aust. J. Biol. Sci., 1959 12, 282-293. Tonin, C; Aluigi, A; Varesano, A; Vineis, C. Keratin-based nanofibres in Nanofibers, Kumar, A. 2010. Tonin, C; Zoccola, M; Aluigi, A; Varesano, A; Montarsolo, A; Vineis, C; Zimbardi, F. Study on the conversion of wool keratin by steam explosion. Biomacromolecules, 2006 7, 3499-3504. Tsai, PP; Schreuder-Gibson, H; Gibson, P. Different electrostatic methods for making electret filters. J Electrostat, 2002 54, 333-341. Varesano, A; Aluigi, A; Vineis, C; Tonin, C. Study on the shear viscosity behaviour of keratin /PEO blends for nanofibre electrospinning. J Polym Sci Pol Phys, 2008 46, 1193-1201. Vasconcelos, A, Freddi, G; Cavaco-Paulo A. Biodegradabile materials based on silk fibroin and keratin, Biomacromolecules, 2008 9, 1299-1305. Yamauchi, K; Khoda, A. Novel proteinous microcapsules from wool keratins. Colloid Surface B, 1997 9, 117-119.

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Yamauchi, K; Maniwa, M; Mori, T. Cultivation of fibroblast cells on keratincoated substrata. J Biomater Sci-Polym E, 1998 9, 259-70. Yamauchi, K; Yamauchi, A; Kusunoki, T; Kohda, A; Konishi, Y. Preparation of stable aqueous solution of keratins, and physicochemical and biodegradational properties of films. J Biomed Mater Res, 1996 31, 439444. Yamauchi, A.; Yamauchi, K. Formation and Properties of wool keratin films and coating in Protein- Based films and Coating, Gennadios, A, CRC Press, Boca Raton, Florida, 2002. Yin, J; Rastogi, S; Terry, AE; Popescu, C; Self-organization of oligopeptides obtained on dissolution of feather keratins in superheated water. Biomacromolecules, 2007 8, 800-806. Zahan H.; Wool is not keratin only. Plenary Lecture, 6th Internat. Wool Tex. Res. Conf., Pretoria, 1980 1. Zoccola, M; Aluigi, A; Patrucco, A; Vineis, C; Forlini, F; Locatelli, P ; Sacchi, MC; Tonin, C. Microwave Assisted Chemical Free Hydrolysis of Wool Keratin, 7th International Conference on Polymer and textile Biotechnology Milan, 2011. Zoccola, M; Aluigi, A; Vineis, C; Tonin, C; Ferrero, F; Piacentino, MG. Study on cast membranes and electrospun nanofibers made from keratin/fibroin blends. Biomacromolecules, 2008 9, 2819-2825. Zoccola, M; Montarsolo, M; Aluigi, A; Varesano, A; Vineis, C; Tonin, C. Electrospinning of polyamide6/modified-keratin blends. e-Polymer, 2007 105, 1-19.

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Chapter 3

APPEARANCE AND DISTRIBUTION OF CYTOKERATINS 13, 14 AND 18 IN THE LINGUAL EPITHELIUM DURING MORPHOGENESIS OF THE RAT TONGUE Shin-ichi Iwasaki1, Tomoichiro Asami2 and Hidekazu Aoyagi1 Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

1

Advanced Research Center, The Nippon Dental University School of Life Dentistry at Niigata, Japan 2 Gunma PAZ College School of Nursing, Japan

ABSTRACT Appearance and distribution of different kinds of cytokeratins are closely related to morphogenesis of the various organs. On the morphogenesis of the rat tongue during prenatal and postnatal development, we could recognize the specific appearance and distribution of some kinds of cytokeratins, such as cytokeratins 13 (K13), 14 (K14) and 18 (K18). No immunoreactivity specific for K13 and K14 was detected on the dorsal epithelium of the anterior part of the tongue in fetuses on embryonic day 15 after conception (E15), at which time the lingual epithelium was composed of a few layers of cuboidal cells. No lingual papillae are recognizable in this area. K14-specific immunoreactivity was first detected on the lingual epithelium of fetuses on E17 and K13-specific immunoreactivity on E19. The number of layers

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Shin-ichi Iwasaki, Tomoichiro Asami and Hidekazu Aoyagi of cuboidal cells in the lingual epithelium also increased from E17 to E19. At all postnatal stages, K13-specific immunoreactivity became to be gradually evident in the suprabasal cells of the interpapillary cell columns and K14-specific immunoreactivity did in the basal and suprabasal cells of the papillary and interpapillary cell columns according to the development of filiform papillae. On the other hand, no immunoreactivity specific for K13 and K14 was detected in the lingual epithelium on E15, at which time the primitive rudiment of the circumvallate papillae was detectable by the thickening of several layers of cuboidal epithelial cells. On E17 and E19, the developing circumvallate papillae were clearly recognizable. No K13 and K14-specific immunoreactivity was evident in the lingual epithelium around these structures. K14-specific immunoreactivity was first detected in the basal layer of the epithelium of the circumvallate papillae on postnatal day 0 (P0) and K13-specific immunoreactivity was detected on P7. Morphogenesis of the circumvallate papillae progressed significantly from P0 to P14, and K13 and K14-specific immunoreactivity was clearly recognizable after P7. K13-specific immunoreactivity was generally evident in cells of the intermediate layer of the epithelium, while K14specific immunoreactivity was detected in cells of the basal and suprabasal layers. K18-specific immunoreactivity was detectable in the single layer of periderm cells that covered the dorsal epithelium of the tongue of fetuses on E13. The immunoreactivity was sparsely distributed throughout the cytoplasm in some periderm cells. On E15, K18-specific immunoreactivity was also detectable in the periderm cells. The distribution of the K18-specific immunoreactivity was sparsely distributed in the cytoplasm in some periderm cells on E13 and E15. On E17, the K18-specific immunoreactivity was very distinct in the periderm cells, which had become swollen and elliptical and covered the dorsal epithelium of the fetal tongues. The pattern of distribution of the immunoreactivity was different from that on E13 and E15, and K18specific immunoreactivity was compactly distributed over almost the entire cytoplasm in most periderm cells. No K18-specific immunoreactivity was detectable on the dorsal epithelium of the tongue of fetuses on E19. Periderm cells had disappeared completely by E19.

INTRODUCTION Cytokeratins, the intermediate-filament proteins of epithelial cells, can be divided into two groups, type I and type II, and the members of each respective group are expressed in vivo as specific pairs that form

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heteropolymers (Steven, 1990; Albers & Fuchs, 1992). Cytokeratins 5 (K5) and 14 (14) are of particular interest because they are expressed in mitotically active keratinocytes in all types of stratified squamous epithelium (Nelson & Sun, 1983). The expression of these keratins in mitotically active keratinocytes has been demonstrated by in situ hybridization and/or immunohistochemical localization of these proteins in the epidermis (Roop et al., 1987; Lersch & Fuchs, 1988; Moll et al., 1989), the tongue (Dhouailly et al., 1989) and various other types of stratified and pseudo-stratified epithelium (Moll et al., 1989). By contrast, K4 and K13 are associated with differentiating suprabasal cells of the non-keratinized and parakeratinized oral epithelium (Heyden et al., 1992; Barrett et al., 1998; Okazaki et al., 2003), and K13 has also been detected in suprabasal cells of the keratinized oral epithelium (Heyden et al., 1992) and nasal epithelium (Lu et al., 2002). In normal human skin, the pattern of basal staining of K13 in the epidermis was consistent in all samples examined (Okazaki et al., 2003). On the other hand, the specific pair of cytokeratins 8 (K8) and 18 (K18) is found in most simple epithelia and their derivatives in adult mammals (Berkovitz et al., 1997). Many immunohistochemical studies have been performed to identify the types and localizations of keratins and keratin-related proteins in the periderm cells of the skin in both human and other mammalian fetuses (Moll et al., 1982; Dale et al., 1985; van Muijen et al., 1987). Analysis by scanning electron microscopy of the tongues of rats (Iwasaki et al., 1997) and mice (Iwasaki et al., 1996) revealed that filiform papillae form just before birth in these rodents. Light and transmission electron microscopy demonstrated that there are no rudiments of filiform papillae and no signs of keratinization in the dorsal lingual epithelium of rats and mice at the middle and late periods of gestation. By contrast, keratinization of the dorsal lingual epithelium is clearly recognizable in newborn rats and mice, together with the rudiments of filiform papillae (Iwasaki et al., 1999a, b). Thus, the filiform papillae of rats and mice seem to develop rapidly and exclusively during the 2 or 3 days before birth, in parallel with the keratinization of the dorsal epithelium of the tongue. On the contrary, the same analysis by scanning electron microscopy of the tongues of rats (Iwasaki et al., 1997) and of mice (Iwasaki et al., 1996) indicated that the circumvallate papilla, one type of gustatory papillae, was developed between E12 and E16 in rat fetuses and before E15 in mouse fetuses, and AhPin et al. (1989) described the circumvallate papilla in the mouse has been developed from E11 to E18. The more detailed investigation by Hirao et al. (2007) clearly defined that the primordium of circumvallate papilla was observed at E13 in the rat, and

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that by Jitpukdeebondintra et al. (2002) also confirmed that invagination and cell division in the circumvallate papilla of mouse embryos detected from E11.5 to E14. Based on the data described above, the present study was designed to clarify the timing of the appearance and changes in the expression of keratin 14, which is an important marker of epithelial stratification and keratinization, during the morphogenesis of filiform papillae on the rat tongue. We used a novel immunofluorescence method to examine the localization of keratins 13, 14 and 18 on semi-ultrathin section of epoxy resin-embedded specimens.

EXPERIMENTAL METHODS OF IMMUNOHISTOCHEMISTRY Details of the total experimental methods used in the present chapter were already published in the articles by Iwasaki and Aoyagi (2010, 2011a, b). The actual methods employed in the present chapter are as follows.

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Antibodies Mouse monoclonal antibodies specific for K13, which had been purified from human esophagus, were purchased from Progen Biotechnik GmbH (Heidelberg, Germany). Mouse monoclonal antibodies specific for K14, which had been raised against a synthetic peptide of 15 amino acids that corresponded to the carboxy-terminal region of human keratin 14, were purchased from Cymbus Bioscience (Southampton, UK). A mouse monoclonal antibody specific for K18, which had been purified from the cytoskeletal proteins of human HeLa cells, was purchased from Progen Biotechnik (Heidelberg, Germany). Biotin-conjugated rabbit antibodies against mouse IgG, IgA and IgM were purchased from Nichirei Biosciences (Tokyo, Japan).

Preparation of Tissues Tongues, taken from fetuses on E15, E17 and E19 and from juveniles on day P0, P7 and P14, were fixed in 4% formaldehyde titrated from paraformaldehyde in 0.1M phosphate buffer (pH7.4) at 4 oC for 5 h. After rinsing in 0.1 M phosphate buffer, all samples were dehydrated and embedded

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in epoxy resin (Epon 812, TAAB, Reading, UK), which was polymerized overnight at 60 oC. Epoxy resin-embedded samples were cut into 500 nm sections with a diamond knife on an ultramicrotome (MT-XL; RMC, Tucson, AZ, USA). The sections were mounted on glass slides and incubated in 10% sodium methoxide for 3 min at 37 oC to remove the epoxy resin (Mayor et al., 1961). After passage through an acetone series, they were transferred to phosphate-buffered saline (PBS; pH 7.4).

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Immunofluorescence Labeling After retrieval of antigens by heating sections in a microwave oven (500W) for 2 min in 10 mM sodium citrate buffer, pH 6.0 (Shi et al., 1991), sections on slides were allowed to cool at room temperature for 8 min and were then transferred to PBS at room temperature. Thereafter, sections were incubated with the monoclonal antibodies specific for K13, K14 and K18 overnight at 4 oC. To determine the optimum working dilutions of the preparations of K13-, K14- and K18-specific antibodies, we tested dilutions from 1:25 to 1:800, respectively. The optimum dilutions of all preparations of antibodies were estimated to be 1:50 and 1:100. After washing in PBS, each section was incubated with biotin-conjugated rabbit antibodies against mouse IgG, IgA and IgM (Nichirei Biosciences, Tokyo, Japan) for 30 min at room temperature and then, incubated for 30 min at room temperature, with streptavidin-Alexa Fluor® 543 (Molecular Probes, Eugene, OR, USA) for detection of K13, with streptavidin-Alexa Fluor® 488 (Molecular Probes, Eugene, OR, USA) for detection of K14 or with streptavidin-Alexa Fluor® 633 for detection of K18. Sections were then mounted with FluoroGuardTM Antifade Reagent (Bio-Rad Laboratories, Hercules, CA, USA). The specificity of immunoreactions was checked by preparation of controls under the following conditions: (a) omission of the primary antibody; (b) replacement of the primary antibody with normal mouse serum at the same dilution; and (c) pre-absorption of the primary antibody with the corresponding antigen at 10100 g/ml for 24 h at 4oC.

Confocal Laser-Scanning Microscopy All specimens were examined with a confocal laser-scanning microscope (LSM510 or LSM710; Carl Zeiss, Jena, Germany) that was equipped with an

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argon laser or a helium–neon laser. The dimensions of all images displayed on the monitor were 1024 x 1024 pixels. For single scanning for detection of the fluorescence of Alexa Fluor® 546, we used a 543-nm laser wavelength filter; a 543-nm primary dichroic beam-splitter; and a 560-nm low-pass filter. For single scanning for detection of the fluorescence of Alexa Fluor® 488, we used a 488-nm laser wavelength filter; a 488-nm primary dichroic beam-splitter; and a 505- to 530-nm band-pass filter. For single scanning for detection of the fluorescence of Alexa Fluor® 633, we used a 633-nm laser wavelength filter; a 514/633 primary dichroic beam-splitter; and a 650-nm low-pass filter. Combinations of pixel sizes from 0.12 m x 0.12 m to 0.17 m x 0.17 m and a 40 x objective with a numerical aperture (NA) of 0.75 were used for observations. Immediately after recording immunofluorescent images, we also recorded the differential interference contrast (DIC) images of each specimen. Furthermore, after staining of specimens with 0.2% toluidine blue (Waldeck GmbH, Munster, Germany) in 2.5% Na2CO3, we examined the corresponding images by laser-scanning microscopy in the transmission mode. Finally, images, showing the immunoreactivity of K13 or of K14 and the microanatomy, recorded in transmission mode, were stacked on top of one another by computer and analyzed (Photoshop 6.0, Adobe, San Jose, CA, USA) after an examination of the same respective DIC images and images obtained in transmission mode. Images, showing the immunoreactivity of K18, were stacked directly on the same respective DIC images.

IMMUNOHISTOCHEMICAL LOCALIZATION OF CYTOKERATIN 13 DURING MORPHOGENESIS OF FILIFORM PAPILLAE The laser-scanning micrographs that showed the localization of immunoreactive K13 and the images in transmission mode that revealed the histology and morphology of cells in semi-ultrathin sections of the anterior dorsal epithelium of the tongue, where the filiform and fungiform papillae are formed during the embryonic and postnatal development of rats, yielded the following information. A single layer of periderm cells formed a smooth covering on the apical side of the cuboidal epithelial cells. No immunoreactive K13 was detectable on the dorsal epithelium of the tongue of fetuses on E15 (Figure 1A1, 2) and E17 (Figure 1B1, 2).

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Figure 1. Combinations of laser-scanning micrographs that show the localization of immunoreactive keratin 13, examined at 543 nm, and images obtained by confocal laser-scanning microscopy in transmission mode, which show the histology and cellular morphology of semi-ultrathin sections of the lingual body. A1: sagittal section from a fetus on E15, A2: higher magnification of a part of the epithelium in A1; B1: sagittal section from a fetus on E17, B2: higher magnification of a part of the epithelium in B1; C1: sagittal section from a fetus on E19, C2: higher magnification of a part of the epithelium in C1; D1: sagittal section from a juvenile on P0, D2: higher magnification of a part of the epithelium in D1; E1: sagittal section from a juvenile on P7, E2: higher magnification of a part of the epithelium in E1; F2: sagittal section from a juvenile on P14, F2: higher magnification of a part of the epithelium in F1. E: dorsal lingual epithelium; PE: periderm cell; CT: connective tissue; M: muscle; FP: filiform papillary cell column; IP: interpapillary cell column; A: anterior cell column of filiform papilla; P: posterior cell column of filiform papilla; KL: keratinized cell layer; arrow: connective tissue papillae. Scale bars: 10 m.

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The corresponding images in transmission mode indicated that the lingual epithelium was composed of one or two layers of the same type of cell on E15 and of several layers of the same type of cell on E17. Immunoreactivity specific for K13 was first detected in the suprabasal cells of the papillary and interpapillary cell columns of fetuses on E19. The reactivity was concentrated in the narrow interpapillary cell columns rather than in the papillary cell columns. However, the immunoreactivity specific for K13 was generally weak overall (Figure 1C1, 2). Immunoreactivity specific for K13 was stronger in the suprabasal cells of the narrow interpapillary cell columns of newborns on P0 than in those of the papillary cell columns, reflecting the appearance of a keratinized layer in the papillary epithelium. The immunoreactivity in immunopositive cells in the interpapillary cell columns seemed to be more densely distributed in the cytoplasm than that on E19. Immunoreactivity specific for K13 in the suprabasal cells of the interpapillary cell columns was denser than it was in the suprabasal cells of the papillary cell columns (Figure 1D1, 2). The pattern of distribution of immunoreactivity specific for K13 in juveniles on P7 was similar to that on P0, in spite of the progression of morphogenesis of filiform papillae (Figure 1E1, 2). The distribution of immunoreactivity specific for K13 in juveniles on P14 was more extensive in the entire deep intermediate layer of the interpapillary cell columns that contained suprabasal cells. The immunoreactivity was stronger throughout the cytoplasm of immunopositive cells, with none in nuclei, as compared to that on P7. By contrast, immunoreactivity specific for K13 in the suprabasal cells of the papillary cell columns disappeared completely with the development of the thick keratinized layer of filiform papillae (Figure 1F1, 2).

IMMUNOHISTOCHEMICAL LOCALIZATION OF CYTOKERATIN 14 DURING MORPHOGENESIS OF FILIFORM PAPILLAE Similar analysis to that described above yielded the following information about K14 in the anterior dorsal epithelium of the rat tongue. No immunoreactive K14 was detected on the dorsal epithelium of the tongues of fetuses on E15 (Figure 2A1, 2). Immunoreactivity specific for K14 was apparent mainly in the basal and suprabasal cells of the papillary cell columns in fetuses on E17.

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Figure 2. Combinations of laser-scanning micrographs that show the localization of immunoreactive keratin 14, examined at 488 nm, and images obtained by confocal laser-scanning microscopy in transmission mode, which show the histology and cellular morphology of semi-ultrathin sections of the lingual body. A1: sagittal section from a fetus on E15, A2: higher magnification of a part of the epithelium in A1; B1: sagittal section from a fetus on E17, B2: higher magnification of a part of the epithelium in B1; C1: sagittal section from a fetus on E19, C2: higher magnification of a part of the epithelium in C1; D1: sagittal section from a juvenile on P0, D2: higher magnification of a part of the epithelium in D1; E1: sagittal section from a juvenile on P7, E2: higher magnification of a part of the epithelium in E1; F2: frontal section from a juvenile on P14, F2: higher magnification of a part of the epithelium in F1. E: dorsal lingual epithelium; PE: periderm cell; CT: connective tissue; M: muscle; FP: filiform papillary cell column; IP: interpapillary cell column; A: anterior cell column of filiform papilla; P: posterior cell column of filiform papilla; KL: keratinized cell layer; arrow: connective tissue papillae. Scale bars: 10 m.

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The immunoreactivity was particularly strong in the basal cells along the connective tissue papillae and was very weak in the suprabasal cells. The immunoreactivity extended over the entire cytoplasm, with none in the nucleus, in most of the basal cells of the papillary cell columns along the connective tissue papillae, but it was not as widely distributed in the suprabasal cells (Figure 2B1, 2). On E19, very weak immunoreactivity specific for K14 was detected clearly in the basal and suprabasal cells of the interpapillary cell columns, and it was restricted to a very narrow region between the rudiments of filiform papillae. No immunoreactive cells were detected in the intermediate and surface layers in both the papillary and interpapillary cell columns (Figure 2C1, 2). In newborns on P0, immunoreactivity specific for K14 was detected in basal and suprabasal cells of the papillary and interpapillary cell columns of the lingual epithelium. The immunoreactivity was particularly strong in the basal cells of the papillary cell columns along the connective tissue papillae. By contrast, it was less distinct in the suprabasal cells. The immunoreactivity in the basal and suprabasal cells of the interpapillary cell columns was still very weak but somewhat stronger than that in fetuses on E19. The immunoreactivity extended over a large part of the cytoplasm, with none in the nucleus, in the strongly immunopositive cells of the papillary cell columns along the connective tissue papillae. No immunoreactive cells were detected in the deep and shallow intermediate layers or in the keratinized or surface layers of both the papillary and the interpapillary cell columns (Figure 2D1, 2). Immunoreactivity specific for K14 was concentrated in the basal and suprabasal cells of the papillary and interpapillary cell columns of juveniles on P7. There was no specific difference in terms of the distribution of immunoreactivity specific for K14 between the anterior and posterior cell columns of filiform papillae. However, the immunoreactivity of the interpapillary cell columns remained very weak. The immunoreactivity of cells of the papillary cell columns along the connective tissue papillae, which were located in the center of each filiform papilla, extended over a large part of cytoplasm but was not evident in the nuclei. No immunoreactive cells were detected in the intermediate and keratinized or surface layers of both the papillary and the interpapillary cell columns (Figure 2E1, 2). Immunoreactivity specific for K14 in juveniles on P14 was distinct in the basal and suprabasal cells not only in the papillary cell columns along the connective tissue papillae but also in those in the interpapillary cell columns, which were clearly wider than the interpapillary cell columns on P7. However, it was very distinct in the basal and suprabasal cells of the papillary cell columns. There was still no clear difference in

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immunoreactivity specific for K14 between the anterior and posterior cell columns of filiform papillae. The immunoreactivity extended over the entire cytoplasm, with none in the nucleus, in most immunopositive cells. No immunoreactive cells were detected in the intermediate and keratinized or surface layers of both papillary and interpapillary cell columns (Figure 2F1, 2).

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IMMUNOHISTOCHEMICAL LOCALIZATION OF CYTOKERATIN 13 DURING MORPHOGENESIS OF CIRCUMVALLATE PAPILLA In the posterior area of the tongue where a single circumvallate papilla is forming on the median line, the epithelium extended relatively deeply into the underlying connective tissue (lamina propria) in fetuses on E15, however, at this stage this connective tissue was not seen forming an invagination into the basal side of the epithelial thickening. Each epithelial cell had a centrally located large round nucleus. A single layer of periderm cells also formed a smooth covering on the apical side of the cuboidal epithelial cells that formed the rudiment of the circumvallate papillae. Mesenchymal connective tissue was widely scattered beneath the epithelium. Lingual muscle tissue was not visible in the posterior dorsal region of the tongue. No K13-specific immunoreactivity was detectable in the epithelium of the posterior side of the tongue that included the rudiment of the circumvallate papillae (Figure 3A). On E17, the epithelium of each developing circumvallate papilla became somewhat thicker than in earlier stages, and developed into several layers of cuboidal cells. In particular, the basal side of the circular sulcus around the central bulge was distinctly swollen and composed of several layers of cells. However, most of the epithelium in the region close to the developing papillae still consisted of a single layer of cuboidal cells, and the total thickness of the epithelium in this area remained unchanged. At this stage, the connective tissue began to penetrate deep into the central part of the developing circumvallate papilla from the basal side. Each epithelial cell still had a large round nucleus. Periderm cells were clearly recognizable, but there were no recognizable signs of keratinization of the epithelium that formed the developing circumvallate papillae. Mesenchymal connective tissue was scattered beneath the epithelium, and myogenesis just began beneath the developing papillae, but no K13-specific immunoreactivity was detectable in

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the epithelium in this region (Figure 3B). On E19, the epithelium of the circumvallate papilla, most of which had developed into a few layers of cuboidal cells, had thickened considerably. The thickening on the basal side of the circular sulcus around the central bulge was particularly distinct, and the epithelium around the circumvallate papillae now consisted of a few layers of cuboidal cells. Each epithelial cell still had a large round nucleus. Periderm cells were more dispersely scattered than on E17 and had disappeared from most of the surface of the developing tongue.

Figure 3. Combinations of laser-scanning micrographs that show the localization of immunoreactive keratin 13, examined at 543 nm, and images obtained by confocal laser-scanning microscopy in transmission mode, which show the histology and cellular morphology of semi-ultrathin sections of the developing circumvallate papilla. A: vertical section from a fetus on E15; B: vertical section from a fetus on E17; C: vertical section from a fetus on E19; D1: section from a juvenile on P0, D2: higher magnification of a part of the epithelium in D1; E1: verticalsection from a juvenile on P7, E2: higher magnification of a part of the epithelium in E1; F1: vertical section from a juvenile on P14, F2: higher magnification of a part of the epithelium in F1. E: dorsal lingual epithelium; PE: periderm cell; CT: connective tissue; CP: connective tissue papilla; M: muscle; CS: circular sulcus; SD: salivary duct; TB: taste buds; arrow: connective tissue papillae. Scale bars: 10 m.

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There were still no recognizable signs of keratinization of the epithelium that formed the developing circumvallate papillae. Condensed connective tissue was widely distributed under the epithelium, and it was also densely packed in the central bulge of the circumvallate papillae. Myogenesis had progressed significantly beneath the connective tissue. No K13- or K14specific immunoreactivity was detectable on the epithelium around this region (Figure 3C). On P0, the epithelium of both the circumvallate papillae and the region close to the papillae became completely stratified and squamous. Epithelial cells located closest to the surface were now significantly flattened, while those from the most basal stratum were round to cuboidal and had a large rounded nucleus. The total thickness of the epithelium had increased in the entire region around the circumvallate papillae. Periderm cells were completely absent at this stage, but there were still no recognizable signs of keratinization of the epithelium in the developing circumvallate papillae. Welldeveloped, condensed connective tissue was widely distributed under the epithelium, and myogenesis had continued to progress beneath the connective tissue that included the papillary region, and the lingual muscle was significantly more developed than on E19. There was still no K13-specific immunoreactivity detectable in the epithelium in this region (Figure 3D1, 2). On P7, the total thickness of the stratified squamous epithelium of both the circumvallate papillae and the region close to the papilla increased, and the surface layer was more distinct than on P0. While cells from the basal epithelial layer still were cuboidal and had a large round nucleus, those from the superficial layer were of the squamous type. However, there were still no clear signs of keratinization in the circumvallate papillae. A few rudiments of taste buds were visible in the epithelium of the basal portion of the circular sulcus. Well-developed, condensed connective tissue was widely distributed under the epithelium, and was also densely packed in the central bulge of the circumvallate papilla. Myogenesis continued to progress beneath the connective tissue of the papillary region, and the lingual muscle developed significantly. K13-specific immunoreactivity was scattered from the upper intermediate layer to the surface layer of the epithelium of both the circumvallate papilla and the region close to the papilla (Figure 3E1, 2). On P14, the total thickness of the stratified squamous epithelium of both the circumvallate papilla and the region close to them was greater than on P7, and the surface layer was more distinct than on P7. Each cell on the basal side of the epithelium had a large round nucleus. In the surface layer, the cells and their nuclei were of the squamous type. However, there were still no

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recognizable signs of keratinization of the epithelium covering the circumvallate papilla. Many taste buds were visible on the epithelium of both the inner and the outer regions facing the basal portion of the circular sulcus. Well-developed, condensed connective tissue was widely distributed beneath the epithelium including the central bulge of the circumvallate papilla. Myogenesis continued to progress beneath the connective tissue covering the papillary region, and the development of the lingual muscle progressed significantly. K13-specific immunoreactivity was clearly distributed from the upper intermediate layer to the surface layer of the epithelium of both the circumvallate papilla and the region close to the papilla (Figure 3F1, 2).

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IMMUNOHISTOCHEMICAL LOCALIZATION OF CYTOKERATIN 14 DURING MORPHOGENESIS OF CIRCUMVALLATE PAPILLA No K14-specific immunoreactivity was detectable in the epithelium of the posterior side of the tongue that included the rudiment of the circumvallate papillae in fetuses on E15 (Figure 4A). On E17, no K14-specific immunoreactivity was detectable in the epithelium in this region (Figure 4B). On E19, no K14-specific immunoreactivity was detectable on the epithelium around this region (Figure 4C). On P0, K14-specific immunoreactivity was faintly recognizable in the basal cells of the epithelium of both the circumvallate papillae and the outer region of the papillae (Figure 4D1, 2). On P7, K14-specific immunoreactivity was more distinctly evident on the basal cells of the epithelium of both the circumvallate papilla and the outer region of the papilla. However, K14-specific immunoreactivity was not clearly visible on the basal cells of the epithelium in the deep basal region of the circular sulcus (Figure 4E1, 2). On P14, K14-specific immunoreactivity was more distinctly recognized and more widely distributed on the basal cells of the epithelium of both the circumvallate papillae and the region close to the papillae. However, K14specific immunoreactivity was indistinct in the basal cells of the epithelium facing the bottom of the circular sulcus, in which taste buds were clearly recognizable (Figure 4F1, 2).

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IMMUNOHISTOCHEMICAL LOCAKIZATION OF CYTOKERATIN 18 DURING PRENATAL DEVELOPMENT

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Immunoreactivity specific for K18 was detectable in the periderm cells, which were slightly swollen and covered the dorsal epithelium of the tongue on E13. The immunoreactivity was sparsely distributed throughout the cytoplasm in some, but not all, periderm cells, and there was none in the nuclei. The lingual epithelium was composed of one or two layers of the same type of cuboidal cell (Figure 5A).

Figure 4. Combinations of laser-scanning micrographs that show the localization of immunoreactive keratin 14, examined at 488 nm, and images obtained by confocal laser-scanning microscopy in transmission mode, which show the histology and cellular morphology of semi-ultrathin sections of the developing circumvallate papilla. A: vertical section from a fetus on E15; B: vertical section from a fetus on E17; C: vertical section from a fetus on E19; D1: vertical section from a juvenile on P0, D2: higher magnification of a part of the epithelium in D1; E1: vertical section from a juvenile on P7, E2: higher magnification of a part of the epithelium in E1; F1: vertical section from a juvenile on P14, F2: higher magnification of a part of the epithelium in F1. E: dorsal lingual epithelium; PE: periderm cell; CT: connective tissue; CP: connective tissue papilla; M: muscle; CS: circular sulcus; SD: salivary duct; TB: taste buds; arrow: connective tissue papillae. Scale bars: 10 m.

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Figure 5. Combination of laser-scanning micrographs that show the localization in the fetal tongue of immunoreactive keratin 18, examined at 633 nm, and DIC images that show the histology and cellular morphology of semi-ultrathin sections. A: sagittal section from a fetus on E13; B: sagittal section from a fetus on E15; C: sagittal section from a fetus on E17; D: sagittal section from a fetus on E19.E, Dorsal lingual epithelium; PE, periderm; CT, connective tissue; FP, rudiments of filiform papillae; KL, keratinized cell layer. Scale bars: 10 m.

Immunoreactivity specific for K18 was detectable in the periderm cells, which were slightly swollen and covered the dorsal epithelium of the tongue on E15. The immunoreactivity was sparsely distributed throughout the cytoplasm in some, but not all, periderm cells, and there was none in the nuclei. The lingual epithelium was composed of one or two layers of the same type of cuboidal cell (Figure 5B). On E17, the immunoreactivity specific for K18 was very distinct in the periderm cells, which had become swollen and elliptical and covered the dorsal epithelium of the fetal tongues. The pattern of distribution of the immunoreactivity was different from that on E15, and immunoreactivity specific for K18 was compactly distributed over almost the entire cytoplasm in most periderm cells. The periderm cells had become somewhat swollen and elliptical, and the lingual epithelium was composed of several layers of cuboidal cells (Figure 5C). No immunoreactivity specific for K18 was detectable on the dorsal epithelium of the tongue of fetuses on E19. The periderm cells had disappeared completely by E19 and that the lingual epithelium was composed of papillary and interpapillary cell columns. The appearance of a layer of keratinized cells was evident (Figure 5D).

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SPECIFICITY OF LOCATION OF CYTOKERATINS 13 AND 14 AND THEIR RELATION TO MORPHOGENETIC PROCESS OF FILIFORM PAPILLAE In adult mammals, the dorsal epithelium of the anterior part of the tongue is generally composed of regularly ordered columns of cells with different degrees of keratinization, namely, the anterior and posterior cell columns of the filiform papillae and the interpapillary cell columns (Wojcik et al., 2001; Jonker et al., 2004). As recently reported (Aoyagi et al., 2008; Iwasaki et al., 2003, 2006a), K13 and K14 are expressed with specific timing and in specific regions of the lingual epithelium during the morphogenesis of filiform papillae. K14 is located exclusively in the basal and suprabasal cells, and K13 is located in the intermediate layer of each interpapillary cell column. K14, in particular, is considered to be a marker of mitotically active cells in the oral epithelium. Thus, the localization of K13 and K14 has been clarified in the anterior part of the tongue during the morphogenesis of the filiform papillae, in parallel with the keratinization of the dorsal epithelium of the tongue (Aoyagi et al., 2008; Iwasaki et al., 2003, 2006a). K4 and K13 together are associated with differentiating suprabasal cells of the non-keratinized and para-keratinized oral epithelium (Morgan et al., 1987; Heyden et al., 1992; Barrett et al., 1998). Heyden et al. (1992) also detected the expression of K13 in suprabasal cells of the keratinized oral epithelium. The above-cited studies clearly indicate that a relationship exists between each subtype of cytokeratin and the differentiation of epithelial cells. Thus, the various cytokeratin subtypes might be important markers of cell differentiation of the oral epithelium during the development and growth of mammals. We detected immunoreactivity specific for K13 in the suprabasal cells of both the papillary and interpapillary cell columns on E19, P0 and P7, with immunoreactivity being more distinct in the interpapillary cell columns of the lingual epithelium than in the papillary cell columns. At these stages, each interpapillary cell column was restricted to a very narrow region and the keratinized layer of the papillary cell columns was not fully developed. In juveniles on P14, immunoreactivity specific for K13 was restricted exclusively to the deep intermediate layer only of the interpapillary cell columns, and immunoreactivity had disappeared from the papillary cell columns. At these stages, the width of interpapillary cell columns increased, and the orthokeratinized layer of the papillary cell column was well developed. Thus, the difference between the cells in the papillary and interpapillary cell columns

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was distinct after P14. Our results are very similar to those in previous reports (Morgan et al., 1987; Heyden et al., 1992; Barrett et al., 1998), which demonstrated that K13 is associated with differentiating suprabasal cells of only the non-keratinized and para-keratinized oral epithelium in adult mammals. However, we found immunoreactivity specific for K13 distributed over the entire deep intermediate layer that contained suprabasal cells. As shown by Dhouailly et al. (1989), K5 is expressed only in mitotically active basal keratinocytes of the lingual epithelium of adult rats. Furthermore, K5 and K14 are expressed simultaneously in mitotically active basal keratinocytes of all types of stratified squamous epithelium (Nelson & Sun, 1983). In the present study, K14 was not detectable in the lingual epithelium of rat fetuses at E15 and E17, when this epithelium is composed of a single or several layers of non-keratinized, cuboidal cells. K14 began to appear in basal and suprabasal keratinocytes of the lingual epithelium in parallel with the initiation of morphogenesis of filiform papillae and of keratinization of the epithelium, just before birth. Initially, immunoreactive K14 was restricted to the basal and suprabasal keratinocytes of the lingual epithelium along the connective tissue papillae, which penetrate into the center of the rudiments of filiform papillae. This immunoreactive area of the epithelium corresponds to the basal layer of the anterior and posterior parts of the filiform papillae. The basal and suprabasal layers, in which immunoreactivity specific for K14 was barely detected, corresponded to the interpapillar area, which was very narrow until the postnatal stage at P14. Our results suggest that the basal and suprabasal keratinocytes of the anterior and posterior parts of filiform papillae begin to proliferate with the initiation of morphogenesis of filiform papillae and of keratinization of the epithelium. Furthermore, after P14, the basal and suprabasal keratinocytes in the interpapillar area continue to be mitotically active to provide a supply of keratinocytes to the interpapillar cell columns after adequately proliferation of cells in the papillar area. In an earlier study, Iwasaki et al. (2003) detected immunoreactivity specific for K14 first on basal cells of the dorsal epithelium of fetus rats on E17. Immunoreactivity was conspicuous in juveniles on P14, and the immunoreactivity of the basal cells of the interpapillary cell columns was weaker than that of the cells in papillary cell columns. However, it is necessary to define exactly the developmental stage at which immunoreactivity specific for K14 appears if we are to clarify the relationship between the role of keratins and the morphogenesis of the lingual papillae. In the present study, K13 and K14 were undetectable in the lingual epithelium of fetuses on E15, when the epithelium was composed of one layer of non-

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keratinized, cuboidal cells. There were no rudiments of filiform papillae, and no evidence of ortho- and para-keratinization of the epithelium was recognizable at these stages. Both K13 and K14 began to appear in the lingual epithelium of fetuses on E17 or on E19, before birth, in parallel with the initiation of morphogenesis of filiform papillae. Thus, the first appearance of K13 and K14 seems to be related to the morphogenesis of filiform papillae rather than to the beginning of keratinization of the epithelium. Takaishi et al. (1998) detected K13 and K14 in the mucosal epithelium of the lower lip of mice 16.5 days post-coitus. Thus, they detected K13 and K14 at an earlier stage than we did in the present study. The difference might be due to differences between species and/or location, namely lingual epithelium versus lip mucosa.

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SPECIFICITY OF LOCATION OF CYTOKERATINS 13 AND 14 AND THEIR RELATION TO MORPHOGENETIC PROCESS OF CIRCUMVALLATE PAPILLA As indicated by the previous section of this chapter, Aoyagi et al. (2008) reported that K14 began to appear in basal cells of the dorsal epithelium of the lingual body in fetal rats on E17, in parallel with the initiation of invagination of the connective tissue papillae into the epithelium, which was composed of non-keratinized cuboidal cells. However, the morphogenesis of filiform papillae had not yet begun, and no keratinization of the epithelium was recognizable at this stage. Subsequently, and particular postnatally, K14specific immunoreactivity became distinct on the basal cells. K13-specific immunoreactivity appeared on E19 in the suprabasal cells of both the papillary and interpapillary cell columns of the lingual body, after the initiation of morphogenesis of filiform papillae. However, Iwasaki et al., (2011) has demonstrated that no K14 was detectable in the epithelium around the circumvallate papilla-forming region from E15 to E19. At these stages, the lingual epithelium of this region consisted of one or two layers of nonkeratinized cuboidal cells. Moreover, no K13 was detectable in the epithelium around the circumvallate papilla-forming area from E15 to E19. The differentiation of epithelial cells to the squamous type around the circumvallate papilla-forming area starts somewhat later than that around the filiform papilla-forming area, although the morphogenesis of circumvallate papillae starts earlier than that of filiform papillae. These phenomena indicate

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that the cellular differentiation of the lingual epithelium might be synchronized with its squamous differentiation. Furthermore, the morphogenesis of the gustatory and non-gustatory papillae might be controlled by a mechanism that is completely different from the differentiation of the epithelial cell.

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SIGNIFICANCE OF CYTOKERATIN 18 LOCALIZED IN PERIDERM CELLS OF THE TONGUE DURING THE EMBRYONIC DVELOPMENT The periderm cells were already present on the lingual epithelium of fetal rats on E15, when these cells were very thin and sparsely scattered. Subsequently, these cells increased in volume and then a layer of periderm cells formed a close-fitting cover over the entire surface of the epithelium. These periderm cells had disappeared completely by E19; their disappearance parallels the squamous stratification of the lingual epithelium and the appearance of the keratinized layer (Iwasaki et al., 2006b). The changes in the periderm cells were very similar to those observed in human skin and in the skins of other experimental animals. According to McGowan and Coulombe (1998), periderm cells can be recognized in the fetal skin of mice from E14.5 to E16.5, but are absent at birth. The timing of the appearance and disappearance of periderm cells in the fetal skin of mice seems to coincide developmentally with that on the lingual epithelium of fetal rats. The morphological changes in the periderm cells of the lingual epithelium of rats revealed a process similar to that in human fetal skin, as reported by Holbrook and Odland (1975). Both K8 and K18 are found in the periderm cells of developing human skin (Moll et al., 1982; Dale et al., 1985; van Muijen et al., 1987). The presence of K18, which is specific for the simple epithelium, is also demonstrated in the periderm of the lingual epithelium of fetal rats. There seem to be no significant differences in the pattern of distribution of K18 in periderm cells between fetal skin and the epithelium of the tongue. Van Muijen et al. (1987) reported that K4 and K13 were also expressed in the periderm of the skin of human fetuses. No information is available, to our knowledge, about the temporal details of the expression of keratins other than K18 in the oral epithelium.

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CONCLUSION As conclusion of this chapter, the patterns of immunoreactivity of K13, K14 and K18 significantly differed as the morphogeneses of the tongue progressed. The significance of appearance and localization of each keratin has been literally discussed in relation to morphogenetic mechanism of the lingual epithelium.

ACKNOWLEDGEMENTS The authors are grateful to Professor M. Tsuchimochi, Department of Oral and Maxillofacial Radiology, The Nippon Dental University School of Life Dentistry at Niigata, for his encouragement and helpful support. This research was supported by Research Promotion Grants (no. NDUF-11-10) from the Nippon Dental University.

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REFERENCES AhPin, P., Ellis, S., Arnott, C. & Kaufman, M. H. (1989). Prenatal developmental and innervation of the circumvallate papilla. Journal of Anatomy, 162, 33-42. Albers, K. & Fuchs, E. (1992). The molecular biology of intermediate filament protein. International Review of Cytology, 134, 243-279. Aoyagi, H., Asami, T., Yoshizawa, H., Wanichanon, C. & Iwasaki, S. (2008). Newly developed technique for dual localization of keratins 13 and 14 by fluorescence immunohistochemistry. Acta Histochemica, 110, 324-332. Barrett, A. W., Selvarajah, S., Franey, S., Wills, K.-A. & Berkovitz, B. K. B. (1998). Interspecies variations in oral epithelial cytokeratin expression. Journal of Anatomy, 193, 185-193. Berkovitz, B. K. B., Whatling, R., Barrett, A. W. & Omar, S. S. (1997). The structure of bovine periodontal ligament with special reference to the epithelial cell rests. Journal of Periodontology, 68, 905–912. Dale, B. A., Holbrook, K. A., Kimball, J. R., Hoff, M. & Sun, T.-T. (1985). Expression of epidermal keratins and filaggrin during human fetal skin development. Journal of Cell Biology, 101, 1257–1269.

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Dhouailly, D., Xu, C., Manabe, M., Schermer, A. & Sun, T.-T. (1989). Expression of the hair-related keratin in a soft epithelium: subpopulation of human and mouse dorsal tongue keratinocytes express keratin markers for hair-, skin- and esophageal-types of differentiation. Experimental Cell Research, 181,141-158. Heyden, A., Huitfeldt, H. S., Koppang, H. S., Thrane, P. S., Bryne, M. & Brandtzaeg, P. (1992). Cytokeratins as epithelial differentiation markers in premalignant and malignant oral lesions. Journal of Oral Pathology and Medicine, 21, 7-11. Hirao, T., Saga, T., Kusukawa, J. & Yamaki, K. (2007). Angiogenesis and developmental expression of vascular endothelial growth factor in rat lingual papillae. Kurume Medical Journal, 54, 9-24. Holbrook, K. A. & Odland, G. H. (1975). The fine structure of developing human epidermis: light, scanning, and transmission electron microscopy of the periderm. Journal of Investigative Dermatology, 65, 16–38. Iwasaki, S., & Aoyagi, H, (2010). Fluorescence immunohistochemistry in combination with DIC and transmission images of confocal LSM for studies of semi-ultrathin specimens of epoxy resin-embedded samples. In: A. Méndez-Vilas, & J. D az (Eds.), Microscopy: Science, Technology, Applications and Education Vol. 3 (Microscopy Series No.4, pp. 20962102). Badajoz, Spain: Formatex. Iwasaki, S., & Aoyagi, H, (2011a). Fluorescence immunohistochemistry in combination with differential interference contrast microscopy for studies of semi-ultrathin specimens of epoxy resin-embedded samples. In: H. Chiarini-Garcia, & R. C. N. Melo (Eds.), Light Microscopy Methods and Protocols (Methods in Molecular Biology, Vol. 689, pp. 229-240). New York, USA: Humana Press, c/o Springer Science + Business Media. Iwasaki, S., & Aoyagi, H, (2011b). Fluorescence immunohistochemistry by confocal laser-scanning microscopy for studies of semi-ultrathin specimens of epoxy resin-embedded samples. In: C.-C. Wang (Ed.), Laser Scanning, Theory and Applications (pp. 195-214). Rejeka, Croatia: InTech. Iwasaki, S., Yoshizawa, H. & Kawahara, I. (1996). Study by scanning electron microscopy of the morphogenesis of three types of lingual papilla in the mouse. Acta Anatomica, 157, 41-52. Iwasaki, S., Yoshizawa, H. & Kawahara, I. (1997). Study by scanning electron microscopy of the morphogenesis of three types of lingual papilla in the rat. The Anatomical Record, 247, 528-541.

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Iwasaki, S., Okumura, Y. & Kumakura, M. (1999a). Ultrastructural study of the relationship between the morphogenesis of filiform papillae and the keratinization of the lingual epithelium in the mouse. Cells Tissues Organs, 165, 91-103. Iwasaki, S., Yoshizawa, Y. & Kawahara, I. (1999b). Ultrastructural study of the relationship between the morphogenesis of filiform papillae and the keratinization of the lingual epithelium in the rat. Journal of Anatomy, 195, 27-38. Iwasaki, S., Aoyagi, H. & Yoshizawa, H. (2003). Immunohistochemical detection of the expression of keratin 14 in the lingual epithelium of rats during the morphogenesis of filiform papillae. Archives of Oral Biology, 48, 605-613. Iwasaki, S., Yoshizawa, H. & Aoyagi, H. (2006a). Immunohistochemical expression of keratins 13 and 14 in the lingual epithelium of rats during the morphogenesis of filiform papillae. Archives of Oral Biology, 51, 416426. Iwasaki, S., Aoyagi, H. & Asami, T. (2006b). Expression of keratin 18 in the periderm cells of the lingual epithelium of fetal rats: visualization by fluorescence immunohistochemistry and differential interference contrast microscopy. Odontology, 94, 64-68. Iwasaki, S., Aoyagi, H. & Yoshizawa, H. (2011). Localization of keratins 13 and 14 in the lingual mucosa of rats during the morphogenesis of circumvallate papillae. Acta Histochemica, 113, in press. Jitpukdeebondintra, S., Chai, Y. & Snead, M. L. (2002). Developmental patterning of the circumvallate papilla. International Journal of Developmental Biology, 46, 755-763. Jonker, L., Kist, R., Aw, A., Wappler, I. & Peters H. (2004). Pax9 is required for filiform papilla development and suppresses skin-specific differentiation of the mammalian tongue epithelium. Mechanisms of Development, 121, 13-22. Lersch, R. & Fuchs, E. (1988). Sequence and expression of a type II keratin, K5, in human epidermal cells. Molecular and Cellular Biology, 8, 486493. Lu, D.-P., Tatemoto, Y., Kimura, T. & Osaki, T. (2002). Expression of cytokeratins (CKs) 8, 13 and 18 and their mRNA in epithelial linings of radicular cysts: implication for the same CK profiles as nasal columnar epithelium in squamous epithelial lining. Oral Diseases, 8, 30-36.

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Mayor, H. D., Hampton, J. C. & Rosario, B. (1961). A simple method for removing the resin from epoxy-embedded tissue. Journal of Cell Biology 9, 909-910. McGowan, K. M. & Coulombe, P. A. (1998). Onset of Keratin 17 Expression Coincides with the Definition of Major Epithelial Lineages during Skin Development. Journal of Cell Biology, 19, 469–486 Moll, R., Moll, I. & Wiest, W. (1982). Changes in the pattern of cytokeratin polypeptides in epidermis and hair follicles during skin development. Differentiation, 23, 170–178. Moll, R., Dhouailly, D. & Sun, T.-T. (1989). Expression of keratin 5 as a distinctive feature of epithelial and biphasic mesotheliomas. Virchows Archiv B Cell Pathology, 58, 129-145. Morgan, P. R., Leigh, I. M., Purkis, P. E., Gardner, I. D., van Muijen, G. N. P. & Lane, E. B. (1987). Site variation in keratin expression in human oral epithelia - an immunocytochemical study of individual keratins. Epithelia, 1, 31-43. Nelson, W. & Sun, T.-T. (1983). The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies. Journal of Cell Biology, 97, 244-251. Okazaki, M., Yoshimura, K., Suzuki, Y. & Harii, K. (2003). Effects of subepithelial fibroblasts on epithelial differentiation in human skin and oral mucosa: heterotypically recombined organotypic culture model. Plastic and Reconstructive Surgery, 112,784-792. Roop, D. R., Huitfeldt, H., Kilkenny, A. & Yuspa, S. H. (1987). Regulated expression of differentiation-associated keratins in cultured epidermal cells detected by monospecific antibodies to unique peptides of mouse epidermal keratins. Differentiation, 35, 143-150. Shi, S.-R., Key, M. E. & Karla, K. L. (1991). Antigen retrieval in formalinfixed, paraffin-embedded tissues: an enhanced method for immunohistochemical staining based on microwave heating of tissue sections. Journal of Histochemistry and Cytochemistry, 39, 741-748. Steven, A. C. (1990). Intermediate filament structure. In: R. D. Goldman, & P. M. Steinert (Eds.), Cellular and molecular biology of intermediate filaments (pp. 233-263). New York, USA: Plenum Press. Takaishi, M., Takaya, Y., Kuroki, T. & Huh, N.-h. (1998). Isolation and characterization of a putative keratin-associated protein gene expressed in embryonic skin of mice. Journal of Investigative Dermatology, 111, 128-132.

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van Muijen, G. N. P., Warnaar, S. O. & Ponec, M. (1987). Differentiationrelated changes of cytokeratin expression in cultured keratinocytes and fetal, newborn, and adult epidermis. Experimental Cell Research, 171, 331-345. Wojcik, S. M., Longley, M. A. & Roop, D. R. (2001). Discovery of a novel murine keratin 6 (K6) isoform explains the absence of hair and nail defects in mice deficient for K6a and K6b. Journal of Cell Biology, 154, 619-630.

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In: Keratin: Structure, Properties and Applications ISBN 978-1-62100-336-6 Editors: Renke Dullaart et al. pp. 89-111 ©2012 Nova Science Publishers, Inc.

Chapter 4

WATER SORPTION OF HUMAN KERATINIZED FIBERS: EFFECT OF WOOL KERATIN PROTEINS AND PEPTIDES Clara Barba1, Meritxell Martí1, Alisa Roddick-Lanzilotta2, Albert M. Manich1, Josep Carilla1, Jose L. Parra1 and Luisa Coderch1 Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

1

IQAC (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain Agresearch, Private Bag 4749, Christchurch, New Zealand

2

INTRODUCTION Water produces changes in the properties of human keratinized fibers, such as hair and nails, and is therefore of fundamental interest. Water diffusivity in wool, horn, and the corneocyte phase of stratum corneum considerably increases with increased water content in the tissue [1]. However, water sorption of wool is well documented [2] whereas there are few data on human hair and nails. Human hair is a keratinized fiber which is divided into three structural zones: medulla, cortex, and cuticle [3]. Reactive cosmetic treatment of hair often impairs the fiber structure. The resulting damage has an adverse effect on hair water absorption at ambient humidities and leads to an increase in swelling or to liquid retention on wetting [4]. Like hair, the nail plate consists of hard keratin and lipids.

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The nail plate is an indicator of overall health [5]. The degree of hydration is thought to be the most important factor influencing the physical properties of the nail [6]. The use of nail care products and procedures to beautify and groom the nails is extremely common. Unfortunately, when improperly used, nail cosmetics can lead to nail diseases [7]. Brittleness in the nail may be caused by trauma, such as repeated wetting and drying, repeated exposure to detergents and water, and excessive exposure to harsh solvents, such as those found in nail polish remover [8]. There is a growing consumer trend toward natural actives that have the potential to maintain hair and nails with a healthy, youthful appearance [9]. Wool proteins are mild, natural, biodegradable and sustainable with multiple functionalities and have potential for use in the personal care and detergent markets [10, 11]. In this work the effect on hair and nails of two keratin proteins isolated from wool has been investigated, an intact keratin intermediate filament protein extract (K-protein) and a low molecular weight keratin peptide from intermediate filament proteins (K-peptide). Both keratins have cystine present in the active S-sulphonated form. This unique chemistry might enable the keratin peptide and protein to reform disulphide bonds in damaged hair and nails and to replenish the natural disulfides bonds of the hair fibres, directly affecting their properties. The determination of water sorption isotherm by isothermally applying discrete, cumulative humidity changes involves dynamic and static aspects from which diffusion coefficients and equilibrium water contents have been deduced. Time/absorption isotherms provide a complete description of the absorption phenomenon under particular conditions such as initial regain of the sample, temperature and relative humidity [12]. A number of equations have been proposed for modeling sorption isotherms, being in our case the GAB (Guggenheim-Anderson-deBoer) equation successfully applied [13]. The main aim of this work is to apply two different S-sulfonated wool keratins, K-protein and K-peptide to untreated hair and nail and to hair and nail subjected to different chemical cosmetic treatments. The moisture absorption/desorption isotherms curves for untreated and treated hair and nail and the kinetics of these processes are studied in this work. The effectiveness of these keratin ingredients at restoring the water sorption characteristics of the keratin tissues is determined.

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METHODOLOGY Materials Acetone and Hydrogen Peroxide 30% was supplied by Merck (Darmstadt, Germany), Ammonium Persulfate was supplied by Amresco (Ohio) and Keratin peptide (MW 0.999). The values obtained are shown in Table II. Monolayer capacity values and energy constant, which are much higher for human hair related to human nails, confirm the differences in water sorption between these two human keratinized tissues. Estimation of the kinetics of the moisture uptake and loss is a good strategy for obtaining more detailed information about the structure integrity of a given sample. There were significant differences in the rate at which human hair and human nails reached equilibrium. Curves are depicted in Figure 2. Nails

0,31

Hair

0,21 0,16 0,11 0,06 0,01 -0,04 0

10

20

30

40

50

60

70

80

90

100

Relative Hum idity (%)

Hair Moisture absorption/desorption at the equilibirum (%)

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g Water/g Sample

0,26

8,80 8,00 7,20 6,40 5,60 4,80 4,00 3,20 2,40 1,60 0,80 0,00

nails

15 25 35 45 55 65 75 85 95 85 75 65 55 45 35 25 15

5

Relative Hum idity (%)

Figure 2. Moisture uptake as a function of time and time of half absorption/desorption in min for human hair and nails.

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Results show that nails take much longer to reach equilibrium, especially in the absorption process, than human hair. The nail plate is composed of highly compacted keratinized cells [31], making it difficult for water to be absorbed and desorbed through the nail structure. This low permeability is more marked at low humidity values. Differences in the keratinized structure of hair and nails are also confirmed when the apparent diffusio coefficients (DA) (Table II) and the time of half absorption/desorption (t1/2) (Figure 2) are evaluated. The lower nail permeability with respect to hair fibers is demonstrated by a considerable decrease in its apparent diffusion coefficient and by significantly higher values of the t1/2 in all the RH steps, mainly at low RH values.

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TREATED HAIR SORPTION MEASUREMENTS The hair sample was subjected to a common cosmetic treatment (bleaching) which is based on an oxidation process that affects the keratin structure of the hair [32]. Then bleached hair was treated either with the keratin protein or peptide [33]. Furthermore, a study was performed to evaluate the restorative capacity of the wool keratin samples when applied on nails previously deteriorated with acetone. Acetone treatment was carried out due to the association of this product with nail brittleness and discoloration [34]. Sorption isotherms of virgin and bleached hair were evaluated and are shown in Figure 3. Differences can be seen between the sorption isotherms of untreated and bleached hair samples. Evaluation of the moisture absorbed and desorbed at different relative humidity demonstrates that the bleach treatment damages the hair fibers, slightly reducing their moisture sorption capacity with respect to the untreated ones. This decrease in the moisture sorption for the bleached hair may be attributed to the chemical treatment which affects mainly the fiber surface and slightly decreases its ability to retain water. Moisture sorption isotherms for the bleached hair after being treated with either the keratin protein or keratin peptide are also shown in Figure 3. It can be seen that application of both keratin types lead to higher levels of moisture absorbed and desorbed across the range of relative humidity studied, when compared to the nontreated bleached sample. This change in the moisture absorbed and desorbed, reaching values similar to that of the virgin hair sample, indicates a possible restoration of the fibers due to the keratin treatments. This is in accordance with an earlier study where an improvement of the moisture content of bleached hair due to the application of wool keratin peptide and protein was demonstrated [35].

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Water Sorption of Human Keratinized Fibers

Figure 3. Water sorption isotherms for virgin (UT) and bleached hair, and bleached hair treated with the keratin samples.

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Table III. Maximum moisture regain, GAB monolayer capacity (Wm), GAB energy constant (Cg), GAB energy constant (K) and GAB determination coefficient (R2) for Virgin (UT) and Bleached (B) human hair, and Bleached hair treated with both keratin samples (B-Kpep and B-Kpro)

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UT B B-Kpep B-Kpro

Regain at 95% RH (%) 25.87 24.60 24.69 25.09

Wm (%) 9.875 8.537 10.682 9.717

Cg 10.956 11.198 9.361 10.905

K 0.657 0.688 0.594 0.650

R2 0.999 0.999 0.999 0.999

The regression of the experimental desorption isotherms data by the GAB model gives values of the monolayer capacity (Wm) and the energy constants (Cg and K) [30]. A good fit of the GAB model to the uptake and desorption data was achieved (R2>0.999), the values obtained are shown in Table III. Results show that both keratin treatments improve the water sorption characteristics of bleached fibers. There is an increase in the amount of water absorbed in the monolayer when the bleached hair sample is subjected to the wool keratin treatments reaching higher monolayer capacity values than that obtained for the virgin hair sample, although for the keratin peptide the bonding is weaker as indicated by lower values of the energy constant. This modification indicates an increase of water binding sites in hair which can be attributed to the presence of the keratin peptide and the keratin protein in/on the hair fibers. Energy constant K results, which show the amount of water on the secondary upper sorbed layers, demonstrate the deterioration effect of the bleach treatment, which may lead to a more porous fiber with an increase of the amount of water reaching the upper layers. Furthermore, there is a decrease of the value of the energy constant K for the keratin treated fibers thus indicating a possible restoration of the fiber integrity due to the keratin treatment. The rate at which equilibrium is achieved is a good indicator of the sample condition. The kinetics of moisture sorption was evaluated for all hair samples studied and curves are shown in Figure 4. Virgin hair reaches equilibrium more slowly than bleached hair. Virgin hair is more hydrophobic than bleached hair because has less anionic surface groups, thus has less driving force for water adsorption. Evaluation of the kinetics of moisture uptake and loss at each RH step used to build up the isotherm demonstrate that both keratin treatments altered

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the rate at which the bleached hair reached equilibrium moisture content at any given RH. When the bleached hair is subjected to either wool keratin treatment the time to reach equilibrium is increased, on both the absorption and desorption cycle. Thus, the wool keratins may limit the quantity of moisture absorbed when hair is exposed to environments with high levels of humidity over short time periods, therefore preventing styling problems associated with limp over-hydrated hair.

Figure 4. Moisture uptake for virgin (UT) and bleached (B) human hair, and bleached hair treated with both keratin samples (B-Kpep and B-Kpro) as a function of time.

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In addition, the wool keratin samples increased the absolute amount of water absorbed into bleach-damaged hair over a wide range of moderate RH conditions which may prevent problems associated with dry hair, such as poor texture, increased tendency for split-ends, and poor tensile/bending strength [36]. The moisture diffusion kinetics for the hair fibers have also been evaluated and the apparent diffusion coefficients (DA) and the total time to reach equilibrium (tT) have been calculated as detailed in the experimental section. They are summarized in Table IV. The time of half absorption/desorption (t1/2) for each RH step has also been calculated, results are shown in Figure 5. Table IV. Apparent diffusion coefficient (DA) and total time to reach equilibrium (tT) for virgin and bleached hair and bleached hair treated with both keratin samples (mean value ± SD).

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UT B B-Kpep B-Kpro

DA (min-1) 0.0160 ± 0.007 0.0203 ± 0.010 0.0179 ± 0.009 0.0173 ± 0.008

tT (min) 3204,22 2895,12 2906,21 3042,05

Figure 5. Time of half absorption/desorption in min for virgin (UT) and bleached (B) human hair and beached hair treated with both keratin samples (B-Kpep and B-Kpro) as a function of time.

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There is an inverse relationship between the time parameter and the diffusion coefficient, i.e. a higher time is needed to reach equilibrium for fibers with small water permeability and therefore a small diffusion coefficient. Deterioration of hair due to bleaching is characterized by a marked decrease in the time needed to reach equilibrium and by an increase in its water permeability. Results demonstrate that when the keratin samples are applied to bleached hair they appear to induce an improvement in the fiber integrity, as evidenced by an increase on the t1/2 on the majority of the RH steps (Figure 5) and on the total time the sample needs to reach equilibrium (Table IV). In addition there is a decrease in the apparent diffusion coefficient demonstrating a decrease of the fiber water permeability. Furthermore, it can be seen that this decrease in the water permeability is much more marked for hair treated with the higher molecular weight wool keratin protein. As described in previous work [35], the keratin protein has the ability to form a cohesive film in the hair surface; this keratin coating may act to prevent/decrease water diffusion through the hair fibers. The overall effect of the K-protein treatment is to restore the damaged hair to its original water permeability levels.

TREATED NAILS SORPTION MEASUREMENTS A study was performed to evaluate the restorative capability of the wool keratin samples when applied on nails previously deteriorated with acetone. The water sorption isotherms of untreated nails (UT), acetone damaged nails (Ac) and acetone damaged nails treated with the keratin peptide (AcKpep) or the keratin protein (Ac-Kpro) were determined and are visualized in Figure 6. Comparison of the isotherms shows only minor differences, the most important ones being for the keratin protein treated nails which present a slight diminution in the moisture absorbed and desorbed in all the RH steps. It is known that the nail plate becomes soft and tends to be double layered when its water content exceeds 20% [37-39]. Furthermore, in previous work it has been demonstrated that when nails are submitted to a deterioration treatment an increase on the nail water content is found [14]. Minimum differences on the monolayer capacity and energy constants values were found between the different nail treatments, indicating that no clear conclusions can be obtained from the GAB results.

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Figure 6. Water sorption isotherms for nails non-treated (UT) deteriorated with acetone (Ac) and treated with both keratin samples after acetone deterioration (Ac-Kpro and Ac-Kpep).

Table V. Maximum moisture regain, GAB monolayer capacity (Wm), GAB energy constant (Cg), GAB constant K and GAB determination coefficient (R2) for nails non treated (UT), deteriorated with acetone (Ac) and treated with both keratin samples after acetone deterioration (Ac-Kpro and Ac-Kpep)

UT Ac Ac-Kpep Ac-Kpro

Regain at 95%RH (%) 23.13 22.28 22.99 22.76

Wm (%)

Cg

K

R2

7.024 7.098 6.929 7.177

8.1862 7.6514 7.4160 6.1055

0.7476 0.7351 0.7416 0.7513

0.999 0.999 0.999 0.999

As in the hair study modifications of the rate at which the nails reach equilibrium were studied (Figure 7). Evaluation of the rate curves didn‟t show much difference between untreated nails and nails damaged with acetone, possibly indicating that the acetone treatment had not been very aggressive.

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Figure 7. Moisture uptake for nails non treated (UT), deteriorated with acetone (Ac) and treated with both keratin samples after acetone deterioration (Ac-Kpro and AcKpep) as a function of time.

Application of the keratins to acetone damaged nails appears to alter the rate at which the nail reached equilibrium moisture content at any given RH. When acetone damaged nails are subjected to either wool keratin treatment the time to reach equilibrium is increased, on both the absorption and desorption cycles. This behaviour is also found when evaluating the total time needed to reach equilibrium, shown in Table VI.

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Table VI. Apparent diffusion coefficient (DA) and total time to reach equilibrium (tT) for nails non treated (UT), deteriorated with acetone (Ac) and treated with both keratin samples after acetone deterioration (Ac-Kpro and Ac-Kpep) (mean value ± SD)

UT Ac Ac-Kpep Ac-Kpro

DA (min-1) 0.0043 ± 0.004 0.0047 ± 0.005 0.0042 ± 0.005 0.0036 ± 0.004

tT (min) 4885.24 4894.98 5072.78 5322.98

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It can be seen that the keratin treatments significantly increase this time parameter, reaching higher values than for the untreated nails, demonstrating thus the structure of the nail recovery previously damaged with acetone. The apparent diffusion coefficients of the nail samples were also evaluated (Table VI). The results show that the acetone treatment deteriorated the nail structure with an increase in its diffusion coefficient thus an increase in the water permeability. For keratin treated nails, there is a clear decrease in the values of diffusion coefficient indicating a decrease on the water permeability of this nails samples.

Figure 8. Time of half absorption/desorption in min for nails non-treated (UT) deteriorated with acetone (Ac) and treated with both keratin samples after acetone deterioration (Ac-Kpro and Ac-Kpep) as a function of time.

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Water Sorption of Human Keratinized Fibers

It can be seen that this decrease in the water permeability is much more marked for nails treated with the higher molecular weight wool keratin protein Furthermore, in the evaluation of the time of half absorption and desorption in each step of relative humidity it can be seen that, acetone damaged nails treated with both keratin sample have longer times to reach equilibrium in the majority of the RH steps evaluated (Figure 8). These results demonstrate the improvement of the nail structure integrity, previously deteriorated with acetone, thus the restorative capacity of both wool keratin samples.

WOOL PROTEIN AND PEPTIDE EFFECT ON HAIR AND NAILS

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Comparison of results for hair and nail leads to relevant differences on the behaviour of both human keratinized tissues (Table VII). Hair samples present higher water sorption when compared with nails as demonstrated by the maximum regain and monolayer capacity values. Table VII. Maximum moisture regain, GAB monolayer capacity (Wm), apparent diffusion coefficient (DA) and total time to reach equilibrium (tT) for virgin and bleached hair and bleached hair treated with both keratin samples and for nails non-treated (UT), deteriorated with acetone (Ac) and treated with both keratin samples (Ac-Kpro and Ac-Kpep)

HAIR

NAILS

UT B B-Kpep B-Kpro UT Ac Ac-Kpep Ac-Kpro

Regain at 95%RH (%) 25.87 24.60 24.69 25.09 23.13 22.28 22.99 22.76

Wm (%)

DA (min-1)

tT (min)

9.875 8.537 10.682 9.717 7.024 7.098 6.929 7.177

0.0160 ± 0.007 0.0203 ± 0.010 0.0179 ± 0.009 0.0173 ± 0.008 0.0043 ± 0.004 0.0047 ± 0.005 0.0042 ± 0.005 0.0036 ± 0.004

3204,22 2895,12 2906,21 3042,05 4885.24 4894.98 5072.78 5322.98

In addition, there is a significant difference in the rate at which hair and nails reach equilibrium as shown by the values obtained for the tT. It can be clearly seen that nails take much longer to reach equilibrium than human hair.

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The nail plate is composed of highly compacted keratinized cells [31], making it difficult for water to be absorbed and desorbed through the nail structure. Finally, when evaluating the values for the diffusion coefficients it can be demonstrated that the nail has lower permeability with respect to hair fibers evidenced by a much lower apparent diffusion coefficient. However, it is important to emphasize that application of both wool keratin samples on human hair and nails lead to similar results, with a decrease on the water permeability (lower water diffusion). Thus an improvement of the keratinized structures can be deduced, and this effect is much more pronounced for the wool keratin protein treatment. As described in a previous study [35], the keratin protein has the ability to form a cohesive film on the keratinized tissue surface; this keratin coating may act to prevent/decrease water diffusion through the hair and nails fibers.

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CONCLUSIONS A lower moisture regain and a much lower diffusion coefficient were obtained for human nails with respect to human hair. Modification of hair and nails water sorption properties following a conventional cosmetic treatment has been demonstrated with a reduction in the hair moisture sorption capacity for bleached hair. Permeability, directly related to the diffusion coefficient, tends to increase with both cosmetic treatments on hair and nails. Application of the wool keratin peptide and protein was demonstrated to improve the moisture absorption/desorption capacity of damaged hair and nails. Keratin peptides and proteins were shown to restore the integrity of chemically damaged human keratinized tissues, inducing a decrease in its diffusion coefficient, which in turn indicates improved (decreased) permeability of hair and nails. This is more marked for the keratin protein due to its fiber coating properties.

REFERENCES [1] [2]

Gunt, H.B. and Kasting, G.B., Equilibrium water sorption characteristics of the human nail. Int. J. Cosmet. Sci. 29 (2007) 487. Watt, I.C., Sorption of water vapour by keratins, Macromol. Chem. 18 (1980) 169–245.

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Water Sorption of Human Keratinized Fibers [3] [4] [5]

[6]

[7] [8] [9] [10]

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[11]

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Ruetsch, S.B., International wool textile research conference (1995). Wolfram, L.J., Human hair: a unique physicochemical composite. J Am Acad Dermatol., 48 (2003) 106–14. Egawa, M., Ozaki, Y., and Takahashi, M., In vivo measurements of water content of the fingernail and their seasonal change, Skin Res. Technol. 12 (2006) 126–132. Van de Kerkhof, P.C., Pasch, M.C., Scher, R.K., Kerscher, M., Gieler, U., Haneke, E., and Fleckman, P., Brittle nails syndrome: a pathogenesis-based approach with a proposed granding system, J. Am. Acad. Dermatol. 53 (2005) 644–651. Gawkrodger, D.J., Dermatology: An Illustrated Colour Text, Edinburgh, NY, Churchill Livingstone, 1992, 64. Fitzpatrick, B., et al, Dermatology in General Medicine, New YorK, McGraw-Hill, 1993 Vol I, 699. Gleason-Allured, J.B., The blossoming of naturals. Part 1: the buying public. Cosmet Toilet. 121 (2006) 54–5. Roddick-Lanzilotta, A., Kelly, R., Scott, S. and Chahal, S., New keratin isolates: actives for natural hair protection, J Cosmet Sci 58 (2007) 40511. Barba, C., Mendez, S., Roddick-Lanzilotta, A., Kelly, R., Parra, J.L., and Coderch, L., Cosmetic effectiveness of topically applied hydrolysed keratin peptides and lipids derived from wool, Skin Res Technol 14 (2008) 243-8. Manich, A.M., Maldonado, F., Carrilla, J., Catalina, M., and Marsal, Moisture adsoprtion/desorption kinetics of bovine hide powder, A J. Soc. Leath. Technol. Chem. 94 (2010) 15-20. Ansari, F., and Majzoobi, M., Effect of glycerol on the moisture sorption isotherms of figs, J of Food Engineering 93 (2009) 468-473. Barba, C., Martí, M., Manich, A.M., Carilla, J., Parra, J.L., Coderch, L.: Water absorption/desorption of human hair and nails. Thermochim Acta 2010: 503-504: 33-39. Keis, K., Huemmer, C.L., and Kamath, Y.K., Effect of oil films on moisture vapour absorption on human hair, J Cosmet Sci., 58 (2007) 135-145. Tonon, R., Baroni, C., Brabet, O., Guibert, D., Pallet, M., and Hubinger, J., Water sorption and glass transition temperature of spray dried açai (Euterpe oleracea mart) juice, Food Eng, (2009).

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[17] Zografi, G., Kontny, M., Yang, A., and Brenner, G., Surface area and water sorption of microcrystalline cellullose, Int. J. Pharm. 18 (1984) 99–116. [18] Jonqueries, A., and Fane, A., Modified BET models for modeling water sorption in hydrophilic glass polymers and systems deviating strongly from ideality, J. Appl.Polym. Sci. 67 (1998) 1415–1430. [19] Lewicki, P., The applicability of the GAB model to food water sorption isotherms, Int. J. Food Sci. Technol. 32 (1997) 553–557. [20] Pradas, M., Samchez, S., Ferrer, G., and Ribelles, J., Thermodynamics and statistical mechanics of multilayer adsorption, J. Chem. Phys. 121 (2004) 8524–8531. [21] Brunauer, S., Emmet, P., and Teller, E., Adsorption of gases in multimolecular layers, J. Am. Chem. Soc. 60 (1938) 309–319. [22] Anderson, R., Modifications of the Brunauer, Emmet and Teller equation, J. Am. Chem. Soc. 68 (1946) 686–691. [23] Manich, A.M., Maldonado, F., carilla, J., Catalina, M., and Marsal, A., Moisture sorption/desorption of collagen, J Am Leath Chem As (2010) (In press) [24] Arslan, N., and Togrul, H., The fitting of various models to water sorption isotherms of tea stored in a chamber under controlled temperature and humidity, .J. Stored Prod. Res. 42 (2006) 112–135. [25] Vickerstaff, T., The Physical Chemistry of Dyeing, Oliver and Boid, London, 1954. [26] Heldman, D.R., Hall, C.W., Hedrick, T.I., Vapor equilibrium relationships of dry milk. J Dairy Sci. 48 (1965) 845-852. [27] Al-Muhtaseb, A.H., McMinn, W.A., Magee, T.R.A, Water sorption isotherms of starch powders Part 1: mathematical description of experimental data, J Food Eng. 61 (2004) 297-307. [28] Wortmann, F.J., Stapels, M., Elliott, R., Chandra, L., The effect of water on the glass transition of human hair, Biopolymers. 81 (2006) 371-5. [29] Barba, C., Méndez, S., Martí, M., Parra, J.L., Coderch, L.: Water content of hair and nails. Thermochim Acta 494 (2009) 136-140. [30] Timmermann, E.O., Multilayer sorption parameters: BET or GAB values? Colloids Surf A. 220 (2003) 235–260. [31] de Berker, D.A., Andre, J., and Baran, R., Nail biology and nail science, Int. J. Cosmet. Sci. 29 (2007) 241–275. [32] Zvik C., The science of hair care. New York: Marcel Dekker Inc., 1986. [33] Barba, C., Martí, M., Roddick-Lanzilotta, A., Manich, A., Carilla, J., Parra, J.L., Coderch, L.: Water sorption of nails treated with wool

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Water Sorption of Human Keratinized Fibers

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keratin proteins and peptides. J Therm Anal Calorim 2011: 104: 323329. Kechijian, P., Nail polish removers. Are they harmful? Semin. Dermatol. 10 (1991) 26–28. Barba, C., Scott, S., Roddick-Lanzilotta, A., Kelly, R., Manich, A.M., Parra, J.L., Coderch, L.: Restoring important hair properties with wool keratin proteins and peptides. Fiber Polym 11(7) (2010) 1055-1061. Challoner, N.I., Chahal, S.P., and Jones, R.T., Moisture regulation of hair using cosmetic proteins. Croda NC 04910 (1999) 1–12. Sugawara, T., Kawai, M., and Suzuki, T., The relationship between moisture content of human fingernails and the mechanical properties of the fingernails J. Soc. Cosmet. Chem. Jpn. 33 (1999) 283–289. Sugawara, T., Kawai, M., and Suzuki, T., The relationship between moisture content of human fingernails and the mechanical properties of the fingernail (Part 2). J. Soc. Cosmet. Chem. Jpn. 33 (1999) 364–369. Sugawara, T., The relationship between moisture content and mechanical properties of fingernail (Part 4). J. Soc. Cosmet. Chem. Jpn. 37 (2003) 282-286.

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In: Keratin: Structure, Properties and Applications ISBN 978-1-62100-336-6 Editors: Renke Dullaart et al. pp. 113-131 ©2012 Nova Science Publishers, Inc.

Chapter 5

LIPID STRUCTURES OF VARIOUS STRATUM CORNEUM INVESTIGATED BY ELECTRON PARAMAGNETIC RESONANCE Kouichi Nakagawa

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Department of Radiological Life Sciences, Graduate School of Health Sciences, Hirosaki University, Hirosaki, 036-8564 Japan

INTRODUCTION Stratum corneum (SC) is the outermost layer of skin and the skin barrier against chemicals, surfactants, UV irradiation, and environmental stresses. The SC has a heterogeneous structure composed of corneocytes embedded in the intercellar lipid lamellae as illustrated in Figure 1. The morphology of the SC lipids is closely associated with the main epidermal barrier. Knowledge of the lipid structure is important in understanding the mechanism of irritant dermatitis and other SC diseases. The structural information of the SC lipid is obtained by the analysis of aliphatic spin probes incorporated into intercellar lamella lipids using EPR (Electron Paramagnetic Resonance) [1-6]. EPR in conjunction with spin probe method non-destructively measures the ordering of the lipid bilayer of SC.

Phone & Fax: 0172-39-5921, E-mail: [email protected]

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EPR (or ESR: Electron Spin Resonance) utilizes spectroscopy, which measures the freedom of an unpaired electron in an atom or molecule. The principles behind magnetic resonance are common to both EPR and nuclear magnetic resonance (NMR), but there are differences in the magnitudes and signs of the magnetic interactions involved. EPR probes an unpaired electron spin, while NMR probes a nuclear spin. EPR can measure 10-9 M (moles per liter) concentration of the probe and one of the most sensitive spectroscopic tools. Therefore, EPR is able to elucidate skin lipid structures as well as dynamics. It is important to know the composition of SC lipid as well as its structure in relation to depth. The various components, such as ceramides, cholesterol, and free fatty acids of SC lipids have been investigated by TLC (thin-layer chromatography) [7, 8]. Structural information organized by the components is essential for knowing the detailed functions of SC. The role of the intercellular SC lipid bilayer in relation to barrier function has been investigated by IR (infrared) spectroscopy [9, 10] and X-ray diffraction [11]. IR examination showed that the outer layers were less cohesive and the intercellular lipids are more disordered compared with the deeper membrane, based on the C-H stretching absorbance of the methylene groups of the lipid acyl chains. The X-ray approach is somewhat limited to model lipid membranes containing water or in vitro SC specimens, and it is difficult to obtain information about depthrelated changes of the SC. On the other hand, the EPR probe method can provide insight into the SC lipid organization as well as its dynamics. Intercellular route

Sebaceous secretion

SC Corneocytes Intercellular space

Fatty acid

Lipid Bilayer

Figure 1. Schematic representation of the modified “Brick and Mortar” model of the stratum corneum (SC) is shown. Also, there is shown the most likely probe location in the lipid bilayer and pathways of drug (or spin probe) permeation through intact stratum corneum.

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The physicochemical properties of intercellar lipids of SC as a function of various surfactants [1, 2], water contents [3], various kinds of spin probes [4], and ordering (or fluidity) change of the SC lipid [1, 4-6] were investigated. EPR is a reliable, sensitive, and non-destructive technique to measure the probe in the lipids at ambient temperature. An introduction on EPR spectroscopy and its application in conjunction with slow-tumbling simulation to elucidate the organization of SC lipids are discussed next. This technique provides confirmatory and complementary information about structure and physicochemical properties on a molecular level. The advantage of using the spin probes is that not only the structure but also the acyl chain motion in the stratum corneum (SC) lipid can be determined. These studies provided the fluidity related behaviors of SC at the different conditions by measuring EPR signals. EPR measurements and the simulation analysis can potentially provide further quantitative insight into the skin-lipid structures.

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APARATUS EPR apparatus consists of a klystron to generate microwaves, electromagnet, resonant cavity, microwave detector, amplifier, A/D converter, and PC as shown in Figure 2. The microwaves from the klystron have a constant frequency, and those microwaves reflected from the resonant cavity are detected, changed to an electronic signal, amplified and then recorded. In contrast to NMR, substances which contain unpaired spin can be observed by EPR. Paramagnetic substances including transition metal complexes, free radicals, macromolecules, and photochemical intermediates are observed. Approximately 10-13 mole of a substance gives an observable signal, thus EPR has great sensitivity. Momentum of electron spin in a magnetic field orients only two quantum states: ms = ½ and - ½. Application of an oscillating field perpendicular to a steady magnetic field (H) induces transitions between the two states provided the frequency ( ) of the oscillating field satisfies the resonance condition:

E

E1 2

E

1 2

g H,

Thus,

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(1)

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h

E

g H,

(2)

where E is the energy-level separation, h is Planck‟s constant, g is a dimensionless constant called the g-value, and is the electron Bohr magneton, and H is the applied magnetic field. Phase Shifter

Circulator Microwave Source

Detector Signal

Sample

Magnet

Amplifier A/D

S

N Cavity

mI = 1

Energy Levels

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Figure 2. Block diagram of EPR spectrometer.

0 -1

E1/2 ms = 1/2

mI = -1

ms = -1/2

0

E-1/2

1

A

mI = 1

0

0

-1

Magnetic Field

Figure 3. Hyperfine levels and transitions for a nitroxide nitrogen nucleus (14N) of I = 1 with positive coupling constant. An observable EPR observable spectrum is shown.

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The interaction of an electron spin in resonance with a neighboring nuclear spin in a molecule is called hyperfine coupling. In the case of nitroxide spin probe, 14N of the probe has three quantum states: mI = +1, 0, and -1. Each quantum state interacts with an electron spin and further splits into two sets of energy states as shown in Figure 3. The selection rules for transitions in hyperfine coupling are ms = 1 and mI = 0. Thus, one can observe three transition (resonance) lines for fast tumbling nitroxide spin probe in a spectrum. The interval of the resonance lines is called the hyperfine coupling constant (A). The EPR spectra are usually recorded as the first derivative of the absorption spectrum as shown in lower part of Figure 3.

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Stratum Corneum Cyanoacrylate Glue Stripping The sampling method was first utilized by Marks and Dawber [12] to obtain SC sheets. Recently, Yagi, Nakagawa, and Sakamoto developed a process to study SC properties [6]. The SC specimens were successively removed from the mid-volar forearm and shank of the volunteers, who had given informed consent to the procedure [6]. All subjects had normal skin, as judged by visual assessment. A glass plate (7 mm x 37 mm; Matsunami Glass Ind., Ldt., Tokyo, Japan) on which a single drop (~1.2 mg) of a commercially available cyanoacrylate resin had been uniformly spread was used to strip the SC sheet as depicted in Figure 4. Only approximately 1 mg of SC sample is required for the studies.

Mid-volar forearm

EPR

A glass plate

Cyanoacrylate

Incubation with probe

Figure 4. Schematic representation of SC sample procedures and the EPR spectrum.

Once the glue has solidified, no significant signal arise from the cured resin or from the spin probe dissolved in the resin; the only signal observed

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arise from the spin probe in the attached SC sheet. This method has the advantage of avoiding prior exposure of the SC to enzymes. EPR intensity slightly depends on how thick a sample is removed by each stripping, but it can be adjusted by the amount and areas of glue on the glass plate.

Preparation of SC Sheets for EPR Measurements One piece of stripped SC (~ 5 x 22 mm2) was incubated in ~50 M of a spin probe (Figure 5) aqueous solution for about 60 minutes at 37 C (Figure 4). The probe solution was dropped on the SC sheet. The SC sheet repels the aqueous solution but the probe goes into the lipid phase during the incubation. After rinsing with distilled water to remove excess spin probe, the SC sample was mounted on an EPR cell. O 1

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O

5-DSA

5 O

N

O

CHL

O N O

H

Figure 5. Chemical structures of 5-doxylstearic acid (5-DSA) and 3β-doxyl-5αcholestane (CHL) spin probes.

Spin Probes Organic free radicals containing the nitroxide group are called spin probes or spin labels. The ordering (or fluidity) of the lipid bilayer is obtained with doxylstearic acid (DSA) which most commonly used. Commercially available

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spin probes, 5-doxylstearic acid (5-DSA) and 3β-doxyl-5α-cholestane (CHL), were used to obtain the ordering of the SC lipid. The chemical structures of 5-DSA and CHL are depicted in Figure 5. Changes of the lipid chain ordering are able to monitor using various probes. The orientation of spin probe reflects the local molecular environment and should serve as indicator of conformational changes in lipid bilayers.

EPR Line-Shapes due to Spin Probe Motion

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The line-shapes and line-widths can vary under certain spin probe environments. When line broadening arises from incomplete averaging of the g-value and the hyperfine coupling interactions within the limit of rapid tumbling in a medium, EPR line-shape starts changing from the triplet pattern. EPR spectra of nitroxide radicals for different tumbling times as well as different order parameters are presented in Figure 6. Schematic illustration of lipid bilayer structures and corresponding EPR spectra is also shown in Figure 7. If a spin probe is oriented (immobilized) in a lipid membrane, EPR spectrum is an anisotropic pattern which clearly shows parallel (2A ) and perpendicular (2A ) hyperfine coupling structures (the top spectrum in Figure 6). Description of spectra

Approx. tumbling time (ns)

Approx. order parameter

Immobilized

0.5

0.7

Moderately Immobilized

2.5

0.3

Weakly Immobilized

5.0

0.1

2A

2A

Figure 6. Nitroxide EPR line-shape as a function of tumbling time and order parameter. The parallel and perpendicular hyperfine couplings, 2A and 2A , are also indicated for an anisotropic (immobilized) EPR spectrum.

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Kouichi Nakagawa Disordered structure

Ordered structure

Order Parameter: S ≈ 0

: Lipid

Order Parameter: S ≈ 1

: Spin probe

mobile

Immobilized

Figure 7. Schematic representation of lipid bilayer structures as a function of lipid ordering. The corresponding EPR spectral patterns were also indicated.

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The order parameter is approximately 0.7 or higher. If a spin probe tumbles relatively fast (weakly immobilized) in a lipid membrane, EPR spectrum is a triplet pattern with unequal intensities. The order parameter is usually very small (~0.1).

Qualitative order Parameter (S) The inclination of the principal axis of the nitroxide radical to the rotational axis of the long-chain probe molecule represents a measure of the order-disorder of the molecular assemblies of a membrane. The order parameter indicates the membrane chain dynamics and microenvironment of the medium in which the spin probe is incorporated. The conventional order parameter (S) is determined from the hyperfine coupling of the EPR signals according to the following relations [13]:

S AZZ

a'

AII

AII A 1 AXX 2

AYY

a , a'

2A , 3

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(3)

(4)

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where a is the isotropic hyperfine value, (AXX + AYY + AZZ)/3; AXX, AYY, and AZZ are the principal values of the spin probe. The following principal components were used for 5-DSA [14].

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AXX, AYY, AZZ = (0.66, 0.55, 3.45) mT

(5)

The experimental hyperfine couplings of 2A and 2A are obtained from the experimental spectrum (as shown in Figure 6). The order parameter indicates that the S value increases with increasing anisotropy of the probe site in the membrane. On the other hand, the S value becomes zero for completely isotropic motion of the nitroxide radical. Since the spin probe is incorporated into the highly oriented intercellular lipid structure in normal skin, in which the probe cannot move freely due to the rigidity of lipid structure, its EPR spectrum represents the microscopically oriented profile as depicted in Figure 7. When the normal structure is completely destroyed by chemical and/or physical stress, the EPR spectral profile changes to three sharp lines because the probe mobility is unrestricted. Thus, the EPR spectral profile reflects the rigidity of the environment of the probe moiety. However, conventional analysis measuring 2A and 2A from the observed spectrum gives limited information concerning the probe moiety in the membrane, and may not reveal subtle differences in the overall experimental spectra related to the membrane chain ordering [6].

Quantitative order Parameter (S0) by Slow-Tumbling Spectral Simulation The slow-tumbling motions on the order of 10-7 s of the aliphatic spin probes in membranes were evaluated by using the nonlinear least-squares fitting program NLLS to calculate the EPR spectra based on the stochastic Liouville equation [15, 16]. The EPR spectra for spin probes incorporated into the multilamellar lipid bilayer were calculated according to various distribution of the probe in the membrane. The spectrum of a sample can be regarded as the superposition of the spectra of all of the fragments. The lipid and 5-DSA molecules in the lipid bilayer experience ordering potentials, which restrict the amplitude of the rotational motion. The ordering potential in a lipid bilayer determines the orientational distribution of molecules with respect to the local ordering axis of the bilayer [17]. The

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overall orientation of the probe can be expressed by the order parameter (S0), which is defined as follows [16, 18] S0

1 (3 cos2 2

1)

d exp( U / kT ) D002 d exp( U / kT )

,

(6)

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which measures the angular extent of the rotational diffusion of the nitroxide probe moiety. Gamma ( ) is the angle between the rotational diffusion symmetry axis and the z-axis of the nitroxide axis system as shown in Figure 8. The = ( , , ) are the Euler angles between the molecular frame of the rotational diffusion tensor, U is the ordering potential, and D is a Winger rotation matrix element. In addition to S0, the simulation calculates slowtumbling motions of the probe in the bilayer, providing rotational diffusion coefficients, as described in detail elsewhere [19]. The values of the rotational diffusion coefficients (dynamic values) are in relation to the S0 values. The A and g of the principal components were used for the simulation of 5-DSA [14]. AXX, AYY, AZZ = (0.66, 0.55, 3.45) mT

(7)

gXX, gYY, gZZ = (2.0086, 2.0063, 2.0025).

(8)

Bilayer Surface

Z z O O

N

y

x

Figure 8. A schematic representation of a conformation of DSA spin probe in the SC membrane, where Z-axis of the acyl chain is parallel to z-axis of the nitrogen 2Pz orbital.

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The local or microscopic ordering of the nitroxide probe in the multilamellar lipid bilayer is characterized by the S0 value. A larger S0 value indicates highly ordered structure (rigid) and a smaller S0 shows less ordered structure (less rigid or mobile). Changes of the lipid structural ordering of SC are able to be monitored using the aliphatic probes. The orientation of spin probe reflects the local molecular environment and should serve as indicator of conformational changes in lipid bilayers of the SC. The modern simulation takes into account overall experimental intensities, line-widths, and hyperfine coupling values and provides the quantitative information regarding the probe environment. Therefore, S0-value reflects the local ordering of the lipid structure in the membrane. The error of the spectral simulation is a few percent in the case of the dipalmitoylphosphatidylcholine membrane [19]. In the presence of fast motion of the probe in the SC, the simulation may result in the deviation from the experimental spectra.

RESULTS AND ANALYSES

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Qualitative order Parameter (S) and Quantitative order Parameter (S0) of SC Lipids The modified “Brick and Mortar [20] model of the SC is illustrated in Figure 1. SC intercellular lipids arrange themselves into bilayer and pack into lamellae. The single-chain 5-DSA normally dissolves into lipids and fat phases. The most likely location of the single-chain probe in the SC. The aliphatic probe will be located in the lipid phase and fat like sebaceous secretion of the SC. Figure 9 shows the experimental and simulated EPR spectra of 5-DSA in the SC. The reasonable agreement of the experimental and simulated spectra suggests that simulation analysis can provide detailed information regarding the SC lipids. The S0 value changes from 0.61 to 0.96, while the S value is in the range of 0.56 to 0.59. The conventional S value was obtained by the Eq. 3 measuring the hyperfine values from the observed spectrum. There are significant differences between the conventional and simulated order parameters. Because the slow-tumbling simulation calculates the total line-shape of the spectrum, it is able to extract more detailed information about the SC structure than the conventional analysis, which is normally ambiguous in distinguishing the two hyperfine components (parallel and perpendicular)

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from the experimental spectrum due to the presence of weak and broad signals [5]. Thus, the S0 values (0.2 ~ 0.9) obtained by the simulation suggest that the outermost SC layers are less rigid (or more mobile, S0 ~ 0.2), while the deeper lipid layers (S0 ~ 0.9) have more rigid and oriented structures. The arrow in the spectrum indicates the characteristic peak, which is prominent only for the first strip (Figure 9). This peak diminishes in intensity with increasing depth in the SC. The marked peak appears near the center of the spectrum because the probe embedded in the first sample stripped has greater freedom of motion. The other two lines of the nitroxide probe overlaid the central region of the spectrum. Further investigation of the characteristic peak was performed. Figure 10 (a) shows the EPR spectrum of the first strip from SC. The strong and broad peak observed for the SC sheet from the human forehead is shown in Figure 10 (b). The peak intensity decreases after washing the SC with soap (Figure 10 (c)). Thus, the characteristic signal can be attributed to sebaceous secretion [6]. The strength of the signal is considered to reflect the abundant sebaceous secretion at the forehead compared with that of the forearm.

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Stripping number

Simulated Order Parameter

S0

1

0.61

3

0.96

5

0.96

1 mT Figure 9. Experimental (solid line) and simulated (dashed line) EPR spectra of 5-DSA probe. Stripping numbers show consecutively stripped SC from the surface downwards. The arrow of stripping number 1 indicates the characteristic peak. The EPR spectra were obtained with the single scan.

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Figure 10. Experimental EPR spectra of 5-DSA in the first stripped SC from human mid-volar forearm (a), the first stripped SC from human forehead pre-washing (b), and the first stripped SC from human forehead after-washing (c). The short dashed line corresponds to the characteristic signal. The long dashed line corresponds to the probe incorporated into the SC lipids.

Quantitative order Parameter (S0) Related to SC Lipid Structure One can calculate the angle (γ in Figure 8) between the rotational diffusion symmetry axis (the lipid in SC) and the z-axis of the nitroxide axis system. Figure 11 represents the schematic illustration of the bilayer distance in relation to the angle. The simulated S0 value of 0.61 can be the angle of 30°. The value of 0.96 is the angle of 9.4°. The angle suggests that the SC lipids align nearly perpendicularly to the bilayer surface. The larger S0 value yields larger distance between the lipid bilayer. The analysis implies that the longer distance of the lipid bilayer can be related to the well-oriented SC structure. Figure 12 shows that human SC stripped from lower-leg presents typical EPR spectra of 5-DSA incorporated in the SC lipids. The EPR spectrum about stripping number 1 is slightly different from that of number 3.

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Lipid Bilayer Simulated value S0 = 0.61 ( = 30 º)

Distance

Figure 11. Schematic illustration of relative lipid bilayer distances and the values of simulated order parameter (S0) related to the angles (γ) between the bilayer surface and the single-chain probe.

Stripping number

1

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S0 = 0.28

3 S0 = 0.60

2 mT Figure 12. Experimental (solid line) and simulated (dashed line) EPR spectra of 5-DSA in the first and the third stripped human SC from lower-leg. The EPR spectrum was obtained with the single scan.

The characteristic peak indicated by the arrow in the spectrum is prominent for the first strip. The reasonable agreement of the simulated and experimental spectra suggests that simulation analysis can provide

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comprehensive information regarding the SC lipids. The S0 value changes from 0.28 to 0.60, while the S value is in the range of 0.63 to 0.64. The S0 values of 0.28 and 0.60 are the angle of 44° and 31°, respectively. The higher S0 value implies that the lower SC lipids have better-ordered structure than those of the upper SC lipids. Satisfactory agreement between the experimental and calculated spectra can provide a quantitative S0, which reveals the microscopic ordering in association with the structure of the SC lipids. The EPR simulation can potentially provide further insight into skin-lipid structures. The order parameter (S0) of spin probe will provide the useful index about structural dependence as a function of the SC depth. It is notable that the value is not the absolute index for living animals. The value may differ from sample to sample. However, the relative value of the particular SC sample as a function of the depth could provide a useful index of the SC. Next, interaction between keratin solution from human epidermis and 5DSA was examined. Figure 13 shows EPR spectra of the keratin/5-DSA and 5-DSA stock solutions. EPR spectrum of 5-DSA stock solution shows typical nitrogen triplet pattern of the probe in H2O solution as presented in Figure 13 (A). The EPR spectrum of keratin/5-DSA solution also shows the triplet pattern (Figure 13 (B)) and stays the same after one hour. The similar spectra for both experiments provide that 5-DSA probe does not strongly interact with human keratin in the solution. The results suggest that 5-DSA probe most likely do not permeate keratin in the period [21].

(A) 5-DSA/H2O

(B) Keratin/5-DSA/H2O

2 mT Figure 13. EPR spectra of (A) 5-DSA stock solution and (B) keratin/5-DSA solution are shown. EPR spectra were taken at ambient temperature.

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Other Applications of the EPR Method Effects of Mild Surfactants on SC Lipids EPR in conjunction with a slow-tumbling simulation was utilized for examining the effect of diluted detergent on stratum corneum (SC) lipid structure. SC from the back of hairless mouse (HOS:HR-1) was stripped consecutively from one to three times. EPR spectrum of 5-DSA incorporated in the control SC demonstrated a characteristic peak for the first strip. A slowtumbling simulation for 5-DSA showed slight differences in ordering values (S0) of the SC for the control and detergent treated SC. The S0 values were 0.15 and 0.32, respectively. EPR spectra of the detergent treated SC showed that the characteristic component was eliminated. Thus, the EPR method along with the simulation analysis revealed the differences in ordering of the detergent treated SC. Different types as well as mixtures of surfactants change the SC structure of the lipid bilayer differently. Kawasaki et al. examined the influence of anionic surfactants, sodium lauryl sulfate (SLS) and sodium lauroyl glutamate (SLG), on human SC by the EPR spin label method [1]. The qualitative order parameter obtained by 1.0% wt SLS-treated cadaver SC was 0.52. On the other hand, the high S value of 0.73 for 1.0% wt SLG was obtained. The results suggest clear surfactant effects on the ordering of the lipid bilayer. In addition, a reasonable correlation between the qualitative order parameters and human clinical data (visual scores and transepidermal water loss values) was demonstrated. Effects of Skin Penetration Enhancers on SC Lipids Interaction of skin penetration enhancer correlates with the ordering of the intercellular lipid bilayers. Nakagawa and Anzai investigated the effects of terpenes, -terpineol and (+)-limonene, on SC lipids utilizing the EPR spin probe method [23]. The EPR spectra of -terpineol treated SC were totally different from those of untreated SC. The results suggest that -terpineol increases in the penetration of local bilayers surrounding 5-DSA. The -terpineol enhanced permeation of the single chain 5-DSA about three times than that of the control. However, EPR spectra of CHL in the SC did not show a clear difference for each strip, except for the signal intensity. The results imply that CHL permeates into SC lipid differently from 5-DSA. The enhancement of the 5-DSA is more significant than that of CHL.

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Therefore, the present results can be useful for various drug administrations via the skin.

CONCLUSION EPR along with a modern computational analysis provides quantitative insight into the SC structure as a function of the depth. The EPR spectral pattern contains important information regarding the probe mobility as well as the SC lipid structure. Satisfactory agreement between the experimental and calculated spectrum can provide a quantitative S0, which gives the microscopic lipid ordering of the SC lipid. The SC lipid structures can be related to the SC barrier functions. In addition, the EPR method recognizes sebaceous exudates [6], detergents [22], and penetration enhancers [23]. Therefore, the EPR technique could in turn provide more comprehensive information, which would further the understanding of various SC. The relative value of various SC can provide a useful index regarding the SC.

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REFERENCES [1]

[2]

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Kawasaki, Y., Quan, D., Sakamoto, K., Cooke, R., & Maibach, H.I. (1999) Influence of surfactant mixtures on intercellular lipid fluidity and skin barrier function. Skin Res Technol, 5, 96-101. Mizushima, J., Kawasaki, Y., Tabohashi, T., & Maibach, H.I. (2000) Effect of surfactants on human stratum corneum: electron paramagnetic resonance. Int J Pharm, 197, 193-202. Alonso, A., Meirelles, N.C., Yushmanov, V.E., et al. (1996) Water increases the fluidity of intercellar membranes of stratum corneum: correlation with water permeability, elastic and electrical resistance properties. J Invest Dermatol, 106, 1058-1063. Nakagawa, K. (2010) Electron Paramagnetic Resonance Investigation of Stratum Corneum Lipid Structure, Lipids, 45, 91-96. Nakagawa, K., Mizushima, J., Takino, Y., Kawashima, T., & Maibach, H.I. (2006) Chain ordering of stratum corneum lipids investigated by EPR slow-tumbling simulation. Spectrochimi Acta Part A Mol & Biomol Spectroscopy, 63, 816-820.

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[7]

[8]

[9]

[10]

[11]

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[12]

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[15]

[16]

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Kouichi Nakagawa Yagi, E., Sakamoto, K., & Nakagawa, K. (2007) Depth dependence of stratum corneum lipid ordering: A slow-tumbling simulation for electron paramagnetic resonance. J Invest Dermatol, 127, 895-899. Bontė, F., Saunois, A., Pinguet, P., & Meybeck, A. (1997) Existence of a lipid gradient in the upper stratum corneum and its possible biological significance. Arch Dermatol Res, 289, 78-82. Weerheim, A., & Ponec, M. (2001) Determination of stratum corneum lipid profile by tape stripping in combination with high-performance thin-layer chromatography. Arch Dermatol Res, 293, 191-199. Bommannan, D., Potts, R.O., & Guy, R.H. (1990) Examination of stratum corneum barrier function in vivo by infrared spectroscopy. J Invest Dermatol, 95, 403-408. Zhang, G., Moore, D.J., Mendelsohn, R., & Flach, C.R. (2006) Vibrational microspectroscopy and imaging of molecular composition and structure during human corneocytes maturation. J Invest Dermatol, 126, 1088-1094. Pilgram, G.S.K., Engelsma-Van Pelt A.M., Bouwstra, J.A., & Koerten, H.K. (1999) Electron diffraction provides new information on human stratum corneum lipid organization studied in relation to depth and temperature. J Invest Dermatol, 113, 403-409. Marks, R., & Dawber, R.P. (1971) Skin surface biopsy: an improved technique for the examination of the horny layer. Br J Dermatol, 84, 117-123. Hubbell, W.L., & McConnell, H.M. (1971) Molecular motion in spinlabeled phospholipids and membrane. J Am Chem Soc, 93, 314-326. Ge, M., Rananavare, S.B., & Freed, J.H. (1990) ESR studies of stearic acid binding to bovine serum albumin. Biochim Biophys Acta, 1036, 228-326. Schneider, D.J., & Freed, J.H., Calculating slow motional magnetic resonance spectra. In: Berliner LJ & Reuben J (eds) Biological Magnetic Resonance Vol. 8, New York: Plenum Press, 1-76, 1989. Budil, D.E., Lee, S., Saxena, S., & Freed, J.H. (1996) Nonlinear-leastsquares analysis of slow-motion EPR spectra in one and two dimensions using a modified Levenberg-Marquardt algorithm. J Magn Reson Ser A, 120, 155-189. Meirovitch, E., Igner, D., Igner, E., Moro, G., & Freed, J.H. (1982) Electron-spin relaxation and ordering in smectic and supercooled nematic liquid crystals. J Chem Phys, 77, 3915-3938.

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[18] Crepeau, R.H., Saxena, S., Lee, S., Patyal, B.R., & Freed, J.H. (1994) Studies on lipid membranes by two-dimensional Fourier transform ESR: enhancement of resolution to ordering and dynamics. Biophys J, 66, 1489-1504. [19] Ge, M., & Freed, J.H. (1998) Polarity profiles in oriented and dispersed phosphatidylcholine bilayers are different. An ESR study. Biophys J, 74, 910-917. [20] Elias, P.M. (1983). Epidermal lipids, barrier function and desquamation. J Invest Dermatol, 80(suppl), 44-49. [21] Nakagawa, K. (2011) Elucidated Lipid Structures of Various Human Stratum Corneum Investigated by EPR Spectroscopy, Skin Res Technol, 17, 245-250. [22] Nakagawa, K., & Anzai, K. (2011) Stratum Corneum Lipid of Hairless Mouse Investigated by Electron Paramagnetic Resonance, Appl Magn Reson, in press. [23] Nakagawa, K., & Anzai, K. (2010) Stratum Corneum Lipid Structure Investigated by EPR Spin-Probe Method: Application of Terpenes, Lipids, 45, 1081-1087.

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In: Keratin: Structure, Properties and Applications ISBN 978-1-62100-336-6 Editors: Renke Dullaart et al. pp. 133-147 ©2012 Nova Science Publishers, Inc.

Chapter 6

KERATIN EXPRESSION IN THE HUMAN PITUITARY GLAND AND ITS APPLICATION TO NEOPLASTIC PITUITARY CELLS Hidetoshi Ikeda

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Research Institute for Pituitary Disease, Southern Tohoku General Hospital. 7-115 Yatsuyamada, Koriyama, Fukushima 963-8563, Japan,

ABSTRACT The detection of specific keratins may be of diagnostic value in terms of dividing epithelial tissues into various classes depending upon their cellular origin and morphologic and/or growth features. Therefore, this review will focus on the normal patterns of keratin expression in the developing human pituitary gland, in non-neoplastic pituitary cells, and in several pathological conditions such as Rathke‟s cleft cyst and pituitary adenomas and its associated pathology (i.e. fibrous bodies and Crook‟s hyaline change).

INTRODUCTION Keratin filaments are a member of the intermediate filaments (IMF) family (keratin, glial fibrillary acidic protein (GFAP), vimentin, neurofilament E-mail: [email protected] or [email protected]

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proteins (NFPs), and desmin) and are a characteristic component of epithelial cells. Expression of most keratins follows well-defined rules that are mainly related to three factors: (a) epithelial cell type, (b) the differentiation programs of stratified epithelia, and (c) the state of cellular growth. The existence of these rules suggests that the detection of specific keratins may be of diagnostic value in terms of dividing epithelial tissues into various classes depending upon their cellular origin and morphologic and/or growth features. In general, the immunologic features of tumor cell IMFs are the same as those of their tissue of origin. Therefore, in cases of neoplastic transformation, there are no major changes in the synthesis and type of intermediate filament proteins compared with normal tissues. Expression of different IMF subunits varies at different stages of neoplastic progression, and in cells maintained under different growth conditions. IMF class switches are known to occur during embryogenesis in the central nervous system, and phenotypic changes in IMF are also correlated with tumor malignancy [1]. Keratin may also play an important role in migration, via specific interactions with the extracellular environment, thereby influencing cell shape. In this review, special attention is paid to the expression of keratin subtypes during the development of neoplastic pituitary cells, and to the clinical application of keratins as distinct histochemical markers.

1. KERATIN EXPRESSION IN THE DEVELOPING HUMAN PITUITARY GLAND When crown-rump length or ovulation age are used as a time axis for development, a profound bias due to individual variability is introduced, which makes these factors fundamentally unreliable as temporal indices of pituitary morphological development and differentiation. Therefore, I have classified the stages of development according to characteristic morphological and developmental features of the primordial organ [2,3]. The early development of the pituitary gland can be classified into the following stages: Stage I (before 3rd fetal week); the cell groups that will form the pituitary primordium are located within the stomodeal epithelium. Stage II (around the 4th and 5th fetal week); Rathke‟s diverticulum begins to form with a dorsally directed evagination from the stomodeal epithelium; however, at this stage the diencephalon diverticulum is not formed. Stage III (around the 6th fetal week); this stage begins with the formation of the diencephalon

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diverticulum and lasts until the continuity between Rathke‟s pouch and the primitive oral cavity is lost. Stage IV (between the 7th and 13th fetal week) this stage lasts until the contact between Rathke‟s pouch and the brain is limited to the superior posterior wall of the pouch and its bilateral external surface. I believe that consideration of these stages is the most appropriate way in which to study the nature of the environmental factors and cellular interactions involved in organogenesis. A total of 19 cytokeratin polypeptides have been identified on the basis of isoelectric pH and molecular weight [4]. Of these, cytokeratins 7, 8, 18, and 19 are characteristic of simple epithelial cells [4,5]. Among IMFs, keratin is the first to appear in the anterior and intermediate lobes of the pituitary, followed by GFAP and vimentin. Only cytokeratin is observed (in the form of simple epithelial type keratins 8 and 19) during morphogenesis of the pituitary gland, with cytokeratin 8 being the earliest to appear [6]. Although cytokeratin 8 is observed within Rathke‟s diverticulum at stage II, cytokeratin 19 is observed within Rathke‟s pouch only after stage III [6].

Figure 1. During the early stages of development of Rathke‟s pouch, cytokeratin 19 was observed at stage IV. Note that the distribution of positive immunoreactivity for cytokeratin 19 and ACTH are similar within the pouch.

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During the early stages of development of Rathke‟s pouch, the epithelium, though derived from the simple epithelium of the stomodeum, is a pseudostratified and not simple. Since the epithelium is positive for cytokeratins 8 and 19, this pseudo-stratified epithelium retains the cytokeratin characteristics of simple epithelium. The immunoreactivity of cytokeratins 8 and 19 varies from place to place, according to the degree of cellular differentiation. Cells immunoreactive for cytokeratin 8 are present throughout Rathke‟s diverticulum during stage II; however, during stage III, strongly immunoreactive cells become confined for the first time to cells that differentiate into endocrine cells [7]. Cytokeratin 19 appears during stage III, when its distribution on immunoreactive cells is almost the same as that of cytokeratin 8 [6] (Figure 1). Furthermore, at sites of contact between the pituitary primordium and the diencephalon, which maintains their epithelial characteristics and high proliferative activity [3,6,7], both the appearance of cytokeratin and the differentiation of early adrenocorticotropic hormone (ACTH) cells are delayed. Thus, heterogeneous patterns of cytokeratin 8 and 19 distribution appear to reflect the degree of cellular proliferation and differentiation.

2. KERATIN EXPRESSION IN NON-NEOPLASTIC PITUITARY CELLS Cytokeratin immunoreactivity is observed in most of the epithelial cells within the pituitary gland, including the endocrine cells in the anterior lobe [8]. Among endocrine cells, cytokeratin staining is positive in some somatotrophs and lactotrophs and in most corticotrophs [9]. In addition, the cells of the pars tuberalis, most of which are follicle stimulating hormone/luteinizing hormone (FSH/LH) immunoreactive, show significant immunoreactivity for cytokeratin (Figure 2). Therefore, there seems to be no clear correlation between hormone production and cytokeratin immunoreactivity. Squamous cell nests found in the pars tuberalis and, occasionally, in the pars anterior, increase with age, suggesting that they may represent metaplasia [10]. Squamous metaplastic cells in the pars tuberalis (Figure 3), the columnar and ciliated epithelia that form follicular structures, and anterior lobe cells infiltrating the posterior lobe [11-13] also show cytokeratin immunoreactivity. Cytokeratin and NFPs may be co-expressed in endocrine cells in the pituitary anterior lobe [13,14].

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(a). Pars tuberalis of pituitary gland.

(b). HE staining

(c) keratin staining

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Figure 2. Pars tuberalis of the pituitary gland. Keratin-positive pituitary endocrine cells were observed all along the anterior surface of the stalk.

(a) HE b) Keratin Squamous metaplastic cells in the pars tuberalis.

Figure 3. Squamous cell nests were observed in the pars tuberalis, and most cells showed positive keratin immunoreactivity. (a) HE stain. (b) Keratin stain.

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However, Halliday et al [9] could not demonstrate NF immunoreactivity in the anterior lobe of the formalin-fixed pituitary tissue; although this may have been due to a problem with the fixation process. Ogawa used 10% phosphate-buffered formaldehyde and absolute ethanol solutions for fixation, but observed NF-positive staining only in ethanol-fixed material. Thus, alcohol fixation offers some advantages over the formalin fixation with regard to the preservation of IMF antigenicity [15].

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3 KERATIN EXPRESSION BY NEOPLASTIC PITUITARY CELLS In general, the immunologic features of tumor-cell intermediate filaments (IF) are the same as those in the tissue of origin [16]. In addition, tumors do not acquire additional IF types [17]. Pituitary adenomas express four types of IMF, namely keratin, vimentin, GFAP, and NF [13,18], and the expression profile of IMF in pituitary adenomas is the same as that in normal pituitary tissue. Adenoma cells secreting growth hormone (GH), prolactin (PRL), and ACTH frequently contain cytokeratin filaments, while those secreting glycoprotein hormones show negative expression [19]. A variable proportion of cells within PRL adenomas contain cytokeratins, usually showing diffuse cytoplasmic staining. GH adenomas show two patterns of cytokeratin staining; namely, diffuse cytoplasmic staining or staining of intra-cytoplasmic aggregates. No relationship between cytokeratin immunoreactivity and hormone levels was found in patients with GH adenoma either in vitro or in vivo [19]. Keratin subtypes and IMF expression levels are different between the solid portion and cystic epithelial portions of adenomas [18], suggesting that the expression of keratin subtypes, as well as IMF types, changes in accordance with cellular differentiation (even within the same tumor tissue). Kasper et al. [20] described an unusual “neo-expression” of cytokeratin 19 never observed in endocrine cells of the normal human pituitary gland. However, cytokeratin 19 expression has been shown in both the developing and adult human pituitary gland [5]. a) Fibrous bodies and their implications Fibrous bodies, which are intra-cytoplasmic aggregates [21], belong to the cytokeratin family [22]. However, some fibrous bodies can be stained with

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antibodies to vimentin or NF [13,23]. The functional significance of fibrous bodies is unknown. Fibrous bodies appear to occur exclusively in sparsely granulated GH hormone cells and acidophil stem cells [24]. However, it seems that fibrous bodies are non-specific structures, which mostly contain cytokeratins, and occur in a variety of neoplasms (such as GH adenoma (Figure 4a), prolactinoma (Figure4b), paraganglioma, bronchial adenomas, insulinomas, chemodectomas, basal cell carcinomas, carcinoids of various organs, and Merckel cell tumors), which secrete peptide hormones [19,25]. The cytokeratin reactivity in glycoprotein hormone-producing cells within non-neoplastic tissue and adenomas is weak [25].

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Normal adenophypophysis Cushing's adenoma

Keratin

Keratin

a) Crook‟s hyaline change in patients with Cushing‟s disease.

HE

Keratin

b) Crook‟s cell adenoma in Cushing‟s adenoma

Figure 4. (a) Fibrous body formation in a patient with acromegaly. (b) Fibrous body formation in a patient with prolactinoma.

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Pituitary adenomas associated with acromegaly can be divided into two different types according to the pattern of cytokeratin distribution: either dotlike cytokeratin immunoreactivity (type 1 adenoma) or a perinuclear filamentous distribution (type 2 adenoma). Type 1 adenomas are more aggressive than type 2 adenomas, showing a larger tumor size, a higher incidence of suprasellar extension and cavernous sinus infiltration, and are associated with higher numbers of re-operations and incomplete resections [26]. b) Implication of Crook‟s hyaline change

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Crooke‟s hyaline change is present in a significant number non-tumorous corticotroph cells in Cushing‟s disease patients (Figure 5) and in neoplastic cells in some cases of corticotroph adenomas [27]. Crook‟s change comprises IMF of the keratin type. Ultrastructural features include a thick ring composed of densely packed filaments (8 nm in diameter) surrounding the nucleus, and a perinuclear cluster of organelles. High levels of glucocorticoids may stimulate of the production of cytokeratin in basophils as they suppress the production and release of ACTH [28].

Figure 5. (a) Crook‟s hyaline changes in patients with Cushing‟s disease. (b) Crook‟s cell adenoma in Cushing‟s adenoma.

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Recently, cytokeratin 20 was found to be a sensitive and specific marker for Crook‟s cells and also for the previously unrecognized, subtle, cytokeratin changes that occur in corticotrophs in response to hypercortisolism [29]; although the mechanisms by which cytokeratin filaments accumulate under conditions of hypercortisolism are still unknown. Massive accumulation of cytoplasmic cytokeratin may have significant effects on cellular function by suppressing the cytoplasmic transport of secretary products from the Golgi complex to the plasmalemma [27,30]. a) Follicullo-stellate cells or Rathke‟s cleft cysts Folliculo-stellate (FS) cells in the anterior pituitary gland are characterized by their star-like appearance [31] and their ability to form follicles [32]. Although FS cells do not produce pituitary hormones, their tendency to surround endocrine cells with their long cytoplasmic processes (Figure 6) suggests that they regulate endocrine cells through intercellular communication. Cytokeratin was first demonstrated in the folliculo-stellate cells of the human adenohypophysis by Tachibana and Yamashima [33].

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Rathk’s cyst within pituitary adenoma

Figure 6. Electron microscopic observation of Rathke‟s epithelium. FS cells (arrow) tended to surround endocrine cells with their long cytoplasmic processes.

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Keratin, the S-100 protein [34-36], GFAP [37], vimentin [38], and fibronectin [39] are all markers for FS cells. Co-expression of cytokeratin, vimentin, and GFAP is characteristic of Rathke‟s cleft cysts [18,40], in which focal staining of the simple epithelial cytokeratins 7, 8, 18 and 19 is observed [41]. Keratin, vimentin, and GFAP may also be co-expressed in epithelial cells forming follicles in the pars intermedia [6,13,41,43], in which there is an inverse relationship between GFAP/S100 protein positivity and ACTH/MSH positivity. It is well established that FS cells secrete many different growth factors, including FGF, VEGF, and IL-6. FS cells are thought to be a type of stem cell, which has the potential to differentiate into endocrine cells [44].

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a) Fibrous body formation in patient with prolactinoma

b) Fibrous body formation in patient with Acromegaly

a) Fibrous body formation in patient with prolactinoma

b) Fibrous body formation in patient with Acromegaly

Figure 7. Rathke‟s cyst formation within a pituitary adenoma in a patient with acromegaly.

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Sometimes pituitary adenomas form Rathke‟s cleft cysts within adenoma tissues [45], suggesting that adenoma cells have the capacity to differentiate into Rathke‟s epithelial cells (Figure 7). b) Oncogene activation by cytokeratin 18 Cytokeratins 8 and 18 are unique in terms of their genetic localization, since they are the only keratin pair that has both genes on the same chromosome [46]. Activated H-Ras, as well as activated Src, Lck, or Raf, stimulates the transcription of cytokeratin 18, suggesting that it is a direct target for the Ras signal transduction pathway [47]. Cytokeratin-positive neuroectodermal tissue exhibits a specific cytokeratin pattern: expression of the primary cytokeratin pair 8/18 [20]. However, the persistent and sometimes unexpected expression of cytokeratin 18 in a variety of tumors may be explained by oncogenic activation of cytokeratin 18 transcription.

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Ikeda H, Yoshimoto T. Immunohistochemical Study of Anaplastic Meningioma with Special Reference to the Phenotypic Changes in Intermediate Filament Proteins. Annals of Diagnostic Pathology, 2003; 7:214–222. Ikeda H, Niizuma H, Suzuki J, Sasano N. Stereographic aspect of the developmental primordium of the human pituitary gland with special reference to the connection with the diencephalon. In: Yoshimura F, Gorbman A (Eds.) Pars distalis of the pituitary gland. Elsevier, Amsterdam. 1986; pp. 16–19. Ikeda H, Suzuki J, Sasano N, Niizuma H. The development and morphogenesis of the human pituitary gland. Anat Embryol. 1988; 178:327–336. Moll R, Werner W, Franke W, Schiller DL. The catalog of human cytokeratins: Patterns of expression in normal epithelia, tumors and cultured cells. Cell, 1982; 31:11–24. Cooper D, Schermer A, Sun TT. Classification of human epithelia and their neoplasms using monoclonal antibodies to keratins: Strategies, Applications, and Limitations. Laboratory Invest. 1985; 52:243–256.

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[19] Ironside JW, Royds JA, Jefferson AA, Timperley WR. Immunolocalization of cytokeratins in the normal and neoplastic human pituitary gland. J Neurol Neurosurg Psychiatry. 1987; 50:57–65． [20] Kasper M. Cytokeratins in intracranial and intraspinal tissues. Adv Anat Embryol Cell Biol. 1992; 126:1–82. [21] Racadot J, Olivier L, Porcile E, Debrye C, Klotz HP. Mixed pituitary adenoma with acromegalic symptoms. A light microscopic and electron microscopic study. Ann Endocrinol (Paris). 1964; 25:503–7. [22] Neumann PE, Goldman JE, Horoupian DS, Hess MA. Fibrous bodies in growth hormone-secreting adenomas contain cytokeratin filaments. Arch Pathol Lab Med. 1985; 109:505–508. [23] Kasper M, Karsten U, Stosiek P, Moll R. Distribution of intermediatefilament proteins in the human enamel organ: unusually complex pattern of co-expression of cytokeratin polypeptides and vimentin. Differentiation, 1989; 40:207–214. [24] Horvath E, Kovacs K. Morphogenesis and significance of fibrous bodies in human pituitary adenomas. Virchows Arch B Cell Path. 1978; 27:69– 78. [25] Hoefler H, Denk H, Walter GF. Immunohistochemical demonstration of cytokeratins in endocrine cells of the human pituitary gland and in pituitary adenomas. Virchows Arch A Pathol Anat Histol, 1984; 404:359–368. [26] Mazal PR, Czech T, Sedivy R, Aichholzer M, Wanschityz J, Klupp N, et al. Prognostic relevance of intracytoplasmic cytokeratin pattern, hormone expression profile, and cell proliferation in pituitary adenomas of acromegalic patients. Clinical Neuropathol. 2001; 20:163–171. [27] Felix IA, Horvath E, Kovacs K. Massive Crooke’s hyalinization in corticotroph Cell adenomas of human pituitary: A histological, immunocytological, and electron microscopic study of three cases. Acta Neurochirurgica, 1981; 58;235–243． [28] Neumann PE, Horoupian DS, Goldman JE, Hess MA. Cytoplasmic filaments of Crooke’s Hyaline change belong to the cytokeratin class. Am J Pathol. 1984; 116:214–222. [29] Eschbacher JM, Coons SW. Cytokeratin CK20 is a sensitive marker for Crooke’s cells and the early cytoskeletal changes associated with hypercortisolism within pituitary corticotrophs. Endocr Pathol. 2006; 17:365–376.

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[30] Ikeda H, Yoshimoto T, Ogawa Y, Mizoi K, Murakami O. Clinicopathological study of Cushing's diseases with a large pituitary adenoma. Clinical Endocrinol (Oxford). 1997; 46:669–679. [31] Rinenhart JF, Farquhar MG. Electron microscopic studies of the anterior pituitary gland. J Histochem Cytochem 1953; 1:93–113． [32] Kageyama M. The follicular cell in the pars distalis of the dog Pituitary gland; an electron microscope study. Endocrinology, 1965; 77:1053– 1060. [33] Tachibana O, YamashimaT. Immunohistochemical study of folliculostellate cells in human pituitary adenomas. Acta Neuropathol (Berlin), 1988; 76:458–464. [34] Nakajima T, Yamaguchi H, Takahashi K. S-100 protein in folliculostellate cells of the rat pituitary anterior lobe. Brain Res, 1980; 191:523–531. [35] Cocchia D, Miani N. Immunohistochemical localization of the brainspecific S100 protein in the pituitary gland of the adult rat. J Neurocytol. 1980; 9:771-782. [36] Ikeda H, Yoshimoto T. Clinicopathological study of Rathke's cleft cyst. Clin Neuropathology. 2002; 21:82–91. [37] Velasco ME, Roessmann U, Gambetti P. The presence of glial fibrillary acidic protein in the human pituitary gland. J Neuropathol Exp Neurol, 1982; 41:150–163. [38] Marine F, Boya J, Lopez-Carbonell A, Borregon A. Immunohistochemical localization of intermediate filaments and S-100 proteins in several non-endocrine cells of the human pituitary gland. Arch Hist Cytol, 1989; 52:241–248. [39] Liu YC, Tanaka S, Inoue K, Kurosumi K. Localization of fibronectin in the folliculo-stellate cells of the rat anterior pituitary by the double bridge peroxidase-anti-peroxidase method. Histochemistry, 1989; 92:43–45. [40] Ikeda H, Yoshimoto T, Suzuki J. Immunohistochemical study of Rathke's cleft cyst. Acta Neuropathol, 1988; 77:33–38. [41] Kasper M, Karsten U, Stosiek P, Moll R. Distribution of intermediatefilament proteins in the human enamel organ: unusually complex pattern of co-expression of cytokeratin polypeptides and vimentin. Differentiation, 1989; 40:207–214. [42] Kovacs K, Horvath E. Cytology. In: Hartmann WH (Eds) Tumors of the pituitary gland, 2nd series, Fasc. 21. Armed Forces Institute of Pathology, Washington. 1986, pp16–55.

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[43] Kasper M, Karsten U. Co-expression of cytokeratin and vimentin in Rathke’s cysts of the human pituitary gland. Cell Tissue Res. 1988; 253:419–424. [44] Inoue K, Couch EF, Takano K, Ogawa S. The structure and function of folliculo-stellate cells in the anterior pituitary gland. Arch Histol Cytol. 1999; 62;201–218. [45] Voit D, Saeger W, Luedeck DK. Folliculo-stellate cells in pituitary adenomas of patients with acromegaly. Pathology, 1999; 195:143–147. [46] Waseem A, Alexander CM, Steel JB, Lane EB. Embryonic simple epithelial keratins 8 and 18: chromosomal location emphasizes difference from other keratin pairs. New Biol, 1990; 2:464–478. [47] Pankov R, Umezawa A, Maki R, Der CJ, Hauser CA, Oshima RG. Oncogene activation of human keratin 18 transcription via the Ras signal transduction pathway. Proc Natl Acad Sci USA. 1994; 91:873– 877.

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In: Keratin: Structure, Properties and Applications ISBN 978-1-62100-336-6 Editors: Renke Dullaart et al. pp. 149-211 ©2012 Nova Science Publishers, Inc.

Chapter 7

KERATIN FIBERS FROM CHICKEN FEATHERS: STRUCTURE AND ADVANCES IN POLYMER COMPOSITES

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Ana Laura Martínez-Hernández and Carlos Velasco-Santos Instituto Tecnológico de Querétaro, División de Estudios de Posgrado e Investigación. Santiago de Querétaro, Querétaro, México

ABSTRACT Natural fibers have been involved in an emerging kind of polymer composites taking advantage of the outstanding properties that nature confers them. Thus, several types of biofibers have recently attracted increasing interest of engineers and scientists since mimic their structures or make use of all their potential is a motivating challenge in polymeric materials field. Biofibers can be obtained from different renewable natural sources, those from vegetal origin have been the most exploited, however alternative raw materials, such as keratin biofibers are coming into view. Keratin, as fiber, can be found in hair and feathers. Human hair keratin and wool have been studied since many years ago, due to textile and medical implications, whereas keratin from feathers has not been highlighted enough. Keratin fiber has a hierarchical structure, with a highly ordered conformation, is by itself a biocomposite, product of a large evolution of animal species. Keratin fibers from feathers are non-

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Ana Laura Martínez-Hernández and Carlos Velasco-Santos abrasive, eco-friendly, biodegradable, insoluble in organic solvents, and also have good mechanical properties, low density, hydrophobic behavior and finally low cost. These characteristics make keratin fibers from chicken feather a suitable material to be used as a high structural reinforcement in polymer composites. However the development of keratin fibers as a new reinforcement must be based on a complete characterization in order to know their features, advantages and restrictions. The analysis of keratin fiber from chicken feather include spectroscopic analysis using both Fourier transform infrared and Raman, thermal studies including differential scanning calorimetry and thermogravimetric analysis, also contact angle determinations reflecting the hydrophobic behavior are shown. Morphological details are observed with optical, transmission and scanning electron microscopy. During the last decade these new fibers have been studied by different research groups as reinforcement for several synthetic polymers; their results are reviewed together by first time in concerned literature. Thus, keratin fibers from chicken feathers are shown as a novel eco-friendly material that must be adequately applied in the development of green composites. Finally, the studies reviewed in this chapter can be the scaffolding to increase the use of this protein taking as base the knowledge of its properties and scope, and therefore the current and future research in keratin could be related to high importance areas as nanotechnology or environmental decontamination, opening interesting gates in multidisciplinary areas that could take advantage of the high performance that nature confers to keratin fibril protein.

1. INTRODUCTION Keratin, a natural protein, can be considered one of the most abundant biopolymers over the world. Its importance is due to not only by this plenty but also by its high organizational structure reflected in diverse properties and related with distinct tissues for several species. Keratin can be found in varied organisms totally dissimilar, such as humans, horses, snakes and birds, among others, this biopolymer has specific functions in each one according with the related tissue. This chapter deals just one of these: keratin from chicken (Gallus gallus) feathers. The first part describes briefly the origin and evolution of this tangled structure and some interesting findings in fossils are mentioned. Later the most important features of keratin are explained, mentioning for instance its microstructural arrangement, composition and important functional groups, thermal and mechanical properties. In the third part, the most studied application of keratin from feathers is discussed; it

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covers polymer composites reinforced with feather keratin fibers, quill and whole feather. Thus, some of the most important contributions of several authors are reviewed. The last part of this chapter provides information about one of the most promising areas to change and improve the properties of keratin: grafting. This procedure can be done using the keratin fiber as the main biopolymer chain and joining some synthetic polymer or using the keratin polypeptide chain to attach it over a principal synthetic polymer. In both cases keratin is a versatile compound that imparts diverse properties, worthy to be studied. It is important emphasize that these applications: reinforcement in composites and modification by grafting, have been studied more seriously only since the last decade and they are very promising. However, several scientific interests are converging toward keratin, since it can be exploited much more in order to take advantage of their properties in high interest research areas such as environmental remediation or nanotechnology.

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2. FEATHERS, A HIGH EVOLVED MATERIAL Fibril proteins, such as keratin or collagen, are a common feature in vertebrate organisms. Their production with a particular conformation is undoubtedly caused by the adjustment process to the environmental conditions to survive. Thus, the creation of feather keratin implies a chain of mutations in the evolution process. Feathers are probably one of the most complex derivatives of the integument in any vertebrate. In fact, the presence of feathers is not always related with birds, since there are probes of feather before bird‟s origin. This has been proved by recently discovered fossil evidence. Several research groups has published the presence of non-scale skin appendages, however the homology between these structures and bird feathers is still on open debate. Nevertheless, it is worthy of mention that primitive feathers or similar skin appendages showed in actual poultry were already present in large dinosaurs 130 million years ago (Ortega et al, 2010). An interesting review of important fossil feather discoveries is presented by Davis and Briggs (1995), but in spite of this is a complete summary, it only gives information from Late Jurassic age to Late Pliocene and recently some findings show non avian feathers that were found in an archosaur fossil named Longisquama insignis, which lived 220 million years ago, these appendages antedate the feathers of Archaeopteryx, the first known bird from the Late Jurassic (145 million years ago) (Jones et al, 2000). On the other hand,

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according with Turner et al, (2007) some nonavian theropod dinosaurs were partially covered in feathers or filamentous protofeathers, which could have been used for display, in shielding nests for thermal control, or for creating negative lift during incline running. In this sense there are different hypotheses regarding the origin and early evolution of feathers. The main discussion focuses on whether the feather evolved on an aerodynamic background or a nonaerodynamic environment related with purposes of insulation, weight reduction, heat shields, waterproofing or display (Zhang and Zhou, 2000). Actually feathers are complex hierarchical arrangements of a three level branched structure composed of a rachis (primary shaft), barbs and barbules (secondary and tertiary branches respectively). Taking into account developmental theories, modern feathers probably evolved through the following stages: a) elongation of scales, b) appearance of a central shaft, c) differentiation of vanes into barbs, and d) appearance of barbules and barbicel (Zhang and Zhou, 2000; Perrichot et al, 2008). Figure 1 shows graphically this evolution process, which is divided in several phases according to developmental patterns summarized by Norell and Xu, (2005).

Figure 1. Phases of feather evolution. (Adaptation from Norell and Xu, 2005; some parts are also licensed under the Creative Commons Attribution-Share Alike License 3.0 Unported (CC-BY-SA).

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First, the feather originates as a hollow tube (phase 1) that changes into a series of barbs (phase 2). In the phase 3 the barbs self organize along a rachis. The phase 4 is characterized by the origin of barbules, which are close related with the self organization of the feather, in this stage the organizational structure looks as a modern feather, in addition to the phase 5, distinguished by development of the asymmetrical vanes on the flight feathers, which is not shown in figure 1. An elegant description of modern feathers was given by Norell and Xu, (2005). They classify feathers in five main types: contour feathers, down feathers, semiplumes, filoplumes and bristles. All of them have a common organization: a central hollow tube, called calamus (at initial length) or rachis (over a certain distance), the second structures are called barbs, these are branches joined to the rachis, and finally barbules, microscopic self-organizing structures. These components are illustrated in figure 2, where it is possible to observe the well defined structures described above. On the other hand figure 3 shows fossil feathers from the Early Cretaceous, conserved in amber (Perrichot et al, 2008); these images show great similarities between optical micrograph shown in figure 2(b), both were taken using transmitted light microscope.

Figure 2. Images of feather structure: a) semiplume feather, b) optical micrograph, c) and d) Scanning electron micrograph.

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Figure 3. Optical micrograph of fossil feathers from Early Cretaceous, conserved in amber. (Reprint with permission of Perrichot et al, 2008, original figure page 1198, Proc. R. Soc. B).

The intricate development and structural arrangement of feathers have attracted the attention of several scientific disciplines, besides many biological materials, such as feathers; have pathways that have been highlighted by different research areas like nanotechnology, materials science, medicine, environmental remediation, among others. Meyers et al, (2011) mentioned some of these unique features: a) Natural structures are assembled from the bottom-up process using surrounded components. This means “self assembly” is dictated by nature to have the necessary conformation to survive and evolved.

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b) The ability to serve more than one purpose, for instance feathers involves diverse functions: mechanosensory, ornament, flight, thermal regulation, etc. This kind of structure is called “multifunctional”, which is worthy to be imitated and could be the basis for new and high developed materials or devices. c) The properties are highly dependent on the level of water in the structure. Specifically, keratin fiber has three different types: free water, loosely bounded water and chemically bounded water. This last contributes to the conformational stability of protein due to interactions with negatively and positively charged groups of the polypeptide chains and to its ability to form hydrogen bonds, thus water produces a three dimensional network of interactions within the keratin structure, which together with the disulfide bond of the cystine, stabilizes the secondary structure (Martínez-Hernández et al, 2005a). d) The morphology and properties of natural structures are determined by the evolution, environmental restrictions and availability of materials and elements. e) The synthesis of almost all biological materials is realized in an aqueous environment at ambient temperature and 1 atm of pressure. f) The structures are hierarchical, which means they are ordered by different scale levels. Hierarchy confers them distinct and unique properties. Scientists are interested in understand these pathways in order to take advantage and use the capabilities and potential of these characteristics by mimic or improving the applications of biological materials in diverse areas. Feathers have relation with all the features described above; thus, represent an interesting material to be studied deeply. Their characteristics initiate, as can be seen in figure 2, at the morphological level, which represents a self-organized hierarchical structure. This characteristic has been studied by several authors at different levels. Recently Meyers et al, (2011) reported hierarchy in Falco sparverius primary remiges. These authors described that the feather rachis has a cellular core limited by a solid wall, both constituted by keratin. The internal structure, similar to foam, has an intricate arrangement. The observed cells has an approximate diameter of 10 m, but higher magnification in scanning electron microscopy (SEM) shows that cells are not solid but are formed by a network of fibers with diameters around 200 nm (figure 4).

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Figure 4. Scanning electron micrographs of macaw feather, (a) Rachis, barb and barbules; (b) Hollow section of macaw feather rachis. (Reprint with permission of Meyers et al, 2011, Elsevier).

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Figure 5. Scanning electron micrographs of falcon feather rachis. (Reprint with permission of Meyers et al, 2008, Elsevier).

In addition, Meyers et al, (2008) illustrated the closed-cell foam within the rachis and barb structures (figure 5). According to Reddy and Yang (2007), the presence of honeycomb structures is the main reason for the low density of barbs, also provides air and heat insulating capabilities different to any other natural fiber. In fact, until 2007, none of the natural or synthetic fibers commercially available have a density as low as that of chicken feathers, around 0.8 g/cm3. On the other hand, also Martínez-Hernández et al, (2005a) observed by SEM that central barbs are hollow and have an irregular, non-cylindrical shape, which is detailed in figure 2(c). In this article Keratin fibers from chicken feathers were characterized by transmission electron microscopy (TEM), the micrographs are shown in figure 6, where microfibrils are immersed within the amorphous matrix, identified by its dark color. Figures 6(a) and 6(b) show the twisted microfibrils forming a helix that is related with the high mechanical strength from fiber. In figures 6(c) and 6(d) the presence of two different structures inside the keratin fibers: microfibrils and protofibrils, is confirmed. The first has a more order and crystalline structure than the matrix, being this amorphous and with a high content of amino acid cystine. The protofibrils are inside the microfibrils and are also surrounded by the matrix, as can be appreciated in the figures 6(c) and 6(d).

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Figure 6. Transmission electron microscopy of chicken feather fiber; a-b) longitudinal sections, c-d) cross section.

All the components of feathers are constituted by keratin, but it is important notice that there are differences in composition between feather and other avian keratins, for instance the molecular weight of keratin for feather was measured as 10,500 g/mol, whereas for claw or beak keratin was determined a higher value from 13,000 g/mol to 14,500 g/mol (Meyers et al, 2008). The next section of this chapter deals with keratin composition, hierarchical organization of protein arrangement and certain properties useful for practical applications.

3. FEATHER KERATIN STRUCTURE AND PROPERTIES Proteins are the most versatile example of biopolymers due to the flexibility of their structures and functions. Proteins are usually composed of 20 different natural amino acids, which are joined in sequential chains. The

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specific construction of the poly (amino acid) chain allows the formation of a plenty quantity of proteins with diverse properties. This sequence or chain is known as the primary structure. Once defined this primary structure, the second level of hierarchical array appears: secondary structure. This is the local spatial arrangement of the polypeptide chain, usually defined as helices or -sheets. After these two initial levels, proteins have the three dimensional conformation of the secondary structures, called tertiary structure, and the quaternary structure formed by the assembly of individual proteins in supramolecular complexes (Heim et al, 2010). By themselves proteins have complicated structures, but precisely because of this intricate organization, they have innumerable functions in living organisms. Keratin is a fibril protein; it consists of polypeptide chains formed by the condensation of different amino acids. The amino acid content of feathers depends on the breed, food and environment, but generally prevail serine, proline, glycine, valine and cystine (Walker and Rogers, 1976; Schmidt, 1998; Martínez-Hernández et al, 2005a). The primary structure of feather keratin has been studied by several authors since some decades ago. Walker and Rogers in 1976 asseverate that in down feather keratin, most keratin chains possessed amino-terminal and carboxylterminal regions, rich in half-cystine, and a large internal region devoid of half-cystine and rich in hydrophobic amino acids. On the other hand, Fraser and Parry (2008) elucidate the importance of certain amino acids in the conformational arrangement, for instance proline, one of the most abundant in keratin, has an important influence on protein conformation due to the rigid constraints caused by its ring structure on rotation of the N- C bond and to the cis-trans isomerism about the preceding peptide bond. Secondary structure of keratin feather has been a discussion topic since many years ago. Arai et al, (1983) were based on primary structure of feather keratin to predict its secondary conformation, as well as Fraser and Parry (2008). Figure 7 shows a representative scheme of both proposals. As can be seen -sheet structure and coil are common for both predictions. Besides these reports, feather keratin secondary structure has been studied using infrared and Raman spectroscopy, X-Ray diffraction and electron microscopy by many other research groups. Several authors consider that feather keratin presents -sheet, turn and random coil structure, whereas others identify -helix, or helical array of structure, so there is not a definitive conclusion about this structural arrangement. A summary of the reported keratin types in feathers is shown Table 1.

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Table 1. Keratin secondary structure found in feathers from different species Feather part Avian species Barbules Cockatoo

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Barbs

30 % β - sheet, turn and random coil King Penguin, Wood β - keratin Stork, American Crow Chicken α-keratin type Fowl

Quill

Seagull Pigeon

Rachis

Non specified Fowl Seagull Chicken

Calamus Non specified

Proposed Secondary Structure - sheet

Seagull Goose Non specified Non specified Non specified Non specified Emu Chicken

Chicken

Reference Akhtar and Edwards, 1997. Arai et al, 1983 Yu et al, 2004

Reddy and Yang, 2007 - type, feather keratin structure Berg et al, 2002 - sheet as dominant conformation Pauling and Corey, - helix 1951 α - helix and pleated sheet layers Mercer, 1961 Feather keratin structure Dove et al, 2007 Resembled α - keratin - sheet, 18 % helical from Schor and Krimm, twisted sheet 1961 Remaining of turn and other arrangements Helical array of β- crystallites Church et al, 1998 β - configuration and Takahashi et al, α - protein 2004 Ambrose and Elliot, 1952* - sheet and 4 % α - helix Rintoul et al, 2000 - helix Filshie and Rogers, 1962 Helical structures Ramachandran and Dweltz, 1962† -sheet Yu, 2005 - sheet, Sun et al, 2009 - turn and 11.18 % random. 41 % α - sheet and Fraser et al, 1971 21 % Random

* According with Ambrose and Elliot, 1952, Proc. Roy. Soc. A206, 206, cited by Mercer, 1961. † Proposed by Ramachandran and Dweltz, 1962, 147, Collagen Proc. Symp., Univ. Madaras, India, cited by Takahashi et al, 2004.

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Figure 7. Amino acid sequence and prediction of secondary structure of keratin from a) fowl feather (adapted from Arai et al, 1983) and b) emu feather (Adapted from Fraser and Parry, 2008).

The polypeptide chain of keratin has been extensively studied by infrared and Raman spectroscopies, because of that, it is possible to identify the intrinsic functional groups in feather keratin. Figures 8 and 9 show FTIR and Raman spectra of keratin fibers from chicken feathers (Martínez-Hernández et al, 2005a). It was found that the Raman spectra obtained from the rachis, calamus, barbs and barbules from chicken feathers were the same (Church et al, 2010). The moieties found by these vibrational spectroscopies are important since they are suitable to be attached to other biological or synthetic materials. In addition structural details on secondary conformation are suitable to study due to amide bands. The infrared spectra of proteins show nine normal modes of Amide bands, of these, Amide I, II, III, A and B are very sensitive to hydrogen bonding, dipole-dipole interactions and peptide backbone geometry, thus can be useful indicators of secondary structural changes. The signals of Amides are assigned as follows: Amide I and A are mainly the CO and NH bond stretch, respectively, whereas the Amide II and III are mixtures of the CN stretch and HNC bend (Hayashi and Mukamel, 2008; Xu et al, 2009). Specifically the Amide I mode is principally the peptide carbonyl on the v(CONH) unit together with an out of phase CN stretching component and contribution from the CCN deformation, whereas the Amide II corresponds with δ(NH) in plane and v(CN), with contributions from v(CC), plane and v(NC). The amide III mode originates from in-plane combination of -plane and v(CN), with contributions from the v plane (Akhtar et al, 1997). On the other hand the Amide A band and Amide B originate from a Fermi resonance caused by the first overtone of Amide II and the v(NH). FTIR and Raman spectra of feather fiber can be analyzed by defined wavenumber range:

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Ana Laura Martínez-Hernández and Carlos Velasco-Santos a) Region from 3500 to 2500 cm-1. The signals around 3300 and 3150 cm-1 correspond to the Amide A and B bands. The bands next to 3300 cm-1 have been associated with ordered regions in -helix protein structure and correspond with the v(NH) (symmetrical stretching) modes (Pielesz et al, 2000; Akhtar and Edwards, 1997). The Amide B band is reported from 3056 to 3075 cm-1 (Wojciechowska et al, 1999). Additionally the range from 3100 to 2700 cm-1 was assigned as characteristic for dipolar ion amino acids RCH(NH3+)COO- (Pielesz et al, 2000). The NH3+ group corresponds to a wide band around 3100–2700 cm–1 with v(NH) and va(NH) (asymmetrical stretching vibrations). Edwards et al, (1998) report a shoulder around 2962 cm-1, that can be assigned to va(CH3). A signal at 2930 cm-1 has been assigned to the v(CH3) for different keratin samples, but also this signal has been considered as a Fermi resonance doublet through the interaction of the overtone of the v(CH3) mode for the methyl groups at the acyl chain termini (Pielesz et al, 2000).

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b) Region from 1700 to 500 cm-1. The strong signal at 1652 cm-1 was assigned to the v(C=O) Amide I, with -helix conformation, whereas the signal at 1666 cm-1 corresponds to the v(C=O) Amide I for -sheet conformation. The band at 1531 cm-1 was assigned to the NH in plane bending for sheet conformation (Edwards et al, 1998). Wojciechowska et al, (1999), also reported that the Amide I band is close related to v(C=O) of the carbonyl groups and is observed around 1600-1690 cm-1; Amide II appears at 1480-1580 cm-1 and is related with (NH) (symmetrical bending vibration) and v(CN). According to Akhtar et al, (1997) and Long (1997) the signal at 1455 cm-1 corresponds with δ(CH2) or δ 3). Also the band near 1375 cm-1 can be assigned to δ(CH). The bands around 1230-1240 cm-1 have been related to the β-sheet structure from amide III and the band close to 1174 cm-1 is produced by v(CC) from side chain aminoacids. The region from 1050 to 1150 cm-1 also corresponds to skeletal v(CC). Finally, the v(CS) from alkylthiols is localized approximately at 730-620 cm-1, this group is originated from amino acid cystine (Edwards et al, 1998).

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Figure 8. FTIR spectrum of keratin fiber from chicken feather.

Figure 9. Raman Spectra of keratin fiber from chicken feather.

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3.1. Thermal Properties Thermal behavior of keratin from feathers depends on chemical composition and structural arrangement. Many applications for this material are restrained by this performance. In spite of this, the studies regarding this aspect are few. Martínez-Hernández et al, (2005a) presented thermal results, where single keratin fiber from chicken feather was characterized by means of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The DSC results show an endothermic peak at 69°C, which implies the loss of “free water”; it coincides with the first mass loss in TGA, a decrease of 5% in fiber mass from around 25°C to 55°C (figure 10(a)). In the same article, the last DSC endothermic peak at 418°C was assigned to a decomposition process, which also is reflected in TGA from 222°C to 392°C as a decreasing from 10% to 78% in fiber mass. This decomposition is associated with disulphide bonds destruction and elimination of H2S, also involves the denaturation of the helix structure and the thermal pyrolysis of the chain linkages, peptide bridges and the skeletal degradation. Several chemical reactions occur in this region where protein compounds are decomposed to lighter products and volatile compounds such as H2S, CO2, H2O, HCN (Popescu and Augustin, 1999). Recently, an interesting thermal study on keratin was developed by Brebu and Spiridion, (2011). Their results show a comparison of the thermal behavior of sheep wool, human hair and chicken feathers.

Figure 10. Thermogravimetrical curves of keratin materials: a) keratin fibres of chicken feathers; b) Wool, Hair and feathers (Reprint with permission of Brebu and Spiridion, 2011, Elsevier).

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Figure 10(b) distinguishes the TGA curves for these three keratin materials, all follow a similar pattern in decreasing mass, as can be observed. These authors also studied the pyrolysis products, founding only few differences, mainly in the distribution of compounds in aqueous phases for these three keratin materials. The thermal decomposition involves two main steps; in both there are several overlapped periods. The first degradation step takes place from 170°C to 300°C, it initiates with formation of NH3 (167°C) and CO2 (197°C), with maximum evolution for these compounds at 273°C and 287°C respectively. After, the process continues with formation of sulphur containing inorganic compounds: SCS (240°C), SCO (248°C), H2S (255°C) and SO2 (253-260°C). The final compounds in this stage are water (255°C) and thiol (257°C). The second stage, above 300°C, involves a second formation of thiol (320°C), a maximum evolution of nitriles from 340°C to 480°C and the formation on phenol (370°C) and 4-methylphenol (400°C), these last represent the most important degradation compounds. In figure 11 the FTIR peaks assigned by Brebu and Spiridion (2011) to the most important degradation compounds can be appreciated.

Figure 11. FTIR spectrum of the evolved gases from TGA analysis of chicken feathers (Reprint with permission of Brebu and Spiridion, 2011, Elsevier).

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3.2. Hydrophobic Behavior of Feather Keratin

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Hydrophobicity has been understood as the tendency of non-polar molecules to form aggregates in order to reduce their surface of contact with polar molecules such as water. The importance of hydrophobic interactions is due to they are often the driving force in a variety of physical and biological phenomena (Sarkar and Kellog, 2010). In fact, important effects in proteins, such as self assembly or secondary structure transitions are dictated by hydrophobic interactions between the amino acids (Bowerman et al, 2009). Protein fibers such as silk, wool or hair have been studied as hydrophobic materials. For instance two kind of fibroin were compared by Acharya et al, (2009), founding that fibroin from Antheraea mylitta is more hydrophobic than the fibroin from Bombix mori (mean contact angles are 91.1° and 67.2° respectively). On the other hand, keratin from wool shows contact angle of around 109° and wetting time of 100 seconds, these results reveal the hydrophobic nature of keratin (Gaffar Hossain et al, 2010). Hair, other example of keratin fiber, also shows hydrophobic behavior since a contact angle of 108° was calculated using Wilhelmy Force Profiles (Wortmann et al, 2010).

Figure 12. Advancing and receding contact angles of feather keratin fibers and the corresponding contact angles hysteresis.

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Similar results are shown by Lodge and Bhushan, (2006) and Molina et al, (2001). Taking into account the above results is not surprising that feather keratin also show hydrophobicity. Sun et al, (2009) report that chicken feather is a hydrophobic material with a water contact angle of 138°. But this was also reported before by Martínez-Hernández et al, (2005a), since feather fibers do not change appreciably neither advancing nor receding contact angles after repeated immersions of the same fiber in water. The difference between these measured angles, which is known as contact angle hysteresis, is very small as can be observed in figure 12, and this fact could be taken as a hydrophobic indicator. Lam et al, (2001, 2002) demonstrated that liquid sorption and/or retention is a probable cause of the time dependence of contact angle hysteresis (as well as advancing and receding contact angles), In addition the advanced contact angle remains near 90°, thus it can be considered that fiber has a hydrophobic behavior (Wortmann et al, 2010 and Molina et al, 2001).

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3.3. Mechanical Properties of Feather Fiber The different amino acids in keratin play an important role in several properties of feathers; hydrophobic behavior is a clear example, but not the only one, since also mechanical properties can be affected by the crosslinking between hydrogen bonds, dipole attractions or S-S linkages due to cysteine. Another important contribution is the self assembly hierarchical microstructure (Martínez-Hernández et al, 2003b). Thus, the highly cross-linked structure gives feathers good mechanical properties. Recently, Zhan and Wool, (2011), have studied the mechanical properties of the chicken feather barbs, including the tensile properties and dynamical mechanical properties. These authors observed that the stress-strain curve, shown in figure 13, began with an initial non-Hookean, J-shaped curve, after a linear region is presented, followed by the yield point and the break point. This mechanical behavior can be explained in terms of structural changes, taking into account the assumptions of Martínez-Hernández et al, (2005b). The linear region is due to changes in bond angles and bond spacing but without affect the secondary configuration in the helical structure of the microfibrils. During the continuous development of strain, the helical structure is unfolded due to realignment in the hydrogen bonds in relation to keratin components; this causes a change in secondary structure, which in wool and hair is called - transition (Hearle, 2000). In this zone, the mechanical properties of fiber may be recovered if the fiber is

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released before its rupture. However, in the vicinity of break point, the fiber loses its mechanical properties and finally is broken due to hydrogen and disulphide bonds are disrupted, thus denaturing the protein structure. Zhan and Wool, (2011) also show the effect of diameter variance in chicken feather barbs through their length and from the barb location on the feather, therefore causing a large discrepancy on tensile modulus results. The authors conclude that the strain at break of chicken feather barb has a mean value of 6.93%, the tensile modulus was 3.59 ± 1.09 GPa and average tensile strength was 203 ± 74 MPa.

Figure 13. Tensile stress-strain curve of a chicken feather barb (Reprint with permission of Zhan and Wool, 2011, John Wiley and Sons).

On the other hand, Reddy and Yang, (2007), measured the tensile properties of chicken feather barbs. These were compared with turkey barbs and wool in table 2. As can be observed in turkey barbs also the kind of feather plays an important role in the mechanical properties, the difference probably is due to variance in diameter and characteristics inherent to the feather function. Another interesting comparison in mechanical properties of feathers was done by Cameron et al, (2003). These authors studied the Young‟s modulus of three species of bird: goose, swan and ostrich.

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Table 2. Tensile properties of chicken feather barbs, turkey feather barbs and wool (Adapted from Reddy and Yang, 2007)

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Fiber Chicken barbs Turkey barbs* Turkey barbs† Wool * Pennaceous feather. † Plumalaceous feather.

Strength (MPa) 187.2 ± 59.8 107.9 46.8 156-234

Elongation (%) 7.7 ± 0.85 7.96 16.43 30-40

Modulus (MPa) 4628 ± 1449.5 2021.5 581.1 3900-5850

For each one three specific areas of the rachis were evaluated at 0, 50 and 75 % of total rachis length from calamus to tip. Primary flight feathers were chosen for swan and goose, whereas for ostrich were wing feathers. Their results are shown in figure 14, where it is possible to observe that swan and goose rachis have an increase in Young‟s modulus along the rachis, nevertheless in ostrich rachis this behavior was not observed, since a considerable increasing was not visible. It is interesting to observe that Young‟s modulus for rachis is higher than that for barbs. In turn, figure 15 (from Zhan and Wool, 2011) shows the storage modulus for chicken feather barbs, evaluated by Dynamical Mechanical Analysis. This curve manifest a gradual decreasing as temperature increases, but the values have significant deviations due to the heterogeneity of the keratin protein structure and the geometrical irregularity of the barbs.

Figure 14. Mean tensile Young‟s modulus for swan, goose and ostrich feather rachis. (Reprint with permission of Cameron et al, 2003, Elsevier).

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Figure 15. Storage modulus for chicken feather barb. (Reprint with permission of Zhan and Wool, 2011, John Wiley and Sons).

This decreasing is common for almost all polymers and represents a tendency to be a less rigid material.

4. FEATHER KERATIN FIBERS; A HIGH STRUCTURAL REINFORCEMENT IN POLYMER COMPOSITES One way to take advantage of the feather keratin features described above is their use as high structural reinforcements in polymer composites. Thus, between the different bonds that stabilize the hierarchical structure in keratin fibers there are some susceptible to be used as possible chemical reaction points and then have a chemical interface. The stable thermal behavior of feather keratin, in which decomposition process does not start before 200°C, allows establishing a possible range of temperature for its use. In addition, morphological characterization through optical and scanning electron microscopy show some interesting features, such as surface roughness, flexibility, and high length to diameter ratio. It was found that feather keratin fibers have an apparent diameter ranging from 4 to 8 μm with dispersion

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measured by its standard deviation of 1.4 μm. The contact angle measurements support a hydrophobic character in feather fibers, which suggest a good interface with polymers, in contrast to cellulose hydrophilic nature that causes poor distribution in synthetic polymers. Beside these features, mechanical properties are also very interesting compared with those for cellulose natural fibers (shown in table 3). In spite of tensile strength and elastic modulus are not very high, this last is comparable to cotton and coir, these two natural fibers have also a higher value for elongation percentage, other natural fibers are below; however chicken feather density (less than 1.0 g/cm3) is a great advantage of chicken feather is its compared to cellulose fiber density. Taking into account these characteristics, feather fibers are being successfully employed by different authors to reinforce polymeric composites with some advantages over other reinforcing materials such as plant (cellulose) and synthetic fibers. These advantages include low cost, low density and the high specific properties mentioned above, together with their biodegradable and nonabrasive nature. Besides these, feather fibers are effectively a selfsustainable and continuously renewable source of fiber. Therefore these fibers represent an alternative source for keratin, which could produce new and strong engineering materials from non petrochemical origin. The proper utilization of keratin fibers from chicken feather opens research possibilities in materials field and, at the same time, contributes to diminish the squandering of natural resources. Table 3. Mechanical properties of natural cellulose fibers (Adapted from Ku et al, 2011) Fiber

Density (g/cm3) Cotton 1.5-1.6 Jute 1.3 Flax 1.5 Hemp 1.47 Kenaf 1.45 Sisal 1.5 Coir 1.2 Softwood Kraft 1.5 Pulp

Elongation (%) 7.0-8.0 1.5-1.8 2.7-3.2 2-4 1.6 2.0-2.5 30 4.4

Tensile Strength (MPa) 400 393-773 500-1500 690 930 511-635 593 1000

Elastic Modulus (MPa) 5.5-12.6 26.5 27.6 70 53 9.4-22 4.0-6.0 40

Thus, keratin materials obtained from feathers as barbs, barbules and quill have been incorporated to different polymer matrices in order to verify the

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potential application of the diverse feather parts in the development of polymer composites and their features related with the lightness, structural, mechanical and thermal properties. In addition, acoustic and electrical properties have been studied in the keratin feather composites due to the interesting sound absorption and insulated properties that these materials possess. Therefore, this section focuses to present the researches related with the synthesis and characterization of polymer matrices composites reinforced with different feather parts. Next the mechanical, morphological, thermal, acoustic and electrical properties of feather keratin polymer composites (FKPC) are reviewed.

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4.1. Mechanical Properties of Feather Fiber Polymer Composites The mechanical performance is one of the most studied properties in FKPC. Different mechanical parameters have been evaluated when feather keratin is incorporated to various synthetic polymer matrices such as: Polypropylene (PP), Polyethylene (PE), Poly(methylmethacrylate) (PMMA) and Epoxy. Keratin has been incorporated to these polymer matrices such as fiber using the bars and barbules of the feather, the quill, quill powder and the whole feather, also combinations using other kind of fibers with chicken feathers have been included in synthetic polymer matrices. Next, the mechanical properties related with the incorporation of keratin materials obtained from chicken feathers are described. Some researches focus to obtain FKPC have used barbules obtained according to the patent US 5750030 1998. The process allows separate almost completely the quill and thus, it is possible to use barbules, although some parts of barbs are also found, short uniform keratin fibers are successfully obtained by this method. The mechanical properties of the composites reinforced with this kind of keratin fiber are reviewed next. Martinez–Hernandez et al, (2005b) synthesized polymer composites reinforced with keratin fiber utilizing PMMA as matrix. For these composites, 1wt % to 5wt % of fiber (taking as reference the weight of methyl methacrylate monomer) was incorporated to polymer matrix during monomer polymerization. Keratin biofiber produces a positive effect in PMMA, since the Young‟s modulus of this rigid polymer is modified with the incorporation of fiber. Table 4 shows the Young‟s Modulus obtained by tensile testing for each concentration of fiber used in this research, in comparison with the

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theoretical Young‟s modulus calculated by the Halpin-Tsai equation for short fiber reinforced composites. Table 4. Young’s modulus of Keratin Fiber Polymer Composites (Polymer Matrix PMMA) (Adapted from Martínez-Hernández et al, 2005b)

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Keratin Fiber Weight Percent 0 1 2 3 4 5

Theoretical Young‟s Modulus (GPa) (Halpin-Tsai) 5.00 4.98 4.95 4.93 4.91 4.89

Young´s Modulus (GPa) (Tensile Testing) 5.05 5.50 5.66 5.97 6.17 6.50

It is evident that keratin biofiber form a composite with PMMA, because the experimental values of Young‟s Modulus are superior to the theoretical. Also the interface produced between keratin fiber and PMMA was evaluated by SEM in the samples after fracture in tensile test. The figure 16 shows some images obtained in this research, where the fiber is broken and covered by the polymer after mechanical stress, indicating load transfer in the composite. Barone and Schmidt, 2005, used also the keratin fiber obtained by the process described in the patent US 5705030 1998. Fibers with different lengths were incorporated to polyethylene (PE) through mechanical mixing in order to evaluate mechanical properties by tensile test. Elastic and specific moduli of KFPC increase with respect to PE matrix at different loads; except to the composite with 0.5 volume fraction of keratin fiber; authors did not observe a good dispersion in this composite. Figure 17 shows both; elastic modulus and specific modulus obtained with 0.1 cm length keratin fibers; it is possible to observe in this figure that keratin fiber contributes efficiently modifying PE, since not only the Young‟s Modulus increases its values with keratin fiber contents, in addition, the lightness of fiber produces important increments on specific modulus in composites taking as reference PE matrix. Also, in this research the elastic modulus and yield stress at constant concentration and variable fiber aspect ratio were evaluated. Figure 18 shows the obtained results, it is clear that keratin fiber has functional lengths and therefore adequate aspect ratios to be included in polymer composites, also considering low aspect ratios, the good interactions at interface level produce higher values in the modulus and yield stress, better than in PE. Thus, considering these results, it is possible to assume that the surface nature of fiber contributes more

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than aspect ratio in the mechanical properties of these composites; since, both mechanical parameters (elastic modulus and yield stress) do not change significantly with higher aspect ratios of the fiber.

Figure. 16. Scanning Electron Microscopy of fractured composites (PMMA-keratin fiber). Reprint with permission of Martínez-Hernández et al, 2005b, Elsevier).

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Figure. 17 Elastic modulus and specific modulus of composites as function of keratin fiber (0.1 cm) (Reprint with permission of Barone and Schmidt, 2005, Elsevier).

Figure 18. Elastic modulus and yield stress of composites as function of fiber aspect ratio at 20 wt % of fiber loading. (Reprint with permission of Barone and Schmidt, 2005, Elsevier).

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Figure 19. Agglomerate fraction (af) vs. dispersion time at different speeds and simultaneous melt temperatures. (Reprint with permission of Barone et al, 2005, Elsevier).

Barone et al, 2005 developed polymer composites by mechanical mixing using High Density Polyethylene (HDPE) and as reinforcement keratin fiber; authors used only one concentration of fiber (20 wt %) in order to evaluate the influence of processing parameters on mechanical properties of composites. The variables studied were processing time, temperature, speed and state of fiber distribution. Agglomerate fraction of fiber in composites was obtained in the compounding, relating that the break-up of an agglomerate is described as a strain-induced process where the strain ( ) required is the strain at the time for complete dispersion ( td). Figure 19 shows the fiber agglomerate fraction as function of dispersion time and speed obtained during the mixing process. Authors point out that the smallest total strain is found at 75 rpm, thus, it is suggested that this is the most efficient mixing speed of the three used. Results also show that the separation between the 50 rpm and 75 rpm curves is larger than the split between the 75 rpm and 100 rpm curves indicating the non-linear viscoelastic nature of the HDPE/fiber composite melt.

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The results of other variables in compounding of composites in this research are shown in figure 20, where the effect of processing speed on the tensile properties of composites is shown. It is clear that the compounding speed influences the rigid behavior of the composites, because the elastic modulus is higher in the composites than in the pure HPDE. Also the strain for composites is lower than the strain found in HDPE and decreases notably with the compounding speed. Tensile properties as function of processing time and melt temperature also were evaluated in this research. In subsequent research, Barone (2005) studied the influence of feather fiber in the crystalline structure of diverse polyethylene (PE) samples. Keratin feather fibers with 0.1 cm length were incorporated in polyethylene of different crystallinity and fiber was included at 20 wt %.

Figure 20. Tensile properties of composites (HDPE-20 wt% 0.1 cm keratin fiber). Processing conditions Tset = 140 C for 15 min at different speeds and molded at 160 C for 3 min. Test speed =2.5 cm/min. (Reprint with permission of Barone et al, 2005, Elsevier).

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Results indicate that mechanical properties and interface in composites depend on polymer crystallinity; since composites present good interface between polymer and fiber when the crystallinity of polymer is low, however, in the composites with PE matrix with high crystal structure the interface results weak and composites show voids between polymer and fibers. Authors also conclude that the keratin fibers inhibit crystallinity in low ordered polyethylenes but enhance crystallinity in high ordered polyethylenes. Keratin fiber (free of quill) has been incorporated in epoxy polymer matrix. Mechanical properties such as erosion wear rates as function of impingement angle were evaluated in a composite with 20 wt % of reinforcement (Rao et al, 2007). In all evaluated angles, composite shows lower erosion wear rates than epoxy, authors mentioned that, feather fibers improve significantly the erosion wear performance in this kind of polymer matrix. In further research in the same group Mishra and Nayak, (2010) analyzed the microhardness, density and flexure properties of epoxy–keratin fiber composites at different loadings of feather fibrils. They found that the microhardness decreases significantly with fiber content; since the results were approximately 49 Hv from the composite with 5 wt% until 20 Hv for the composite with 30 wt% of fiber. Also flexure strength increases notably with fiber concentration; since results show approximately 15 MPa for the composite with 5wt% of fiber until almost 40 MPa for the composite with 30 wt% of feather fiber. Thus, in spite of the epoxy used in this research is a rigid polymer and keratin fiber obtained from chicken feather is a fiber with less modulus (2.5-5 GPa) (MartínezHernández et al, 2005b; Barone, 2005) than polymer matrix in this specific case, keratin fiber shows the features to change epoxy behavior and reinforce the polymer matrix; the hydrophobic nature of keratin fiber produces interactions that allow to develop really a polymer composite. In spite of Dynamical Mechanical Analysis is a very effective technique in order to evaluate viscoelastic properties of polymer and transitions, only, one research with this analysis has been reported related to the characterization of KFPC considering synthetic polymer matrix. Martínez-Hernández et al, (2007) reported the Dynamical Mechanical Characterization of PMMA-keratin fiber composites. Figure 21 shows the storage modulus (E´) results obtained for PMMA and KFPC for this research; in the figure is possible to observe that the initial modulus (E´ at 35 C) is increased with 1 wt % and 2 wt % of fibers. However, when more than 3 wt % of fiber is added, E´ decreases gradually at room temperature.

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Figure 21. Storage modulus curves from dynamical mechanical analysis obtained for PMMA and KFPC at different weight concentrations from 1 to 5 wt %. (Reprint with permission of Martínez-Hernández et al, 2007, Elsevier).

Figure 22. Tan delta curves from dynamical mechanical analysis obtained for PMMA and KFPC at different weight concentrations from 1 to 5 wt %. (Reprint with permission of Martínez-Hernández et al, 2007, Elsevier).

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Nevertheless, the composite behavior at higher temperatures is outstanding. E´ reaches 258 MPa at 80 C for PMMA without reinforcement, whereas for the rest of materials with keratin biofiber storage modulus is quite larger: from 140% until 283% with 988 MPa for composite 4. Figure 22 shows the Tan delta results obtained for the same composites and PMMA. Damping values are related with the fiber and matrix adhesion level; a weak fiber/matrix bonding will reflect in higher values. It is possible to observe in the figure that the reinforced composites have smaller energy dissipation coefficients. This indicates a good interface between the keratin biofiber and the matrix. Damping in composites decreased considerably, due to polymer chains attached in keratin biofibers diminish their mobility, decreasing the friction among them. Thus, in spite of feather fiber has shown different progress when is incorporated in some synthetic polymer matrices, specifically in mechanical properties, other researchers have been focus to use other parts of feather. The studies involve the incorporation of quill, whole feather and the comparison with feather fiber. Next the mechanical properties obtained in these works are reviewed. Huda and Yang, (2008) synthesized composites with polypropylene (PP) reinforced with ground chicken quill. These materials were compared with jute-PP composites also produced in this research. Authors estimated a rough determination of outer and inner voids in the composite considering the nature of reinforcements (quill and jute) and evaluated the mechanical properties of both composites taking into account these parameters and density. The inner voids calculated for the composites reinforced with ground quill increase with the concentration, since, inner voids depend directly on reinforcement voids, and however outer voids practically were constant in all composites reinforced with quill. In the case of jute inner voids increase lightly with reinforcement concentration because the jute fiber does not contain alot of voids. Mechanical properties, flexure strength, impact resistance, tensile strength and tensile modulus were evaluated in both composites as function of some conditions such as holding temperature, reinforcement concentration and density. Also the influence of these parameters was evaluated in sound absorption, but this is discussed in other section of this chapter, and only in this part the mechanical properties are reviewed. Figure 23 shows the effect of holding temperature on mechanical properties of composites. In this context, the mechanical behavior, flexure strength, impact resistance and elastic modulus change positively when temperature is increased from 175 C to 185 C with constant concentration of quill (35 wt%), however when the

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temperature increases above 185 C the same effect was not observed. The mechanical properties for these composites obtained with other concentrations at constant holding temperature (185 C) are shown in the table 5. It is possible to observe that impact resistance tends to increase with quill concentration, however, tensile strength and tensile modulus decrease with quill concentration; authors attributed this effect due to a decrement in density of composite materials, and because quill is a very short fiber. As it was mentioned the research also compared the mechanical properties of quill composites with jute composites, nevertheless in the case of mechanical properties due to the different nature and structure of both materials (origins, aspect ratios, density) and also taking into account that composites were developed with different concentrations of fiber, results are not comparable at the same concentration. Jute-PP composites show higher mechanical behavior than quill-PP composites for all properties. The most interesting properties related with this research are the superior acoustic properties than quill composites show (analyzed in other section). Thus, although a good interface was exhibited in keratin composites, and other properties have been found as good opportunity to take advantage of keratin obtained from chicken feather in this kind of composites; there is an open gate for improve mechanical properties, even if it is considered that processing of feather in polymers is a recent topic. In addition, despite mechanical properties are not extremely high in some researches are superior to polymer matrix. Unfortunately, the results shown by these authors do not allow observing this fact.

Figure 23. Effect of holding temperature on mechanical properties of composites PPquill at 35 wt%. (Reprint with permission of Huda and Yang, 2008, Elsevier).

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Table 5. Mechanical Properties of ground quill-PP composites held at 185 C. (Adapted from Huda and Yang, 2008)

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Quill contain (wt %) 20 30 35 40 50

Impact Resistance (J/m) 27.6 ± 2.0 63.6 ± 4.8 64.4 ± 5.0 73.5 ± 5.1 67.8 ± 8.8

Tensile strength (MPa) 5.0 ± 0.1 4.4 ± 0.1 3.4 ± 0.4 3.1 ± 0.3 3.0 ± 0.1

Tensile Modulus (MPa) 211.5 ± 3.2 192.3 ± 9.0 159.6 ± 9.1 135.2 ± 9.6 125.3 ± 9.3

Huda and Yang, (2009) evaluated the mechanical properties of different composites using as reinforcements quill, feather fiber and jute again, as comparison material; the matrix used this time was fibers of High Density Polyethylene/Polypropylene (HDPE/PP). Keratin and jute, as fiber and powder, were evaluated as reinforcement materials. Processing parameters and mechanical properties were evaluated as the authors describe in their previous study (Huda and Yang 2008). Composite thickness and holding temperature were also evaluated. Table 6 shows the features and mechanical properties obtained in this research for composites with keratin-HDPE/PP. In the table is possible to observe that quill composites present superior mechanical properties than fiber composites and this is independently if the reinforcement materials are complete or as powder. Quill is a more rigid material than fiber and it‟s like honeycomb structure is responsible of flexure properties without break, instead of this structure with voids, fiber has a solid structure. Table 6. Comparison of mechanical properties of keratin-HDPE/PP and jute-HDPE/PP composites with 35 wt % of reinforcement material. (Adapted of Huda and Yang, 2009) Composite

Thickness Density Flexural (mm) (g/cm3) strength (MPa) Feather fiber 4.2 0.36 4.2 ± 0.2 Feather fiber 3.2 0.47 5.6 ± 0.7 Quill 3.2 0.47 9.8 ± 1.0 Jute 3.2 0.47 9.0 ± 0.8 Feather fiber 2.0 0.75 9.8 ± 1.1 powder Quill powder 2.0 0.75 14.4± 1.0 Jute powder 2.0 0,75 11.2 ± 1.1

Modulus of elasticity (MPa) 380 ± 31 548 ± 82 805 ± 48 1315 ± 42 893 ± 85

Offset yield load (1.3 mm) (N) 16.6 ± 0.7 9.0 ± 1.4 20.4 ± 1.2 20.0 ± 1.2 9.5 ± 0.9

1399 ± 144 13.2± 0.6 1197 ± 124 9.0 ± 0.8

Impact resistance (J/m) 41.0 ± 7.6 30.1 ± 4.1 55.6 ± 5.0 82.1 ± 5.0 25.4 ± 5.6 25.9± 5.4 27.1 ± 7.4

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In addition, the different arrangement of keratin in each material is substantial. On the other hand the other material presented as comparison is jute. As we mentioned before, jute is a material with different composition, nature, aspect ratio and surface behavior. However, it is interesting to analyze that quill powder composites possess higher mechanical properties than jute powder composites at same concentration with exception of impact resistance. Thus, the changes in processing parameters and the changes in the forms of feather keratin allow knowing more options for this kind of FKPC. In the same research group, Reddy and Yang, (2010) developed PP composites reinforced with three forms of keratin from feathers: whole feather, quill powder and fiber. Research shows that in spite of fiber has shown important features when is incorporated to polymer matrices, whole feather with an adequate processing method and optimized conditions allows to obtain keratin polymer composites with significant mechanical properties. Parameters such as thickness and density of composites and keratin concentration were evaluated to verify their inference in mechanical properties. The tensile strength and modulus shown a clear tendency to increase with whole feather concentration for the composites with 35 wt %, 40 wt% until the composites with 50 wt%, however, both strength and modulus decrease, from 15.6 MPa in strength for the composite 50/50 (feather/PP) until 13.4 MPa for the composite 60/40 (feather/PP). In the case of tensile modulus, this decreased from 1461 MPa until 1166 MPa for the composites 50/50 and 60/40 respectively. Mechanical properties of these composites were dependent on density and thickness. For instance, elastic modulus increases since 603 ± 240 MPa until 1900 ± 166 MPa for composites with a density of 1000 kg/m3 and 2000 kg/m3 respectively. Also, the elastic modulus changes since 800 ± 77 MPa until 1400 ± 156 MPa, for composites with thickness of 4.2 mm and 2.8 mm respectively. Table 7 shows the results of mechanical properties obtained for keratin composites with 35 wt % of different keratin type as reinforcement. In spite of powdered quill-PP composite shows higher flexural strength than fiber-PP and whole feather-PP composite; whole feather-PP composite presents superior tensile properties than composites developed with processing feather (quill or fiber). Thus, the research proves that whole feather with adequate matrix and processing shows interesting properties as potential reinforcement material and in this case improves the mechanical behavior in comparison with processing feather-composites. It is known, that several reinforcements of polymer composites are modified chemically in order to get good interface between polymer matrix and the reinforcement involved.

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Table 7. Comparison of mechanical properties of keratin-PP composites with 35 wt % of reinforcement material. Composite thickness = 3.2 mm. (Adapted from Reddy and Yang, 2010) Reinforcement type

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Whole feather Powdered quill Feather fiber

Flexural strength (MPa) 7.8 ± 0.8 10.2 ± 12 5.6 ± 0.7

Tensile strength (MPa) 9.0 ± 1.5 3.4 ± 0.4 3.2 ± 0.6

Tensile modulus (MPa) 7.8 ± 0.8 7.8 ± 0.8 7.8 ± 0.8

Composites synthesized with keratin materials as reinforcement, analyzed until this section do not involve any treatment over keratin structure. Basically, two treatments have been development over the chicken feather keratin fibers: polymer grafting (Martínez-Hernández et al, 2003a, 2008, Jin et al, 2011) discussed in other section and alkali treatment. This later approach has been used in order to improve compatibility between epoxy matrix and feather keratin fiber. Mishra et al, (2010) modify feather fiber with 1N solution of sodium hydroxide for three days. 20 wt % of unmodified and modified fibers were incorporated to epoxy polymer matrix. Density of composites decreases with respect to epoxy polymer, since 1.28 g/cm3 for epoxy until 0.829 g/cm3 for the composites. This allows developing light composites with promissory mechanical properties. Inasmuch as, the flexural strength and elastic modulus of the composite made with alkali treated feather are 24.192 and 4.339 N/mm2 respectively and both are higher than strength and modulus of the composite prepared with untreated feather, 18.816 and 1.216 N/mm2, respectively. If well, more studies are necessary in order to analyze the influence of chemical treatments in composite properties, these results are promissory due to the improved interface is reflected in mechanical properties reached after modification of keratin fibers. Other studies have incorporated feather fiber in polypropylene (PP) but in combination with cellulose fibers, for instance the researches developed by Bullions et al, (2004), (2006). They varied the compositions of feather fiber (Ff) with cellulose fibers. In one study keratin fiber was mixed with recycled kraft pulp fiber (Pf), recycled news pulp fiber (Nf), and retted kenaf bast fiber (Kf) in PP matrix. The results of these composites developed in different 11 concentrations show that the contributions of the four fibers used to the composite strength were found to be from highest to lowest: Pf > Nf > Ff > Kf; in addition two factors are thought to play major roles in determining the contributions of each fiber to composite strength: fiber aspect ratio and fiber

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strength. In other study of Bullions et al, (2004) feather fiber and kraft pulp fiber were incorporated in PP but adding also Maleic Anhydride (Ma). In this case the incorporation of Ma was realized directly in PP matrix instead of be applied directly over fiber surface. Both PP and MaPP were used in order to make composites using wetlay process using different plies of wetlay prepreg. Authors found that the MaPP added to the wetlay prepreg increased the strength and modulus of the composite panels compression molded from these prepregs, demonstrating that the fiber, PP, and MaPP do not need to be melted blended to achieve improvements in strength in modulus. Table 8 shows the percent of increment reached in different mechanical properties for the composites where MaPP was used with feather fiber, pulp fiber and the combination of both reinforcements. Table 8. Percent Increase in mechanical properties using 8 wt% of MaPP. (Adapted of Bullions et al, 2004).

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Reinforcement type Ff Ff/Pf Pf

Tensile Modulus 31.1% 37.0% 44.5%

Flexural Modulus 19.2% 19.3% 47.2%

Tensile Strength 21.0% 30.2% 37.2%

Flexural Strength 27.7% 25.7% 47.0%

4.2. Thermal Properties of Feather Fiber Polymer Composites Thermal behavior of polymer composites is always a fundamental source of information about compatibility between fibers and matrix and thermal restrictions for applications. In this section a brief discussion concerning some synthetic polymers reinforced with keratin fibers from chicken feathers is included. Martínez-Hernández et al, (2007), studied the effect of keratin fiber reinforcement on thermal behavior of poly(methyl methacrylate) (PMMA). The DSC shows that glass transition temperature (Tg) in the composites increased with the keratin fiber content due to an enhance in the rigidity at molecular level, caused by joining the polypeptide chains of keratin with PMMA. The PMMA matrix has a Tg value of 72°C, whereas Tg for the composite with 5 % (in weight) is 109°C. This difference is because of keratin fibers and PMMA have good compatibility, reflecting thus a proper interface. The complete values of Tg for these composites are shown in table 9. Besides

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DSC, Martínez-Hernández et al, (2007), also reported the TGA of pure PMMA and composites. TGA curves, depicted in figure 24, show an increase in thermal stability when the keratin fiber content is enhanced. This also is observed in the decomposition temperature when composites have lost 30% of their weight (table 9). As can be observed pure PMMA losses 30% weight at 269°C, but composite with 5 wt % of keratin fiber losses this same weight percentage at 368°C. This variation also is caused by the good dispersion between keratin fiber and polymer matrix. Table 9. Thermal data of keratin fiber-PMMA composites obtained by DSC and TGA.

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Composite 0 1 2 3 4 5

Tg (°C) 72 95 97 100 101 109

30% weight loss temperature (°C) 269 276 276 278 305 308

Figure 24. TG curves of pure PMMA and keratin fiber-PMMA composites with 1 to 5 wt %. (Reprint with permission of Martínez-Hernández et al, 2007, Elsevier).

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The importance of thermal behavior analysis involves not only the characterization of the Tg values or the weight loss in a temperature range, but also the explanation of molecular changes that causes these transitions and degradations. Thus, thermal decomposition of keratin fiber-PMMA composites involves two stages: first from 125 to 200°C, in this first phase the head to head bonds and the unsaturated end groups from the synthetic polymeric chains included during the starting and finishing reactions are decomposed. As can be observed in figure 24, the weight loss at this range is minimum, only between 6 and 8% of the total weight; therefore it is possible to suppose that there are only few chains with these weak linkages. The second stage begins around 225°C and has different temperatures for ending, at 310°C for pure PMMA and at 360°C for sample with 5 wt % of keratin fiber. This phase is defined by the pyrolysis of C-C and C-H bonds, involved in the PMMA depolimerization, which was initiated in the labile thermal groups mentioned before. Therefore the weakest bonds are broken with temperature producing volatile monomer units in a process inverse to the polymerization. On the other hand Barone, (2005), studied the thermal properties of Low and High Density Poly(ethylene) (LDPE and HDPE respectively) reinforced with 20 wt % of keratin fiber from chicken feather. The thermal behavior of the polymers and composites were evaluated by DSC according to the method described in ASTMD3417 and D3418. Pure polymers and composites were tested from 30 to 200°C at 10°C/min, heating cycles provide information about the state of the polymers and composites as a function of the processing conditions. For the first cycle, the heat of fusion ( Qm,1) and the melting temperature (Tm,1) were calculated. Later, the crystalline fraction of polymers and composites were determined according to:

X1

Qm,1 Q f (1 m f )

(1)

where Qf = 290J/g, is the theoretical heat of fusion of 100% crystalline PE, and mf is the mass fraction of fiber. Table 10 shows the results of Tm, X1 and density of polymers and composites. From these results the authors concluded that the presence of the keratin fibers affects the crystallinity in the PE matrix, since fibers restrain crystallinity in LDPE LD133A and LLDPE 2045, but enhances crystallinity in HDPE HD5502SA and HDPE HD7760. The general trend shows that PE matrices in the composites have thinner and easier melting crystals than the bulk polymers when they were processed in a similar way.

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Table 10. Properties of PE and PE-keratin fiber composites (Adapted from Barone, 2005). PE

Source

LDPE LD133A LLDPE 2045 LLDPE 2037 HDPE HD5502SA HDPE HD7760

Tm,11 (°C) 112

X1 1

0.45

Density1 (g/cm3) 0.911

123 128 132

0.44 0.52 0.69

0.914 0.940 0.972

123 127 131

0.39 0.57 0.71

132

0.71

0.963

130

0.77

X1 pol

Dow

Density Tm,1 pol (g/cm3) (°C) 0.923 113

Dow Dow BP

0.920 0.934 0.954

Exxon

0.952

0.37

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4.3. Acoustic Properties Attenuation of sound by proteins has been demonstrated by different authors from diverse scientific areas (Wagner et al, 2001; Almagor et al, 1992; Povey et al, 2011). This effect has been used in many studies in medicine, food, biology and chemistry among others. Povey et al, (2011) studied bovine serum albumin, and they conclude that velocity and attenuation relate to different features of the protein molecule. Velocity is sensitive to changes in conformation, secondary structure, and any significant variations in elastic or shear properties, attenuation is affected by relaxation processes and possibly by aggregation of polypeptide chains. A report on ultrasound absorption for a protein was made previously by Almagor et al, (1992); these authors observed that absorption of ultrasonic energy by proteins are associated with volumedependent relaxation process and also is close related with protein hydration. Thus, the lubricating water is an important factor on the internal friction of the protein molecule, specifically the energy dissipation associated with internal motions of the polypeptide chain. Therefore, lubricating water can be considered as a responsible condition for the ultrasound absorption since exist a coupling between water and protein that stabilizes its structure. The authors supported this conclusion considering some findings where the ultrasound absorption of denatured proteins is markedly low; this fact suggests that protein ultrasound absorption is related to tertiary structures. Wagner et al, (2001) demonstrated that sound attenuation of polymerizing actin reflects 1

These properties were determined in PE reinforced with 20 wt % of keratin fibers.

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supramolecular structures, since fibrillar bundles of actin have been found to be regions of high acoustic attenuation, which depends on packing density and homogeneity of the actin network. They considered also that attenuation is also related to filament length. The sound attenuation by proteins, specifically by keratin, can be applied in materials area. Interesting results on sound absorption of polymer composites reinforced with keratin fibers were found by Huda and Yang, (2009) and Reddy and Yang (2010). Feather fiber, feather quill and jute (cellulose fiber) were used in order to make High Density Polyethylene/Polypropylene (HDPE/PP) composites, which were characterized by sound dampening (Huda and Yang, 2009). The sound dampening curves of these composites do not show a clear trend, but the authors relate the feather fiber content, the thickness and the resultant dampening. For instance, composites with 30 and 40% of feather fiber have similar sound dampening curves up to 1.5 kHz, after which these composites present an inconstant behavior. For composites with 35 and 50% of feather fiber, intense increases are observed around 1.2 and 1.0 kHz respectively, but after the maximum values were reached the sound damping decreases, as if the composite were saturated by ultrasound energy, for this evaluation, authors include only feathers of 4.2 mm. After, the effect of thickness is observed considering a constant 35% concentration of feather fiber but varying fiber thickness. The results show that for 4.2 and 5.8 mm thickness the sound dampening is higher than composites with 2.0 and 3.2 mm in thickness, nevertheless only curves for 3.2 and 5.8 follow an almost linear behavior, the others reached maximum values but also minimum depending on both, thickness and frequency. Authors report that macro voids and continuous air channels through the cross section allow part of the sound energy to pass through the composite without being absorbed leading to decreased damping below 2.3 kHz. This article also shows that chicken feather fiber or quill composites have superior sound absorption than jute composites. The authors conclude that the amount of voids, composite density, formation of air channels and composite thickness are important factors in the sound dampening. Thus, they relate sound absorption with microscopic structure of the feather fiber or quill, in fact the authors report that the superior sound absorption of feathers takes place from microscopic and morphological characteristics, mainly by inner voids, which is only a partial explanation since they did not consider the classic sound absorption intrinsic in the proteins. A similar study was realized by Reddy and Yang, (2010), but in this case they used PP as matrix and the whole feather as reinforcement. The sound

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absorption properties were measured by the noise reduction coefficient (NRC) according to ASTM standard C423-99A. Table 11 summarizes the effect of ratio Feather/PP, density and thickness of composites on their sound absorption. Besides the authors also report absorption coefficients changing the test frequency since 1 to 5 kHz and comparing also proportion of feathers/PP, density and thickness. In spite the results are very interesting and useful for future applications of chicken feathers, there is a lack of explanation that justify the sound absorption of keratin fibers, since again in this article, the authors based all the sound absorption on the presence of voids, it means in the hollow microstructure of feathers, but the important contribution of protein by itself and the thermodynamic process involved are not discussed. Therefore this is a great area to research in order to understand completely this complex process in feather fibers composites.

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Table 11. Noise Reduction Coefficient of whole feather/PP composites. (Adapted from Reddy and Yang, 2010). Parameter Ratio of feather/PP (% w/w) 35/65 40/60 50/50 60/40 Density of composite (g/m2) 1000 1250 1500 1750 2000 Thickness of composite (mm) 2.8 3.2 3.6 4.2

Noise Reduction Coefficient 0.26 0.23 0.21 0.35 0.19 0.28 0.26 0.29 0.27 0.18 0.26 0.26 0.28

4.4. Electric Properties As it was demonstrated above, chicken feather fiber composites have interesting mechanical and sound absorption properties, but less attention has been paid to obtaining information on thermal and electric features of such

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composites. In agreement with Mishra and Nayak, (2010), advances on low kdielectric materials can be considered as one of the most important objectives in modern high-speed microelectronics. For instance printed circuit boards, such as cell phones, televisions, computers, keyboards, etc, require high electrical resistance and relatively low dielectric constants (Zhan et al, 2011). Printed circuit board bases can be made of synthetic polymers such as polyethylene, polyetheretherketone, polyimide, epoxide or phenolic resins. The developments of these bases include also composites. Therefore developing low dielectric materials from locally available and natural resources, such as keratin fibers, could be of great interest from economic, environmental and technological point of view. As it was observed in figures 4 and 5, feather quill and barbs have hollow structures, so these fibers contain a significant volume of voids. In these cavities the air is an ideal dielectric material, having a minimum dielectric constant of 1.0, thus feather fibers can act also as dielectric material (Mishra and Nayak, 2010). Mishra and Nayak, (2010) studied the dielectric behavior in epoxy matrix reinforced with chicken feather fibers. In this study samples were coated with graphite paint on the opposite faces and heated for 5 min in an oven for drying. Dielectric measurements were carried out at a frequency of 1 Hz to 1 MHz, the temperature is controlled with a programmable oven at a rate of 5°C at a frequency of 100 Hz. Dielectric data are collected at intervals of 5°C, for dielectric analysis each sample was placed between two gold electrodes. The results of this study shows that dielectric constant decrease from 4.5 to 2.1 with an increase in keratin fiber concentration, also decrease with increasing frequency, as is expected in most dielectric materials due to dielectric relaxation. However increasing temperature causes a slight increment in dielectric constant, this is possibly due to alignment of dipoles when the composite softened gradually with temperature. On the other hand, recently Zhan et al, (2011) published an elegant report of electrical properties of chicken feather fiber reinforced epoxy composites. In this case the composites were made with EPON 862 (Diglycidyl ether of bisphenol-F) and curing agent Epikure W, both were obtained from Hexion Specialty Chemicals, Inc. (Houston, TX). Chicken feather fibers (average aspect ratio is 200) were provided by Feather fiber Corp. The authors evaluated volume resistivity and surface resistivity; both are shown in figure 25. Both parameters decreased as fiber content increased, this means that feather fibers have lower resistivity than the matrix.

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Figure 25. Volume and surface resistivity of EPON/feather fiber composites. (Reprint with permission of Zhan et al, 2011, Elsevier).

These properties are important because of printed circuit board materials must have high volume and surface resistance in order to prevent leakage of current through the material or across the surface. Permittivity or dielectric properties were other important factor studied by Zhan et al, (2011). A low dielectric constant is an important property for printed circuit board, given that it increases the signal speed, reduces the crosstalk effects between signal lines, and diminishes the power consumption. Figure 26 shows the results obtained by Zhan et al, where dielectric constant is depicted for different composites taking into account a range in frequency from 10 kHz to 5 MHz. As it can be observed dielectric constant decreases as the frequency increases. This is expected in most polymer dielectrics due to relaxation phenomena. Dielectric relaxation is related with dipolar (rotational) polarization, it depends on the molecular structure of the material. If the frequency has a high value, the rotational motion of the molecules lags behind the electric field, causing a reduced dielectric constant. Composites with keratin fibers from chicken feathers have dielectric constants lower than 4.6, whereas the EPON/7628 has values around 5.4, as can be observed in figure 26.

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Figure 26. The dielectric constant of the composites with feather fiber. (Reproduced with permission of Zhan et al, 2011, Elsevier).

These authors coincide with Mishra and Nayak, (2010) and assume that dielectric constants of the keratin fiber composites decrease because of the hollow structure of feather parts, which incorporate air into the composite structure.

4.5. Keratin as Reinforcement in Biopolymers Nowadays the importance of biopolymers is emerging gradually due to the environmental problems caused by synthetic polymers, as their long time of decomposition and the high cost of petroleum, principal non renewable raw material for synthetic polymers. One of the most important uses of synthetic polymers is composites, so that alternative sources of petroleum-based composites become important and biocomposites involving biodegradable polymer as matrix and natural fibers as reinforcement have attracted great

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interest. Thus, some authors have tried to combine biodegradable polymers and feather keratin in order to study diverse properties in these composites. Hong and Wool, (2005), developed an interesting composite from soybean oils and keratin feather fibers. Acrylated epoxidized soybean oil (AESO, Ebecryl 860, UCB Chemical Co., Atlanta, GA) and soybean oil pentaerythritol glyceride maleates (SOPERMA) were used with 33 wt % of styrene comonomer (Aldrich) as liquid molding resins. In this study composites were prepared with 0, 5, 10, 20, 30 and 45 wt % of keratin fibers. These authors conclude that keratin fibers are compatible with AESO resin. Some of their most relevant results are summarized next. The density of the composites decreased with increasing keratin fiber content, thus density values of AESO and SOPERMA resins are 1.08 g/cm3 and 1.13 g/cm3 respectively, whereas for AESO - keratin fiber, 20 wt % is 1.02 g/cm3 and for SOPERMA - keratin fiber, 20 wt % is 1.06 g/cm3. The dielectric constant of composites with AESO and keratin fibers was also evaluated, in this case the dielectric constant decrease linearly from 2.7 to 1.7 while fiber content is increased, in consequence dielectric constant values were lower than that of usual semiconductor insulator material, for instance silicon dioxide, epoxies or polyimides. The coefficient of thermal expansion of the AESO - keratin fiber composite was also studied, the results show that this coefficient decreases with increasing keratin fiber content, from 127.2 m/m°C for AESO resin to 67.4 m/m°C with 30 wt %, which was similar to that of silicon material or polyimides. The thermomechanical characterization shows that the storage modulus of AESO composite was considerably improved from 1.313 GPa for AESO resin to 2.085 GPa for composite with 30 wt % of keratin fiber, both at 40°C, whereas the Tg values were not affected. On the other hand the fracture toughness for AESO resin was 1.458 MPa/m2 and for the composite with 30 wt % of keratin fibers was 1.768 MPa/m2, the fracture energy was 1.420 and 1.945 KJ/m2 for AESO resin and the composite with 30 wt % of keratin fibers, respectively, therefore both characteristics were increased with increasing fiber content. Accordingly with these results the properties of the new composites are satisfactory to be applied. The production of composites using Poly (lactic acid) (PLA) as matrix and keratin fibers from chicken feathers was reported by Zhao et al, (2008). PLA and keratin fibers composites were fabricated by melt compounding with a Thermo Haake MiniLab twin-screw micro extruder at 180°C. The content of keratin fibers in these composites was 2, 5, 8 and 10 wt %. In this study, the authors show that the tensile strength of PLA decreases as keratin fiber is

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incorporated, but the tensile modulus for all the biocomposites is higher than that of only PLA, for the composite with 5 wt % a maximum value of 4.2 GPa were evaluated, which is 16% higher than PLA‟s corresponding value. In spite of the elongation at break shows a decreasing trend with increasing of keratin fiber content, a maximum value of 5.3 % is reached for composites with 2 wt %, which represents an increasing of 56% with respect to pure PLA. This behavior means that the ductility of PLA matrix was improved by keratin fiber addition. This interesting study was continued and reported by Cheng et al, (2009). In this report, the authors included dynamic mechanical analysis (DMA), thermomechanical analysis (TMA) and thermogravimetric analysis (TGA), besides mechanical and thermal properties of pure PLA and PLA – keratin fiber composites. In their conclusions, the authors assert that due to the effectiveness of keratin fiber reinforcement the mechanical and thermomechanical properties are improved. Thus, the incorporation of keratin fiber increases considerably the storage modulus and reaches a maximum enhancement of 73% compared to PLA. At the same time the tan values decreases due to the applied stress can be effectively transferred from PLA to keratin fibers, thus the mobility and deformation of the matrix is reduced and the mechanical loss to overcome inter-friction between molecular chains is reduced by the keratin fibers. The TGA results show that composite with 5 wt % of keratin fiber has the best thermal stability compared to PLA and the other composite, so that PLA – keratin fiber composites are suitable to offer good mechanical and thermomechanical properties. An important feature of composites with biopolymers and keratin fibers from chicken feathers is the biodegradability, which was evaluated by Ahn et al, (2011). In this report the biodegradability of three types of bioplastic was evaluated by measuring carbon dioxide produced from lab-scale compost reactors containing mixtures of bioplastic fragments and compost inoculum held at 58°C for 60 days. Table 12 shows the components of the three bioplastics and their corresponding biodegradability percentages. The authors report that biodegradability of bioplastic A was very low compared to literature values for other PLA materials, this could be due to PLA undergoes chemical structural changes during polymer extrusion and injection molding and this may inhibit biodegradation. However, they observed by near infrared spectroscopy that poultry feather was not degraded during composting.

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Table 12. Composition and biodegradability of bioplastic composites. Components* Type A 100 13 ± 3

PLA Keratin feather fiber Starch Urea Glycerol Biodegradability§ * All components are in wt %. § Biodegradability values are percentages.

Bioplastic composites Type B Type C 80 5 50 15 25 25 53 ± 2 39 ± 3

4.6. Cellulose – Keratin Feather Composites

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Different efforts to include keratin feather and cellulose fibers have been made for several authors, in spite of the basic difference between these two natural biopolymers. Winandy et al, (2003) made a series of cellulose fibre panels adding different quantities of chicken feather fiber, cellulose fiber and 5 % of phenol formaldehyde resin; table 13 shows the components, structure and mechanical properties of these panels. Table 13. Composition of cellulose-keratin fiber composites (Adapted from Winandy et al, 2003). Cellulose Fiber Board Structure keratin fiber (%) 95-0 Single Layer 75-20 Single Layer 75-20 3 Layer keratin core 75-20 3 Layer keratin faces 47.5-47.5 Single Layer 0-95 Single Layer * IBS: Internal Bond Strength. ME: Modulus of Elasticity. BS: Bending Strength. TS: Two hour Thickness Swell. WA: Water Absorption.

Evaluated Properties* IBS ME BS TS 1.00 1.00 1.00 1.00 0.93 0.96 0.90 0.58 1.11 1.06 0.71 1.14 1.31 0.73 0.95 1.30 1.36 0.73 0.82 0.38 0.98 0.49 0.61 0.27

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WA 1.00 0.57 1.02 1.12 0.48 0.36

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The authors conclude that addition of chicken feather keratin fiber had very little negative effect on internal bond strength. Modulus of elasticity and bending strength are similar between single layer board with 20% of keratin fiber and board with 95% of cellulose, whereas single layer and three layers have differences in modulus of elasticity and bending strength, according to the authors these are probably due to remnants of processing procedure, thus they are not related to differences between the varying board structures. On the other hand Two-hour thickness swell test and water absorption test revealed that board with 47.5 % of keratin fiber has superior resistance values than the other composites. As well, Barone (2009) reported the use of lignocellulose fibers and feather keratin, but in this case keratin biopolymer was used as matrix. This was prepared by using poultry feather keratin, glycerol, water and sodium sulfite. Lignocellulose fibers were from corn stalk, wheat straw, banana fiber, coffee chaff, hemp, flax and kenaf in order to evaluate different sources, length and mass fraction. Their elastic modulus results show a positive reinforcement when kenaf (0.048), hemp (0.057), coffee chaff (0.060) and flax (0.057) were used (The reported values are in GPa and represent the absolute elastic modulus, 0.046 was the observed value for the control sample without cellulose fiber). In addition TGA showed that lignocellulosic fibers increased the thermal stability of these composites. FTIR results in this article indicated some chemical interactions between keratin matrix and cellulose fibers that could enhance reinforcement behavior, however, as the author affirm; there is not a definitive trend between the chemical nature of the fibers and their reinforcing capacity.

5. GRAFTING USING FEATHER KERATIN In order to increase and diversify the uses of biopolymers and natural fibers several studies have been accomplished to modify their properties through different approaches, such as grafting. The aim of grafting is to introduce synthetic polymers into the main chain of biopolymer through chemical modifications. This coverage confers additional properties to the biopolymer itself, without diminishing its intrinsic characteristics (Bianchi, 2000). The modification of natural protein by grafting is an attractive technique because of it allows the improvement of some naturally poor features associated to the natural material. For instance dimensional stability, rub

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resistance, photo yellowing, oil and water repellency, tensile properties, felting shrinkage, abrasion and pilling resistance, hygroscopicity, chemical resistance, dying behavior, durability and bacterial resistance have been dramatically improved in silk and wool by means of grafting (Das and Saikia, 2000; Tsukada et al, 1997; 1998; Giri and Samal, 1991, Mishra et al, 1982). Grafting is based on heterogeneous reactions produced by free radical formation on the selected biopolymer. Free radicals can be produced by either chemical or physical methods including the use of redox systems, oxidant agents or low and high energy radiations. The elected (synthetic) monomer polymerizes starting from free radicals on biopolymer. Different studies on wool fibers show that the favorite grafting sites are the thiol groups on cystine amino acid, however amine and hydroxyl moieties have been found also to act as reactive sites (Tsukada et al, 1997, Sarac 1999, Freddi et al, 1999). In addition table 14 include a summary of the chemical groups found in feather, the quantities are calculated from their amino acid composition (Schmidt, 1998; Martínez-Hernández et al, 2008). In redox systems, numerous reducing agents such as alcohols, thiols, ketones, aldehydes, amines, amides or acids (malic, tartaric, citric, lactic, among others), combined with oxidizing metal ions, for instance Mn(III), Mn(VII), Ce(IV), V(V), Co(III), Cr(VI) or Fe(III), have been employed. These metal ions react with reducing agents to produce free radicals in aqueous medium, which initiate the polymerization or the grafting. Martínez-Hernández et al, (2003a, 2008) used the redox system in order to graft methyl methacylate monomer (MMA) on feather keratin fiber. The redox system included MMA, KMnO4, malic acid and sulfuric acid aqueous medium. Table 14. Chemical reactive groups in feather keratin. Reactive Group Feather Keratin*. Free carboxyl 27-44 Amide 78 Carboxyl plus amide 105-122 Phenolic hydroxyl 11-12 Aliphatic hydroxyl 134-174 Amino 7-12 Aromatic nuclei 13-14 Half disulfide 57-68 Oxidisable 309-376 * Content as grams equivalents per 105 g of keratin.

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First the feasibility of grafting PMMA on keratin protein structure was evaluated (Martínez-Hernández et al, 2003a). Original and grafted feather keratin fibers were characterized by IR and Raman spectroscopies and their thermal behavior was studied by DSC and TGA. The modified surface was observed through SEM. The possible reaction mechanism is depicted in figure 27. The reaction starts by forming the free radicals, as soon as are formed, they can either initiate the polymerization to form PMMA chains or they can attach the keratin active sites to produce grafting. FTIR spectra of feather keratin fiber and PMMA grafted keratin fiber are depicted in figure 28, several changes in the grafted fiber spectrum are observed. For instance the band at 3080 cm-1 is increased with respect to the signal at 2878 cm-1, this change is attributable to the aliphatic chain extension attached to the NH-R group.

Figure 27. Reaction mechanism proposed in the production of free radicals from malic acid and main backbone of keratin with KMnO4 redox system.

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In the beginning the keratin fiber has a CO-NH-R group and the amine bond is a preferred grafting site. The peak at 2970 cm-1 changes when it is compared to the 2933 cm-1 band. This effect is because of aliphatic chain is increased as consequence of the synthetic grafted polymer. PMMA spectrum shows usually the 2970 cm-1 signal (Gangopadhyay and Ghosh, 2000). In the other hand, considering as reference the band at 1537 cm-1, the peak at 1666 cm-1 is diminished due to CO-NH- is affected during the grafting reaction, figure 27). At 1171 cm-1 a shoulder in non-grafted fiber disappears in the PMMA-grafted fiber, this is assigned to the C-C=O bond, modified during the reaction in keratin carboxyl groups. A new signal appears around 930 cm-1, this corresponds to the C-O-C moiety of PMMA. Raman spectra are shown in figure 29, the most significant changes include increments in the signals at 2930 and 1110 cm-1, and these correspond to variations in the aliphatic chain due to polymer grafted. The peak at 1110 cm-1 is also affected by v(C-O-C) that is related with PMMA molecules. The increased shoulder at 3050 cm-1 can be due to either v(CH3) of –C-CH3 or by aliphatic chain enlargement of NH-R group. The signals at 1663 and 1234 cm-1 correspond to CO-NH(amide I) and v(CN)- (NH) (amide III) respectively; which are affected by the grafting reaction.

Figure 28. Infrared spectra of keratin fiber and PMMA-grafted fiber.

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Figure 29. Raman spectra of keratin fiber and PMMA-grafted fiber.

Figure 30. SEM images of keratin fibers: a) and b) ungrafted, c) and d)PMMA-grafted.

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DSC analysis of these samples also showed some changes in the thermal behavior when PMMA was grafted in keratin fiber, for instance, between 240 and 280°C a denaturation of the helix structure caused by destruction of disulfide bonds was observed in fibers, however this undergoes a slight shift to 306°C with PMMA grafting. In addition a new endothermic signal originated by thermal degradation of PMMA is observed at 327°C. On the other hand TGA results are very close between the two fibers (grafted and unmodified fibers), only a slight thermal resistance is observed for PMMA-grafted keratin fiber (Martínez-Hernández et al, 2003a). Surface characteristics of ungrafted and grafted keratin fibers were observed by SEM. Figure 30-a shows well defined nodes in ungrafted keratin fiber, these are covered by the polymer on the surface of PMMA-grafted keratin fiber. At the same time, the cleft lines or striations (figure 30-b) are also covered in the grafted fiber (Figure 30c-d). In 2008, Martínez-Hernández et al, reported the effect of different concentrations of MMA monomer, oxidizing and reductive agents on percent yield of grafting. The maximum percentages reached and the corresponding conditions are summarized in table 15. All the components have important effects on grafting yield. For instance increasing in monomer concentration leads to a consistent enhance in the percentage of graft yield. This linear increase is probably due to high rate of diffusion of the monomer molecules from solution phase to the active sites at protein backbone which are not hindered at the initial concentrations. Dissimilar results were obtained increasing KMnO4 concentrations, since 3.0 X 10-3 M was the optimum condition for grafting yield (75%), beyond this, with 5 X 10-3 M the grafting decreases until 65% and with 8 X 10-3 M the grafting grows again until almost 70%. Table 15. Maximum percentages of grafted MMA on keratin fiber and their corresponding conditions. Component MMA monomer Malic acid KMnO4 H2SO4

Best concentration (M) 0.7 X 10-3 50 X 10-3 3.0 X 10-3 150 X 10-3

Maximum percentage of MMA grafted. 80 79 75 74

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This unusual behavior could be consequence of the high concentration on Mn, because at this condition the free radicals produced on the main chain of polypeptide might be oxidized or ended, as was observed in similar protein systems (Samal et al, 1981). In addition, a higher quantity of metal ions can interact with more monomer molecules giving rise the amount of homopolymer, decreasing thus the grafting. Totally opposed was the behavior with H2SO4, it begins with 100 X 10-3 M that produces the lowest grafting yield for this series (66%), after with 150 X 10-3 M reaches its maximum (74%), but decreases again until 72% with 200 X 10-3 M. This performance is due to at lower concentrations of acid media the free radical production is limited, and after the optimum condition was reached, some recombination or disproportionation reactions are induced by the increased H+ concentration. Interestingly, these results are in agreement with other protein systems (Samal et al, 1981; Misra et al, 1982). Finally, the variation in malic acid concentration was from 10 X 10-3 M to 50 X 10-3 M, the behavior shows a constant increase of the grafting yield as more organic acid was added. Thus, as the number of carboxyl moieties is raised, also the number of free radical sites on the polypeptide backbone increases, therefore there are more opportunities for the MMA molecules to react with free radicals causing an increase in the percentage grafting yield. Grafting route has been employed for other research groups in order to modify feather keratin not only as fiber, but also using keratin solutions. Sastry et al, (1997), reported the use of hydrolyzed feather keratin that was successfully graft copolymerized with 2-hydroxyethyl methacrylate (HEMA). This study was continued and years later Kavitha et al, (2005) included hydrolyzed keratin mixed with gelatin in a grafting procedure with HEMA to achieve better physico-chemical properties for the called hydrogels. The results show that hydrolyzed keratin-gelatin grafted with HEMA has better mechanical properties compare to hydrolyzed keratin-gelatin or hydrolyzed keratin grafted with HEMA. In turn Wang and Wei, (2006) reported a transamidification reaction utilized to prepare low molecular polypeptides from chicken feathers, which were acylated with maleic anhydride and finally co-polymerized with acrylic acid. Other kind of process used to change the inherent characteristic of feather keratin is the chemical modification through the insertion of different chemical moieties in the main structure of keratin polypeptide. Recently Reddy et al, (2011) described the chemical modification of feather keratin by reaction with acrylonitrile and sodium carbonate as catalyst. This process implies an etherification, specifically a cyanoethylation that provides better

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thermoplasticity to the keratin. The authors assume that the reaction between the hydroxyl groups of keratin and acrylonitrile is a typical nucleophilic addition reaction. The possible mechanism of this reaction is shown in the figure 31. These authors conclude that cyanoethylation of keratin was a suitable way to provide thermoplasticity to feather keratin. The increment in weight percentage was related with increasing ratio of catalyst to keratin. Cyanoethylation was confirmed by FTIR, since the spectra show the presence of a new absorption peak at 2260 cm-1 that corresponds with nitrile groups.

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Figure 31. Possible mechanism in the cyanoethylation reaction of feather keratin. (Adapted from Reddy et al, 2011).

Keratin protein is extremely versatile, given that not only it can be grafted or chemically modified, in addition could be used to give some desirable properties to synthetic polymers through grafting of keratin on polymeric fibers or films. Recently Wu et al, (2011) studied the effect of grafting feather keratin on polypropylene films (PP) with two different processes: air plasma graft (APG) and post-plasma grafting (PPG). The graft performance was evaluated by comparison of contact angles, printability, ageing test and surface chemical composition analysis. PP films shown a more hydrophilic surface and printing properties after both process of grafting, but the best results were obtained with PPG as can be observed in figure 32. Our research group has demonstrated that feather keratin grafted on synthetic polymers can be favorable for the degradation process; in addition an environmental non agresive procedure can be used to produce free radicals on synthetic polymers. Thus, Martínez-Sánchez, (2007) used an eco-friendly method to generate free radicals in nylon through UV irradiation, after these nylon fibers were grafted with hydrolyzed feather keratin by a redox reaction. The insertion of keratin on nylon structure was observed by FTIR and SEM, besides their mechanical and thermal properties, as well as their behavior under accelerated degradation were evaluated. Figure 33 shows nylon fibers

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before and after UV irradiation, it is possible to observe the surface damages caused by the treatment, with some cracks where keratin can be introduced into the fiber. Keratin grafted nylon fibers are observed in figure 34, both images show a complete dispersion of keratin over the nylon surface. This grafting procedure does not affect significantly nor the thermal behavior neither the mechanical properties before the accelerated degradation.

Figure 32. Contact angles as a function of time on PP films, untreated and modified with air plasma graft (APG) and post-plasma graft (PPG). (Reprint with permission of Wu et al, 2011, Elsevier).

Figure 33. Nylon fibers, a) before UV irradiation, b) after 24 hours of UV irradiation, c) after 48 hours of UV irradiation.

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Figure 34. Nylon fibers grafted with hydrolyzed keratin.

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CONCLUDING REMARKS Feather keratin has attracted the interest of diverse scientific areas, since understand the evolution of these complex structures until research on useful applications based on their specific characteristics. In this context, an active area as materials field is the adequate boundary to involve keratin in high technological and scientific advances. This fact takes into account that materials field is evolving continuously and the importance of develop new and better composites, polymers, fibers or films is evident every day. Therefore, this common material: feathers, is an outstanding source of probable developments because of its interesting characteristics derived from its fibrillar protein condition. The applications mentioned in this chapter were briefly discussed, but the reinforcement of polymers and biopolymers, together with grafting on or grafting with this protein are subjects of extensive and deserved research. A worthy mention must be done in the environmental remediation and nanotechnology areas, in which keratin from feather has also promising advances. In the case of environmental remediation, the functional moieties of keratin are the base for adsorption of recalcitrant heavy metal contaminants (Saucedo-Rivalcoba, 2011a, 2011b). On the other hand important nanostructures like carbon nanotubes or graphene, both extensively studied in nanotechnology field, can be chemically modified with this protein in order to give them multifunctional properties. However, both applications merit an extensive and apart review mainly due to the structural implications and the possibility to be adequately exploded. Thus, in this chapter the features, properties, recent researches in materials field and other applications related with the keratin obtained of chicken feather are exposed, given an

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evidence of the potential that offer this material as research field and possible applications.

ACKNOWLEDGEMENTS We thank to Dr. Víctor M. Castaño and Dr. Miguel de Icaza from CFATA-UNAM for their valuable and kind collaboration in all time of our research. The kind authorization for using some figures from Elsevier Science, Creative Commons Attribution-Share Alike, John Wiley and Sons and The Royal Society is gratefully acknowledged.

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INDEX

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A abuse, 10 accessibility, 111 acetic acid, 57 acetone, 79, 106, 114, 120, 121, 122, 123, 124, 125, 126 acid, 44, 45, 47, 48, 51, 52, 54, 56, 58, 59, 63, 65, 69, 137, 138, 151, 183, 185, 231, 233, 237 acidic, 3, 29, 50, 51, 54, 57, 58, 154, 169 acromegaly, 161, 165, 167, 169 acrylic acid, 238 acrylonitrile, 238 ACTH, 156, 157, 159, 162, 164, 166 active site, 232, 236 additives, ix, 43, 55 adenoma, 159, 160, 161, 162, 165, 167, 168 adhesion, 37, 41, 42, 59, 209 adhesion level, 209 adjustment, 173 adrenocorticotropic hormone, 156 adsorption, 64, 71, 117, 129, 241 age, 4, 106, 154, 157, 174 aggregation, 15, 219 agriculture, 67 alcohol abuse, 10 alcoholic liver disease, 33 alcohols, 231 aldehydes, 231

algorithm, 151 alkaline hydrolysis, 52 alters, 16 amine, 231, 233 amines, 231 amino, 44, 45, 50, 52, 55, 78, 182, 183, 186, 187, 191, 193, 231 amino acid, 44, 45, 50, 52, 55, 78, 182, 183, 186, 187, 191, 193, 231 amino acids, 45, 50, 52, 55, 78, 183, 186, 191, 193 ammonia, 48 ammonium, 105 amplitude, 141 anisotropy, 140 anti-apoptotic role, 21 antibody, 20, 26, 80 antigen, 80 antigenicity, 159 apoptosis, viii, 2, 9, 18, 19, 20, 22, 36, 38, 39, 40 aqueous solutions, 58, 62 arginine, 36, 52 argon, 80 aspartic acid, 50, 54 assessment, 136 ATP, 33 attachment, 54, 57, 58, 66, 109 avian, 174, 182

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Index

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B basal cell carcinoma, 160 basal layer, x, 76, 94 base, xiv, 65, 172, 241 basement membrane, 40 basophils, 162 behaviors, 133 bending, 118, 187, 229 bias, 154 bile, vii, 1, 7, 14, 33 bile duct, 7, 33 biliary tract, 9 Biocompatibility, 63, 68 biodegradability, 54, 68, 228 biodegradation, 228 biologically active compounds, 64 biomass, 68 biomaterials, 69, 71, 72 biomedical applications, ix, 44, 68 biopolymer, 173, 230, 231 biopolymers, 172, 183, 225, 228, 229, 230, 241 biopsy, 32, 151 biosynthesis, 41, 70 biotin, 79 birds, vii, 44, 45, 173, 174 bisphenol, 223 bleaching, 105, 114, 120 bleeding, 65 blend films, 71 blends, 56, 58, 63, 64, 69, 73, 74 bonding, 116, 186, 209 bonds, ix, xi, 43, 44, 46, 47, 48, 50, 51, 52, 53, 60, 64, 65, 67, 104, 189, 193, 197, 217, 236 bone, 61 bone form, 61 brain, 155, 168 breakdown, 39 breast cancer, 19, 23, 40, 41 breast carcinoma, 24, 42 brittleness, 114 building blocks, 50 by-products, 22

C cadaver, 148 calcium, 30, 60, 61, 71 cancer, 6, 24, 25, 41, 42 cancer cells, 24, 25, 42 carbon, 228, 241 carbon dioxide, 228 carbon nanotubes, 241 carbonyl groups, 187 carboxyl, 55, 61, 232, 233, 237 carcinoma, 40, 42 caspases, 21, 38 casting, 57 catalyst, 238 C-C, 217, 233, 234 cell biology, 26 cell culture, 57, 101 cell cycle, 18, 38 cell death, 18, 20, 22, 38, 39, 40 cell differentiation, 23, 93 cell division, 78 cell fate, 18 cell line, 4, 22, 23, 24, 27, 40, 42 cell lines, 22, 24, 40, 42 cell phones, 222 cell surface, 24 cellulose, 69, 197, 198, 214, 220, 229, 230 cellulose fibre, 229 central nervous system, 154 challenges, 60 chaperones, 9, 33 chemical, ix, xii, 43, 44, 45, 50, 56, 58, 62, 64, 71, 72, 105, 114, 138, 140, 189, 197, 214, 228, 230, 231, 238, 239 chemical interaction, 230 chemical properties, 44, 238 chemical reactions, 189 chemical reactivity, 72 chemical structures, 138 chemicals, xii, 59, 64, 131 chicken, xiv, 172, 173, 181, 182, 185, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 207, 209, 210, 214, 215,

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Index 218, 220, 221, 222, 223, 224, 227, 228, 229, 238, 242 childhood, viii, 2, 7 chitosan, 57, 73 cholesterol, 132 chromatography, 132, 150 chromium, 55 chromosome, 26, 165 chronic diseases, 9 cirrhosis, viii, 2, 7, 11 classes, xiii, 101, 153, 154 classification, 44 cleaning, 45, 63 cleavage, ix, 20, 38, 43, 46, 48, 51, 53 clinical application, 154 closure, 24 clustering, 22 CO2, 189, 190 coffee, 230 collaboration, 242 collagen, 44, 53, 55, 57, 59, 129, 173 colon, 6 color, 181 commercial, 65, 66, 67 communication, 163 compatibility, 62, 214, 215, 216 complexity, 27, 37 composites, xiv, 172, 173, 198, 199, 200, 202, 203, 204, 205, 206, 207, 209, 210, 211, 212, 213, 214, 216, 217, 218, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 241 composition, 7, 10, 45, 50, 52, 70, 132, 151, 173, 182, 189, 212, 231, 239 compost, 228 composting, 228 compounds, 189, 190 compression, 58, 60, 71, 215 computer, 81 conception, ix, 75 condensation, 20, 183 conference, 127 configuration, 185, 193 connective tissue, 82, 85, 86, 88, 89, 91, 92, 94, 95

consensus, 27 constituents, 8, 21 construction, 183 consumption, 224 contamination, 45 contour, 175 control group, 65 COOH, 20 copper, vii, 1, 7, 10, 55 correlation, 40, 149, 150, 157 cortex, xi, 45, 46, 103 cosmetic, xi, xii, 66, 67, 103, 105, 114, 127, 130 cosmetics, xi, 104 cost, xiv, 172, 198, 225 cotton, 197 covering, 81, 86, 89 cracks, 239 Croatia, 99 crown, 154 crystal structure, 206 crystalline, 51, 181, 205, 218 crystallinity, 205, 218 crystallites, 185 crystals, 218 cultivation, 59, 61, 73 culture, 57, 101 cuticle, xi, 45, 46, 53, 103 cycles, 124, 218 cyst, xiii, 153, 165, 167, 169 cysteine, 44, 47, 52, 54, 60, 64, 193 cystine, ix, xi, 43, 44, 45, 46, 48, 49, 51, 52, 53, 65, 67, 104, 178, 182, 183, 187, 231 cytochrome, 19, 22 cytoplasm, x, 19, 38, 76, 83, 85, 86, 91 cytoskeleton, 20, 25, 29, 31, 32, 33, 41 cytotoxicity, 40

D damages, 114, 239 damping, 220 decomposition, 189, 197, 216, 225 decomposition temperature, 216 decontamination, xiv, 172

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defects, 62, 65, 102 deficiencies, 25 deformation, 186, 227 degradation, viii, 2, 7, 15, 30, 38, 51, 58, 189, 190, 239 degradation process, 239 denaturation, 189, 236 density values, 226 deposition, 41 depth, 132, 143, 147, 149, 151 derivatives, 65, 77, 174 dermatitis, xii, 131 desmosome, 24 desorption, xii, 64, 66, 67, 105, 106, 107, 108, 109, 110, 111, 112, 113, 116, 117, 119, 123, 125, 127, 128, 129 destruction, 50, 52, 189, 236 detachment, 20 detectable, x, 9, 76, 81, 86, 87, 89, 90, 91, 94, 96 detection, xiii, 11, 33, 37, 39, 52, 80, 100, 153, 154 detergents, xi, 104, 149 developmental theories, 174 deviation, 142 dialysis, 52, 58, 60, 65, 67 dielectric constant, 222, 224, 225, 226 dielectrics, 224 diet, 7, 10, 14, 30 differential scanning, xiv, 172, 189 differential scanning calorimetry, xiv, 172, 189 diffraction, 151, 184 diffusion, xii, 104, 109, 111, 113, 118, 119, 120, 124, 126, 127, 141, 145, 236 diffusivity, xi, 103 disease progression, 15 diseases, vii, xi, xii, 1, 7, 26, 36, 104, 132, 168 disorder, 139 dispersion, 197, 200, 204, 216, 239 displacement, 47 distilled water, 47, 58, 59, 137

distribution, ix, x, 3, 22, 24, 38, 40, 75, 76, 83, 85, 92, 97, 141, 156, 161, 166, 190, 197, 204 DNA, 19 DNA damage, 19 donors, 16 draft, 26 drug delivery, 58 drug release, 65 drug withdrawal, 10 drugs, 11, 61, 64, 106 drying, xi, 57, 58, 60, 67, 71, 104, 222 DSC, 189, 216, 218, 232, 236 ductility, 227 durability, 231 dyes, 64, 109 dynamic mechanical analysis, 227

E electric field, 61, 224 electrical properties, 198, 223 electrical resistance, 150, 222 electrodes, 222 electron, xiii, 78, 132, 134, 135, 150, 167, 168, 176, 180, 181, 182, 184 electron microscopy, 78, 182, 184 Electron Paramagnetic Resonance, v, xii, 131, 132, 150, 152 electrospinning, 61, 62, 63, 72, 73 elongation, 174, 197, 227 embryogenesis, 154 enamel, 167, 169 encapsulation, 64 encouragement, 97 endocrine, 156, 157, 158, 160, 163, 164, 166, 167, 169 endothermic, 189, 236 energy, 111, 112, 116, 121, 134, 136, 209, 219, 220, 226, 231 engineering, 58, 59, 198 enlargement, 31, 234 environment, 15, 59, 60, 64, 138, 140, 142, 154, 174, 178, 183 environmental conditions, 173

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Index environmental factors, 155 environmental stress, xii, 131 environmental stresses, xii, 131 enzyme, 67 enzymes, 51, 137 epidermis, 77, 98, 101, 102, 147 epidermolysis bullosa, 36 epidermolysis bullosa simplex, 36 epithelia, vii, 1, 4, 6, 9, 24, 28, 29, 33, 77, 101, 154, 157, 165, 166 epithelial cells, vii, viii, x, 2, 3, 4, 19, 20, 21, 25, 28, 39, 44, 59, 76, 77, 81, 86, 93, 96, 154, 155, 157, 164, 165, 166 epithelium, ix, x, 23, 28, 37, 39, 75, 76, 77, 81, 82, 84, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 155, 156, 163 epoxy polymer, 207, 214 EPR, xii, 132, 133, 134, 135, 136, 137, 138, 139, 140, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152 equilibrium, xii, 18, 48, 49, 104, 107, 108, 110, 111, 112, 113, 117, 119, 122, 124, 125, 126, 129 erosion, 207 esophagus, 78 ESR, xii, 132, 151, 152 estrogen, 38, 40 ethanol, 59, 159 etherification, 238 ethylene, 57, 62, 218 ethylene glycol, 57 ethylene oxide, 62 eukaryotic, 21 evagination, 155 evaporation, 56, 65 evidence, viii, 2, 22, 25, 27, 33, 95, 174, 242 evolution, xiii, 172, 173, 174, 175, 178, 190, 241 exposure, xi, 65, 104, 137 extracellular matrix, 23, 55 extraction, vii, ix, 44, 46, 47, 48, 49, 50, 52, 53, 58 extrusion, 228

F fabrication, 56, 58, 59, 60, 61, 73 Fabrication, 59, 70, 73 fat, 64, 143 fatty acids, 132 fetus, 82, 84, 88, 91, 92, 95 fiber content, 200, 207, 216, 220, 223, 226, 227 fibrillation, 56, 72 fibrin, 44 fibroblasts, 23, 101 fibrosis, 37 filament, xi, 4, 21, 22, 26, 27, 28, 29, 30, 39, 42, 46, 77, 98, 101, 104, 154, 166, 167, 169, 220 filiform, x, 76, 77, 78, 81, 82, 83, 85, 86, 92, 93, 94, 95, 99, 100 film thickness, 57 films, ix, 44, 52, 55, 56, 57, 58, 63, 68, 71, 72, 73, 74, 128, 239, 240, 241 filters, 61, 63, 73 filtration, ix, 44, 55, 65, 67, 68, 70 financial, 68 financial support, 68 fixation, 159 flexibility, 25, 183, 197 flight, 175, 178, 195 fluorescence, 80, 98, 100 follicle, 45, 157 follicle stimulating hormone, 157 follicles, 163, 164 food, 108, 129, 183, 219 force, 117, 191 formaldehyde, ix, 44, 55, 64, 79, 159, 229 formation, vii, 1, 5, 7, 9, 10, 11, 12, 16, 18, 28, 30, 31, 32, 33, 34, 35, 50, 51, 52, 53, 56, 60, 62, 63, 155, 161, 164, 165, 183, 190, 220 fossils, 173 fracture toughness, 226 fragility, 36 fragments, 141, 228 free radicals, 134, 137, 231, 232, 233, 237, 239

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freedom, xii, 132, 143 freezing, 59 friction, 209, 219, 227 FTIR, 185, 186, 188, 190, 191, 230, 232, 238, 239 fusion, 218

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G gel, 54, 68 gene expression, 4, 9, 27, 30 gene regulation, 27 genes, 3, 4, 11, 22, 26, 36, 66, 165 genetic background, 10, 33 geometry, 186 Germany, 78, 80, 105 gestation, 77 gland, 160, 165, 167, 168 glass transition, 128, 129, 216 glass transition temperature, 128, 216 glial cells, 3 glue, 136 glutamate, 148 glutamic acid, 50, 54 glutamine, 50 glycerol, 57, 58, 128, 230 glycine, 46, 53, 70, 72, 183 glycosylation, 4, 5, 37 goose, 194, 196 granules, 3 graphite, 222 growth, viii, xiii, 2, 3, 19, 42, 57, 60, 68, 93, 98, 153, 154, 159, 164, 167 growth factor, 19, 98, 164 growth hormone, 159, 167

H hair, vii, x, xi, xii, xiii, 44, 58, 65, 66, 67, 68, 69, 72, 98, 101, 102, 103, 104, 105, 106, 109, 110, 111, 112, 113, 114, 116, 117, 118, 119, 120, 122, 125, 126, 127, 128, 129, 130, 171, 189, 191, 193 hair follicle, 101

hairless, 148 HDPE, 204, 205, 206, 211, 212, 218, 219, 220 HE, 158, 159 healing, 65 health, xi, 5, 29, 42, 104 heat shield, 174 heat shock protein, 33 heavy metals, 68 helium, 80 hemp, 230 hepatitis, 11, 32, 36, 37 hepatitis a, 11, 32 hepatocellular carcinoma, viii, 2, 7 hepatocytes, vii, 1, 6, 7, 9, 10, 11, 12, 14, 15, 16, 17, 18, 21, 22, 24, 25, 30, 31, 32, 33, 34, 35, 41, 42 hepatoma, 22, 27 hepatotoxicity, 34 heterogeneity, 195 hexane, 47 histology, 81, 82, 84, 88, 91, 92 histone, 29 histone deacetylase, 29 homeostasis, 18, 25 homogeneity, 220 hormone, 157, 159, 160, 168 hormone levels, 159 hormones, 159, 160, 163 horses, 173 human genome, 3, 26 humidity, xii, 57, 104, 106, 107, 111, 113, 114, 117, 125, 129 hyaline, xiii, 6, 153, 161, 162 hybrid, 61, 73 hydrogels, ix, 44, 65, 66, 68, 238 hydrogen, 47, 178, 186, 193 hydrogen bonds, 47, 178, 193 hydrolysis, ix, 43, 45, 50, 51, 53, 67 hydrophobicity, 66, 192 hydroxyapatite, 61, 73 hydroxyethyl methacrylate, 238 hydroxyl, 231, 232, 238 hydroxyl groups, 238 hyperplasia, 34

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Index hypothesis, 23 hysteresis, 110, 192

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I ideal, 222 identification, 17, 167 IFNγ, 16 images, 80, 81, 82, 84, 88, 91, 92, 99, 175, 200, 236, 239 IMF, 154, 159, 162 immobilization, 71 immunofluorescence, 11, 27, 29, 78 immunohistochemistry, 98, 99, 100 immunoreactivity, ix, x, 75, 76, 81, 83, 85, 86, 88, 89, 90, 91, 93, 94, 95, 96, 97, 156, 157, 159, 161 improvements, 215 in situ hybridization, 12, 26, 27, 77 in vitro, 28, 30, 32, 41, 42, 57, 58, 132, 159 in vivo, 32, 41, 54, 57, 61, 65, 77, 150, 159 incidence, 161 India, 185 indirect effect, 22 induction, 7, 10, 33, 35, 41 industries, 68 infection, 37 inflammation, 34 informed consent, 136 infrared spectroscopy, 8, 31, 151 ingredients, xii, 105 inhibition, 16, 24 inhibitor, 32 initiation, 11, 21, 28, 94, 95 injure, 21 injuries, viii, 2, 17, 18 injury, 11, 22, 31, 32, 33, 36, 37, 65, 69 inoculum, 228 insertion, 238, 239 insulation, 174 insulin, 18, 35, 160 insulin resistance, 35 integration, 28 integrity, viii, 2, 17, 23, 24, 25, 37, 66, 112, 117, 120, 125, 127

integument, 174 interface, 65, 197, 200, 201, 206, 209, 210, 213, 216 interference, 31, 35, 80, 99, 100 intermediate filaments (IFs), vii, 1 intoxication, 12, 34 involution, 29 ionizing radiation, 19 ions, 48, 50, 61, 67, 231 irradiation, 65, 239, 240 isolation, 21 isotherms, xii, 104, 105, 107, 109, 110, 114, 116, 120, 128, 129 Italy, 43, 68

J Japan, 79, 80, 131, 136, 153 justification, 108 juveniles, 79, 83, 85, 86, 93, 95

K keratinocytes, 4, 77, 94, 98, 102 ketones, 231 kinetic model, 108, 111 kinetics, xii, 66, 69, 105, 107, 112, 117, 118, 128 KOH, 67

L lactic acid, 227 lamella, xii, 132 leaching, 60 lead, xi, 10, 16, 55, 104, 114, 116, 126 leakage, 224 lens, 3 lesions, 29 leucine, 54 ligament, 98 light, 10, 58, 98, 167, 176, 214 light transmission, 58

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lipids, xi, xii, 67, 69, 104, 128, 131, 132, 133, 143, 145, 146, 149, 150, 152 liver, vii, 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 22, 25, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 65, 69 liver cells, 32 liver disease, vii, 1, 5, 6, 7, 9, 10, 17, 25, 28, 33, 37 liver failure, 37 local order, 141, 142 localization, 22, 24, 27, 41, 42, 77, 78, 81, 82, 84, 88, 91, 92, 93, 97, 98, 165, 168, 169 low sulphur content, 44 lower lip, 95 lung cancer, 22 luteinizing hormone, 157 lysine, 30, 52 lysozyme, 60, 71

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M machinery, 20 macromolecules, 134 magnetic field, 134 magnetic resonance, xiii, 132, 151 majority, 120, 125 malignancy, 41, 154 Mallory-Denk bodies (MDBs), vii, 1 mammalian cells, 3 mammals, vii, 3, 44, 45, 77, 93 man, 52 management, 65 mapping, 39 mass, 45, 107, 109, 189, 190, 218, 230 mass loss, 189 materials, 50, 55, 61, 67, 68, 70, 73, 177, 178, 186, 190, 191, 197, 198, 199, 209, 210, 211, 214, 220, 222, 223, 228, 241 materials science, 177 matrix, 23, 24, 46, 48, 53, 65, 66, 141, 181, 199, 200, 206, 207, 209, 211, 213, 214, 215, 216, 218, 221, 222, 223, 226, 227, 230 matter, 45

MDB formation, 7, 9, 10, 11, 16 measurement, 107 measurements, 48, 107, 128, 133, 197, 222 mechanical properties, xiii, 52, 55, 56, 57, 63, 66, 130, 172, 173, 193, 194, 197, 199, 200, 204, 206, 209, 210, 211, 212, 213, 214, 215, 228, 229, 238, 239 mechanical stress, viii, 2, 25, 200 media, 51, 70, 72, 237 median, 86 mediation, 22 medical, xiii, 171 medicine, 64, 177, 219 medulla, xi, 103 melanoma, 23, 40, 41 melt, 204, 205, 227 melting, 63, 218 melting temperature, 218 membranes, 59, 74, 132, 140, 150, 151 memory, 33, 35 mercury, 55 metabolism, vii, 1, 7, 10 metal ion, ix, 43, 55, 63, 71, 231, 237 metal ions, ix, 43, 55, 231, 237 metastasis, 23 metastatic cancer, 23 methyl group, 187 methyl methacrylate, 199 mice, viii, 2, 7, 8, 10, 11, 13, 14, 15, 16, 17, 21, 22, 25, 26, 30, 31, 32, 34, 35, 36, 37, 41, 57, 66, 77, 95, 96, 102 microcrystalline, 129 microelectronics, 222 microhardness, 207 microscope, 80, 168, 176 microscopy, 27, 29, 81, 82, 84, 88, 91, 99, 100 microstructure, 193, 221 microwave heating, 101 microwaves, 134 migration, 23, 24, 25, 41, 154 mitochondria, 3, 19, 22, 40 mitosis, viii, 2, 19 mixing, 57, 58, 200, 204 MMA, 231, 236, 237

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Index models, viii, 2, 7, 10, 107, 129 moderates, 21 modifications, vii, 1, 4, 5, 6, 12, 15, 22, 25, 122, 230 modulus, 194, 195, 196, 197, 199, 200, 203, 205, 207, 208, 210, 213, 214, 215, 226, 227, 229, 230 moisture, xii, 66, 67, 105, 108, 109, 110, 111, 112, 114, 116, 117, 118, 120, 121, 123, 126, 127, 128, 130 moisture content, 66, 108, 109, 111, 114, 117, 123, 130 moisture sorption, 67, 111, 114, 117, 127, 128 mole, 134 molecular biology, 98, 101, 244 molecular mass, 51, 53 molecular structure, 9, 28, 31, 70, 224 molecular weight, ix, xi, 8, 31, 43, 50, 51, 52, 53, 54, 55, 63, 66, 104, 120, 125, 155, 182 molecules, viii, 2, 16, 19, 44, 45, 51, 60, 109, 141, 191, 224, 234, 237 monoclonal antibody, 79 monolayer, 107, 109, 111, 112, 116, 121, 125, 126 monomer molecules, 236 morphogenesis, ix, 75, 78, 83, 93, 94, 95, 99, 100, 155, 165 morphology, xii, 23, 24, 81, 82, 84, 88, 91, 92, 131, 178 mortality, 13 moulding, 58, 60 mRNA, viii, 2, 11, 12, 27, 100 mRNAs, 11 mucosa, 95, 100, 101 multicellular organisms, 3 mutant, 15, 20, 21, 24, 28, 34, 36 mutation, 5, 17, 22, 36, 37 mutations, viii, 2, 17, 25, 36, 37, 173 myogenesis, 87, 89

N NaCl, 60

nail polish, xi, 104 nanofibers, 64, 69, 72, 74 nanostructures, 241 nanotechnology, xiv, 172, 173, 177, 241 National Research Council, 43 natural polymers, 62 natural resources, 198, 222 near infrared spectroscopy, 228 nematic liquid crystals, 151 neon, 80 neoplastic tissue, 160 nerve, 65, 69 nervous system, 3 neurofilaments, 3 neurons, 3 neutral, 3, 57, 58 New Zealand, 103, 105 next generation, 41 nitrogen, 68, 135, 142, 147 nitroxide, 135, 137, 138, 139, 140, 141, 142, 143, 145 nitroxide radicals, 138 NMR, xiii, 27, 132, 134 nodes, 236 nodules, 30, 32 non-keratinous proteins, 45 non-polar, 191 novel polymers, 50 nuclear magnetic resonance, xiii, 132 nuclei, 83, 86, 89, 91, 232 nucleus, 20, 23, 85, 86, 87, 89, 135, 162 null, 12, 13, 18, 22 numerical aperture, 80 nutrition, 45

O oil, 64, 128, 226, 231 omission, 80 operations, 52, 161 opportunities, 237 oral cavity, 155 oral lesion, 98 organ, 6, 18, 155, 167, 169 organelles, 3, 162

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Index

organic solvents, xiii, 64, 172 organize, 175 organs, ix, 6, 59, 75, 160 ornithine, 52 ossification, 61 ovulation, 154 oxidation, 46, 53, 54, 63, 65, 73, 114 oxidative stress, 11, 14, 16, 22

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P pancreatic cancer, 29 parallel, 11, 78, 93, 94, 95, 138, 139, 142, 143 pathogenesis, 34, 128 pathology, xiii, 27, 30, 31, 32, 34, 37, 38, 41, 153 pathways, viii, 2, 5, 16, 18, 19, 21, 133, 177, 178 peptide, ix, xi, xii, 43, 50, 51, 53, 54, 66, 67, 78, 104, 105, 106, 114, 116, 120, 127, 160, 183, 186, 189 peptides, 50, 51, 52, 66, 69, 70, 101, 127, 128, 130 periodontal, 98 peripheral nervous system, 166 permeability, 22, 67, 113, 120, 124, 126, 127 permeation, 133, 149 permission, 177, 180, 181, 190, 191, 194, 196, 202, 203, 204, 206, 208, 209, 211, 217, 223, 225, 240 permit, 52, 60 petroleum, 225 pH, 48, 49, 54, 55, 65, 79, 105, 155 phenol, 190, 229 phenolic resins, 222 phenotype, 23 phosphate, 9, 15, 22, 57, 59, 61, 65, 79, 159 phosphatidylcholine, 152 phospholipids, 151 phosphorylation, viii, 2, 4, 5, 6, 8, 14, 15, 17, 19, 22, 29, 32, 35, 36 physical and mechanical properties, 55 physical properties, xi, 104, 110

physicochemical properties, 71, 133 physiology, 40 PI3K, viii, 2 pituitary gland, vii, xiii, 153, 155, 157, 158, 160, 163, 165, 166, 167, 168, 169 plasma membrane, vii, 1 plasminogen, 24, 42 plasticity, 71 plasticized films, 57 plastics, 68 platform, 20, 25 playing, 9, 16 Pliocene, 174 PMMA, 199, 200, 202, 207, 208, 216, 217, 232, 234, 235, 236 point mutation, 22 polar, 52, 55, 64, 111, 191 polar groups, 64 polarization, 224 poly(methyl methacrylate), 215 polyamides, 52 polycondensation, 50 polyethylenes, 206 polyimides, 226 polymer, xiii, 56, 58, 59, 61, 62, 171, 173, 197, 198, 199, 200, 204, 206, 207, 209, 210, 213, 215, 216, 220, 224, 226, 228, 233, 234, 236, 244 polymer chains, 209 polymer composites, xiii, 171, 173, 197, 198, 199, 200, 204, 213, 215, 220 polymer materials, 56 polymer matrix, 199, 207, 210, 213, 216 polymeric chains, 217 polymeric composites, 197 polymeric materials, xiii, 171 polymerization, 199, 218, 231, 232 polymers, ix, 43, 55, 108, 129, 196, 197, 210, 218, 225, 239, 241 polypeptide, 173, 178, 183, 185, 216, 219, 237, 238 polypeptides, 28, 45, 101, 155, 167, 169, 238 polypropylene, 209, 214, 239 population, 18, 68

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Index porosity, 60, 61, 71 positive reinforcement, 230 potassium, 67 poultry, 174, 228, 230 precipitation, 47 preparation, 80 preservation, 20, 159 principles, xiii, 132 probe, xii, xiii, 132, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 149 pro-inflammatory, 16 project, 26, 68 prolactin, 159 proliferation, viii, 2, 18, 57, 58, 66, 94, 157, 168 proline, 183 protection, 13, 15, 39, 40, 128 protective role, viii, 2, 18, 25 protein family, 9 protein kinases, C, 9, 15, 33, 40 protein oxidation, 22 protein structure, 186, 193, 195, 232 proteinase, 24 proteolysis, 35 pulp, 214 purification, 52 pyrolysis, 189, 190, 217

Q quantum state, 134, 135

R radical formation, 231 radicals, 231, 237, 239 Raman spectra, 185, 186, 233, 235 Raman spectroscopy, 184 raw materials, xiii, 171 reaction mechanism, 47, 232 reaction rate, 48 reactions, 47, 217, 231, 237 reactive groups, 231

reactive oxygen, 40 reactive sites, 231 reactivity, 68, 83, 160 receptors, 19 recombination, 237 reconstruction, 58, 72 recovery, 10, 66, 124 recycling, 64 reform, xi, 67, 104 regeneration, 22, 60, 65, 69, 73 regression, 41, 108, 112, 116 reinforcement, xiv, 172, 173, 204, 207, 209, 210, 211, 212, 213, 215, 221, 226, 227, 230, 241 relaxation, 151, 219, 222, 224 relaxation process, 219 relevance, 168 remediation, 173, 177, 241 remodelling, 24 repair, 61, 66 repulsion, 48 researchers, viii, 2, 10, 26, 51, 55, 62, 209 residues, 44, 47, 48, 49, 52, 55, 60, 64 resilience, 25 resins, 226 resistance, 21, 22, 25, 37, 39, 44, 210, 212, 223, 230, 231 resolution, 151 response, 8, 11, 15, 16, 22, 65, 162 restoration, 66, 114, 117 restrictions, xiv, 172, 178, 215 risk, 37 risk factors, 37 RNA, 31, 35 rodents, 77 room temperature, 47, 79, 106, 207 root, 109 roughness, 197 Royal Society, 242 rules, 58, 136, 154

S scanning electron microscopy, xiv, 77, 99, 172, 179, 197

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Index

science, 130 SCO, 190 scope, xiv, 172 secrete, 160, 164 secretion, 45, 143, 144 semiconductor, 226 senses, 17 sensitivity, 12, 17, 20, 22, 34, 38, 134 serine, 29, 38, 50, 52, 54, 183 serum, 80, 151, 219 serum albumin, 151, 219 services, 15 shape, 3, 23, 25, 40, 111, 138, 139, 143, 154, 181 shear, 73, 219 sheep, 45, 72, 189 shock, 21, 32 showing, 5, 81, 159, 161 side chain, 55, 187 signal transduction, 165, 170 signaling pathway, 38 signalling, viii, 2, 5, 16, 18, 19, 21, 23, 25 signals, 133, 139, 143, 186, 234 signs, xiii, 77, 87, 88, 89, 132 silicon, 226 silk, 44, 53, 58, 63, 71, 73, 191, 231 simulation, 133, 141, 142, 143, 147, 148, 150 single chain, 149 siRNA, 24 skeleton, 3 skin, vii, xii, xiii, 17, 36, 44, 66, 67, 77, 96, 98, 100, 101, 102, 131, 132, 133, 136, 140, 147, 149, 150, 174 skin diseases, 17 small intestine, 6 snakes, 173 sodium, 47, 57, 79, 148, 214, 230, 238 sodium hydroxide, 214 software, 110 solubility, 38, 51 solution, 47, 48, 52, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 74, 105, 106, 137, 147, 148, 214, 236 solvents, xi, 104

sorption, xii, 66, 69, 105, 106, 107, 108, 110, 111, 112, 114, 116, 121, 128, 129, 130, 193 sorption experiments, 106 sorption isotherms, xii, 105, 107, 110, 114, 116, 121, 129 sorption process, 107 Spain, 99 species, xiii, 27, 40, 48, 55, 95, 172, 184, 194 spectroscopy, xii, 132, 133 spin, xii, xiii, 132, 133, 134, 135, 136, 137, 138, 139, 140, 142, 147, 148, 149, 151 spin labels, 137 spindle, 45, 46 sponge, 9, 15, 22, 59, 60, 71, 73 squamous cell, 28 squamous cell carcinoma, 28 stability, 5, 60, 178, 230 stabilization, 107 standard deviation, 197 starch, 129 state, 23, 45, 51, 60, 109, 111, 135, 154, 204, 218 states, 134, 136 steel, 47 stem cells, 160 sterilisation, 59 storage, 195, 207, 226, 227 stratification, 78, 96 stress, 7, 9, 10, 13, 14, 17, 18, 22, 25, 29, 33, 46, 140, 193, 194, 200, 203, 227 stretching, 132, 186 structural changes, 186, 193, 228 structural modifications, 62 structural protein, 44, 59 styrene, 226 substrate, 22, 23, 57 substrates, 20, 31, 57 sulfate, 148 sulfuric acid, 231 sulphur, ix, 43, 44, 46, 53, 70, 72, 190 Sun, 77, 94, 98, 101, 166, 185, 192, 245 surface area, 55, 61 surface layer, 85, 86, 89

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Index surface properties, 71 surface tension, 61 surfactant, 47, 149, 150 surfactants, xii, 131, 133, 148, 150 survival, 13, 18, 25 susceptibility, 17, 33 swelling, xi, 57, 58, 64, 104 symmetry, 141, 145 symptoms, 167 syndrome, 128 synthesis, 50, 52, 154, 178, 198 synthetic fiber, 181, 198 synthetic polymers, xiv, 65, 172, 197, 215, 222, 225, 230, 239

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T target, 15, 18, 21, 22, 24, 165 TEM, 181 temperature, xii, 37, 51, 52, 58, 59, 79, 104, 107, 129, 133, 148, 151, 178, 195, 197, 204, 205, 210, 211, 216, 217, 222 tensile strength, 194, 197, 210, 213, 227 terpenes, 149 testing, 108, 199 texture, 118 TGA, 189, 190, 191, 216, 227, 230, 232, 236 thermal decomposition, 190, 217 thermal degradation, 236 thermal expansion, 226 thermal properties, 198, 218, 227, 239 thermal resistance, 236 thermal stability, 63, 216, 227, 230 thermogravimetric analysis, xiv, 172, 189, 227 threonine, 50 time periods, 117 tissue, xi, 3, 8, 17, 18, 22, 25, 37, 42, 58, 59, 61, 65, 66, 83, 85, 86, 88, 89, 91, 100, 101, 103, 126, 154, 159, 160, 165, 173 TNF, 19, 21, 36, 38 toxic effect, 57 toxicity, 16 transcription, 165, 170

transfection, 24 transformation, viii, 2, 23, 31, 154 transition metal, 134 translation, 21 translocation, 19 transmission, xiv, 77, 81, 82, 84, 88, 91, 98, 99, 172, 181 transmission electron microscopy, 77, 99, 181 transport, 3, 163 trauma, xi, 65, 104 treatment, xi, 8, 10, 11, 13, 15, 16, 21, 31, 47, 51, 53, 59, 66, 67, 103, 106, 114, 116, 117, 120, 121, 122, 124, 126, 127, 214, 239 trial, 65, 66 trypsin, 57, 58 tryptophan, 50 tumor, viii, 2, 19, 21, 39, 40, 42, 154, 159, 160, 161 tumor cells, 40, 42 tumor necrosis factor, viii, 2, 19, 21, 39, 40 tumors, 28, 30, 159, 160, 165, 166, 167 Turkey, 195 turnover, 5, 9, 15 tyrosine, 46, 50, 53, 70

U ubiquitin-proteasome system, 8 UK, 79 ultrasound, 219, 220 uniform, 199 unique features, 177 United, 246 United Kingdom, 246 urea, 47, 51, 60 urokinase, 24, 42 USA, 79, 80, 81, 99, 101, 106, 170, 242 UV, xii, 19, 22, 40, 131, 239, 240 UV irradiation, xii, 131, 239, 240

Keratin: Structure, Properties and Applications : Structure, Properties and Applications, edited by Renke Dullaart, and João

226

Index

V validation, 107 valine, 54, 183 variables, 204, 205 variations, 98, 219, 234 vehicles, 58 velocity, 219 vertebrates, vii vibration, 187 viscoelastic properties, 207 visco-elastic properties, 42 viscosity, 62, 63, 73 visualization, 100 volatile organic compounds, ix, 44, 55

water diffusion, 120, 126 water permeability, 66, 120, 124, 126, 150 water sorption, vii, xi, xii, 66, 69, 103, 104, 105, 111, 112, 116, 120, 125, 127, 129 wear, 207 weight loss, 216, 217 weight reduction, 174 wetting, xi, 104, 192 wild type, 11 wood, 67 workers, 48 wound healing, 64

X X-ray diffraction, 44, 132

W Y yield, 47, 49, 64, 193, 200, 203, 212, 236

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Washington, 169 waste, 68, 71, 72 water absorption, xi, 104, 109, 230

Keratin: Structure, Properties and Applications : Structure, Properties and Applications, edited by Renke Dullaart, and João