Silk : materials, processes, and applications 9780128184950, 0128184957

Table of contents :
Content: 1. Sources and classification of silk2. Structure and properties of silk fibers3. New developments in degumming silk4. Regenerated silk fibers5. Electrospun silk fibers6. Applications of silk7. 3D printing silk8. Future trends in the sources, processing and applications of silk

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Silk: Materials, Processes, and Applications

The Textile Institute Book Series Incorporated by Royal Charter in 1925, The Textile Institute was established as the professional body for the textile industry to provide support to businesses, practitioners and academics involved with textiles and to provide routes to professional qualifications through which Institute Members can demonstrate their professional competence. The Institute’s aim is to encourage learning, recognise achievement, reward excellence and disseminate information about the textiles, clothing and footwear industries and the associated science, design and technology; it has a global reach with individual and corporate members in over 80 countries. The Textile Institute Book Series supersedes the former ‘Woodhead Publishing Series in Textiles’ and represents a collaboration between The Textile Institute and Elsevier aimed at ensuring that Institute Members and the textile industry continue to have access to high calibre titles on textile science and technology. Books published in The Textile Institute Book Series are offered on the Elsevier web site at: store.elsevier.com and are available to Textile Institute Members at a substantial discount. Textile Institute books still in print are also available directly from the Institute’s web site at: www.textileinstitute.org To place an order, or if you are interested in writing a book for this series, please contact Matthew Deans, Senior Publisher: [email protected]

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The Textile Institute Book Series

Silk: Materials, Processes, and Applications Narendra Reddy

Center for incubation innovation research and consultancy, Jyothy institute of technology Bengaluru, Karnataka, India



An imprint of Elsevier

Woodhead Publishing is an imprint of Elsevier The Officers’ Mess Business Centre, Royston Road, Duxford, CB22 4QH, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, OX5 1GB, United Kingdom © 2020 Elsevier Ltd. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/ permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-818495-0

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Contents

1

Sources and classification of silk 1.1 Introduction 1.2 Mulberry and non-mulberry silks 1.3 Spider silks 1.4 Marine silks 1.5 Ant and honey bee silks References

1 1 1 2 3 7 12

2

Structure and properties of silk fibers 2.1 Structure of silk fibers produced by Bombyx mori 2.2 Spider silk References Further reading

13 13 34 44 47

3

New developments in degumming silk 3.1 Conventional degumming using alkali 3.2 Degumming with the aid of surfactants/detergents 3.3 Infrared assisted degumming 3.4 Degumming using enzymes 3.5 Removal of sericin using ionic liquids 3.6 Degumming using steam 3.7 Comparison of various degumming approaches 3.8 Demineralization of wild silks before degumming References Further reading

49 49 53 57 61 65 66 66 68 70 71

4

Regenerated silk fibers 4.1 Regenerated silk using ionic liquids as solvents 4.2 Formic acid as a solvent for silk fibroin 4.3 N-Methylmorpholine N-oxide (NMMO) based solvent system for producing regenerated silk fibers 4.4 Regenerated fibers produced from spider silk 4.5 Novel approaches for producing regenerated silk fibers References

73 73 81 85 88 93 106

viContents

5

Electrospun silk fibers 5.1 Electrospun fibers from B. mori silk fibroin 5.2 Silk fiber blends 5.3 New systems of electrospinning 5.4 Electrospun fibers from wild silks 5.5 Electrospun fibers from spider silk proteins References Further reading

109 109 116 118 121 122 129 131

6

Applications of silk 6.1 Medical applications of silk fibroin 6.2 Medical applications of spider silk proteins 6.3 Clinical uses of silk 6.4 Biotechnological applications of silk 6.5 Cosmetic applications 6.6 Miscellaneous applications References Further reading

133 133 155 157 161 162 166 172 177

7

3D printing silk 7.1 Scaffolds developed from silk fibroin and blends with other biopolymers 7.2 3D printing silk with bacterial cellulose and microalgae 7.3 Scaffolds developed from silk fibroin and blends with synthetic polymers 7.4 3D printed silk fibroin and bioactive glass 7.5 Sericin 3D scaffolds 7.6 3D printed scaffolds from spider silks References

179

Future trends in the sources, processing and applications of silk 8.1 New sources and structure of silk 8.2 New approaches in processing silk into biomaterials 8.3 Emerging and futuristic applications of silk References

201 201 201 202 212

8

179 186 190 193 194 197 198

Index 215

Sources and classification of silk

1

1.1 Introduction Unlike any other fiber, silk is produced on land, water and air. More than 23 different silk lineages in 17 insect orders have been recorded and classified (Table  1.1) (Sutherland et  al., 2010). Most of the silk is produced by the Lepidoptera order of insects and specifically from the Bombycidae and Saturniidae species. Extensive studies have been done to further classify the silks produced by the different species. One example of classifying silk, based on the sequence of amino acids, is given in Fig. 1.1. In addition, silk can also be classified based on the gland in which it is produced. Similarly, silk species have been classified based on the differences in the FTIR spectra (Boulet-Audet et al., 2015) which also was able to distinguish silk based on their composition as seen from Fig. 1.2.

1.2 Mulberry and non-mulberry silks The primary source of silk is from the cocoons of the insect Bombyx mori which has been domesticated for over 5000 years. Before B. mori was domesticated and used for silk production, it has been reported that silk was generated from Bombyx mandarina considered to be the wild ancestor of the B. mori silkworm. The two insects differ by one chromosome number with B. mori having 28 and B. mandarina having 27. However, these two species are considered to be infertile and hence produce distinct cocoons and resulting silk fibers. During the process of domestication, B. mori has evolved as the more suitable option to obtain silk fibers and although B. mandarina is prevalent, it is now considered as wild silk. More than 400 phenotypes and 4310 silkworm germplasm strains have been recorded world wide (Zanatta et  al., 2009). The common mulberry silk worm belongs to the Bombycidae family with B. mori being the most common strain. B. mori silk worms feed on mulberry leaves and hence silk produced by B. mori is also called as mulberry silk. In addition to classification based on feed, B. mori silkworms have also been distinguished depending on the number of cocoon producing cycles. For example, univoltine silkworms have only one cocoon producing cycle compared to two cycles for bivoltine and multiple cycles for multivoltine silk (Table 1.2). Silkworms which feed on non-mulberry leaves are called wild silks and mostly belong to the saturniidae family and further classified into the Attacini sub-group (Fig.  1.3). Some of the common saturniidae insects (Fig.  1.4) also produce cocoons and silk as shown in figure (Chen et al., 2014). Tasar, muga and eri are the wild silks reared and commercially sold in relatively large quantities. The wild Silk: Materials, Processes, and Applications. https://doi.org/10.1016/B978-0-12-818495-0.00001-6 © 2020 Elsevier Ltd. All rights reserved.

2

Silk: Materials, Processes, and Applications

Table 1.1  Type of insects that produce silk, the life stage in which they produce the silk and the gland(s) from which the silk is generated. Common name of insect

Life stage

Gland

Jumping bristletails, silverfish Mayflies Dragon flies Webspinners Crickets Book lice Thrips Kaboono montana Evans Water beetles Plant eating beetles Lacewings Lacewings and ant lions Saw flies and parasitic wasps Parasitic wasps Bees, ants and wasps Saw flies Wasps Wasps Fleas Dance flies Glowworms Midges Butterflies, moths, caddisflies

Adult males Larvae Adult female All stages All stages Adult females Larvae and adults Unknown Adult female Larvae Adult female Larvae Larvae Adult female Larvae Larvae Adult females Adult females Larvae Adult males Larvae Larvae Larvae

Type III secretory units Malpighian tubules Unknown Type III secretory units Labial glands Labial glands Malpighian tubules Unknown Colleterial glands Malpighian tubules Colleterial glands Malpighian tubules Labial glands Abdomen secretion Labial gland Labial gland Type III secretory units Labial glands Labial glands Type III secretory units Labial glands Labial glands Labial glands

silks can also be further classified based on their cocoon characteristics or habitat (Padaki et  al., 2015). Wild silks are generally categorized as those that feed on non-mulberry plants. Wild silks can be classified broadly as temperate and tropical. Antheaea pernyi found in China, A. yamamai found in Japan and A. roylei, A. frithi and A. pernyi are prevalent in temperate conditions whereas A. mylitta is found in tropical conditions and mostly in India. Images of some of the cocoons produced by different wild silk worms are given in Fig. 1.4. The wild silks not only differ in terms of their composition and structure but also have to be processed using harsher conditions than mulberry silks.

1.3 Spider silks Archanids to which spiders belong consists of about 37,000 species and are known to produce silk with extraordinary properties. Different species of spiders produce silk from different glands such as ampullate (dragline), flagelliform etc. (Table 1.3, Fig. 1.5). The extraordinary properties of spider silks depend heavily on the species and glands and shows substantial variations.

Sources and classification of silk3

100 90 51 65 100 84

Antheraea pernyi Antheraea yamamai Actias selene Samia cynthia ricini Eriogyna pyretorum Saturnia biosduvalii Manduca sexta

47

52

Chinese B. mandarina

95

Bombyx mori C108

100 100

Bombycidae

Bombyx mori Dazao ♦

Lepidoptera Noctuidae

Hyphantria cunea

61

Bombycoidea

Bombyx mori Xiafang

Helicoverpa armigera

100 93

Sphingidae

Janpanese B. mandarina

100

60

Saturniidae

Noctuoidea

Ochrogaster lunifer

Notodontidae

Phthonandria atrilineata

Geometridae

Geometroidea

Pyralididae

Pyraloidea

Tortricidae

Tortricoidea

Ostrinia nubilalis

100

Chilo suppressalis

100 100

Diatraea saccharalis Adoxophyes honmai

100 100 73

Grapholita molesta Spilanata jechriaspis Artogeia melete Coreana raphaelis

93

Acraea issoria

91 100

Fabriciana nerippe Locusta migratoria

Pieridae Lycaenidae

Papilionoidea

Nymphalidae Orthoptera (outgroup)

Drosophila yakuba Anopheles gambiae

100

Diptera (outgroup)

0.05

Fig. 1.1  Phylogeny of lepidopteran insects based on the amino acid sequence of the gene 13 PCGs (Liu et al., 2013). Reproduced with permission through Elsevier Open Access Publication.

1.4 Marine silks Unique and distinct silks have been discovered in several marine animals but are probably the least studied among all the sources of silk. For instance, the amphipod (Crassicorophium bonelli) produces fine silk from its legs as an adhesive underwater (Kronenberger et al., 2012). A classification of the possible amphipods that produce marine silks are given in Fig. 1.6. Similar to C. bonelli, caddisflies belonging to the Tricoptera family produces silk based adhesives that are used to prepare structures for storing food. About 12,000 species of Trichoptera have been discovered and classified into sub orders of Annulipalpia, Spicipalpia and Integripalpia. Each species forms distinct cocoons usually for storage of food using stones and debris found underwater. These silks also differ in composition and properties compared to regular silks. Lack of alanine, higher amounts of arginine are distinct features. A mean hydrated net strength of 221 mN/m2 for Hydropsyche siltalia was reported. Ability of these insects to produce underwater silk using various substrates was demonstrated in an aquarium and also in a flow chamber (Ashton et al., 2012) (Fig. 1.7). Ability of the insects to form insoluble silk under water is quiet intriguing and is being studied further.

4

(B)

Sequencing FTIR Caligula simla Caligula simla Caligula cachara Caligula cachara Caligula thibeta Saturnia pavonia Saturnia pavonia Caligula thibeta Saturnia pyri Saturnia pyri Eriogyna pyretorum Actias selene Actias selene Actias luna Actias luna Cricula trifenestrata Graellsia isabellae Eriogyna pyretorum Argema mittrei Opodiphthera eucalypti Argema mimosae Argema mittrei Opodiphthera eucalypti Argema mimosae Cricula trifenestrata Antherina suraka Antheraea suraka Antheraea frithi Antheraea frithi Antheraea pernyi Antheraea yamamai Antheraea mylitta Antheraea pernyi Antheraea yamamai Antheraea mylitta Antheraea roylei Antheraea roylei Antheraea polyphemus Loepa katinka Antheraea polyphemus Graellsia isabellae Antherina suraka Hyalophora cecropia Loepa katinka Rothschildia jacobaea Callosamia promethea Hyalophora gloveri Hyalophora gloveri Hyalophora cecropia Rothschildia jacobaea Callosamia promethea Samia cynthia Samia canningi Samia cynthia Attacus atlas Samia canningi Attacus edwardsii Epiphora bauhiniae Epiphora bauhiniae Attacus atlas Nephila edulis dragline Attacus edwardsii Bombyx mori Bombyx mori Bombyx mandarina Bombyx mandarina Anaphe panda Gonometa postica Gonometa postica Nephila edulis dragline

Group 1 High phenolic content

Antheraea

Group 2 Low phenol, low oxalate Group 3 High oxalate content Saturniini tribe

18442

2709

Saturniidae

(A)

Silk: Materials, Processes, and Applications

Group 4 High sericin content

Attacini tribe

Bombycinae

Group 5 (AG)n b-sheets

Noctuidae Lasiocampidae

0

1200 400 800 Dissimilarity (Euclidean distance)

1600

Fig. 1.2  Classification of silk producing Lepidopteran insects based on the differences in FTIR spectra (Boulet-Audet et al., 2015). Published through open access publication under the terms of the Creative Commons Attribution License.

Table 1.2  Common silkworm varieties and their voltinism. Common name

Scientific name

Voltinism

Mulberry silk Tasar silk (tropical Tasar silk (Temperate) Muga silk Eri silk (Domesticated) Eri silk (wild)

Bombyx mori Antheraea mylitta A. prayeli A. assamensis Philosamia ricini P. Cynthia

Uni, Bi and Multi Uni, Bi and Multi Bi Multi Multi Uni and Bi

Mussels which belong to the Bivalvia class are another species of animals that produce silk under water. Commonly referred to as byssal threads, they help in anchoring of the mussels to various substrates. The byssal threads are quiet unique since one end is stiff and strong whereas the other end is soft and flexible. However, the structure and properties of the mussels from different species (Fig. 1.8) vary considerably. For instance, Pinna noblis generates thousands of fine fibers known as sea silk (Fig. 1.9). Comparatively, Mytilus species produces silk made up to globular proteins organized into nanofibrils (Pasche et al., 2018).

Sources and classification of silk5

92/99 98/99 100 99 100

98/100

Antheraea pernyi domestic Antheraea pernyi wild Antheraea yamamai Actias selene Saturnia pyretorum 100 Caligula boisduvalii Attacus atlas Samia cynthia 100 Samia ricini Manduca sexta Bombyx mandarina Japan Bombyx mori 52/67 Bombyx mandarina China 100

100

Saturniini Saturniidae Attacini

Bombycidae

Thitarodes renzhiensis Drosophila melanogaster

0.05

Fig. 1.3  Classification of silk producing insects based on phylogeny of Bombycidae species (Chen et al., 2014). Reproduced with permission from Elsevier.

A. mylitta

A. assama

A. frithi

A. atlas

P. ricini

G. postica

A. roylei

A. pernyi

II. cecropia A. polyphemus

Fig. 1.4  Images of some of the cocoons produced by different wild silkworms (Kundu et al., 2012). Reproduced with permission from John Wiley and Sons.

6

Silk: Materials, Processes, and Applications

Table 1.3  Classification of spider silk based on the gland in which the silk is produced (Tokareva et al., 2014). Silk gland

Use

Spinneret

Major ampullate Minor ampullate Pyriform Aciniform Tubuliform Aggregate Flagelliform

Dragline, frame threads Dragline reinforcement Attachment disk Swathing prey Cocoon construction Sticky glue for capture Capture spiral

Anterior Median Anterior Median, posterior Median, posterior Posterior Posterior

Reproduced with permission from Elsevier.

Flagelliforme

Aggregate

cylindrical: outer eggsac Cylindricale Minor Ampullate

Piriforme Major Ampullate

Aciniforme

cylindrical: capture core threads aggregate: aqueous coat

aciniform: wrapping silk and packing silk

minor ampullate: auxiliary spiral

major ampullate: structural and drag line silk

piriform: attachment cement

Fig. 1.5  Picture shows the various glands that extrude silk in spiders (N. clavipes), classification of silk and its uses (Tokareva et al., 2014). Reproduced with permission from Elsevier.

Novel silk like proteins were discovered from Nematostella vectensis a sea anemone which produces the silk as a means to capture prey. Like mussels, the nematocysts are capable of withstanding high mechanical stresses and the shape and properties of the silk produced are highly dependent on external factors and stimulations (Fig. 1.10). The proteins in the nematocysts could be made into regenerated and electrospun fibers (Yang et al., 2013) (Fig. 1.11).

Sources and classification of silk7

Hexapoda Miracrustacea Xenocarida

Malacostraca Copepoda

Altocrustacea Pancrustacea

Branchiopoda Oligostraca

Vericrustacea

Thecostraca

Mandibulata Myriapoda Arthropoda Chelicerata Outgroups 0.03

Fig. 1.6  Classification of anthropods that produce silk underwater (Kronenberger et al., 2012). Reproduced with permission from Springer Nature.

Fig. 1.7  Different approaches of forming silk under water in aquariums using PTFE case (A) and between two glass slides (B) (Ashton et al., 2012). Reproduced with permission from John Wiley and Sons.

1.5 Ant and honey bee silks A unique category of silk is produced by ants, wasps and other insects which belong to the insect order Hymenoptera considered to be derived from spiders and silkworms (Fig.  1.12). This insect order consists of 144,000 species and the silk produced is used for building covers from predatory or parasite hosts. Unlike other forms, the Hymenoptera insects produce coiled-coil silks with a unique molecular structure (Sutherland et al., 2010, 2012). Some of the species that produce coiled-coil silk and

8

Silk: Materials, Processes, and Applications

Class

Infraclass

Order

Superfamily

Family

Genus

Mytiloida

Mytiloidea

Mytilidae

Mytilus

Species M.edulis M.californianus

Pinnoidea Pteriomorphia

Atrina

A.pectinata

Pinna

P.nobilis

Pinnidae

Ostreida

P.fucata Pterioidea

Bivalvia

Pteriidae

Pinctada P.margaritifera

Heteroconchia

Pectinida

Anomioidea

Anomiidae

Anomia

A.simplex

Cardiida

Cardioidea

Cardiidae

Tridacna

T.maxima

Fig. 1.8  Classification of the various Bivalvia species that produce “sea silk” (Pasche et al., 2018). Reproduced from Royal Society of Chemistry under Creative Commons Attribution 3.0 Unported Licence.

Fig. 1.9  Morphology of the various types of silk threads produced by Bivalvia class of sea organisms (Pasche et al., 2018). Reproduced from Royal Society of Chemistry under Creative Commons Attribution 3.0 Unported License.

(A) (C)

(D) (B)

GPGNTGYPGQ DPGNTGYPGQ GPGNTGYPGQ GPGNTGYPGQ DPGNTGCPGQ GPGQ DPGNTGYPGQ GSGNTGCPGQ GPGNTGYPGQ

1 41 81 116 156 196 230 270 304

GPGNTGHPGQ DPGNTGYPGQ GPGNTGYPGQ GPGNTGHPGQ GPGNTGCPGQ GPGNTGYPGQ DPGNTGCPGQ GPGQ GPGNTG

GPGNTGYPGQ GPGNTGCPGQ GPSNTGYPWQ GPGNTGYPGQ GSGNTGCPGQ GPGNTGHPGQ GPGNTGCPGQ GPGNTGYPGQ

DPGNTGYPGQ GPGNTGCPGQ GPGNT DPGNTGYPGQ GSGNTGCPGQ GPGNTGYPGQ GSGNTGCPGQ GPSNTGYPGQ

Fig. 1.10  Digital image of the silk producing N. vectensis (A), before (C) and after stimulation (D) and amino acid sequence (B) of the silk produced (Yang et al., 2013). Reproduced with permission through Creative Commons Attribution-NonCommercialNoDerivs 3.0 Unported License.

(A)

(C)

(B)

(D)

Aneroin-30K Aneroin-60K

Stress (MPa)

200 150 100 50 0 0

1

2

3

4

Strain (%)

Fig. 1.11  Images of Aneroin fibers produced by wet spinning (A); SEM image of the fibers (B), fibers made into a skein (C) and properties of the fibers drawn to 30 k and 60 k vectors (Yang et al., 2013). Reproduced with permission through Creative Commons Attribution-NonCommercialNoDerivs 3.0 Unported License.

10

Silk: Materials, Processes, and Applications

their related information are presented in Fig. 1.12 and Table 1.4. Images of the common silks produced by three Hymenoptera are shown in Fig.  1.13 (Kameda et  al., 2014). Silks produced by these species have relatively low molecular weights (30– 50 kDa) with alanine rich core. Ants are another class of insects that produce silk with highly distinct structure, color and properties (Fig. 1.14) (Campbell et al., 2014). For instance, weaver ants were reported to produce silk in the form of nanofibers that were further made into the form of a non-woven web (Reddy et al., 2011). These nanofiber webs had mechanical properties higher than similar webs made from collagen, regenerated silk and other biopolymers. Araneae (spiders) Chrysidoidea

Hornet

> 500 M Yrs ago = 200 M Yrs ago

Social Wasp

Vespoidea

Weaver Ant

Silk with b sheet molecular structure

Australian Bulldog Ant = 280 M Yrs ago

Ponerine Ant Indian Jumping Ant

Coiled coil silk arose*

Lepidoptera (including silkworms)

Honey Bee Bumble Bee

Apoidea Stingless Bee

Fig. 1.12  Evolutionary relationship between silk producing insects (Sutherland et al., 2012). Reproduced with permission from John Wiley and Sons.

Table 1.4  Species of insects that produce coiled coil silk (Kameda et al., 2014). Species

Order/family

Silk gland

Lifestage

Bees, ants, hornets

Hymenoptera/ Vespoidea and Apoidea Hymenoptera/ Tenthredinoidea Mantodea/Mantidae Neuroptera/ Chrysopoidea Siphonaptera/ Multiple

Modified salivary

Larvae

Modified Salivary

Larvae

COllaterial Malphigian tubules

Adult female Larvae

Modified salivary

Larvae

Sawflies Praying mantis Lacewing Fleas

Reproduced with permission from Springer Nature.

Fig. 1.13  Images of the coiled coil silks produced by hornets (A); mantis (B) and lacewings (C) (Kameda et al., 2014). Reproduced with permission from Springer Nature.

Fig. 1.14  Images of silk/cocoons being produced by Oecophylla (top left); Brachymyrmex patagonicus (top right), Leptogenys crustosa (bottom left) and Rhytidoponera victoriae (bottom right) (Campbell et al., 2014). Reproduced with permission from Elsevier.

12

Silk: Materials, Processes, and Applications

References Ashton, N.N., Taggart, D.S., Stewart, R.J., 2012. Silk tape nanostructure and silk gland anatomy of trichoptera. Biopolymers 97 (6), 432–445. Boulet-Audet, M., Vollrath, F., Holland, C., 2015. Identification and classification of silks using infrared spectroscopy. J. Exp. Breiol. 218 (19), 3138–3149. Campbell, P.M., Trueman, H.E., Zhang, Q., Kojima, K., Kameda, T., Sutherland, T.D., 2014. Cross-linking in the silks of bees, ants and hornets. Insect Biochem. Mol. Biol. 48, 40–50. Chen, M.-M., Li, Y., Chen, M., Wang, H., Li, Q., Xia, R.-X., Zeng, C.-Y., Li, Y.-P., Liu, Y.Q., Qin, L., 2014. Complete mitochondrial genome of the atlas moth, Attacus atlas (Lepidoptera: Saturniidae) and the phylogenetic relationship of Saturniidae species. Gene 545 (1), 95–101. Kameda, T., Andrew A. Walker, and Tara D. Sutherland in Chapter 5 Evolution and application of coiled coil silks from insects in T. Asakura and T. Miller (Eds.), Biotechnology of Silk, Biologically-Inspired Systems 5, DOI https://doi.org/10.1007/978-94-007-7119-2 5, © Springer Science CBusiness Media Dordrecht 2014. Kronenberger, K., Dicko, C., Vollrath, F., 2012. A novel marine silk. Naturwissenschaften 99 (1), 3–10. Kundu, S.C., Kundu, B., Talukdar, S., Bano, S., Nayak, S., Kundu, J., Mandal, B.B., et al., 2012. Nonmulberry silk biopolymers. Biopolymers 97 (6), 455–467. Liu, Q.-N., Zhu, B.-J., Dai, L.-S., Liu, C.-L., 2013. The complete mitogenome of Bombyx mori strain Dazao (Lepidoptera: Bombycidae) and comparison with other lepidopteran insects. Genomics 101 (1), 64–73. Padaki, N.V., Das, B., Basu, A., 2015. Advances in understanding the properties of silk. In: Advances in Silk Science and Technology. Woodhead Publishing, pp. 3–16. Pasche, D., Horbelt, N., Marin, F., Motreuil, S., Macías-Sánchez, E., Falini, G., Hwang, D.S., Fratzl, P., Harrington, M.J., 2018. A new twist on sea silk: The peculiar protein ultrastructure of fan shell and pearl oyster byssus. Soft Matter 14 (27), 5654–5664. Reddy, N., Xu, H., Yang, Y., 2011. Unique natural‐protein hollow‐nanofiber membranes produced by weaver ants for medical applications. Biotechnol. Bioeng. 108 (7), 1726–1733. Sutherland, T.D., Young, J.H., Weisman, S., Hayashi, C.Y., Merritt, D.J., 2010. Insect silk: One name, many materials. Annu. Rev. Entomol. 55, 171–188. Sutherland, T.D., Weisman, S., Walker, A.A., Mudie, S.T., 2012. The coiled coil silk of bees, ants, and hornets. Biopolymers 97 (6), 446–454. Tokareva, O., Jacobsen, M., Buehler, M., Wong, J., Kaplan, D.L., 2014. Structure–function– property–design interplay in biopolymers: Spider silk. Acta Biomater. 10 (4), 1612–1626. Yang, Y.J., Choi, Y.S., Jung, D., Park, B.R., Hwang, W.B., Kim, H.W., Cha, H.J., 2013. Production of a novel silk-like protein from sea anemone and fabrication of wet-spun and electrospun marine-derived silk fibers. NPG Asia Mater. 5 (6), e50. Zanatta, D.B., Bravo, J.P., Barbosa, J.F., Munhoz, R.E.F., Fernandez, M.A., 2009. Evaluation of economically important traits from sixteen parental strains of the silkworm Bombyx mori L (Lepidoptera: Bombycidae). Neotrop. Entomol. 38 (3), 327–331.

Structure and properties of silk fibers

2

2.1 Structure of silk fibers produced by Bombyx mori Bombyx mori is the most common silk producing insect. Despite major differences in the habitat, structure and properties of the silkworms and cocoons, silk fibers are typically made up of two proteins, the fibroin and the sericin. Fibroin is the structural protein that provides strength and stability whereas the water soluble sericin acts as glue and is responsible for holding the fibroin filaments together. The silkworm extrudes the fibers in pairs of twisted filaments at a rate of about 6–8 mm/s which are bonded together by sericin. Morphologically, each silk fiber is composed of two separate stands, the bave and the brin with average diameter of the two strands together being 12–15 μm (Guo et al., 2018a,b). Diameter of the fibers usually varies from 10 to 13.7 μm with sericin coating on the outside being about 1–2 μm in thickness (Shen et al., 1998). The silk proteins are made up of two chains, the heavy chain and light chain connected together via a single disulfide bond. The heavy chain has a sequence of 12 crystalline domains interspersed with amorphous regions. In these regions, the proteins exist in the form or random coils/α-helix and β-sheets with transitions between the two forms along the length of the fibers (Johnston et al., 2018). The β-sheets are made up of hydrogen bonds which are considered to be weak and supposed to be responsible for the poor mechanical properties. However, nanocrystals in the β-sheet are highly confined within a few nanometers and provide higher stiffness and strength to the fibers (Keten et al., 2010). In addition, uniform shear deformation and molecular stick-slip within the β-sheets contributes to the extraordinary properties of silk fibers. Unlike fibroin, most of the sericin exists as random coils and the structure is relatively much less influenced by the conditions during fiber extrusion. Interestingly, it has been reported that sericin exists in three layers around the fibroin and that the amino acid composition and solubility of the sericin from these three layers are different (Wang and Yu, 2011). A schematic representation of the three sericin layers is given in Fig. 2.1. Outer layer of sericin accounts for 50%, middle layer for 35% and inner layer for 15%. Amount of sericin and fibroin has also been found to differ from the outer to the inner layer of B. mori silk worm cocoons (Chung et al., 2015). Silkworms spin the cocoon from outside to the inside and in this phenomenon, the fibroin fibers on the outside are mostly open due to the lack of sericin binding the fibers. Extrusion of sericin increases on the fibers spun inside of the cocoon which also decreases the fiber diameter and increases strength. In terms of crystalline structure, silk is classified as silk I and silk II with silk I being amorphous and in the α-configuration compared to silk II which is highly crystalline and in the β-sheet configuration. Structurally, the β-sheets can be further divided to be Silk: Materials, Processes, and Applications. https://doi.org/10.1016/B978-0-12-818495-0.00002-8 © 2020 Elsevier Ltd. All rights reserved.

14

Silk: Materials, Processes, and Applications

Fig. 2.1  Depiction of the distribution and content of the three layers of sericin (Wang and Yu, 2011).

made up of L and H chains. The H chains are the main structural components and contribute to the mechanical properties whereas the L chains are much smaller and do not contain the amino acid sequence required for crystallite formation (Koh et al., 2015). The H chain primarily consists of glycine (43–46%), alanine (25–30%), serine (12%) and tyrosine (5%) and is represented as poly(L-Ala-Gly) and is referred to as the silk I form. This form is usually found in stacked sheets having antiparallel β-sheets. Unit crystal cell in the silk I form is represented as a = 8.94 Å, b = 6.46 Å and c = 11.26 Å. Another form of silk fibroin (silk II) also exists but in antiparallel β-sheets and in a different orientation. Silk I gets converted to silk II during dissolution or after various treatments including exposure to alcohols. Typical unit cell dimensions of silk II are a = 0.944 nm, b = 0.895 nm and c = 0.700 nm. Corresponding d-spacing for the unit cell are given in Table 2.1. In silk I, the Ala side chains are arranged on both side of the β-sheet whereas in silk II, the Ala side chains at every strand are arranged on the same side of the sheet. In addition to the Ala side chains in the β-sheets, the amount and position of tyrosine affects the conformation of the backbone and also the intermolecular chain packing in both the silk forms (Asakura et al., 2004). The β-turn repeats of the alanine residues have an angle of 60 ± 5° for ɸAla and 130 ± 5° for ΨAla and 30 ± 5° for ΨGly.The silk II structure is more stable and has about 1 kcal/mol per residue higher than silk I structure. Considerably different crystal dimensions have

Table 2.1  Observed and calculated d spacings (nm) of silk II (Shen et al., 1998).

d1 d2 d3 d4’ d4 d5 d6 d7 d8 d9

Observed

Calculated

Indices

0.895 0.448 0.370 0.350 0.326 0.301 0.281 0.231 0.209 0.226

0.896 0.448 0.377 0.349 0.325 0.299 0.275/0.285 0.233 0.206/0.209 0.224

(010) (020) (021) (002) (012) or (220) (030) (022) or (130) (003) (023) or (420) (040)

Reproduced with permission from American Chemical Society.

Structure and properties of silk fibers 15

also been suggested for B. mori silk (Drummy et al., 2007). Typical crystalline peaks for most domesticated silk has been detected at 9.2°, 20.0° and 23.9°. Crystal size was measured between 0.8 and 10.8 Å depending on the direction of measurement. Due to the differences in crystallinity (50–60%), the crystalline region of silk is suggested to have a density of 1.35 g/cm3 compared to 1.3 g/cm3 for the amorphous regions. In addition to the silk I and silk II forms, a relatively unknown silk III form has also been reported. This silk III arises due to incomplete transformation from silk I to silk II or is identified as the insoluble portion of the silk proteins (Callone et al., 2016). In terms of the FTIR spectrum, silk III appeared as amide I peak with double signal at 1653 and 1623 cm−1 and a vibrational peak at 1648 cm−1. An NMR peak at 18.2 ppm and shoulder at 261 °C in the DSC curve was also suggested to be from the silk III component. Amount of the β-sheets in the silk fibers was also found to vary depending on which part of the cocoon the fibers were obtained. Inner part of the cocoon contained more crystallized silk than the outside (Chung et al., 2015).

2.1.1 Composition of silk fibers In terms of composition, the B. mori silk consists of two major components fibroin (about 75%) and sericin (typically, 25%) (Shen et al., 1998). Fibroin consists of highly periodic amino acids with glycine (45%), alanine (30%) and serine (12%) being the most repetitive units generally in a ratio of 3:2:1 (Gupta et al., 2007) (Table 2.2). The major protein component in silk is fibroin which has a molecular weight of 416 kDa. Heavy chain in fibroin has a molecular weight of 390 kDa and light chain has molecular weight of 26 kDa. The heavy chain consists of 5242 amino acids compared to only 246 for the light chain. Comparatively, the sericin is mainly composed of glycine, serine and aspartic acid with molecular weights between 10 and 300 kDa (Wang and Yu, 2011). Fig. 2.2 provides a comparison of the amino acids in B. mori and wild silks and representative sequences of the amino acids (Guo et  al., 2018a,b). Amino acid content of sericin has been reported to vary between the three layers. Changes in the amino acid profile of the three layers (Table 2.3) shows that the non-polar amino acids increase from outside to the inside layers. Amino acid content of the sericin resembles that of fibroin as the layers come closer to the fibroin layers (Wang and Yu, 2011).

2.1.2 Wild silks Although wild silks have distinct properties, they have considerable variations in their properties both in silks produced from an individual or different silkworms. For example, silk fibers produced from three separate A. pernyi silkworms showed considerable variations in their mechanical properties (Table 2.4). Wild silks (Antheraea assamensis, Samia ricini and Antheraea mylitta) were studied for their structure and properties in comparison to the most common silk worm B. mori) (Fang et al., 2016a,b). The cocoons were treated with conditions specific to their variety and fibers were collected. Mechanical testing of the fibers was done using a gauge length of 30 mm and crosshead speed of 15 mm/min. Considerable variations were observed in the tensile properties of the fibers. B. mori fibers from both the univoltine and bivoltine varieties

16

Silk: Materials, Processes, and Applications

Table 2.2  Comparison of the major amino acids in four different sources of silk fibroin (Gupta et al., 2007). Amino acid Ala Gly Ser Ile, Leu, Val Arg, His, Lys Asp, Glu Tyr

A. assama

A. pernyi

A. yamamai

B. mori

Residue

%

Residue

%

Residue

%

Residue

%

1193 811 287 35

42.5 28.9 10.2 1.26

1137 720 297 42

43.1 27.3 11.3 1.6

1122 729 292 41

42.3 17.5 11 1.5

1592 2415 635 117

30.2 45.9 12.1 2.2

110

3.9

99

3.8

102

3.8

31

0.6

154

5.3

135

5.1

150

5.7

55

1

138

4.9

139

5.3

136

5.1

277

5.3

Reproduced with permission from Creative Commons Attribution 4.0 International License.

Fig. 2.2  Comparison of the amino acid composition of silks produced by different silkworms (A) and representative amino acid sequences of the silks (B) (Guo et al., 2018a). Reproduced with permission from American Chemical Society.

Structure and properties of silk fibers 17

Table 2.3  Amino acid content and variation between the whole and three layers of sericin in comparison to fibroin (Wang and Yu, 2011). Amino acid

Sericin

Outer layer

Middle layer

Inner layer

Fibroin

Gly Ala Val Leu Ile Phe Met Tyr Pro Tyr Cys Ser Thr Asp Glu His Lys Arg

17.85 6.7 4.05 1.49 1.02 0.67 0.31 – 0.81 3.1 0.38 25.5 7.47 18.38 5.74 1.32 2.08 3.12

16.29 5.2 3.77 1.21 0.79 0.64 0 – 0 2.87 0.69 28 7.78 17.97 6.25 1.32 3.72 3.52

16.35 6.13 4.27 1.77 1.17 0.66 0 – 0.64 3.98 0.95 25.6 8.13 17.08 4.65 1.69 3.16 3.83

17.87 11.58 5.43 4.06 3.76 2.49 0.83 – 0 4.09 0.75 13.32 5.66 15.83 7.34 1.38 2.18 3.41

42.62 33.38 2.58 0.54 0.72 0.81 0.15 – 0.47 5.84 0.26 7.65 0.85 1.79 1.36 0.21 0.33 0.44

had higher strength and modulus but lower elongation than the wild silks (Fang et al., 2016a,b) (Table 2.4). Higher elongation of the wild silks was considered to be essential to adopt to the wild environment. Synchroton FTIR spectrums showed large difference mainly due to the variations in the type, amount and sequences of amino acids. Wild silks are also composed of β-sheets and α-helix or random turns. However, the proportions of the two configurations vary between species and also between studies. It has been reported that the wild silks had β-sheet content ranging from 14% to 23% and α-helix content between 10% and 19% which is opposite to that found in B. mori silks (Fang et al., 2016a,b). In another study, the β-sheet content is reported to be 50% for B. mori compared to 44.9, 45.4 and 42.6 in A. pernyi, S. ricini and A. assamensis, respectively. Corresponding α-helix or random turn content reported was 50%, 55.1%, 54.6% and 57.4%, respectively (Guo et al., 2018a,b). The β-sheet structure in B. mori comes from the sequence Gly-Ala-Gly-Ala-Gly-Ser compared to (Ala)n for the wild silks. In addition, unlike the wild silks, the β-sheets in B. mori are well oriented along the fiber axis. For example, the orientation ratio was 2.94 and orientation factor was 0.924 for B. mori compared to 2.7–2.8 and 0.623–0.873 for the wild silks, respectively. About 5–8% higher % crystallinity was also observed in the domestic silks. Similar differences in diffraction intensities were also observed depending on the species of silk. These differences were also responsible for the higher elongation of wild silks than B. mori silk. Based on artificial fiber spinning and protein regeneration studies, it was suggested that irrespective of the type of silks, the higher level of β-sheet content and better orientation (Table 2.5, Fig. 2.3) would provide higher tensile properties to

18

Table 2.4  Comparison of the properties of wild and domesticated silk fibers (Fang et al., 2016a,b). Sample

Stress, GPa

Strain, %

Breaking energy, MJ/m3

β-Sheet, %

α-Helix, %

Random coil, %

9.6 ± 0.6 8.5 ± 0.4 6.3 ± 0.3 4.5 ± 0.3

0.69 ± 0.02 0.61 ± 0.03 0.56 ± 0.02 0.49 ± 0.02

38.5 ± 6.4 43.8 ± 3.9 47.4 ± 6.1 55.9 ± 5.3

1847 ± 33 176 ± 28 156 ± 24 148 ± 16

– – 23.0 ± 2.0 19.9 ± 2.0

– – 10.1 ± 0.7 18.6 ± 1.2

– – 66.9 ± 1.1 61.5 ± 2.2

3.9 ± 0.2

0.38 ± 0.20

63.5 ± 4.4

139 ± 28

14.7 ± 0.7

13.6 ± 0.9

71.7 ± 1.2

Reproduced with permission from American Chemical Society.

Silk: Materials, Processes, and Applications

B. mori white B. mori yellow Samia ricini Antheraea assamensis, Antheraea mylitta

Modulus, GPa

˙) Unit cell size (A

Nanocrystallite size (nm)

Silk worm species

a

b

c

a

b

c

Crystallinity, %

Orientation factor (f)

B. mori A. pernyi S.c. ricini A. assamensis

9.68 ± 0.20 9.62 ± 0.19 9.63 ± 0.19 9.57 ± 0.18

9.36 ± 0.18 10.60 ± 0.19 10.54 ± 0.21 10.56 ± 0.21

7.02 ± 0.14 6.90 ± 0.13 6.90 ± 0.13 6.88 ± 0.12

3.38 ± 0.07 3.82 ± 0.08 3.74 ± 0.06 3.85 ± 0.07

3.06 ± 0.06 3.06 ± 0.06 3.27 ± 0.06 3.27 ± 0.06

13.36 ± 0.27 4.26 ± 0.08 4.92 ± 0.10 5.13 ± 0.10

45.6 ± 2.5 32.0 ± 1.6 33.2 ± 1.6 31.3 ± 1.5

0.979 ± 0.02 0.945 ± 0.02 0.955 ± 0.02 0.967 ± 0.02

Reproduced with permission from Royal Society of Chemistry.

Structure and properties of silk fibers 19

Table 2.5  Comparison of the physical parameters of different silk worms (Fang et al., 2016a,b).

20

Silk: Materials, Processes, and Applications

Fig. 2.3  Comparison of the diffraction intensities of the five types of silk fibers drawn to different ratios. Panels A to D represent fibers drawn to 1, 2, 4, 6, and 9×, respectively (Fang et al., 2016a). Reproduced with permission from Royal Society of Chemistry.

Fig. 2.4  Correlation between the β-sheet content and mechanical properties of B. mori and common wild silk worms (Guo et al., 2018a). Reproduced with permission from American Chemical Society.

the fibers (Fang et al., 2016a,b). A strong co-relation has been established between the β-sheet content and mechanical properties not just for B. mori but also for the wild silks (Fig. 2.4) (Guo et al., 2018a,b). Chemical composition and mechanical properties of the wild silks have also been found to be different between wild species when compared to B. mori silk (Fig. 2.5). Structure of A. pernyi silk was found to closely resemble that of spider dragline silk. However, the properties of A. pernyi silk were highly dependent on the extrusion conditions, particularly reeling speed. A study was conducted to determine the

Structure and properties of silk fibers 21

Fig. 2.5  Differences in the FTIR spectrum of the five types of silk fibers (Fang et al., 2016a). Reproduced with permission from Royal Society of Chemistry.

Table 2.6  Increase in the structural parameters (S/mol) in A. pernyi silks with increasing reeling (extrusion speeds) (Fang et al., 2017). Reeling rate, mm per sec

β-Sheet

α-Helix

Random coil

Naturally spun 8 15 30 50 75

0.628 0.558 0.566 0.611 0.642 0.692

0.152 0.086 0.124 0.169 0.221 0.245

0.143 0.086 0.128 0.167 0.210 0.224

Reproduced with permission from Royal Society of Chemistry.

effect of reeling rates (8 to 75 mm/s) on the structure and properties of forcibly reeled A. pernyi silk. As seen from Table  2.6, β-sheet content increased from 0.553 S/mol to 0.692 S/mol and increase in α-helix and random coil content was also observed (Fang et  al., 2017) with increase in spinning speed. Hence, substantial increase in tensile strength, elongation and modulus (Fig. 2.6), similar to that of spider silk was acheivable by controlling the reeling speeds. Variations in the structure due to changes in extrusion speeds was suggested to be useful in generating silk fibers with desired properties (Fang et  al., 2017). However it would be practially difficult to tailor the silkworms to extrude at desired speeds. Another common wild silk produced by the insect called Antheraea assama is known for the golden colored fibers (Gupta et al., 2015) (Fig. 2.7). Structural analysis has shown that the fibers contain a highly repetitious crystalline core followed by a non-repetitious end. At the molecular level, a protein having a size of 230 kDa forms the basic structural unit and is composed of polyalanine repeats. These repeats consist of tight β-sheet crystals alternating with non-polyalanine repeats that consist of less orderly antiparallel β-sheets, β-turns and partial α-helices (Gupta et al., 2015).

22

Silk: Materials, Processes, and Applications

Stress (Mpa)

600 500 400 300 200

800 Young’s Modulus Breaking Stress 600

20 400 10 200

100 0

(A)

0

5

10

15

20

25

30

35

40

(B) 0

8

15

30

50

75

0

Reeling rate (mm/s)

Strain (%)

Breaking energy (MJ/m3)

Breaking strain (%)

40

30

20

10

0

(C)

8

15

30

50

Reeling rate (mm/s)

75

120

80

40

0

(D)

8

15

30

50

75

Reeling rate (mm/s)

Fig. 2.6  Changes in the tensile properties of forcibly reeled A. pernyi silk with increasing reeling rates (Fang et al., 2017). Reproduced with permission from Royal Society of Chemistry.

Fig. 2.7  Pictures of V instar A. assama silk worm (A), cocoon (B), silk gland measuring about 30 cm showing the anterior silk gland, middle silk gland and posterior silk gland (C), flourescent images of the silk glands (D) (Gupta et al., 2015).

Breaking stress (MPa)

700

30

Young’s modulus (GPa)

75 mm/s 50 mm/s 30 mm/s 15 mm/s 8 mm/s

800

Structure and properties of silk fibers 23

Higher content of tight β-crystals and denser packing and lower serine content are suggested to be responsible for the higher tensile strength and golden luster of the fibers. Variations in the properties of silk fibers were also found when the mechanical properties were tested in water, ethanol or methanol (Wang et al., 2014). Such variations in properties were suggested to be due to the different disordered structures and hydrogen bond stackings in the amorphous regions of the silk fibers. Some studies have suggested that A. pernyi silk can have tensile properties similar to that to spider silk under optimum extrusion conditions. This is because, the crystal structure of A. pernyi consists mostly of β-sheets made up of polyalanine, similar to that in the major ampullate gland of spider silks. To better understand the structure of A. pernyi silk and avoid the variations during natural fiber formation, fibers were forcibly reeled from three different silkworms and their structure was investigated (Fu et al., 2011). Properties of the silk fibers produced by the three differetn silkworms was considerably different (Wang et al., 2014) (Table 2.7). The crystal structure of A. pernyi silk was found to be predominantly composed of β-sheets based on X-ray crystal structure data and Raman spectroscopy peaks at 1667 cm−1 and 1093 cm−1. Forcibly as-reeled fibers from A. pernyi silk worms had breaking strain of 0.34 to 0.63, much higher than that of both forcibly reeled spider major ampullate silk and B. mori silk. Changes in crystal dimensions and orientation also occur when B. mori silk is extended during production or external stretching. β-Sheet crystals in the silk were deformed and more tightly packed after stretching which lead to lower d-spacings. Although, the % crystallinity of the fibers was found to decrease (42–36%) with increase in stretching ratio (Numata et  al., 2015a,b), the orientation of the β-crystals showed substantial improvement and improved alignment towards the fiber axis. Large changes in the orientation and content of silk was also observed using synchotron FTIR microscopy when A. pernyi silk was subject to stretching (Ling et al., 2013). A combination of highly oriented β-sheets but slightly oriented α-helix and random coils were discovered. However, upon stretching, the orientation of the β-sheets did not show much change but the amount and orientation of random coils increased substantially (Table  2.8). Comparitively, the order of α-helix increased but β-sheet content decreased (Ling et al., 2013). The presence of higher amounts β-sheets with amorphous content enables better reorganization during stretching and provides considerably increased mechanical properties. In addition, unlike B. mori silks, the size of nanocrystallites in the A. pernyi silk β-sheets decreased considerably with increase in strain rates (Fig. 2.8) (Guo et al., 2017). A proposed mechanism for the change in Table 2.7  Variations in the properties of A. pernyi silk fibers produced by three different silkworms under similar conditions (forced reeling) (Wang et al., 2014).

Sample

Initial modulus, GPa

Post-Yield modulus, GPa

Yield stress, MPa

Breaking strain, %

Breaking stress, MPa

Breaking energy, MJ/m3

1 2 3

8.6 ± 0.3 9.9 ± 0.3 11.8 ± 0.4

0.51 ± 0.05 0.75 ± 0.07 1.52 ± 0.09

241 ± 3 268 ± 11 318 ± 27

73 ± 7 40 ± 4 23 ± 2

525 ± 27 549 ± 33 628 ± 29

259 ± 23 154 ± 20 106 ± 9

Reproduced with permission from Royal Society of Chemistry (Great Britain).

24

Silk: Materials, Processes, and Applications

Table 2.8  Changes in the molecular order parameters of different configurations of single A. pernyi silk fibers after subjecting to different strains (Ling et al., 2013). Strain rates Conformations

0

0.1

0.2

0.3

β-Sheet Random coil α-Helix

0.63 ± 0.07 0.07 ± 0.046 0.25 ± 0.040

0.65 ± 0.09 0.10 ± 0.046 0.20 ± 0.05

0.66 ± 0.09 0.15 ± 0.07 0.26 ± 0.10

0.65 ± 0.08 0.18 ± 0.06 0.28 ± 0.09

Conformations

Content, % 0

0.1

0.2

0.3

38 ± 0.07 44 ± 1 18 ± 3

38 ± 2 46 ± 2 17 ± 3

38 ± 4 46 ± 2 15 ± 4

37 ± 3 48 ± 3 15 ± 3

β-Sheet Random coil α-Helix

Reproduced with permission from American Chemical Society.

Fig. 2.8  Decrease in the size of nanocrystallites along the interchain direction (a) and intersheet direction (b) and fiber axis (c) with increase in strain percentage (Guo et al., 2017). Reproduced with permission from John Wiley and Sons.

oreintation of the nanocrystallites with increasing strain rates is given in Fig. 2.9 (Guo et al., 2017). In another study, it has been shown that properties of A. pernyi silk are directly related to the rate of reeling. Both Young’s modulus and breaking stress increased but strain decreased as the rate of reeling increased. Hence, the properties of A. pernyi silk could be tailored to match that of B. mori or spider silk just by varying the extrusion speeds. Silk produced by an insect Antheraea yamamai cultivated in Japan since ancient times was discovered to have structure and properties between that of B. mori and the wild silks. Unlike B. mori silk which contains two protein chains (heavy chain and light chain), A. yamamai silk contains only one chain, the heavy chain which is composed of 42.9% alanine, 27.2% glycine, 11% serine, 5% tyrosine and 4.9% asparagine. Although the silk had only one chain, it was composed of two motifs (Arg-Gly-Asp) also called the RGD peptides which are found in A. pernyi silk and are

Structure and properties of silk fibers 25

Fig. 2.9  Schematic representation of the changes in nanocrystalline structure of A. pernyi silk fibers with increasing strain rates (Guo et al., 2017). Reproduced with permission from John Wiley and Sons.

suggested to promote cell attachment and growth (Numata et al., 2015a). The other motif consists of long poly(alanine) sequences and were found not to contribute to the strength, thermal or structural properties of the silk. Morphologically, the silk fibers had average diameter of 26.2 μm made up to nanofibers of 100 nm in diameter (Giesa et al., 2011) (Fig. 2.22). Contradicting the general acceptance that spider silk is an exceptional material, Porter et al. (2013) demonstrate that fibers with similar strength and toughness could be generated using other common polymers as long as appropriate modulus to diameter ratios are achieved. A high hydrogen bond energy density was also necessary for achieving good toughness (Porter et al., 2013). It was suggested that the nanostructure alone may not be responsible for the fracture mechanics but will also depend on the micron scale dimensions. Also, the properties of spider silk varied between the two glands. Interestingly, it was found that the properties of fibers from the minor ampullate gland are similar between various species of spiders (Guinea et al., 2012). Fibers produced by the minor ampullate gland did not show supercontraction and properties in water were similar to that of B. mori silk. Fibers produced by minor ampullate gland had diameters of 1.8 ± 0.1 μm compared to 5.9 ± 0.2 μm to 3.0 ± 0.3 μm depending on the species of spiders tested. Substantial differences in fiber properties were observed when the testing was done in air or water (Table 2.16). Although spider silk has exceptional mechanical properties, several researchers have shown addition of materials or modifications during fiber production can lead to change properties. In one such attempt,

40

Silk: Materials, Processes, and Applications

Table 2.15  Changes in the orientation and increase in strength of spider silk as the spinning speed varies (Du et al., 2006). Crystallite size (nm)

Reeling speed, mm/s

a

b

c

Stress, MPa

1 2.5 10 25 100

2.4 2.3 2.1 2.2 2.1

3.5 3.4 2.7 2.7 2.7

7.3 7.1 6.5 6.4 6.4

300 500 900 1000 1500

Reproduced from permission from Elsevier.

Fig. 2.22  Hierarchial arrangment of the nanocrystals to fibrils that provide exceptional mechanical properties to the spider silk fibers (Giesa et al., 2011). Reproduced with permission from American Chemical Society.

it was shown that intentional insertion of metals such as zinc, titanium, aluminium into the inner protein structure could improve the toughness of silk fibers (Lee et al., 2009). The infusion was done using atomic layer deposition in the presence of water. It was hypothesized that metal ions can preferably bind to proteins and form metalprotein complexes. Hydrogen bonds that were broken due to presence of water vapor would reform into metal-coordinated or covalent bonds that lead to higher strength. Several other reports have also compared the properties of spider silk from diffferent species and with other silks produced by various insects (Table 2.17). It is evident

Spider Forcibly spun Nephila inaurata Argiope trifasciata Max Contracted Nephila inaurata Argiope trifasciata

Nephila inaurata Argiope trifasciata

Testing condition/ gland In air Major Minor Major Minor In air Major Minor Major Minor In water Major Minor Major Minor

Strength, GPa

Elongation, %

Work of rupture, J/m3

14.2 ± 0.6 11.2 ± 0.7 10.7 ± 0.3 10.0 ± 0.4

1.80 ± 0.06 1.5 ± 0.2 1.3 ± 0.2 1.04 ± 0.06

0.26 ± 0.01 0.46 ± 0.05 0.17 ± 0.02 0.45 ± 0.02

264 ± 5 300 ± 50 90 ± 30 240 ± 20

4.0 ± 0.5 12.0 ± 0.8 3.5 ± 0.1 8.2 ± 0.9

1.46 ± 0.09 1.22 ± 0.07 0.91 ± 0.06 0.7 ± 0.1

0.70 ± 0.02 0.46 ± 0.02 0.80 ± 0.02 0.35 ± 0.07

270 ± 20 245 ± 25 236 ± 7 150 ± 40

1.7 ± 0.1 1.0 ± 0.1 1.42 ± 0.05 1.22 ± 0.02

0.67 ± 0.01 0.53 ± 0.09 0.95 ± 0.03 0.51 ± 0.01

280 ± 10 210 ± 60 185 ± 8 245 ± 25

Modulus, GPa

0.041 ± 0.004 0.39 ± 0.03 0.022 ± 0.001 1.42 ± 0.09

Reproduced with permission from American Chemical Society.

Structure and properties of silk fibers 41

Table 2.16  Properties of spider silk fibers obtained from various species and tested in air or water (Guinea et al., 2012).

42

Table 2.17  Comparison of the approaches used and properties of protein fibers obtained from different species of spiders (Piorkowski et al., 2018) True values

Species

Treatment

Young’s modulus (GPa)

Arachnocampa

>90% RH

0.1

tasmaniensis Arachnocampa

Stress at break (MPa)

ln(mm mm1)

Toughness (MJ m3)

0.02

122.77

0.6

0.04

25.82

0.01

2.4

2.3

159.04

18.38 30% RH

tasmaniensis Mytilus edulis

Strain at break

16.1 12.1

0.02

0.85 In sea water

0.87

Drya

302.4

0.36

9.09

2347.4

0.19

19.8

44.18 0.26

183.73

0.24

–

0.61

–

49.40 Aulacomya ater

In sea water

–

110.20 50.02

Aulacomya ater

In distilled

–

127.78

0.58

–

water Chrysopa carnea

30% RH

5.8

70

0.03

1

Chrysopa carnea

70% RH

3.2

155

2.00

97

Chrysopa carnea

100% RH

1.3

232

4.34

110

Stenopsyche

65% RH

–

–

0.02

–

Silk: Materials, Processes, and Applications

Drya

10.1 31.5

1.75 Aulacomya ater

0.7 12.5

18.69 Mytilus edulis

2.8

Stenopsyche

Water

marmorata

Saturateda

–

–

0.41

–

Antheraea pernyi Antheraea pernyi

10% RH

11.8

–

–

–

70% RH

8.5

–

–

–

Antheraea pernyi

98% RH

0.8

–

–

–

Bombyx mori

Drya

14

570

0.20

–

Bombyx mori

In water

3.8

Argiope aurantia

Weta

0.009

450 0.011

1034

0.008 Argiope trifasciata

a

Wet

0.005

Weta

949

0.0001

534 800

51 Argiope trifasciata

Cleaned

Argiope trifasciata

35% RH

0.3

Argiope trifasciata a

In water

1300

Statistical significance. Reproduced with permission from John Wiley and Sons.

1420

75

65 6 – 0.08

250

0.02

90

0.03

185

50

0.17 50

0.001

0.05

0.76 200

0.022

185

0.5

800 10.7

0.16

99

0.2

100

a

211

1.72 200

Cleaneda

– 0.24

1.44 40

64 Argiope trifasciata

1.57

292

0.001 Argiope argentata

0.27 344

0.95

30 8

Structure and properties of silk fibers 43

marmorata

44

Silk: Materials, Processes, and Applications

that in addition to spiders, many insects such as mussels and lacewing produce silk with distinct properties (Piorkowski et  al., 2018). However, it must be recognized that the properties of the fibers are dependent on the testing conditions, particularly humidity and strain rates. For example, silk threads produced by A. tasmaniensis had higher extensibility and were tougher at higher humidity (90% RH) compared to lower humidity (30% RH). It was suggested that water could induce plasticization and improve properties (Piorkowski et al., 2018).

References Asakura, T., Suita, K., Kameda, T., Afonin, S., Ulrich, A.S., 2004. Structural role of tyrosine in Bombyx mori silk fibroin, studied by solid‐state NMR and molecular mechanics on a model peptide prepared as silk I and II. Magn. Reson. Chem. 42 (2), 258–266. Blamires, S.J., Blackledge, T.A., Tso, I.-M., 2017. Physicochemical property variation in spider silk: Ecology, evolution, and synthetic production. Annu. Rev. Entomol. 62, 443–460. Callone, E., Dirè, S., Hu, X., Motta, A., 2016. Processing influence on molecular assembling and structural conformations in silk fibroin: Elucidation by solid-state NMR. ACS Biomaterials Science & Engineering 2 (5), 758–767. Cao, K., Liu, Y., Ramakrishna, S., 2017. Recent developments in regenerated silk fiber. J. Nanosci. Nanotechnol. 17 (12), 8667–8682. Challis, R.J., Goodacre, S.L., Hewitt, G.M., 2006. Evolution of spider silks: conservation and diversification of the C‐terminus. Insect Mol. Biol. 15 (1), 45–56. Cheng, L., Huang, H., Zeng, J., Liu, Z., Tong, X., Li, Z., Zhao, H., Dai, F., 2019. Effect of different additives in diets on secondary structure, thermal and mechanical properties of silkworm silk. Materials 12 (1), 14. Chung, E.J., Hyung, W.J., Park, H.J., Chan, H.P., 2015. Three‐layered scaffolds for artificial esophagus using poly (ɛ‐caprolactone) nanofibers and silk fibroin: an experimental study in a rat model. J. Biomed. Mater. Res. Part A 103 (6), 2057–2065. Drummy, L.F., Farmer, B.L., Naik, R.R., 2007. Correlation of the β-sheet crystal size in silk fibers with the protein amino acid sequence. Soft Matter 3 (7), 877–882. Du, N., Liu, X.Y., Narayanan, J., Li, L., Lim, M.L.M., Li, D., 2006. Design of superior spider silk: From nanostructure to mechanical properties. Biophys. J. 91 (12), 4528–4535. Eisoldt, L., Smith, A., Scheibel, T., 2011. Decoding the secrets of spider silk. Mater. Today 14 (3), 80–86. Fang, G., Sapru, S., Behera, S., Yao, J., Shao, Z., Kundu, S.C., Chen, X., 2016a. Exploration of the tight structural–mechanical relationship in mulberry and non-mulberry silkworm silks. J. Mater. Chem. B 4 (24), 4337–4347. Fang, G., Huang, Y., Tang, Y., Qi, Z., Yao, J., Shao, Z., Chen, X., 2016b. Insights into silk formation process: Correlation of mechanical properties and structural evolution during artificial spinning of silk fibers. ACS Biomaterials Science & Engineering 2 (11), 1992–2000. Fang, G., Tang, Y., Qi, Z., Yao, J., Shao, Z., Chen, X., 2017. Precise correlation of macroscopic mechanical properties and microscopic structures of animal silks—using Antheraea pernyi silkworm silk as an example. J. Mater. Chem. B 5 (30), 6042–6048. Fernandes, J., Nicodemo, D., Oliveira, J.E., Silva, F.A., Fidelis, M.E.A., Silva, L.E., Tonoli, G.H.D., 2017. Enhanced silk performance by enriching the silkworm diet with Bordeaux mixture. J. Mater. Sci. 52 (5), 2684–2693.

Structure and properties of silk fibers 45

Fu, C., Porter, D., Chen, X., Vollrath, F., Shao, Z., 2011. Understanding the mechanical properties of Antheraea pernyi silk—From primary structure to condensed structure of the protein. Adv. Funct. Mater. 21 (4), 729–737. Giesa, T., Arslan, M., Pugno, N.M., Buehler, M.J., 2011. Nanoconfinement of spider silk fibrils begets superior strength, extensibility, and toughness. Nano Lett. 11 (11), 5038–5046. Gosline, J.M., DeMont, M.E., Denny, M.W., 1986. The structure and properties of spider silk. Endeavour 10 (1), 37–43. Guinea, G.V., Elices, M., Plaza, G.R., Perea, G.B., Daza, R., Riekel, C., Agulló-Rueda, F., Hayashi, C., Zhao, Y., Pérez-Rigueiro, J., 2012. Minor ampullate silks from Nephila and Argiope spiders: Tensile properties and microstructural characterization. Biomacromolecules 13 (7), 2087–2098. Guo, C., Zhang, J., Wang, X., Nguyen, A.T., Liu, X.Y., Kaplan, D.L., 2017. Comparative study of strain‐dependent structural changes of silkworm silks: Insight into the structural origin of strain‐stiffening. Small 13 (47), 1702266. Guo, C., Zhang, J., Jordan, J.S., Wang, X., Henning, R.W., Yarger, J.L., 2018a. Structural comparison of various silkworm silks: An insight into the structure–property relationship. Biomacromolecules 19 (3), 906–917. Guo, Z., Xie, W., Gao, Q., Wang, D., Gao, F., Li, S., Zhao, L., 2018b. In situ biomineralization by silkworm feeding with ion precursors for the improved mechanical properties of silk fiber. Int. J. Biol. Macromol. 109, 21–26. Gupta, M.K., Shama, K.K., Phillips, D.M., Sowards, L.A., Drummy, L.F., Kadakia, M.P., Naik, R.R., 2007. Patterned silk films cast from ionic liquid solubilized fibroin as scaffolds for cell growth. Langmuir 23 (3), 1315–1319. Gupta, A., Mita, K., Arunkumar, K.P., Nagaraju, J., 2015. Molecular architecture of silk fibroin of Indian golden silkmoth, Antheraea assama. Sci. Rep. 5, 12706. Johnston, E.R., Miyagi, Y., Chuah, J.-A., Numata, K., Serban, M.A., 2018. Interplay between silk Fibroin’s structure and its adhesive properties. ACS Biomaterials Science & Engineering 4 (8), 2815–2824. Keten, S., Xu, Z., Ihle, B., Buehler, M.J., 2010. Nanoconfinement controls stiffness, strength and mechanical toughness of β-sheet crystals in silk. Nat. Mater. 9 (4), 359. Koh, L., Cheng, Y., Teng, C., Kin, Y., Loh, X., et al., 2015. Structures, mechanical properties and applications of silk fibroin materials. Prog. Polym. Sci. 46, 86–110. Koski, K.J., Akhenblit, P., McKiernan, K., Yarger, J.L., 2013. Non-invasive determination of the complete elastic moduli of spider silks. Nat. Mater. 12 (3), 262. Lee, S.-M., Pippel, E., Gösele, U., Dresbach, C., Qin, Y., Vinod Chandran, C., Bräuniger, T., Hause, G., Knez, M., 2009. Greatly increased toughness of infiltrated spider silk. Science 324 (5926), 488–492. Ling, S., Qi, Z., Knight, D.P., Huang, Y., Huang, L., Zhou, H., Shao, Z., Chen, X., 2013. Insight into the structure of single Antheraea pernyi silkworm fibers using synchrotron FTIR microspectroscopy. Biomacromolecules 14 (6), 1885–1892. Liu, Y., Shao, Z., Vollrath, F., 2005. Relationships between supercontraction and mechanical properties of spider silk. Nat. Mater. 4 (12), 901. Long, D., Lu, C., Wang, Y., Yan, S., Zhang, Q., Wang, X., 2018. Structure and properties of camphor silk. The Journal of The Textile Institute 109 (9), 1186–1192. Ma, L., Akurugu, M.A., Andoh, V., Liu, H., Song, J., Wu, G., Li, L., 2019. Intrinsically reinforced silks obtained by incorporation of graphene quantum dots into silkworms. Science China Materials 62 (2), 245–255.

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Silk: Materials, Processes, and Applications

Martel, A., Burghammer, M., Davies, R.J., Riekel, C., 2007. Thermal behavior of Bombyx mori silk: Evolution of crystalline parameters, molecular structure, and mechanical properties. Biomacromolecules 8 (11), 3548–3556. Nova, A., Keten, S., Pugno, N.M., Redaelli, A., Buehler, M.J., 2010. Molecular and nanostructural mechanisms of deformation, strength and toughness of spider silk fibrils. Nano Lett. 10 (7), 2626–2634. Numata, K., Sato, R., Yazawa, K., Hikima, T., Masunaga, H., 2015a. Crystal structure and physical properties of Antheraea yamamai silk fibers: Long poly (alanine) sequences are partially in the crystalline region. Polymer 77, 87–94. Numata, K., Masunaga, H., Hikima, T., Sasaki, S., Sekiyama, K., Takata, M., 2015b. Use of extension-deformation-based crystallisation of silk fibres to differentiate their functions in nature. Soft Matter 11 (31), 6335–6342. Oktaviani, N.A., Matsugami, A., Malay, A.D., Hayashi, F., Kaplan, D.L., Numata, K., 2018. Conformation and dynamics of soluble repetitive domain elucidates the initial β-sheet formation of spider silk. Nat. Commun. 9, 2121–2132. Piorkowski, D., Blackledge, T.A., Liao, C.‐.P., Doran, N.E., Wu, C.‐.L., Blamires, S.J., Tso, I.‐.M., 2018. Humidity‐dependent mechanical and adhesive properties of Arachnocampa tasmaniensis capture threads. J. Zool. 305 (4), 256–266. Porter, D., Guan, J., Vollrath, F., 2013. Spider silk: Super material or thin fibre? Adv. Mater. 25 (9), 1275–1279. Randrianandrasana, M., Wu, W.-Y., Carney, D.A., Wagoner Johnson, A.J., Berenbaum, M.R., 2017. Structural and mechanical properties of cocoons of Antherina suraka (Saturniidae, Lepidoptera), an endemic species used for silk production in Madagascar. J. Insect Sci. 17 (1), 17. Reddy, N., Yang, Y., 2010. Morphology and tensile properties of silk fibers produced by uncommon Saturniidae. Int. J. Biol. Macromol. 46 (4), 419–424. Rising, A., Johansson, J., 2015. Toward spinning artificial spider silk. Nat. Chem. Biol. 11 (5), 309. Römer, L., Scheibel, T., 2008. The elaborate structure of spider silk: structure and function of a natural high performance fiber. Prion 2 (4), 154–161. Seydel, T., Kölln, K., Krasnov, I., Diddens, I., Hauptmann, N., Helms, G., Ogurreck, M., Kang, S.-G., Koza, M.M., Müller, M., 2007. Silkworm silk under tensile strain investigated by synchrotron X-ray diffraction and neutron spectroscopy. Macromolecules 40 (4), 1035–1042. Shen, Y., Johnson, M.A., Martin, D.C., 1998. Microstructural characterization of Bombyx mori silk fibers. Macromolecules 31 (25), 8857–8864. Sinsawat, A., Putthanarat, S., Magoshi, Y., Pachter, R., Eby, R.K., 2002. X-ray diffraction and computational studies of the modulus of silk (Bombyx mori). Polymer 43 (4), 1323–1330. Talukdar, B., Saikia, M., Handique, P.J., Devi, D., 2011. Effect of organic solvents on tensile strength of muga silk produced by Antheraea assamensis. Int. J. Pure. Appl. Sci. Technol. 7, 81–86. Wang, Y.J., Yu, Q.Z., 2011. Three-layered sericins around the silk fibroin fiber from Bombyx mori cocoon and their amino acid composition. Advanced Materials Research 175, 158– 163. Trans Tech Publications. Wang, Y., Porter, D., Shao, Z., 2013. Using solvents with different molecular sizes to investigate the structure of Antheraea pernyi silk. Biomacromolecules 14 (11), 3936–3942. Wang, Y., Guan, J., Hawkins, N., Porter, D., Shao, Z., 2014. Understanding the variability of properties in Antheraea pernyi silk fibres. Soft Matter 10 (33), 6321–6331.

Structure and properties of silk fibers 47

Wang, X., Zhao, P., Li, Y., Yi, Q., Ma, S., Xie, K., Chen, H., Xia, Q., 2015. Modifying the mechanical properties of silk fiber by genetically disrupting the ionic environment for silk formation. Biomacromolecules 16 (10), 3119–3125. Wang, X., Li, Y., Liu, Q., Chen, Q., Xia, Q., Zhao, P., 2017. In vivo effects of metal ions on conformation and mechanical performance of silkworm silks. Biochimica et Biophysica Acta (BBA)-General Subjects 1861 (3), 567–576. Xu, M., Lewis, R.V., 1990. Structure of a protein superfiber: Spider dragline silk. Proc. Natl. Acad. Sci. 87 (18), 7120–7124. Yazawa, K., Ishida, K., Masunaga, H., Hikima, T., Numata, K., 2016. Influence of water content on the β-sheet formation, thermal stability, water removal, and mechanical properties of silk materials. Biomacromolecules 17 (3), 1057–1066. Yerra, A., Mysarla, D.K., Siripurapu, P., Jha, A., Valluri, S.V., Mamillapalli, A., 2017. Effect of polyamines on mechanical and structural properties of Bombyx mori silk. Biopolymers 107 (1), 20–27. Zhang, H., Magoshi, J., Becker, M., Chen, J.Y., Matsunaga, R., 2002. Thermal properties of Bombyx mori silk fibers. J. Appl. Polym. Sci. 86 (8), 1817–1820.

Further reading Kim, H.H., Kim, M.K., Lee, K.H., Park, Y.H., Um, I.C., 2015. Effects of different Bombyx mori silkworm varieties on the structural characteristics and properties of silk. Int. J. Biol. Macromol. 79, 943–951. Yoshioka, T., Tashiro, K., Ohta, N., 2016. Molecular orientation enhancement of silk by the hot-stretching-induced transition from α-helix-HFIP complex to β-sheet. Biomacromolecules 17 (4), 1437–1448. Zhang, J., Kaur, J., Rajkhowa, R., Li, J.L., Liu, X.Y., Wang, X.G., 2013. Mechanical properties and structure of silkworm cocoons: A comparative study of Bombyx mori, Antheraea assamensis, Antheraea pernyi and Antheraea mylitta silkworm cocoons. Mater. Sci. Eng. C 33 (6), 3206–3213.

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New developments in degumming silk

3

3.1 Conventional degumming using alkali One of the first processes in the manufacture of silk based materials is to degum the silk to remove the glue protein sericin and obtain fibroin fibers. Raw silk may contain up to 25–30% sericin which needs to be removed to obtain the characteristic luster, feel and processability for the fibroin fibers. Typically, silk was degummed by boiling with mild alkali or soap solutions. This step was considered essential and has been adopted for centuries with the sole intention of removing the gum sericin. Until recently, sericin was considered a byproduct and discarded in the degumming liquid. However, sericin has been found to have unique properties and useful for medical, cosmetic and other applications. In addition, discarding about 25–30% of proteins produced by the silk worms is deemed to be a loss to the silk rearers. Also, researchers have developed various new techniques where degumming is considerably less cumbersome, is environmentally friendly and also preserves the properties of sericin. Numerous new approaches have been developed to degum silk using chemical, physical and biological routes as detailed below. Degumming using sodium carbonate is by far the most widely used practice. Although economical and efficient, degumming with sodium carbonate affects fiber (fibroin properties) unless proper conditions are chosen. A study on the influence of various degumming conditions using sodium carbonate showed that there was a linear relationship between alkali concentration and weight loss and thermal stability. No major effect was observed on the β-sheet content, molecular weight or rheological behavior of silk with increasing alkali concentrations in this study (Dou and Zuo, 2015). However, contradictory results have been published on the effect of sodium carbonate degumming on the properties of the fibers. A comparison was made between the degumming efficiency of sodium carbonate and urea for B. mori silk. Raw silk fibers were treated with sodium carbonate at 100 °C for 30 min using a liquor to silk ratio of 50:1 and the procedure was repeated twice. Urea degumming was done using a concentration of 8 mol/L urea for 3 h at 90 °C. No damage had occurred to the silk fibroin due to the urea treatment. In fact, urea degummed silk (fibroin) had better viscosity, higher thermal stability and larger particle size compared to sodium carbonate degummed silk. Hence, urea degumming was suggested to be preferable for developing membranes, nanoparticles and other regenerated silk products (Wang et al., 2018). In a similar study, it was found that degumming using sodium carbonate and subsequent dialysis using urea affected cell viability, ability of fibroin to form 3D scaffolds but did not affect the β-sheet content (Wray et al., 2011). For sericin removal with alkali, silk worm Silk: Materials, Processes, and Applications. https://doi.org/10.1016/B978-0-12-818495-0.00003-X © 2020 Elsevier Ltd. All rights reserved.

60 mb

30 mb

5 mb

Ladder

60 mb

30 mb

5 mb

Silk: Materials, Processes, and Applications Ladder

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460 kDa 268 238

171 117

71

55 41

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Silk solutions after urea dialysis

Fig. 3.1  Changes in the molecular weight of silk fibroin before and after treating with urea and after boiling for 5, 30 or 60 min (Wray et al., 2011). Reproduced with permission from John Wiley and Sons.

c­ ocoons were immersed in 1 L of boiling 0.02 M sodium carbonate solution for 5, 30 or 60 min. Further, the degummed fibers were treated with 20 mL of 8 M urea solution at 80 °C for 5 min. Considerable variations in the molecular weights (Fig. 3.1) of the fibroin proteins were observed before and after treating with urea (Wray et al., 2011). Differences were also observed in the amino acid composition of the silk fibers before and after degumming (Table 3.1). Another alkali, calcium hydroxide was studied for its potential to be used as a degumming agent. Male and female Chinese B. mori cocoons were degummed by boiling in 0.01–0.1% calcium hydroxide solutions at a ratio of 1:60 for up to 60 min. After degumming, the fibers were washed, dried and the procedure was repeated thrice (Zhao et al., 2018). Similar to degumming with other alkalis, the extent of degumming was highly dependent on conditions including temperature, time and bath ratio. Compared to degumming with neutral soap or sodium carbonate, fibers obtained through calcium hydroxide degumming had higher breaking force and elongation. Molecular weight studies showed that there was considerably less damage to the proteins when calcium hydroxide was used instead of sodium carbonate (Fig.  3.2). In addition, it was reported that dissolved sericin and the alkali could be easily recovered and reused. Using olive oil as a wetting agent, multivoltine B. mori silk cocoons were degummed using three different concentrations (0.5, 1 and 2 g/L) of sodium carbonate or bicarbonate at 90 or 98 °C for 30 min. SEM images clearly showed the presence of distinct layers of fibroin (F) and sericin (S) and progressive removal of sericin with increasing degumming conditions. Most of the sericin was removed when degumming

% Sericin to total protein

Degummed silk solutions, boiling time

Amino acid type

100

50

30

10

1

0.1

0%

Nondegummed

5

30

60

B T S Z G A V I L Y F K H R P S:G

17.7 6.9 24.2 7.5 19 6.4 2.8 1.1 2.0 3.0 0.7 2.9 1.0 3.5 1.4 1.27

6.7 2.8 14.2 3 36.8 23 2.5 0.8 0.9 4.5 0.7 1.2 0.5 1.4 0.7 0.39

3.4 1.4 9.8 1 43.2 28.4 2.4 0.8 0.7 5 0.7 0.5 0.3 0.7 0.5 0.23

2.2 1.1 9.1 1.5 45 30 2.3 0.7 0.6 5.3 0.7 0.3 0.2 0.5 0.5 0.19

1.8 0.9 8.7 1.4 45.7 30.5 2.3 0.7 0.6 5.2 0.7 0.3 0.2 0.5 0.4 0.19

1.8 0.9 8.7 1.4 45.5 30.6 2.3 0.7 0.6 5.2 0.7 0.3 0.2 0.5 0.4 0.19

1.7 0.9 8.8 1.4 45.7 30.6 2.3 0.7 0.6 5.3 0.7 0.3 0.2 0.5 0.4 0.19

4.3 2.3 13.6 2.1 39.0 26.0 2.6 0.1 0.8 0.8 5.1 0.7 0.7 0.4 1.0 0.35

1.8 0.9 8.9 1.4 45.4 30.4 2.3 0.7 0.6 5.3 0.7 0.3 0.2 0.5 0.4 0.19

1.8 0.9 9.1 1.4 45.0 30.6 2.3 0.7 0.6 5.2 0.7 0.3 0.2 0.5 0.5 0.19

1.6 0.8 8.9 1.3 46.3 30.8 2.2 0.6 0.5 5.2 0.6 0.2 0.2 0.3 0.4 0.19

Reproduced with permission from John Wiley and Sons.

New developments in degumming silk51

Table 3.1  Amino acid composition (mol %) of silk fibroin solutions obtained using different degumming conditions (Wray et al., 2011).

52

Silk: Materials, Processes, and Applications

Ladder

Std

NS

CH Na2CO3

200 kDa 150 kDa 100 kDa 60 kDa 50 kDa 40 kDa 30 kDa 25 kDa 20 kDa 15 kDa Fig. 3.2  Comparison of the molecular weights of peptides obtained after degumming silk with three different methods (Zhao et al., 2018). NS is neutral soap and CH is calcium hydroxide. Reproduced with permission from Elsevier.

was 25.7%. Further degumming up to 27.9% removed all the sericin but the fibroin surface was found to be damaged as seen from the extensive fibrillation (Allardyce et al., 2016). Considerable reduction in molecular weight of the fibroin peptides and tensile strength of the fibers was observed when strong degumming conditions were used. A concentration of 2 g/L of sodium carbonate at 98 °C for 30 min was found to provide the most optimum degumming and fibers obtained were considered suitable for medical applications (Allardyce et al., 2016). Instead of using any chemicals, Strongly Alkaline Electrolyzed Water (SAEW) was found to be effective to degum silk fibers. Electrolysis was done to keep water ionized at pH 11.5 and used for degumming with liquid to cocoon ratio of 1:40 for 20 min. This procedure was repeated thrice and the fibers were later washed with distilled water and dried (Cao et  al., 2013). As with the chemical methods, degumming using SAEW was influenced by pH, degumming time, ratio of water to fiber and also on the variety of cocoons used for degumming. Extent of degumming using SAEW was found to be slightly higher or similar whereas the tensile properties and thermal stability was slightly lower or similar compared to neutral soap. No degradation of fibroin was expected due to the treatment with SAEW. However, substantial saving in cost and considerable environmental benefits were possible by replacing conventional alkali degumming with SAEW (Cao et al., 2013). Wild silks are comparatively difficult to degum due to the presence of minerals and impurities. Three types of degumming (normal, intensive and control) were done

New developments in degumming silk53

to determine the effect of degumming conditions on properties of Eri (P. C. ricini) silk fibers (Rajkhowa et al., 2011). Normal degumming was done using 2 g/L sodium ­carbonate, 0.6 g/L SDS at 100 °C for 120 min. In the case of intensive degumming, alkali concentration was 10 g/L at 120 °C compared to 1 g/L alkali, 0.6 g/L SDS at 100 °C for 30 min repeated three times for the control degumming. Substantial difference in tenacity of the fibers was detected between the three degummings with values of 4.5, 3.6 and 0.8 cN/dtex for the control, normal and intensive degumming, respectively. Degradation of the fibers due to excessive degumming also affected the molecular weight but resulted in increase in crystallinity. Ability of the silk to be milled into powder for different applications was also affected by the extent of degumming (Rajkhowa et al., 2011).

3.2 Degumming with the aid of surfactants/detergents In addition to alkali, a detergent SDS was used for degumming and the effect of temperature (60–100 °C), time (5–90 min) with and without mechanical agitation on the yield of sericin and properties of fibroin and sericin formed were studied (Teh et al., 2010). Considerable variations were observed in the tensile properties of the fibroin fibers depending on the treatment conditions used (Table 3.2). Mechanical agitation or refreshing the degumming bath resulted in harsh degumming and damage to the fibers including visual fibrillation. However, degumming using SDS30 at 100 °C resulted in complete sericin removal as indicated by the absence of T-cell mediated hyper-­ sensitivity (Teh et al., 2010). To improve efficiency of degumming and obtain fibers with better properties, a silk peptide based surfactant was developed as an alternative to sodium carbonate. The silk protein surfactant (SPS) was synthesized using silk amino acids and lauroyl chloride to have a Critical Micelle Concentration (CMC) value of 7.5 mmol/L and foaming power of 26.25 mN/m. Degumming with SPS was done at various temperatures and time as seen from Fig. 3.3. The extent of degumming was directly related to the conditions used. A degumming ratio of up to 27% was obtained using SPS. However, the degumming rate of SPS was lower than that of sodium carbonate but considerably higher than that of neutral soap (Wang et al., 2015). Lesser decrease in tensile properties and lower degradation of sericin peptides was observed for the SPS treated fibers. It was suggested that SPS was similar in performance to neutral soap and far more superior than degumming using sodium carbonate (Wang et al., 2015). A natural detergent alkyl polyglycoside (APG) that is commercially available and is environmentally friendly and widely used in agriculture and industrial applications was studied for potential degumming of silk and its effect on properties of fibroin fibers. Yellow male B. mori cocoons and purple female cocoons were used for the study. The APG detergent (0.25%) with two different alkyl chain carbon atoms (8–10, 8–14) were used for degumming at boil for 30 min with a material to liquid ratio of 1:90. After degumming, the fibers were washed and dried and the

54

Table 3.2  Comparison of the properties of silk fibers obtained using various degumming conditions (Teh et al., 2010). Yield Strength, MPa

Yield Strain, mm/min

Breaking Strength, MPa

Breaking strain, mm/min

Na2CO3 90 (100 °C, MA) SDS15 (100 °C, MA) SDS30 (100 °C, MA) SDS60 (100 °C, MA) SDS90 (100 °C, MA) SDS15 (100 °C, static) SDS30 (100 °C, static) SDS60 (100 °C, static SDS90 (100 °C, MA) SDS45 (60 °C, MA) SDS90 (60 °C, MA) SDS30 (75 °C, MA) SDS45 (75 °C, MA)

8.1 ± 1.7 9.5 ± 2.4 10.1 ± 3.1 8.9 ± 1.8 8.5 ± 2.1 10.4 ± 2.2 10.5 ± 2.6 9.1 ± 1.9 8.8 ± 2.0 14.6 ± 1.8 9.8 ± 2.7 9.0 ± 2.8 10.5 ± 1.8

146 ± 43 186 ± 52 172 ± 38 129 ± 64 136 ± 25 197 ± 48 184 ± 32 141 ± 54 132 ± 45 174 ± 17 218 ± 54 159 ± 53 156 ± 33

0.020 ± 0.003 0.022 ± 0.005 0.020 ± 0.006 0.017 ± 0.007 0.019 ± 0.005 0.024 ± 0.008 0.021 ± 0.005 0.018 ± 0.004 0.018 ± 0.003 0.014 ± 0.002 0.026 ± 0.009 0.021 ± 0.010 0.017 ± 0.005

309 ± 51 482 ± 70 466 ± 67 366 ± 101 318 ± 79 502 ± 89 489 ± 73 380 ± 98 320 ± 59 533 ± 104 521 ± 103 471 ± 90 436 ± 40

0.13 ± 0.07 0.19 ± 0.06 0.16 ± 0.04 0.12 ± 0.06 0.11 ± 0.06 0.18 ± 0.07 0.20 ± 0.04 0.13 ± 0.05 0.12 ± 0.03 0.15 ± 0.06 0.19 ± 0.08 0.29 ± 0.13 0.15 ± 0.06

SDS60 (75 °C, MA) SDS90 (75 °C, MA) SDS 7.5 + 7.5 (100 °C, MA) SDS 15 + 15 (100 °C, MA) SDS 30 + 30 (100 °C, MA)

8.9 ± 2.0 9.1 ± 1.7 9.8 ± 1.9 8.3 ± 1.4 7.0 ± 1.7

169 ± 24 175 ± 39 177 ± 39 140 ± 37 166 ± 56

0.022 ± 0.004 0.021 ± 0.004 0.023 ± 0.005 0.019 ± 0.004 0.026 ± 0.006

414 ± 64 394 ± 65 495 ± 67 378 ± 55 367 ± 44

0.18 ± 0.08 0.11 ± 0.04 0.19 ± 0.06 0.17 ± 0.07 0.111 ± 0.02

Reproduced with permission through open license publication.

Silk: Materials, Processes, and Applications

Sample

Modulus, GPa

Degumming Rate (%)

New developments in degumming silk55 30

30

25

25

20

20

15

15

10

10

5

5

0

Degumming Rate (%)

(A)

0.05

1 2 0.10 0.20 0.50 SPS Concentration (%)

5

0

40

40

35

35

30

30

25

25

20

20

15

15

10

10

5

5

70 80 90 Temperature (°C)

100

0

0 30

(C)

60

(B)

60 90 120 Bioling Time (min)

150

1/60

1/80

1/100

Bath Ratio (g/mL)

(D)

Na2CO3

NS

SPS

Degumming Method

(E)

Fig. 3.3  Influence of degumming conditions on sericin removal SPS concentration (A), temperature (B), time (C), bath ratio (D) and method of degumming (Wang et al., 2015). Reproduced with permission from Elsevier.

cycle was repeated three times (Wang and Zhang, 2017). Extent of degumming was highly influenced by the degumming conditions such as APG concentration, temperature, boiling time and bath ratio (Fig.  3.4) and varied from 2% to 28%. Tensile strength of the APG degummed fibers was similar to that of the neutral soap degummed fibers but better than that of the alkali degummed fibers. Similarly, the peptide chains of fibroin were not degraded and had molecular weight of about 200 kDa. Physical structure of fibers degummed using all the three methods did not show any differences (Fig. 3.5). A plant based nonionic surfactant obtained from Sapindus mukurossi was used as a substitute for conventional degumming agents (Sarma et  al., 2012). The pericarp of the seed was soaked overnight and the surfactant dissolved in water was used for degumming. Various concentrations of the soap were used at different temperatures and time to achieve maximum sericin removal. A maximum degumming of 18% was obtained when the silk was soaked in the soap solution for 36 h (Sarma et  al., 2012). Biobased extractants from Citrus limon,

56

Silk: Materials, Processes, and Applications

Fig. 3.4  Effect of degumming conditions on the removal (%) of sericin (Wang and Zhang, 2017). Reproduced with permission from Elsevier.

Dillenia indica and Musa balbisiana were used to degum the wild silk Antheraea assamensis as an alternative to ­alkaline ­degumming (Choudhury et  al., 2016). Images of the plants used to produce the biobased degumming agents are shown in Fig. 3.6. The extractants were used at different t­ emperature and time to degum the fibers. Duration of degumming, pH and concentration of extractants influenced the extent of degumming which varied from about 18% to 22%. Among the three natural materials investigated, the lemon and kolakhar provided similar degumming compared to sodium carbonate whereas elephant apple extract had lower degumming efficiency. Tensile strength and elongation of the fibers were similar but modulus was higher by about 10–15 g/den for fibers degummed using lemon or kolakhar. It was suggested that the biobased agents could be effectively used to remove sericin and replace the alkali used for conventional degumming (Choudhury et al., 2016).

New developments in degumming silk57

Fig. 3.5  No major differences were observed in the diffraction patterns of the Na2SO3, neutral soap (NS) or APG degummed fibers (Wang and Zhang, 2017).

Fig. 3.6  Images of the natural materials used to extract the biobased chemicals for degumming silk. Citrus limon (A), Musa balbisiana (B) and Dillenia indica (elephant apple) (C) (Choudhury et al., 2016). Reproduced with permission from Taylor and Francis.

3.3 Infrared assisted degumming Conventional chemical approach of degumming uses alkali at high temperature to remove the sericin. This process also uses considerably large amounts of water. In addition, the sericin removed gets degraded and is difficult to be separated. To overcome these limitations, a IR assisted degumming process was developed by Gupta et  al.

58

Silk: Materials, Processes, and Applications 30

IR (100°C)

Yield (%)

25

IR (110°C) IR (120°C)

20

HTHP (100°C)

15

HTHP (110°C)

10

HTHP (120°C)

5 0

0

30

60 90 Time (min)

120

150

Fig. 3.7  Influence of time and temperature of extraction on the yield of sericin extracted using IR and HTHP processes (Gupta et al., 2013). Reproduced with permission from Elsevier.

(Gupta et al., 2013). Undegummed silk fabrics were immersed in water and heated in an IR dyeing machine with temperatures ranging from 100 to 120 °C. Sericin separated from the fibers was dried either by spray or freeze drying and used for further analysis. Complete removal (up to 28%) of sericin was possible with the IR approach depending on the time and temperature during degumming (Fig. 3.7). Also, the sericin extracted could be completely recovered without any degradation. Ability to efficiently extract high quality sericin without using any chemicals and with low amounts of water has significant cost and environmental implications for the silk industry. It was suggested that the IR heating method could be adaptable for large scale silk processing (Gupta et al., 2013). A large number of variables such as insect species, rearing conditions etc. determine the amount of sericin in silk. Similarly, the extent of removal of sericin and hence the degumming efficiency is highly dependent on the conditions used. A comparative study was done to understand the effect of degumming conditions on various types of silks (Vyas and Shukla, 2016a,b). Degumming was done using hot water, alkali, detergent, enzymes etc. on mulberry, Eri and tasar silks with the assistance of ultrasonication or IR treatment. Ultrasonic treatment was done at a frequency of 40 kHz and output of 220 W at 60 °C for 30 min. Similarly, microwave irradiation was done for 5 min using 850 W of power and operating frequency of 2450 MHz. In addition to weight loss, tensile properties, whiteness and dye uptake varied considerably depending on the type of silk and degumming conditions (Table 3.3). Although the maximum yield of sericin was low for all the three methods and using all the different conditions, considerably higher differences were noticed in the whiteness of the samples after degumming (Table 3.4). In a similar study, the effects of degumming using ultrasonic, soap and enzymes individually and in combination on the properties of Persian silk were studied (Mahmoodi et al., 2010). Ultrasonication was done using a power of 70 W in the presence of 5 g/L sodium bicarbonate at pH between 8 and 9 and 5 g/L Irgasol (detergent) at 60 °C for 15–90 min with liquid to silk ratio of 30:1. Conventional degumming was done using Marseille soap at 95 °C for 15–30 min. Enzymes used were

Mulberry

Tasar

Eri

Treatment condition

C

U

M

C

U

M

C

U

M

None Hot water 5 g/L soda ash, pH 10.5 1 g/L detergent, pH 7.5 1 g/L soda ash +2 g/L H2O2 (50%), pH 9.6 5 g/L enzyme (papain), pH 6.8

– 3.1 5.6 2.7 3.4

– 4.0 6.0 4.2 5.9

– 2.9 5.4 5.3 7.9

– 2.1 5.6 2.7 3.4

– 5.4 6.9 5.8 7.2

– 6.7 7.6 7.4 11.8

– 4.0 5.9 3.6 4.9

– 4.2 5.9 3.1 5.9

– 4.1 5.9 4.0 7.3

2.8

3.8

5.2

2.8

6.2

8.2

3.7

4.5

6.6

Reproduced with permission from Taylor and Francis.

New developments in degumming silk59

Table 3.3  Ability of three different types of silks to be degummed (% weight loss) at various treatment conditions using conventional (C), ultrasound (U) and microwave (M) approaches (Vyas and Shukla, 2016a,b).

60

Table 3.4  Comparison of the whiteness index of three types of silks degummed using different approaches and treatment conditions (Vyas and Shukla, 2016a,b). Mulberry

Tasar

Eri

C

U

M

C

U

M

C

U

M

None Hot water 5 g/L soda ash, pH 10.5 1 g/L detergent, pH 7.5 1 g/L soda ash +2 g/L H2O2 (50%), pH 9.6 5 g/L enzyme (papain), pH 6.8

81.5 78.1 45.6 63.8 92.0

– 78.2 46.9 64.1 93.5

– 76.2 48.9 68.9 93.8

−1.9 −5.2 −6.1 −4.9 16.9

– −0.9 −1.7 −0.6 17.7

– −2.6 2.6 −0.4 17.6

40.5 32.2 19.0 22.5 51.7

– 33.4 22.1 33.0 51.0

– 27.7 27.9 33.3 51.6

75.6

75.6

73.5

−4.8

−4.0

−8.5

30.6

34.5

31.9

Reproduced with permission from Taylor and Francis.

Silk: Materials, Processes, and Applications

Treatment condition

New developments in degumming silk61

savinase and alcalase at pH between 8 and 9 for 10–30 min with the addition of sodium bicarbonate (5 g/L). Generally, degumming (% weight loss) increased with decrease (up to 32%) in the tensile strength of the fibers as more aggressive conditions, particularly if sonication were used. Up to 22% degumming was obtained with a combination of enzymes and soap. Morphologically, degummed fibers were smooth and removal of sericin provided marginally higher elongation to the fibers (Mahmoodi et al., 2010).

3.4 Degumming using enzymes Substantial efforts have been made to replace conventional chemical degumming with enzymatic processes. An enzyme (cocoonase) generated by the silk worm to soften the cocoon and help the moth escape was isolated and used to study the possibility of degumming (Unajak et al., 2015). The enzyme was extracted from the cocoon and expressed in yeast to obtain recombinant enzymes which were later purified (Rodbumrer et al., 2012; Unajak et al., 2015). The enzyme yield of 10 mg/L and highest activity of 26.4 ISU/mg was obtained. Complete removal of sericin without damaging fibroin was achievable within 24 h for the recombinantly produced enzymes compared to 48 h for the natural cocoonase. In another study, recombinantly produced enzymes had much higher specific activity between 93 and 227 U/mg before and after purification compared to the natural cocoonase (Unajak et al., 2015). For degumming, silk samples were immersed in 50 mmol/L of buffer containing 120 μg/mL of enzyme and incubated at 40 °C for 1 h and the process was repeated twice. A control experiment was also done using conventional Na2CO3 approach. Enzymatic degumming efficiency of 98% was achieved with a short time of 1–2 h. In addition to the gum removal, the enzyme was also able to degrade sericin and bleach the fibers which would help in subsequent processing. Cocoonases were considered to be a viable option for treating silk and combining multiple operations. Similar to cocoonases, proteases have been generated from plant and microbial sources and studied for their potential to remove sericin. Serine proteases from Bacillus sp. C4 SS-2013 (C4) were isolated and purified to obtain an activity of 78 U/ mg and molecular weight of 28 kDa and yield of 6.2%. Degumming was done using the enzyme and complete removal of sericin was possible within 2 h without causing any damage to fibroin. In addition, the enzyme treated silk sheet had a considerably lighter color (Fig. 3.8) which will assist in further processing and dyeing of silk (Suwannaphan et al., 2017). Commercially available proteolytic enzymes were studied for their potential to degum crepe silk fabrics at different degumming conditions. Properties of the enzymes used for degumming are shown in Table 3.5. Degumming was done using the enzymes at different pH, temperature, concentrations and time (Table  3.6). Extent of degumming and the properties of the sericin obtained after degumming was highly dependent on the conditions. A highest degumming loss of 25% was obtained with 1 U/g of 3374-L with an average sericin molecular weight of 12 kDa (Freddi et al., 2003). Proteases extracted from different microbial sources were studied for their ability to degum Chinese bivoltine silk (More et  al., 2013). Four proteases, two from fungal and two from actinomycetes were developed and

62

Silk: Materials, Processes, and Applications

Fig. 3.8  Degumming using enzymes not only provided high sericin removal efficiency but also resulted in a substantially lighter color (Suwannaphan et al., 2017). Reproduced with permission from Elsevier.

used for ­degumming at different temperatures and time. In addition to the extent of degumming, the mechanical properties of the fibers were also affected (Table  3.7). Some of the enzymes resulted in improvement in the properties of fibroin and could be considered for commercial applications. Alkaline proteases produced from Beauveria sp. (MTCC 5184) were found to effectively degum Chinese bivoltine silk fibers even at low temperature of 50 °C and 45 min of treatment when 75 units of enzymes were used per gram of silk at pH 9.0 (More et al., 2018). However, extent of degumming was directly related to the degumming conditions. Sericin removed from the silk has peptides with molecular weights between 616 and 1140 Da. Ability to degum at low temperatures and without any damage to the fibers was considered to be favorable for degumming in the large scale (More et al., 2018). Six different proteases and lipases were evaluated for their ability to degum silk and influence on fiber properties was studied (Sarma, 2015). Isolates obtained from Aspergillus niger provided better protease and lipase enzymes for degumming of muga silk cocoons. Effect of different enzymes on the properties of the silk fibers are provided in Table  3.8. Bromelain, a protease found in pineapple wastes was studied for its potential to degum Thai multivoltine silk (Ninpetch

Table 3.5  Properties of the proteolytic enzymes used for degumming silk fabrics (Freddi et al., 2003). Enzyme

Origin

pH

Temperature, °C

Activity

3374-L GC 897-H 3272-C EC 3.4 23.18

Bacillus subtilis Bacillys lentus Carica papaya Aspergillus saitoi

7.5–12 7–12 3.5–9 2.5–6.5

20–60 40–65 65–78 30–60

55.9 MPU/g 44.7 GSU/ml 58.3 FCCPU/g 1 U/mg

Reproduced with permission from Elsevier.

New developments in degumming silk63

Table 3.6  Conditions used for the degumming of silk fabrics (Freddi et al., 2003). Enzyme

Buffer

pH

T °C

Concentration, U/g

Time, min

3374-L GC 897-H 3272-C

Tris–HCl 0.1 M Tris–HCl 0.1 M CA-Na Phosphate 0.1 M CA-Na Phosphate 0.2 M

10 10 6

60 65 65

0.05–2 0.05–2 0.05–2

5–240 5–240 5–240

3

50

0.05–60

5–240

EC 3.4 23.18

Reproduced with permission from Elsevier.

et al., 2015). Up to 96.5% sericin could be removed when 5 g/L of Bromelain was used for degumming. Silk ­fabrics made from bromelain degummed fibers had 10% higher recovery and 4% higher bending compared to alkali treated samples. With no damage to fibroin, high sericin recovery rate and environmentally friendly aspects were considered beneficial over alkaline degumming (Ninpetch et al., 2015). A comparison was done to determine the degumming efficiency of an enzyme (Alcalase), conventional alkali and with a volcanic stone formed from the shrub Table 3.7  Comparison of the effect of degumming using different enzymes on the tensile strength and elongation of silk fibers (More et al., 2013).

Treatment Untreated Conventional (Alkali and soap) Conidobolous coronatus (PTA-4132) Conidiobolus brefeldianus (MTCC 5185) Alkali tolerant fungus (BOA-10) Beauveria sp MTCC 5184 Actinomycete-1 (BOA-2) Actinomycete-2 (BOA-3)

Weight loss, %

Tensile Strength, g/den

Elongation at break, %

– –

– 21.4 ± 0.8

3.5 ± 0.23 3.8 ± 0.25

19 ± 1.6 15 ± 2.2

100

15.7 ± 0.7

3.5 ± 0.22

16 ± 1.7

20

21.1 ± 0.7

3.6 ± 0.16

17 ± 1.4

100

16.8 ± 0.7

3.6 ± 0.18

18 ± 0.9

40

19.6 ± 1.0

3.8 ± 0.25

17 ± 1.5

10

19.6 ± 1.0

4.0 ± 0.25

17 ± 1.3

25

21.8 ± 1.0

4.1 ± 0.25

17 ± 1.2

Enzyme conc. U/g silk

Reproduced with permission from Taylor and Francis.

64

Silk: Materials, Processes, and Applications

Table 3.8  Comparison of the properties of muga silk fibers degummed using different enzymes (Sarma, 2015). Source

Load, g

% Strain

Young’s modulus

Tenacity, g/den

Toughness, g/den

Aspergillus niger Aspergillus fumigatus Aspergillus flavus Aspergillys tamarii Penacillum pinophylium Na2 CO3 Papaya latex Papaya + A. niger

22.3

28.2

65.8

3.7

0.986

22.1

26.9

56.1

3.7

0.958

20.0

26.9

54.9

3.2

0.984

22.5

20.9

56.6

3.6

0.985

22.1

21.1

39.9

3.7

0.973

20.8 20.3 22.6

17.4 24.0 17.7

46.8 52.7 55.6

3.4 2.8 2.6

0.944 0.885 0.829

Open access publication.

Seidlitzia Rosmarinus (Kelyab) (Talebpour et al., 2013). Enzymatic degumming was done using three different concentrations between 0.5 and 1.5 g/L and pH between 7 and 8 for 30 to 60 min at 55 °C. Up to 19% removal of sericin was possible by the enzyme compared to 26% for the plant based material. In another study, alkaline serine proteases (Subtilisin) was used to degum raw silk fabrics at 0.1 to 4 g/L of enzymes and temperature of 80 °C for 20 min in an infrared dyeing machine (Kim et al., 2016). Extent of degumming, color of the degummed fabric, tensile properties and water absorbency were dependent of the conditions during degumming. Morphological analysis showed that the enzyme treated fibers had a smooth and clean surface without any damage to the fibroin compared to untreated or conventional alkali treated fibers (Fig. 3.9). A halotolerant metalloprotease extracted from the bacterial strain Vibrio sp. ­LA-05 was studied for its potential to degum silk (Zhang et  al., 2019). The degumming was done from temperatures between 15 and 45 °C for 1 to 12 h with weight loss ranging from 2 to highest possible at 25%. A marginal increase in breaking force from 0.76 to 0.81 N but substantial increase of elongation from 17.9% to 21.8% was observed after degumming. Similarly, the fibers degummed using the enzyme had a 10 degree higher degradation temperature (319 °C). More interestingly, the fibroin peptide residues did not show any degradation and the average particle size of fibroin obtained after enzymatic degumming was higher at 16.3 nm (Fig. 3.10) suggesting that the process used was not only environmentally friendly but was also able to preserve the properties of fibroin. In addition, the sericin extracted had antioxidant and ferric ion reducing capacity useful for various medical and environmental applications (Zhang et al., 2019).

New developments in degumming silk65

Fig. 3.9  Comparison of the scanning electron images of the cross-section and longitudinal view of untreated (A), NaOH degummed (B) and savinase treated (C) silk fabrics (Kim et al., 2016). Reproduced with permission from Taylor and Francis.

29.0 20.1 14.3 6.5

M

1

2

3

4

30

5

Na2CO3

25 Number (%)

kDa 200.0 116.0 97.2 66.4 44.3

15°C 25°C

20

35°C 45°C

15 10 5 0

(A)

(B)

0

10

20 30 Size (d.nm)

40

50

Fig. 3.10  Comparison of the molecular weight (A) and particle size (B) of the fibroin peptide fractions obtained after degumming silk at different conditions using crude protease (Zhang et al., 2019). Reproduced with permission from Elsevier.

3.5 Removal of sericin using ionic liquids Another environmentally friendly approach that has been suggested for degumming silk is by using ionic liquids. Two ionic liquids 1-butyl-3-methylimidazolium chloride [BMIM]Cl and 1-butyl-3-methylimidazolium chloride [BMIM]Cl were synthesized in the laboratory and used to degum eri silk at different degumming conditions (Vyas and Shukla, 2016b). The degumming efficiency was considerably low at 8.3% and among the two liquids, [BMIM]Cl provided better absorbency and subsequently considered more suitable for dyeing the fibers.

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Silk: Materials, Processes, and Applications

Fig. 3.11  Differences in the molecular weights of raw silk (A), alkali degummed (B), neutral soap degummed (C) and steam degummed silk (D) (Wang et al., 2018). Reproduced with permission from Elsevier.

3.6 Degumming using steam Steaming was demonstrated to be a more efficient, less energy intensive and environmentally friendly approach to degum silk (Wang et al., 2018). Raw silk was i­ mmersed in deionized water for 30 min and later placed in a pressure cooker (above water level) and treated at 0.14 MPa pressure for 30–120 min. After the treatment, the silk was washed in 60 °C deionized water in an ultrasonication bath and again washed three times and dried. Up to 25% degumming was obtained after steam treatment after 90 min, similar to that obtained for the alkali treatment. Molecular weight analysis (Fig. 3.11) showed that there was degradation of fibroin for both the steam and alkali degumming. Tensile strength did not show any major change after any of the treatments but elongation decreased from 18.7% to 11.5% after steam degumming. Overall, a 10% increase in degumming efficiency and 25% decrease in specific energy consumption was projected for steam degumming when considered as an alternative to alkali degumming (Wang et al., 2018).

3.7 Comparison of various degumming approaches A study was done to compare the effectiveness and influence on the properties of fibroin and sericin obtained using different degumming techniques (Table  3.9). Considerable differences were observed in the amino acid composition (Fig.  3.12), fibroin content and degumming efficiency depending on the type of degumming used. Alkali degumming was found to be the most efficient and quick approach but resulted

Sodium oleate

Trypsin

0.02 M Na2 CO3 120

1%, pH 7

1%, pH 8

1%, pH 8

90% BMIB

60

120

180

420

100

90

37

37

85

Sample

C5

C30

C60

C120

Degumming reagent Degumming time, min Temperature, °C

0.02 M Na2 CO3 5

0.02 M Na2 CO3 30

0.02 M Na2 CO3 30

100

100

100

Reproduced with permission from John Wiley and Sons.

Ionic liquid

New developments in degumming silk67

Table 3.9  Various approaches used to degum B. mori silk cocoons (Nultsch et al., 2018).

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Silk: Materials, Processes, and Applications

Fig. 3.12  Changes in the amino acid composition (A and B), fibroin content (C) and degumming efficiency (C) for B. mori cocoons degummed using various approaches (Nultsch et al., 2018). Reproduced with permission from John Wiley and Sons.

in considerable damage (reduction in molecular weight) to fibroin. Ionic liquids do not cause major damage to the peptides but have lower degumming efficiency. Sodium oleate and trypsin were considered to be unsuitable for degumming. Overall, it was suggested that using alkali (sodium carbonate) would be the most commercially appropriate degumming approach although some compromise on fiber quality was inevitable (Nultsch et al., 2018).

3.8 Demineralization of wild silks before degumming Unlike B. mori silk, wild silks contain considerable amounts of minerals that affect degumming, reeling and even properties of the silk fibers. Degumming of wild silks require considerably harsh conditions which also lead to difficulties in reeling and caused damage to the fibers. To overcome these limitations, the possibility of demineralization of cocoons using chemical and natural agents was

New developments in degumming silk69

studied. Muga silk cocoons were treated with either ethylenediaminetetraacetic acid (EDTA), p­ otassium carbonate, citric acid, lemon and an extract from the plant Musa balbisiana (Kolakhar). EDTA treatment was done using pH 10 solution at 45 °C for 72 h, a pH 10.5 solution at 45 °C for 72 h was used for treating with kolakhar and lemon juice was used at pH 2.5 and treated at 45 °C for 72 h. After demineralization, the cocoons were degummed using 0.3% sodium carbonate solution at 90 °C for 30 min. Demineralized and degummed fibers were reeled and fibers obtained studied for their changes in properties. Compared to conventional sodium carbonate reeling without demineralization which produced average reeled length of 235 m and about 30 breaks, substantially higher reeling lengths (up to 350 m) with much fewer breaks (4–16) was possible when different demineralization techniques were used (Choudhury and Devi, 2018). Morphological studies and compositional analysis clearly showed the removal of calcium oxalate crystals after demineralization. Muga silk fibers with higher strength, better elongation and toughness were obtained when demineralization was done before degumming for most of the approaches as seen from Table 3.10. Based on the results obtained, it was suggested that demineralizing the fibers before degumming is a better approach to obtain wild silk fibers with good properties. Table 3.10  Properties of muga fibers after degumming with and without demineralization compared to conventional degumming (Choudhury and Devi, 2018). Treatment EDTA demineralized Kolakhar demineralized Lemon demineralized Potassium carbonate demineralized Citric acid demineralized EDTA demineralized+degummed Kolakhar demineralized+degummed Lemon demineralized+degummed Potassium carbonate demineralized+ degummed Citric acid demineralized+degummed Sodium carbonate degummed

Tenacity, g/den

Strain, %

Modulus, g/den

Toughness, g/den

4.7 ± 0.2 4.9 ± 0.1 4.6 ± 0.8 4.8 ± 0.1

35.1 ± 0.3 36.0 ± 0.7 34.8 ± 1.8 35.8 ± 1.0

84.2 ± 3.4 85.4 ± 3.9 83.9 ± 4.3 85.1 ± 3.9

1.3 ± 0.3 1.3 ± 0.3 1.2 ± 0.5 1.3 ± 0.3

4.5 ± 0.3 4.1 ± 1.7

34.9 ± 0.9 33.3 ± 0.6

83.8 ± 4.4 75.1 ± 4.0

1.2 ± 0.4 1.0 ± 0.5

4.4 ± 0.7

34.3 ± 0.2

72.0 ± 4.8

1.1 ± 0.3

4.2 ± 0.4

32.8 ± 1.9

73.9 ± 4.1

1.0 ± 1.2

4.3 ± 0.3

32.6 ± 1.0

76.6 ± 3.7

1.1 ± 0.1

4.1 ± 2.0

31.1 ± 1.5

70.1 ± 4.0

1.0 ± 0.5

3.8 ± 2.8

32.7 ± 0.3

75.6 ± 3.4

0.8 ± 0.3

Reproduced with permission from Taylor and Francis.

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Silk: Materials, Processes, and Applications

References Allardyce, B.J., Rajkhowa, R., Dilley, R.J., Atlas, M.D., Kaur, J., Wang, X., 2016. The impact of degumming conditions on the properties of silk films for biomedical applications. Text. Res. J. 86 (3), 275–287. Cao, T.-T., Wang, Y.-J., Zhang, Y.-Q., 2013. Effect of strongly alkaline electrolyzed water on silk degumming and the physical properties of the fibroin fiber. PLoS One 8 (6), e65654. Choudhury, M., Devi, D., 2018. Demineralization of cocoons of Antheraea assamensis Helfer (muga) for effective reeling. J. Text. Inst. 109 (4), 552–559. Choudhury, M., Talukdar, B., Devi, D., 2016. Surface smoothening and characterization of silk fibers of Antheraea assamensis Helfer (muga) using some natural agents. J. Text. Inst. 107 (11), 1347–1356. Dou, H., Zuo, B., 2015. Effect of sodium carbonate concentrations on the degumming and regeneration process of silk fibroin. J. Text. Inst. 106 (3), 311–319. Freddi, G., Mossotti, R., Innocenti, R., 2003. Degumming of silk fabric with several proteases. J. Biotechnol. 106 (1), 101–112. Gupta, D., Agrawal, A., Chaudhary, H., Gulrajani, M., Gupta, C., 2013. Cleaner process for extraction of sericin using infrared. J. Clean. Prod. 52, 488–494. Kim, J., Kwon, M.Y., Kim, S., 2016. Biological degumming of silk fabrics with proteolytic enzymes. J. Nat. Fibers 13 (6), 629–639. Mahmoodi, N.M., Arami, M., Mazaheri, F., Rahimi, S., 2010. Degradation of sericin (degumming) of Persian silk by ultrasound and enzymes as a cleaner and environmentally friendly process. J. Clean. Prod. 18 (2), 146–151. More, S.V., Khandelwal, H.B., Joseph, M.A., Laxman, R.S., 2013. Enzymatic degumming of silk with microbial proteases. J. Nat. Fibers 10 (2), 98–111. More, S.V., Chavan, S., Prabhune, A.A., 2018. Silk degumming and utilization of silk sericin by hydrolysis using alkaline protease from beauveria Sp. (MTCC 5184): A green approach. J. Nat. Fibers 15 (3), 373–383. Ninpetch, Uraiwan, Masahiro Tsukada, and Amornrat Promboon. Mechanical properties of silk fabric degummed with bromelain. J. Eng. Fibers Fabrics 10 (3) (2015): 155892501501000319. Nultsch, K., Bast, L.K., Näf, M., El Yakhlifi, S., Bruns, N., Germershaus, O., 2018. Effects of silk degumming process on physicochemical, tensile, and optical properties of regenerated silk fibroin. Macromol. Mater. Eng. 303 (12), 1800408. Rajkhowa, R., Wang, L., Kanwar, J.R., Wang, X., 2011. Molecular weight and secondary structure change in eri silk during alkali degumming and powdering. J. Appl. Polym. Sci. 119 (3), 1339–1347. Rodbumrer, P., Arthan, D., Uyen, U., Yuvaniyama, J., Svasti, J., Wongsaengchantra, P.Y., 2012. Functional expression of a Bombyx mori cocoonase: Potential application for silk degumming. Acta Biochim. Biophys. Sin. 44 (12), 974–983. Sarma, I., 2015. Degumming of muga cocoon with mycogenic extracellular protease and lipase enzyme—an alternative method for efficient reeling of silk. Adv. Appl. Sci. Res. 6, 7–16. Sarma, Mamata B., Subrata Borgohain Gogoi, Depali Devi, and B. Goswami. Degumming of muga silk fabric by biosurfactant. (2012). Suwannaphan, S., Fufeungsombut, E., Promboon, A., Chim-Anage, P., 2017. A serine protease from newly isolated Bacillus sp. for efficient silk degumming, sericin degrading and colour bleaching activities. Int. Biodeter. Biodegr. 117, 141–149. Talebpour, F., Veysian, S.M., Heidari, G.M.E., 2013. Degumming of silk yarn using alkali, enzyme and Seidlitzia Rosmarinus. J. Tex. Polym. 1 (2), 60–64.

New developments in degumming silk71

Teh, T.K.H., Toh, S.-L., Goh, J.C.H., 2010. Optimization of the silk scaffold sericin removal process for retention of silk fibroin protein structure and mechanical properties. Biomed. Mater. 5 (3), 035008. Unajak, S., Aroonluke, S., Promboon, A., 2015. An active recombinant cocoonase from the silkworm Bombyx mori: Bleaching, degumming and sericin degrading activities. J. Sci. Food Agric. 95 (6), 1179–1189. Vyas, S.K., Shukla, S.R., 2016a. Comparative study of degumming of silk varieties by different techniques. J. Text. Inst. 107 (2), 191–199. Vyas, S.K., Shukla, S.R., 2016b. Degumming of eri silk using ionic liquids and optimization through response surface methodology. J. Text. Inst. 107 (9), 1096–1111. Wang, F., Zhang, Y.-Q., 2017. Effects of alkyl polyglycoside (APG) on Bombyx mori silk degumming and the mechanical properties of silk fibroin fibre. Mater. Sci. Eng. C 74, 152–158. Wang, F., Cao, T.-T., Zhang, Y.-Q., 2015. Effect of silk protein surfactant on silk degumming and its properties. Mater. Sci. Eng. C 55, 131–136. Wang, R., Zhu, Y., Shi, Z., Jiang, W., Liu, X., Ni, Q.-Q., 2018. Degumming of raw silk via steam treatment. J. Clean. Prod. 203, 492–497. Wray, L.S., Hu, X., Gallego, J., Georgakoudi, I., Omenetto, F.G., Schmidt, D., Kaplan, D.L., 2011. Effect of processing on silk‐based biomaterials: reproducibility and biocompatibility. J. Biomed. Mater. Res. B Appl. Biomater. 99 (1), 89–101. Zhang, H., Li, H., Liu, H., Lang, D.A., Xu, H., Zhu, H., 2019. Degumming raw silk by a halotolerant metalloprotease isolated from metabolites of Vibrio sp. LA-05. Int. Biodeter. Biodegr. 142, 124–130. Zhao, Z.-L., Li, W.-W., Wang, F., Zhang, Y.-Q., 2018. Using of hydrated lime water as a novel degumming agent of silk and sericin recycling from wastewater. J. Clean. Prod. 172, 2090–2096.

Further reading Wang, Z., Yang, H., Li, W., Li, C., 2019. Effect of silk degumming on the structure and properties of silk fibroin. J. Text. Inst. 110 (1), 134–140.

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Regenerated silk fibers

4

4.1 Regenerated silk using ionic liquids as solvents Table 4.1 lists the common solvent/solvent systems used to dissolve and regenerate silk fibroin. Among the various solvents, ionic liquids have been extensively used to dissolve silk and develop regenerated fibers. Advantages of ionic liquids are that the silk cocoons can be directly dissolved without the need for degumming and ionic liquids are less corrosive and toxic than solvents such as Hexafluoroisopropanol (HFIP) (Goujon et al., 2012). Although regenerated fibers have been produced using ionic liquids, the properties of the fibers obtained have been considerably weaker compared to native fibers and regenerated fibers obtained using other solvent systems. Among the various ionic liquids, protic ionic liquids (PIL) were able to induce βsheet or α-helix formations. Some of the protic ionic liquids considered included triethylammonium phosphate (TeaH2PO4), triethylammonium lactate (TeaLa), triethylammonium triflate (TeaTf) and triethylammonium mesylate (TeaMs). Structure and properties of the regenerated silk obtained was heavily dependent of the type of PILs and coagulation condition used. It was possible to obtain fibers using the PILs as solvent and coagulation bath or even water as the coagulant (Fig. 4.1). Type of solvent controlled the extent of β-sheet or α-helix in the silk. Hence, fibers with tunable properties could be obtained by controlling the type of PIL and coagulation conditions (Zhang et al., 2014). Changes in the structure and properties of silk fibers were observed when 9.0 M LiBr was used to generate the fibers through dry spinning (Sun et al., 2012). In this approach, the degummed fibers were dissolved in 9.0 M LiBr at 40 °C for 2 h and the solution was centrifuged, filtered, washed and dialyzed in water for 3 days. A 20% concentrated regenerated silk fibroin solution was obtained and mixed with a buffer (pH 4.8) and subsequently CaCl2 was added to obtain a calcium concentration of 0.3 M. Solution prepared was extruded at different draw rates and draw ratios into a 80% ethanol coagulation bath in which fibers were formed after 1 h. Differences in crystallinity, crystal size and crystal orientation were observed depending on the draw ratio and speed. Crystallinity of the as-spun fibers was considerably low at about 20% but drawing increased the crystallinity up to 40%, very similar to that of the conventionally degummed fibers. Regenerated silk fibers had crystallite size ranging from 3.6 to 9.6 nm under highest drawn and oriented conditions (Table 4.2). Fibers with tensile strength similar to that of the natural degummed silk were obtained when the draw ratio was 4.0 and drawing rate was 0.9 m/min (Sun et al., 2012). Using the same solvent (LiBr), regenerated fibers with strength higher than that of the natural silk fibers were developed by Zhou et al. High fiber strength was achieved by using hot ammonium sulphate as the coagulation bath and highly concentrated silk fibroin solution for the extrusion. Protein concentration that provided optimum fibers was found to be 15% protein and 30% ammonium sulphate as coagulant and when Silk: Materials, Processes, and Applications. https://doi.org/10.1016/B978-0-12-818495-0.00004-1 © 2020 Elsevier Ltd. All rights reserved.

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Silk: Materials, Processes, and Applications

Table 4.1  Solvent systems used to dissolve silk (Chen et al., 2001). Solvent system

Ratio

Ca(NO3)2–MeOH-H2O LiBr-EtOH-H2O) LiBr-EtOH CaCl2-EtOH-H2O LiBr-H2O Formic acid

75:25 45:44:11 40:60 1:2:8 9.5 M –

Reproduced with permission from Elsevier.

Fig. 4.1  Schematic representation of the approaches used to produce regenerated silk fibers (Koeppel and Holland, 2017). Reproduce with permission from American Chemical Society.

Sample

Crystallite size (nm)

FTIR secondary structure Area %

Draw ratio

Draw rate

As spun fiber Degummed silk 2.0 2.0 2.0 3.0 3.5 4.0

0.5 0.7 0.9 0.9 0.9 0.9

−1

1666 cm−1 β-sheet

1678 cm−1 β-turn

1693 cm−1 β-turn

Breaking stress, MPa

a

b

c

1639 cm

1655 cm 31-Helix

4.7 3.6

4.3 3.0

9.8 9.6

22 5

12 14

13 40

24 23

29 18

45.7 354

3.3 3.1 3.1 2.3 2.1 1.6

2.7 2.7 2.6 2.5 2.1 1.9

7.4 7.3 7.0 5.8 5.0 4.8

6 6 6 4 3 3

28 26 24 24 20 20

24 26 32 38 41 45

38 32 26 21 26 20

4 10 12 13 10 12

– – 168 300 318 327

−1

Reproduced with permission from Royal Society of Chemistry (Great Britain).

Regenerated silk fibers75

Table 4.2  Effect of draw ratio and drawing rate on the size of crystallites in the fibers in the three crystal lattices corresponding to 200 (a), 020 (b) and 002 (c) lattice planes (Sun et al., 2012).

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Silk: Materials, Processes, and Applications

the solution was maintained at 60 °C. Coagulated fibers were immediately drawn by passing through rollers and also steam annealed. Diameter of the fibers drawn 6× was 10.8 μm, smaller than that of the native B. mori silk. Highest breaking stress obtained for the fibers was 450 MPa compared to 400 MPa for natural silk. Similarly, the elongation of the regenerated fibers was as high as 79%. Although the crystallinity and orientation of the regenerated fibers was lower than that of the natural silk, higher mechanical properties were suggested to be due to the fewer defects in the manufactured fibers (Zhou et al., 2009). Further, studies indicated that the fiber formation conditions, particularly drawing, could be controlled to obtain fibers with desired properties (Table 4.3). Fibers coming out of the coagulation bath were drawn at higher speed ratios and subsequently dipped in the coagulating solution. A solution (dope) concentration between 13% and 19% produced fibers with acceptable properties (Yan et al., 2009). At lower concentrations, fibers obtained had morphology similar to that of spider silk spidroin whereas at high concentrations it was similar to B. mori silk. At low concentrations, there was less protein packing density and hence fiber folds were observed. Increasing draw ratio from 2.0 to 6.0 increased breaking strength from 120 to 390 MPa and even the elongation from 4.8 to as high as 32%. Modulus showed an increase from 6.7 to 15.2 GPa (Yan et al., 2009). These regenerated fibers had properties higher than that of natural silk fibers (390 MPa, 19% elongation). To avoid the multiple step and time consuming process of regenerating silk fibers using HFIP, HFA, formic acid or water/PEO solutions, an organic based ionic solution was used to obtain regenerated silk fibers. Degummed silk (fibroin) was mixed with the ionic solvent 1-ethyl-3-methylimidazoium chloride (EMIC) and heated to 95 °C to form the spinning dope. The dope was extruded through a 26 gauge needle at a rate of about 3 m/min into a coagulation bath consisting of either methanol, acetonitrile, water, ethyl acetate, acetone or hexanes (Phillips et al., 2005). Fibers were successfully obtained using the methanol coagulation bath due to formation of β-sheets and better orientation. Increase in the β-sheet content and higher orientation was observed when the fibers were drawn 2 × in the methanol solution. Table 4.3  Properties of regenerated silk fibers obtained using various draw ratios (Zhou et al., 2009). Sample

Diameter, μm

Stress, MPa

Strain, %

Breaking energy, kJ/kg

Raw B. mori silk Regenerated fiber-1 × Regenerated fiber-2 × Regenerated fiber-4 Regenerated fiber-6 ×

15.3–20.5 30.9 ± 2.0

400 ± 20 –

19.7 ± 1.3 –

42.3 ± 3.3 –

22.6 ± 2.1

90 ± 20

3.1 ± 0.9

1.7 ± 0.3

13.2 ± 2.8

260 ± 10

76.9 ± 4.8

111 ± 8.1

10.8 ± 2.4

450 ± 20

27.7 ± 4.2

74.5 ± 13.4

Reproduced with permission from John Wiley and sons.

Regenerated silk fibers77

To study the effect of degumming and post drawing conditions on properties of regenerated silk fibers, cocoons were degummed and later dissolved using LiBr. Regenerated fibroin obtained was redissolved in formic acid and formed into fibers using methanol as a coagulation bath and the filaments obtained were drawn to different draw ratios (Kim and Um, 2014). Viscosity of the solution decreased as degumming ratio increased suggesting that fibroin in the solution was in a entangled state. A viscosity of 0.08 Pa. S was found to be most suitable for producing continuous regenerated fibers. Irrespective of the extent of degumming, the crystallinity of the fibers increased and an optimum occurred at a region similar to that of the optimum draw ratio. However, presence of fibroin was observed to increase the amount and also accelerate the formation of crystallites. It was suggested that the silk fibroin molecules exist as a random coil but turn into short and long range ordered crystalline structure in the coagulation bath. Drawing of the fibers aligns these ordered regions along the length (axis) of the fibers increasing the mechanical properties (Kim and Um, 2014). A schematic representation of the changes in the orientation of the amorphous and crystalline regions due to drawing shown in Fig. 4.2. Properties of silk fibers are dependent on the extent of drawing during extrusion both in the case of natural and regenerated silk. Typically, regenerated silk fibers were drawn in the cold condition to improve mechanical properties. In a different approach, fibroin dissolved in HFIP was made into films and later hot stretched to various extents. Substantial increase in modulus from 1.3 to about 6 GPa and modulus from 58 to 234 MPa with decrease in elongation from 16% to 7% was observed (Table 4.4). Crystallites in the βsheet also decreased to 80 Ȧ compared to 163 Ȧ in the native silk (Yoshioka et al., 2016). Substantial variations in the properties of the fibers were observed when regenerated silk

Post drawing

Coagulation

A) Dope solution

B) As-spun fiber C) Drawn fiber

Random coil in amorphous region Long-range ordered crystallite Short-range ordered crystallite formed after coagulation Short-range ordered crystallite formed additionally after post drawing

Fig. 4.2  Changes in the orientation of the crystalline and amorphous regions of regenerated silk fibers in solution, coagulation bath and after post drawing (Kim and Um, 2014). Reproduced with permission from Elsevier.

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Silk: Materials, Processes, and Applications

Table 4.4  Tensile properties of the samples showed substantial increase with increase in draw ratio and treating with vapors (Yoshioka et al., 2016). Sample

Modulus, GPa

Strength, MPa

Elongation, %

Undrawn 150% drawn 220% drawn 260% drawn 260% vapor treated

1.3 ± 0.2 2.2 ± 0.2 4.0 ± 0.3 5.0 ± 0.1 6.0 ± 0.4

58.0 ± 2.6 99.0 ± 7.2 174.2 ± 12.0 233.5 ± 10.5 161.0 ± 3.2

16.3 ± 0.0 29.0 ± 9.0 11.0 ± 0.3 7.0 ± 0.2 12.3 ± 1.2

Reproduced with permission from American Chemical Society.

was drawn at various draw-down ratios (Fang et al., 2016). A direct relationship existed between draw ratio and tensile strength and modulus whereas elongation showed an inverse relationship with increasing draw ratio (Table 4.5). Corresponding changes were also observed at the molecular level with the β-sheet content increasing marginally with draw ratio resulting in better orientation of the crystallites along the fiber axis (Fang et al., 2016). Although there was an increase in % crystallinity, the crystal size decreased with increasing draw ratio (Table 4.6). Changes in the crystal dimensions were also observed with change in spinning speeds with corresponding increase in orientation factor between 0.922 and 0.956 (Koh et al., 2015). Instead of using HFIP as the solvent for producing regenerated fibers from fibroin, HFA-hydrate (HFA.3H2O) was used to dissolve fibroin and later extruded through a 0.2 mm diameter and 1.2 mm length orifice into a methanol coagulation bath and allowed to stay overnight. Later, the fibers were drawn 3 times their original length and subject to post spinning treatments (Yao et al., 2002) by steaming the fibers at 100 or 125 °C. Silk fibroin was completely soluble in the HFA solution in 2 h compared to 24 h for HFIP. It was suggested that HFA dissolution would avoid degradation of the polymer chains, provide solutions with prolonged stability and proper viscosity for spinning. 13C NMR studies (Fig. 4.3) showed that regenerated silk fibers had β-sheet structure similar to that found in B. mori silk and steaming caused preferential alignment of the polypeptide backbone and also an increase in crystal size. Post spinning conditions such as drawing and annealing were necessary to crystallize the fibers and orient the crystals along the fiber axis. Regenerated fiber obtained had modulus of 54 cN/tex and fineness between 15 and 25 denier compared to 61 cN/tex and 1–2 denier for the B. mori fibers (Yao et al., 2002). Although dry spinning (9.0 M LiBr and redissolution in CaCl2 buffer solution) provided fibers with good properties, subsequent drawing highly influences the properties of the fibers. For instance, using a custom built equipment (Fig. 4.4), silk fibers were dry spun and collected on a drum. Later, the fibers were immersed in 80% ethanol and drawn to 1× to 3× their original length and further immersed in the solution for up to 120 min (Wei et al., 2011a,b). There was a direct correlation between the draw ratio and mechanical properties with higher draw ratios increasing the strength nearly 10 times (Table  4.7). Based on the birefringence, it was suggested that considerable increase in orientation of the molecules in the fibers should have happened leading to higher

Fiber-draw ratio

Area, μm2

Modulus, GPa

Breaking stress, GPa

Breaking strain, %

Breaking energy, MJ/m3

β-Sheet content, %

1× 2× 4× 6 ×-1 6 ×-2 9× B. mori silk

804 ± 26 514 ± 22 348 ± 21 223 ± 18 236 ± 20 175 ± 17 183 ± 15

– 6.8 ± 1.2 14.2 ± 1.4 17.5 ± 1.2 16.8 ± 1.3 18.9 ± 1.1 11.8 ± 0.9

– 0.09 ± 0.02 0.29 ± 0.02 0.42 ± 0.03 0.37 ± 0.02 0.45 ± 0.03 0.40 ± 0.02

– 3.1 ± 0.9 84.3 ± 5.7 41.7 ± 5.1 48.1 ± 6.2 27.3 ± 4.6 19.7 ± 1.3

– 2.3 ± 1.08 185.7 ± 17.7 154.8 ± 17.6 151.3 ± 14.3 91.0 ± 7.4 57.3 ± 4.7

22.8 ± 0.6 23.1 ± 0.4 24.2 ± 0.4 26.6 ± 0.3 25.9 ± 0.4 28.9 ± 0.6 28.0 ± 0.3

Reproduced with permission from American Chemical Society.

Regenerated silk fibers79

Table 4.5  Relation between draw ratios and tensile properties of regenerated silk fibers drawn to different draw ratios (Fang et al., 2016).

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Silk: Materials, Processes, and Applications

Table 4.6  Crystallinity (%) and crystallite size variations with increasing draw ratio (Fang et al., 2016). Crystallite size (nm)

Fiber-draw ratio

% Crystallinity

L1

L2

L3

Orientation degree, %

1× 2× 4× 6 ×-1 6 ×-2 9× B. mori silk

44.3 46.4 48.9 52.5 51.3 54.7 52.4

3.9 3.4 3.3 3.0 3.2 3.0 3.4

5.9 5.4 5.2 4.8 5.0 3.8 3.8

9.2 7.9 7.1 6.5 6.9 6.1 8.8

– 74.7 80.3 84.0 82.4 85.0 92.8

Reproduced with permission from American Chemical Society.

Tyr Cβ Val Cβ

* *

(A)

(B)

ssb

ssb

(C)

Gly Cα Ala Cα

Gly C=O Ala C=O

Ser Cα

(D)

250

Ser Cβ

ssb Tyr Cδ,γ Tyr Cζ

ssb ssb

200

150

Ala Cβ

ssb

100

50

0

ppm from TMS Fig. 4.3  Changes in the 13C CP/MAS NMR spectra of as spun fibers (A), 3 × steam annealed at 100 °C (B), 3 × steam annealed at 125 °C (C) compared to B. mori silk fibers (Yao, 2002). Reproduced with permission from American Chemical Society.

Regenerated silk fibers81

Regenerated silk fibroin aqueous solution

Dry-spum fiber Fig. 4.4  Schematic of the equipment used to produce regenerated silk fibers (Wei et al., 2011a,b). Reproduced with permission from Elsevier. Table 4.7  Properties of dry spun regenerated silk fibers (RSF) obtained at different draw ratios in 80% ethanol and produced using a pH 4.8 spinning dope (Wei et al., 2011a,b). Sample/ draw ratio

Breaking stress, MPa

Breaking strain, %

Initial modulus, MPa

Breaking energy, kJ/kg

Birefringence Δn

As spun RSF-1 × RSF-2 × RSF-3 ×

29.9 ± 13.5 63.9 ± 15.9 199.8 ± 143.4 301.5 ± 70.6

3.6 ± 1.9 7.5 ± 2.3 55.4 ± 21.3 35.8 ± 21.9

1185 ± 479 1660 ± 672 6229 ± 6060 6232 ± 1675

0.5 ± 0.4 1.5 ± 1.1 58.1 ± 34.0 77.6 ± 28.0

– 0.015 0.033 0.038

Reproduced with permission from Materials Research Society.

strength. The strong absorption peaks at 1085 cm−1, 1232 cm−1, and 1666 cm−1 in the Raman spectra, also suggests that there was considerable transition from α-helix to βsheets similar to the observations in other studies. Also, the pH of the spinning dope did not show any significant influence on the mechanical properties since the conformation ratio of α-helix to β-sheets was not changed (Wei et al., 2011a,b). Regenerated fibers with properties similar to that of degummed silk fibers could be obtained using this approach. To make the dry spinning process simpler, Jin et al. reported that the influence of pH on spinning dope can be avoided by selecting the appropriate Ca2+ concentration. Fibers obtained had similar diameter, β-sheet content and conformation compared to natural silk. After post-treatment, the fibers had higher crystallinity and better crystal orientation leading to better mechanical properties (Jin et al., 2013).

4.2 Formic acid as a solvent for silk fibroin One of the most earliest and common approaches of developing regenerated silk fibers is to dissolve silk fibroin in formic acid and regenerating the solutions into fibers using various coagulation baths. In one of the earliest studies, regenerated silk

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Silk: Materials, Processes, and Applications

fibers were produced from fibroin using formic acid as the solvent (Um et al., 2001). Degummed silk was dissolved using calcium chloride, water and ethanol. After dissolution, the solution was dialyzed and regenerated fibroin powder was obtained. Later, the regenerated fibroin was dissolved in formic acid which resulted in a transparent solution. This solution was formed into fibers and also subject to methanol treatment. Dissolution in formic acid provided fibroin with better rheological properties and also improved the β-sheet content and hence crystallinity (39–56%) and crystallinity index from 54 to 72 (Um et al., 2001). Methanol treatment allowed the formation of long range crystals compared to formic acid which resulted in formation of short range crystallites. However, the properties and structure of the fibers obtained depend on the coagulation and pre and post treatment conditions (Um et al., 2001). In another study, Bombyx mori cocoons were degummed and later dissolved using CaCl2, water and ethanol mixture. Dissolved silk was dialyzed using a molecular weight cut-off of 12,000 to 14,000 kDa. Solution was freeze dried to obtain fibroin sponges which were redissolved in formic acid and extruded in the form of fibers (Um et al., 2004a) into various coagulation baths. Some of the baths that were studied include methanol, ethanol, dimethylacetamide (DMAc), dimethylformamide (DMF), tetrahydrofuran (THF), dioxane and dimethyformamide (DMF) at coagulation bath temperatures varying from 25 to 60 °C. Both type of coagulation bath and coagulation temperature affected the morphology of the fibers. As the size of the R group in the alcohols increased, the diffusion rate into the filaments decreased making the fibers to have a contracted shape. In addition to the alcohols, other organic solvents were shown to precipitate silk and provide fibers with good properties (Um et al., 2004b). Also, the coagulation conditions and drawing the as spun filaments in 70 °C water bath to draw ratios up to 5 considerably influenced fiber properties (Fig. 4.5). Although the crystallinity of the fibers was not affected, the crystalline and amorphous orientations increased with increasing draw ratios but was lower than that of the natural silk fibers 4.0

(e)

3.5

Stress (g/d)

3.0 2.5

(d)

2.0

(c)

1.5

(b) 1.0

(a)

0.5 0.0

0

5

10

15

20

25

30

35

40

45

Strain (%)

Fig. 4.5  Influence of draw ratio on the stress–strain properties of regenerated silk fibers compared to raw silk fibers. Fibers were drawn to 2× (A), 3× (B), 4× (C) and 5× (D) (Um et al., 2004b). Reproduced with permission from Elsevier.

Regenerated silk fibers83

Fig. 4.6  Images of the processes used for conversion of fibers into regenerated fibers and nanosized fibrils (Zhang et al., 2015). Reproduced with permission from Elsevier.

(Um et al., 2004b). At the highest draw ratio, the fibers obtained had tensile strength of 2.2 g/denier and 17% elongation. Formic acid treatment was also recommended for obtaining transparent and stable fibroin solution for developing films and other materials. In this approach, silk was dissolved in CaCl2-FA solution having chloride concentration of 4% in which the fibroin was maintained for 3 h at room temperature (Fig. 4.6). Dissolved fibroin was extruded into a coagulation bath consisting of water or methanol. Additional washing with water and drawing up to 4 times was done to improve the mechanical properties (Zhang et al., 2015). The solvent system used was able to disintegrate the fibroin into macroscale, micro scale and further into nanofibrils. Diameter of the nanofibers ranged from 20 to 200 nm (Zhang et al., 2014). Up to 35% silk could be dissolved depending on the degumming and dissolution conditions. The nanofibrils recombined to form the regenerated silk fibers with excellent luster and mechanical properties. Length and diameter of the fibrils obtained was inversely proportional to the concentration of the CaCl2 in the formic acid solution (Zhang et al., 2014). Silk in the CaCl2 solution was observed to be in the amorphous form but gets transformed into the crystalline state after wet-spinning with the crystallinity increasing with drawing ratio. Such changes were responsible for the properties of the fibers obtained. Unlike previous attempts on wet or dry spinning regenerated silk fibers, the CaCl2-FA solution provided fibers with tensile properties similar to that of the native silk fibers (Table 4.8). Ability of the fibroin solution to form macro, micro and nanofibrillar structure, conversion into insoluble β-sheet during fiber formation and high orientation of the nanocrystals during subsequent drawing was suggested to be the reason for the high mechanical properties. In addition, it was shown that water could be used as a coagulation bath for the fiber formation making the process economical and environmentally friendly. In addition to the coagulation conditions, the molecular weight of the proteins and storage time of the spinning dope were also found to affect the fiber properties

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Silk: Materials, Processes, and Applications

Table 4.8  Comparison of the properties of native and regenerated silk fibroin fibers produced at different draw ratios (Zhang et al., 2015). Production condition

Fiber diameter, μm

Strength, MPa

Elongation, %

Modulus, GPa

Breaking energy, kJ/k

As spun 2× 3× 4× Native silk

48.5 ± 8.3 32.1 ± 6.3 23.4 ± 6.5 12.8 ± 4.6 13.6 ± 1.2

95.1 ± 16.8 195 ± 24.9 309 ± 36.5 470 ± 53.5 377 ± 44.5

15.3 ± 5.8 30.6 ± 8.6 45.5 ± 7.1 38.6 ± 6.3 19.1 ± 4.7

3.9 ± 0.7 4.5 ± 1.2 5.8 ± 1.9 6.9 ± 2.1 6.5 ± 1.8

15 ± 3.7 38 ± 6.1 55 ± 7.5 78 ± 11.5 39 ± 5.3

Reproduced with permission from Elsevier.

(Cho et al., 2012). Type of solvent and dissolution conditions used affected the molecular weight and hence the viscosity of the spinning dope. Viscosity of the fibroin solution dissolved using 98% formic acid decreased steadily with increasing storage time (Fig. 4.7). Although degradation of the polymer was observed with increasing storage time, the spinnability of the solutions was maintained for up to 3 days. A viscosity range between 160 and 190 Cps was found to provide most optimum fiber properties (Cho et al., 2012). A new method termed solution blow spinning was developed to obtain regenerated silk fibers in bulk quantities (Magaz et al., 2018). In this approach, B. mori cocoons were degummed using 0.02 M sodium carbonate and the fibroin obtained was dissolved using LiBr. Dissolved proteins were collected by freeze drying and later redissolved using 95% formic acid at concentrations between 8% and 20% to form the spinning dope. To form the fibers, the spinning dope was extruded using a feed rate of 50–60 μL/min and pressure of 30–40 PSI. Fibers coming out of the spinneret were

Viscosity (cps)

10000 SFL (14% SF) SFL (11% SF) SFC3 (14% SF) SFC30 (14% SF) SFC180 (14% SF)

1000

100

10 0

50

100

150 200 Storage time (h)

250

300

350

Fig. 4.7  Changes in the viscosity of the silk fibroin solutions prepared using 98% formic acid (Cho et al., 2012) and storing the fibroin solution for 3, 30, or 180 h of ageing. Reproduced with permission from Elsevier.

5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

Dissolution

Unable to dissolve fibroin

Dissolution & Swelling

Dissolution power

Molar concentration of methanol

Regenerated silk fibers85

Complete dissolution

Swelling

0

2

4

6

Molar concentration of water

8

10

0

1

2

3

4

5

Hydration Number of Calcium Nitrate

Fig. 4.8  Relationship between molar concentration of solvents and dissolution of silk fibroin (left); effect of hydration number on dissolution power (Ha et al., 2003). Reproduced with permission from American Chemical Society.

exposed to vapors of different solvents such as water, absolute ethanol or methanol. Further alignment of the fibers was done by passing the fibers through a channel and aligning the fibers before collection (Magaz et al., 2018). No changes were observed in the chemical composition of the fibers but the β-sheet content increased, particularly for the fibers exposed to water vapor. Methanol or ethanol treatment converted the silk structure partially into silk I and II forms compared to predominantly silk II structure for water vapor exposed fibers. The fibers formed into mats had modulus of 1.13 MPa, strength of 0.3 MPa after treating in water vapor compared to 11 MPa and 3.5 MPa for the fibers spun from the formic acid dope (Magaz et al., 2018) A mixed solvent system that combined calcium nitrate and methanol was used to dissolve silk fibroin and form fibers in a coagulation bath consisting of methanol, acetone or isopropyl alcohol (Ha et al., 2003). Extent of dissolution was dependent on the molar ratio of methanol to water (Fig. 4.8) and on the hydration level of calcium ions with increasing hydration power increasing the dissolution power (Fig. 4.8). Most of the fibers obtained were considered to have a random coil conformation (α-form) and the size and orientation of the β-sheets were also lower than the raw silk. The mechanical properties of fibers obtained using the calcium nitrate-methanol system was not reported and drawing may be necessary to obtain fibers with good properties. Variations in the properties of regenerated fibers were also discovered when silk from eight different varieties was dissolved using CaCl2/H2O/EtOH system at 85 °C for 3 min using a liquor ratio of 1:20 (Chung et al., 2015). Solution obtained was dried into powder and redissolved using formic acid and the properties of the silk fibers developed were studied. Mechanical properties and viscosity of solution were affected but amino acid composition and β-sheet content did not show major differences in the regenerated fibers obtained from the various silk worm varieties (Fig. 4.9).

4.3 N-Methylmorpholine N-oxide (NMMO) based solvent system for producing regenerated silk fibers NMMO is a common solvent for cellulose and other biopolymers. The possibility of dissolving B. mori and other silk in NMMO was investigated. Although up to

Silk: Materials, Processes, and Applications

Shear viscosity (Pa s)

86

1

N74 Geumgwangju SK Hansang II Imbakgalwon

0.1

(A)

0.1

Wonjam 125 Baekokjam Hongbak Wonwon 126

1

10

100

–1

Shear rate (s )

N74

Silkworm variety

SK Geumgwangju Hansang II Imbakgalwon Baekokjam Wonjam 125 Hongbak Wonwon 126

(B)

0.0

0.2

0.4

0.6

0.8

Shear viscosity (Pa s)

Fig. 4.9  Variations in the viscosity and steady state flow of regenerated fibroin solution obtained from different silkworms (Chung et al., 2015). Reproduced with permission from Elsevier.

10% protein could be dissolved, severe degradation of proteins was observed since it was necessary to use temperatures as high as 110 °C for the dissolution to occur. However, studies have shown that regenerated silk fibroin can be dissolved (10–25%) in NMMO and regenerated into fibers using temperatures between 85 and 100 °C. Single regenerated silk fibers could be obtained with strength of about 3 cN/tex (Xu et al., 2005). Similar results were also obtained when silk dissolved in aqueous NMMO was wet spun into ethanol solution and drawn to various ratios (Corsini et al., 2007). Increasing draw ratio improved crystal orientation and decreased the fiber diameter leading to substantially improved tensile properties (Table 4.9). However, tensile strength of the fibers obtained was highest at about 127 MPa with corresponding elongation of 12.7% which were lower than that of B. mori silk at 300–600 MPa and elongation of 19%. Relatively poor orientation of the crystals, presence of undissolved granules in the cross-section were some of the reasons suggested for the poor mechanical properties of the NMMO regenerated silk fibers (Corsini et al., 2007).

Draw ratio 1

Draw ratio 2

Draw ratio 3

Diameter, μm

Modulus, GPa

Strength, MPa

Elongation, %

2.0 2.0 2.0 7.0 7.0 17.0 17.0 17.0 17.0 23.0 35.0 55.0

1.0 1.8 3.8 1.0 2.8 1.0 1.4 1.7 2.0 1.0 1.0 1.0

2.0 3.6 7.6 7.0 19.6 17.0 23.8 28.9 34.0 23.0 35.0 55.0

88 ± 3.0 108 ± 12 73 ± 8 54 ± 30 40 ± 10 44.5 ± 10 35 ± 10 30 ± 8 28 ± 10 36 ± 2 28 ± 5 22 ± 2

3.8 ± 0.4 3.2 ± 0.9 5.3 ± 0.2 4.2 ± 0.1 5.6 ± 0.8 5.6 ± 0.9 7.1 ± 1.2 7.0 ± 1.3 7.2 ± 0.1 4.9 ± 0.0 5.7 ± 1.2 4.8 ± 0.4

43 ± 61 32 ± 8 127 ± 8 38 ± 1 99 ± 7 65 ± 9 115 ± 9 117 ± 5 116 ± 10 64 ± 18 75 ± 17 77 ± 7

1.1 ± 0.1 0.9 ± 0.1 12.7 ± 1.9 1.2 ± 0.1 14.0 ± 3.5 1.2 ± 0.2 2.8 ± 0.3 8.2 ± 2.2 8.6 ± 1.2 1.4 ± 0.4 1.6 ± 0.5 1.7 ± 0.1

Reproduced with permission from John Wiley and Sons.

Regenerated silk fibers87

Table 4.9  Changes in the diameter and mechanical properties of regenerated silk fibers at various drawing ratios (Corsini et al., 2007).

88

Silk: Materials, Processes, and Applications

Similar to the other solvent systems, fiber obtained using NMMO system were found to be influenced by conditions during expansion and post processing. Properties of NMMO regenerated fibers were particularly susceptible to water and a substantial decrease in modulus was observed. Fibers obtained were considerable brittle but drawing in water decreased the brittleness and provided ductile fibers (Table 4.10) (Plaza et al., 2008). However, the regenerated fibers had considerable lower properties compared to the native B. mori silks. Silk fibroin having degree of polymerization ranging from 200 to 250 kDa was first dissolved in LiBr and dry sponge like proteins were collected. Fibroin obtained was later redissolved using commercially available NMMO-H2O (50/50) solution and concentrated to various levels of water content (Marsano et  al., 2005). Fibers were extruded into a coagulation bath (methanol solution) to remove NMMO and later dried and maintained at standard conditions. To obtain fibers with good properties, it was necessary to maintain the high molecular weight of fibroin and the fibroin had to be freeze dried and later dissolved in NMMO and coagulated. In this process of dry-jet-wet system of fiber formation, fibers were first drawn in the coagulation bath and later in air before winding. Similar to the observations in other forms of regenerating silk fibers, drawing was found to increase degree of molecular orientation and hence tensile strength. However, fibers obtained in this process had considerably lower strength and elongation than the native silk fibroin fibers (Table 4.11). Investigations at the nanoscale using atomic force microscopy (AFM) (Fig.  4.10) have revealed that the regenerated fibers have similar nanoglobular structure but the nanoglobules in the regenerated fibers are much smaller and not oriented along the macroscopic axis of the fiber. Size of nanoglobules in B. mori silk were at an average of 23 nm compared to 10–15 nm in the regenerated fibers. In addition, two distinct sizes of the nanoglobules exist and are interdispersed along the fiber axis (Fig. 4.10) (Perez-Rigueiro et al., 2009). It was suggested that unlike natural fiber production, conditions during regeneration are not conducive for the formation of large size crystallites. Although post-­ treatment to the fibers such as drawing and annealing increase the tensile properties of the fibers, the size of the nanocrystals decreases even further. However, drawing improves the orientation of the nanocrystals along the fibers and the number of defects in the nanocrystal alignment decreases leading to improvement in properties, particularly elongation (Perez-Rigueiro et al., 2009). These oriented regions occur intermittently along the fiber and hence the properties of regenerated fibers are unable to match that of the B. mori silk fibers.

4.4 Regenerated fibers produced from spider silk Similar to B. mori silk, proteins from the spider Nephila clavipes were formed into regenerated fibers by dissolving in HFIP (2.5%). Protein solution was added into a syringe and extruded into a acetone bath using needles of inner diameter ranging from 150 to 250 μm. Fibers were allowed to coagulate into the acetone bath for 1 h and later drawn up to 3.5 times their original length (Seidel et al., 2000). As spun fibers were

Draw ratio 1.0 1.1 2.0 3.8 Bombyx mori

Tensile behavior Brittle Ductile

Dry

In water

Modulus, GPa

Strength, MPa

Elongation, %

Modulus, GPa

Strength, MPa

Elongation, %

3.8 ± 0.4 4.2 ± 0.1 7.2 ± 0.1 5.3 ± 0.2 14 ± 1

43 ± 6 38 ± 1 120 ± 10 127 ± 8 570 ± 60

1.1 ± 0.1 1.2 ± 0.1 8.6 ± 1.2 12 ± 2 20 ± 3

0.09 ± 0.03 0.14 ± 0.03 0.10 ± 0.01 0.07 ± 0.01 3.8 ± 0.3

10 ± 1 10 ± 2 25 ± 1 25 ± 1 450 ± 20

124 ± 2 70 ± 14 31 ± 5 28 ± 3 27 ± 3

Reproduced with permission from John Wiley and Sons.

Regenerated silk fibers89

Table 4.10  Extent of improvement in properties of regenerated silk fibers produced using NMMO solvent system with increasing draw ratio or presence of water (Plaza et al., 2008).

90

Table 4.11  Properties of silk fibers regenerated using NMMO and drawn to different ratios in the dry-jet-wet spinning system (Marsano et al., 2005). Draw ratio 1

Draw ratio 2

Diameter, μm

Birefringence

Strength, MPa

Elongation, %

Modulus, GPa

1 2 3 4 5 6 7 Native SF

1.0 15 18 23 15 15 15 –

1.0 1.0 1.0 1.0 1.5 2.3 2.7 –

133 ± 12 26.4 ± 1.0 26.1 ± 0.9 22.2 ± 0.4 22.7 ± 1.4 19.5 ± 0.8 18.5 ± 0.8 11.5 ± 0.2

– 0.018 0.017 0.018 0.023 0.027 0.030 0.060

43 73 77 67 70 104 120 610–690

2.0 1.0 1.5 1.1 4.0 29 35 15

2.6 7.4 7.5 8.6 8.3 8.7 7.2 15–17

Reproduced with permission from Elsevier.

Silk: Materials, Processes, and Applications

Sample

Regenerated silk fibers91

Fig. 4.10  Atomic force image of regenerated silk fibers show the presence of two distinct size of nanoglobules (demarked by the dotted line) (Perez-Rigueiro et al., 2009). Reproduced with permission from Elsevier.

sponge-like and had poor mechanical properties. Drawing aligns the molecules, increased orientation and resulted in formation of fibrils. A highest strength of 320 MPa and modulus of 8 GPa was obtained for the regenerated spider silk which was much lower than that of the native spider silk (875 MPa and 10.9 GPa). Improvement in the strength of the fibers after drawing was suggested to be due to the reinforcement of the fibers by the strong poly(alanine) crystallites. In terms of their crystalline structure, the fibers had d-spacings of 4.4 and 8.1 Å with the position and spacing at the 4.4 Ȧ belonging to the non-crystalline phase of silk. Also, the poly(alanine) crystals were suggested to be formed only after the second drawing. In an interesting study, instead of directly using the silk proteins, the ability of recombinantly produced spider silk proteins to enhance the structure and properties of regenerated silk fibers was studied (Zhu et al., 2010). Since it was found that the properties of native spider silk were related to the protein chain length and molecular weights, attempts were made to produce recombinant proteins with molecular weights between 250 and 320 kDa (Xia et al., 2010). Synthetically designed genes having molecular weight between 100 and 289 kDa similar to that found in natural MaSp1 of major ampullate silk gland of Nephila clavipes and having high glycine content between 43% and 45% were expressed in E. coli. Proteins expressed were collected and purified to specifically obtain the 96-mer silk protein. A protein yield of 1.2 g and purity of 90% was obtained. The proteins were dissolved in HFIP and extruded into fibers and later drawn 5 times their original length to improve mechanical properties. Fibers obtained had tensile strength of 508 MPa, elongation of 15% and modulus of 21 GPa, similar to that of native spider silk fibers (Fig. 4.11). However, the properties of the regenerated fibers were directly dependent on the length of the protein chain (Xia et al., 2010).

92

Silk: Materials, Processes, and Applications

Fig. 4.11  Steps involved in obtaining the recombinant dragline silk proteins (A); SDS electrophoresis and western blot stained proteins (B) mechanical properties (C–F) of the fibers obtained from different fragments of the proteins (Xia et al., 2010). Reproduced from National Academy of Sciences of the United States of America.

In was also found that two types of proteins found in spider silks could be explicitly reproduced in E. coli. These two proteins were added into a fibroin solution prepared using either HFIP or HFA. One of the proteins had a sequence of {DGG(A)6CGA}4 and the other had a sequence of {(GPGGSGPGGGY)2(GPGGAS)}4 which represents the crystalline region in dragline silk of Nephila calvipes and repeats sequence found in flagelliform silk of Nephila clavipes, respectively. Regenerated fibers obtained using HFIP as the solvent and drawn 3 × had higher strength than native silk fibers whereas the HFA fibers had lower strength due to the difference in the long range orientation in the crystalline region. Further, to overcome the limitations of expression in E. coli, the recombinant proteins were introduced into transgenic silkworms and the properties of the fibers obtained was studied. A marginal increase in strength at 395 MPa compared to 322 MPa for native silk was noticed. However, the amount of silk proteins introduced in the silk worms was only between 1% and 6%. Substantial increase in mechanical properties and productivity could be achieved by adding higher level of proteins into the silkworms (Zhu et al., 2010). In a similar approach, a particular construct W3 AcSp1 from acniform spider silk was reconstructed and used to develop regenerated fibers (Weatherbee-Martin et al., 2016). The protein powders were dissolved (8%) in 70% 1,1,1,3,3,3-hexafluoro-2-proponol (HFIP)/30% H2O) solution at 37 °C for 48 h to obtain a spinning dope viscosity of 15.6 mPa.s. The spinning dope was extruded into a coagulation bath made up to 95% ethanol/5% water at an extrusion rate of 16 μL/ min and were called the as-spun fibers. Post modifications to the fibers was done

Regenerated silk fibers93

Table 4.12  Comparison of the properties of undrawn and drawn acniform silk with other silks (Weatherbee-Martin et al., 2016). Fiber type

Strength, MPa

Extensibility, %

Toughness, MJ/m

Modulus, GPa

Diameter, μm

As spun W3 2 × drawn W3 4 × drawn W3 Hand-drawn W2 Hand-drawn W3 Hand-drawn W4 AcSp1

36 ± 12 48 ± 7 92 ± 8 67 ± 16 79 ± 28 115 ± 24 687 ± 56

3.1 ± 12 2.5 ± 1.1 2.6 ± 0.6 31 ± 11 21 ± 10 37 ± 11 86 ± 3

0.5 ± 0.2 0.6 ± 0.2 1.3 ± 0.3 18 ± 10 148 34 ± 14 376 ± 39

1.4 ± 0.8 2.5 ± 1 4.5 ± 0.5 1.7 ± 0.7 2.8 ± 0.8 2.4 ± 0.5 10 ± 4

23 ± 1 12 ± 1 9 ± 1 1.5 ± 0.1 1.8 ± 0.1 3.4 ± 0.3 0.35 ± 0.01

Reproduced with permission from American Chemical Society.

by subjecting to post-spin stretching in water up to 2 × or 4 × their original length. Post ­treatment decreased the diameter and improved tensile properties of the fibers (Table 4.12) due to improved extension in the amorphous regions and better orientation of β-sheets. However, unlike other spider silks, the fibers developed from W3 AcSp1 showed increased β-sheet content but with corresponding decrease in α-helix content which was not observed in other silks. Also, fibers obtained from the acniform silk had lower elongation even after drawing compared to native spider silk or regenerated silks obtained from other sources (Weatherbee-Martin et al., 2016). As discussed above, several studies have been done on regeneration of spider silk. Properties of the fibers obtained are dependent on the conditions during dissolution, particularly pH and molecular weight of the proteins. At low pHs, gel formation and decrease in spinning concentration of the silk protein solutions and the filaments obtained were irregular and had poor properties. At the specific pH, conformational transformation occurs from random coil to the β-sheet and silk fibers with excellent properties could be obtained (Cao et al., 2017). A summary of the previous attempts made to produce regenerated spider silk and corresponding properties are given in Tables 4.13 and 4.14.

4.5 Novel approaches for producing regenerated silk fibers Regenerated silk fibers (composite fibers) with high mechanical properties were obtained using the recombinant approach (You et  al., 2018). In this study, a series of peptide sequences containing either 2, 12, or 16 times the number or repetitive units in MaSp1 or 24 times the repetitive units in MaSp2 were obtained from the black widow spider L. hesperus. Using DNA manipulations and cloning techniques and transgenic modifications, the protein fragments were inserted at various positions into B. mori silkworms to develop heterozygous and homozygous transgenic lineages. Cocoons

94

Silk: Materials, Processes, and Applications

Table 4.13  Comparison of the mechanical properties of regenerated spider silk fibers produced using various solvents (Cao et al., 2017). Spinning dope Silkworm/Ca(NO3)2-CH3OH, 18% Nephila clavipes/HFIP, 2.5% Silkworm/water, 15% Silkworm/H3PO4 Silkworm/HFA, 10% Silkworm/TFA, 13% Silkworm/ Ca(NO3)2-4H2O2-MeOH Silkworm/LiBr.H2O-EtOHH2O, 20% Silkworm/EmimCl, 10% Silkworm/NMMO. H2O, 20% Silkworm/NMMO. H2O, 13% Silkworm/NMMO. H2O, 17%

Coagulation bath

Post spin draw

Strength, GPa

Elongation, %

Acetone

NO

NO

NO

Acetone (NH4)2 SO4 Na2SO4 and (NH4)2 SO4 MeOH MeOH MeOH

9 4 9.3

0.32 0.26 0.15

4–8 78.9 10.1

3 3 No

0.18 0.92 No

16 18.2 1.5

MeOH

3.2

0.08

11

MeOH MeOH EtOH EtOH

– 3.6 2.7 2.8

0.0 0.40 7.2 0.13

– – 35 14

obtained from the modified silk worms were treated with water at various conditions and single fibers were extracted for determining the mechanical properties (You et al., 2018). It was observed that the fibers obtained not only differed between the two ­transgenic lineages but also among the same lineage depending on the point of insertion of the vectors (Table 4.15). A very high linear relationship was found between the protein chain length and mechanical properties without any effect on the morphology or other properties of the fibers. A maximum stress of 336 MPa and strain of 27% was obtained for the composite fibers. A protein with many single functional motifs and sufficiently long protein chains were considered to be necessary to obtain regenerated protein fibers with exceptional mechanical properties (You et al., 2018). A biomimetic microfluidic system (Fig. 4.12) was built to replicate the silk glands in silkworm and obtain regenerated silk (Peng et  al., 2015; Luo et  al., 2014). Silk fibroin solution was prepared using a pH 4.8 buffer and calcium chloride. The solution was injected into the microchip device and allowed to flow at a rate of 2 μL/min through a 250 μm width orifice. Fibers were further drawn through a 100 μm width orifice to improve mechanical properties. As spun fibers coming out of the 250 μm orifice had a diameter of 12 μm similar to that of degummed silk fibers. However, these fibers had lower strength than the natural fibers. Comparatively better fibers were obtained using the 100 μm channel with fiber diameter of 2 μm after drawing which also provided considerably higher mechanical properties than the natural silk fibers. A Young’s modulus of 19 GPa, elongation of 27% and strength of 614 MPa was obtained for the regenerated fibers which was considerably higher than the natural

Regenerated silk fibers95

silk fibers (5 GPa, 16% and 354 MPa, respectively). Higher mechanical properties of the regenerated fibers was suggested to be due to the higher levels of β-sheet content, smaller crystal size and better crystal orientation (Luo et al., 2014). Properties of fibers obtained using the microfluidic system were also affected by the relative humidity (RH) during fiber formation (Table 4.16). Diameter of the fibers obtained at lower RH (40%) were found to be higher than that obtained at 50% RH. Also, fibers obtained at lower humidity had lower amounts of β-sheet structures, low degree of chain and crystalline orientation resulting in inferior properties (Luo et al., 2014). In another study, a biomimetic microfluidic spinning device was developed to replicate the natural production of silk by spiders and obtain regenerated spider silk fibers with excellent properties (Peng et al., 2017). In this approach, recombinant spider silk proteins were obtained using Escherichia coli. Proteins produced had a molecular weight of 47 kDa, 16 monomer repeats and were water soluble. To obtain the spinning solution, the recombinant proteins were dissolved using sodium chloride in water at a concentration of 42%. Spinning was done an a microfluidic device with channel width of 2065 μm at the start and tapered down to 265 μm at the end. Fibers formed were extruded into an ethanol coagulation bath at a rate of 3 cm/s and collected. In another option, fibers from the coagulation bath were reeled in air at a speed of 3 cm/s and collected. Mechanical properties of the regenerated fibers obtained using this approach were better than that of degummed fibers due to the better orientation of the crystallites along the fiber axis. In a similar approach, a capillary spinning equipment was developed to produce regenerated silk fibers. Degummed B. mori silk was dissolved using 9.0 M LiBr at 40 °C for 2 h. Silk solution was adjusted to various pHs and various salts were added to mimic the metal ion concentrations present during natural spinning of the silk fibers. To form the fibers, the solution was forced through a syringe pump at a rate of 1 μL/min. Fibers formed had a diameter of about 12 μm and were air dried and collected for further analysis (Wei et al., 2011a,b). Fiber forming conditions such as concentration of the spinning solution, extrusion and take-up speed and draw ratio played a critical role in determining the properties of the fibers produced. Under the experimental conditions studied, an optimal aspect ratio of 133 for the capillary, capillary length of 20 mm and take-up velocity of 30 mm/s was determined. Although the breaking strength of the as spun fibers was only between 20 and 63 MPa, drawing the fibers 3 × in 80% ethanol solution increased the strength to 298 MPa and elongation up to 16%. Increase in β-sheet conformation, crystallinity and orientation were suggested to be the reasons for the increase in mechanical properties after drawing (Wei et al., 2011a,b). Differences in the peaks at 1253 and 1105 cm−1 representing the random coil or α-helix conformation and the small peaks at 1085 and 1230 cm−1 produced by the β-sheet conformations confirmed the findings. Considerable variations in the properties of the fibers were also observed due to post-treatments using various types and ratios of alcohols (Wei et al., 2012). Based on Raman spectroscopy, it was concluded that post-treatment methods significantly increased the absorption bands at 1085 cm−1, 1232 cm−1, and 1666 cm−1 due to the increase in β-sheet conformations. Consequently, substantial increase in the mechanical properties were also noticed (Table 4.17). Fibers with strength lower than degummed silk but >3 times elongation

96

Silk: Materials, Processes, and Applications

Table 4.14  Table List of references and properties of regenerated silk fibers developed using various approaches (Koeppel and Holland, 2017). Processing parameters

RSF wet spinning

Mw, kDa

Protein conc., wt% or (w/v)%

–

n.s.a

–

12

–

20

–

10

–

10

– – –

15.6 15.6 13

– – – –

13 13 15.6 17

–

10

– –

12.3 12 (w/v)

– –

29 17

–

17

– –

15 15

–

16

Solvent, –

Coagulant, –

Concentrated magnesium nitrate 85% Phosphoric acid +5.7 wt% dimethylformamide 40 wt% LiBr-H2O in ethanol; ethanol with different water contents Hexafluoroacetone hydrate (HFA) Hexafluoro-isopropanol (HFIP) 98% Formic acid 98% Formic acid Aqueous NMMO monohydrate +0.7% n-propyl gallate Formic acid Trifluoroacetic acid (TFA) 98% Formic acid Aqueous NMMO monohydrate +0.7% n-propyl gallate Hexafluoro-isopropanol (HFIP) 98% Formic acid Hexafluoro-isopropanol (HFIP) PEG/LiBr Aqueous NMMO monohydrate +0.7% n-propyl gallate Aqueous NMMO monohydrate +0.7% n-propyl gallate Water Hexafluoro-isopropanol (HFIP) Water

Saturated ammonium solution 25% Aqueous sodium sulfate Methanol, ethanol, isopropanol with 10% aq. LiBr Methanol Methanol Methanol Methanol Ethanol

Methanol Methanol Methanol Ethanol

Ethanol/methanol Methanol Methanol Methanol/water Methanol

Methanol

Aqueous ammonium sulfate Methanol Aqueous ammonium sulfate

Regenerated silk fibers97

Fiber properties Draw ratio, –

Strength, MPa

Extensibility, %

Stiffness, GPa

Toughness, MJ/m3

Diameter, μm

n.s.

2.5 g/den

20–25

n.s.

n.s.

n.s.

9.3

2.1 g/den

10.1

n.s.

n.s.

n.s.

3.2

130b

11

6.7b

12.9c

118.5

3

321.2b,c

16.1c

5.3b,c

37.6b,c

40-50b

3

193c

19c

5.2c

28.2c

40-50b

2 5 2.7

103.8b,c 257.5b,c 120

40b,c 16.4b,c 35

4.1b,c 5.5b,c 7.2

38b,c 30.6b,c 38.9c

189c 119c 18.5 ± 0.8

3 3 4.5 2

1077.3 ± 173b 959.0 ± 149.1b 269.4b,c 127 ± 8

29.3 ± 11.9b 18.1 ± 6.8b 19.5b,c 12.7 ± 1.9

39.9 ± 6.1b 43.2b 4.9b,c 5.3 ± 0.2

257.8c 156.7c 38.7b,c 20.3c

35b 21b 220–270 73 ± 8

n.s.

109.7b

25

n.s.

n.s.

68b

5 3

285.1 ± 10.7b 400.5c

14.0 ± 1.7 20.7c

7.2b,c 4.3c

30.4b,c 51.3c

100c 40c

1.1 n.s.

128.8b,c 313.6d

7.6b 8.5d

6b 13.4d

6.8b,c 20.5d

20–50 41

7.2

172.4d

48.4d

5.1d

55.5d

47

6 3

450 ± 20 408 ± 80

27.7 ± 4.2 21 ± 3

12.5c 73 ± 0.2

100.6 ± 6.3b 51.5c

10.8 ± 2.4 n.s.

6

390 ± 50

32.1 ± 5.8

15.2 ± 3.3

109.1 ± 18.8b

n.s. Continued

98

Silk: Materials, Processes, and Applications

Table 4.14  Continued Processing parameters

RSF dry spinning

Recombinant wet spinning

Mw, kDa

Protein conc., wt% or (w/v)%

–

17

–

17

– – – – – – – –

20 15 12 15 13 15 13 20

–

20

–

50

–

40–60

– –

50 20 and 25

–

44

60 62

>23% 25–30 (w/v)

71

10 to 12%

284.9

20 (w/v)

appr. 50 70

n.s. 30 (w/v)

Solvent, –

Coagulant, –

Aqueous NMMO monohydrate +0.7% n-propyl gallate Aqueous NMMO monohydrate +0.7% n-propyl gallate Water Water CaCl2-FA Water Water Water Water Water + (MES)-(Tris) buffer (pH adjustment) + CaCl2 (Ca2+ adjustment) Water + (MES)-(Tris) buffer (pH adjustment) + CaCl2 (Ca2+ adjustment) Water + (MES)-(Tris) buffer (pH adjustment) + CaCl2 (Ca2+ adjustment) Water + CaCl2 (Ca2+ adjustment) Water Formic acid + CaCl2 (Ca2+ adjustment) Water + CaCl2 (Ca2+ adjustment)

Methanol

Hexafluoro-isopropanol (HFIP) Hexafluoro-isopropanol (HFIP) Hexafluoro-isopropanol (HFIP) Hexafluoro-isopropanol (HFIP) Hexafluoro-isopropanol (HFIP)

Methanol

Aqueous ammonium sulfate Aqueous ammonium sulfate Water Aqueous ammonium sulfate Aqueous ammonium sulfate Aqueous ammonium sulfate Aqueous ammonium sulfate –

–

–

– – – – Methanol and water 90% Isopropanol Isopropanol 90 vol% Methanol in water Isopropanol Isopropanol

Regenerated silk fibers99

Fiber properties Draw ratio, –

Strength, MPa

Extensibility, %

Stiffness, GPa

Toughness, MJ/m3

Diameter, μm

n.s.

336.4d

7.38d

18.5d

20.3d

n.s.

5.3

257.6d

35.3d

7.4d

51.9d

18.4

4 9 4 9 4 9 4 3

221 ± 64 314 ± 19 470.4 ± 53.5 450 ± 30 98c 450 ± 30 98c 301.5 ± 70.6

30 ± 4 37 ± 4 38.6 ± 6.3 27.3 ± 4.6 58.9c 27.3 ± 4.6 58.9c 35.8 ± 21.9

11.2c 10.4c 6.9 ± 2.1 18.9 ± 1.1 37.8c 18.9 ± 1.1 37.8c 6.2 ± 1.7

46.4c 105.3 ± 10b 105.3 ± 15.5b 91.0 ± 7.4 53.5c 91.0 ± 7.4 53.5c 104.8 ± 37.8b

100c n.s. 12.8 ± 4.6 15 ± 4.7b ~25 15 ± 4.7b ~25 5.7

n.s.

295.2 ± 92.2

74.8 ± 47.4

5.8 ± 4

155.9 ± 94.5b

6.4 ± 1.5

4

337.7c

24.6c

11.1c

55.8c

10c

4

357.3 ± 84.3

34.1 ± 8.1

8.8c

86.5c

6.3 ± 2.3

2 2

614 333c

27 35.1c

19 8.8c

136.4b 90.9c

2 20–30

4

541.3 ± 26.1

19.3 ± 4.8

9.4 ± 1.2

76.4 ± 22.8b

9.0 ± 1.3

5 n.s.

269.6b 49.6 ± 19.4

43.4b 15.8 ± 6.1

13.2b 1.1 ± 1.0

101.4b 10.6 ± 10.2

20b 15.8 ± 6.1

0

49.5 ± 7.8

3.6 ± 2.6

0.4 ± 0.3

4.7c

74.1 ± 33.9

5

508 ± 108

15 ± 5

21 ± 4

81.5c

n.s.

5

246.7d

50.6d

4.5d

91.7d

46 ± 2

n.s.

132.5 ± 49.2

22.8 ± 19.1

5.7 ± 2.4

23.7 ± 18.5

17.4 ± 5 Continued

100

Silk: Materials, Processes, and Applications

Table 4.14  Continued Processing parameters

a

Mw, kDa

Protein conc., wt% or (w/v)%

58

26–27 (w/v)

62

26–27 (w/v)

66/48

30 (w/v)

66/48

30 (w/v)

45

20 (w/v)

45

20 (w/v)

66

15 (w/v)

378 dimer 86.5

8 to 10%

8.6

45–60 (w/v)

65

25 (w/v)

50–75 286

12 (w/v) 10–17 (w/v)

47 47 33

12 (w/v) 10–17 (w/v) 50 (w/v)

45–60 (w/v)

Solvent, –

Coagulant, –

Hexafluoro-isopropanol (HFIP) Hexafluoro-isopropanol (HFIP) Hexafluoro-isopropanol (HFIP) Hexafluoro-isopropanol (HFIP) Hexafluoro-isopropanol (HFIP) Hexafluoro-isopropanol (HFIP) Hexafluoro-isopropanol (HFIP) Hexafluoro-isopropanol (HFIP) Hexafluoro-isopropanol (HFIP) Hexafluoro-isopropanol (HFIP) Hexafluoro-isopropanol (HFIP) + >88% formic acid in 4:1 ratio Water Water + Tris/HCl or Naphosphate buffer NaCl/water NaCl/water Aqueous buffer at pH 8

90% Isopropanol/10% water

n.s., not specified. Units converted. Values extracted from graphs/images. d Values converted from true stress/strain into engineering stress/strain. Reproduced with permission through Creative Commons Attribution (CC-BY) License. b c

90% Isopropanol/10% water Isopropanol Isopropanol 95% Isopropanol 95% Isopropanol Isopropanol ZnCl2 and FeCl3 in water Isopropanol Isopropanol Isopropanol

Isopropanol Water + isopropanol Ethanol Ethanol Aqueous solution (sodium acetate, pH 2.5–7.5)

Regenerated silk fibers101

Fiber properties Draw ratio, –

Strength, MPa

Extensibility, %

Stiffness, GPa

Toughness, MJ/m3

Diameter, μm

2–25

127.5 ± 23.0

52.3 ± 23.6

4.4 ± 1.0

54.6 ± 23.6

28.3 ± 6

2–25

96.2 ± 28.8

29.6 ± 20.5

3.8 ± 2.1

22.6 ± 15.7

14.0 ± 8.7

3

37.6 ± 20.4

53.9 ± 68.0

3.4 ± 1.1

17.4 ± 20.1

29.1 ± 5.4

3

59.6 ± 19.2

4.8 ± 8.6

4.3 ± 0.9

2.5 ± 5.4

29.1 ± 5.4

6

121.9 ± 5

18 ± 1

3.9c

17.4 ± 1.2

24.5 ± 0.3

3.5

95.1 ± 3.3

25 ± 4

2.6c

20.7 ± 3.8

30.5 ± 0.5

3

150.6 ± 31.3

84.5 ± 37.8

4c

89.1 ± 23.9

15.1 ± 1.3

5

308 ± 57

9.6 ± 3

9.3 ± 3

24.4c

10

4

53.5 ± 18.0

18.0 ± 21.6

2.90 ± 1.1

9.3 ± 10.9

31.5 ± 4.5

4

39.0 ± 7.4

181.3 ± 103.5

1.6 ± 0.4

59.3 ± 37.2

36.0 ± 5.9

1.5/2

221.7 ± 11

56 ± 6.6

n.s.

102.5 ± 13.6

29.0 ± 1.1

2–2.5 6

192.2 ± 51.5 370 ± 59

28.1 ± 26 110 ± 25

8.3c 4 ± 1

33.8 ± 33.6 189 ± 33

n.s. 27 ± 10

n.s. n.s. 0

62.3 ± 17.2 286.2 ± 137.7 162 ± 8

3.5 ± 1.2 18.3 ± 12.8 37 ± 5

4 ± 2.8 8.4 ± 4.3 6 ± 0.8

1.6 ± 0.9 37.7 ± 28.8 45 ± 7

34c 14c 12 ± 2

102

Table 4.15  Comparison of the properties of composite silk fibers obtained using transgenically modified silkworms using different vector lengths and insertion positions (You et al., 2018). Number of repetitive units

Maximum Stress, MPa

Maximum Strain, %

Young’s modulus, MPa

Toughness, MJ/m2

Lan10-G4 MASP1–2 G4 MASP1–12 G4 MASP1–16 G4

Wild type 2xre-MaSp1 12xre-MaSp1 16xre-MaSp1

190 ± 45 214 ± 72 296 ± 91 327 ± 86

20.5 ± 7.5 72 ± 23 91 ± 27 86 ± 28

1283 ± 568 2553 ± 772 3515 ± 1250 3603 ± 1215

27 ± 14 34 ± 14.9 56 ± 18 63 ± 20

Lan10-G4 MASP1–2 G4 MASP1–12 G4 MASP1–16 G4

Wild type 2xre-MaSp1 12xre-MaSp1 16xre-MaSp1

202 ± 52 241 ± 37 321 ± 72 326 ± 62

21 ± 7.4 23 ± 5.3 25 ± 3.1 27 ± 3.2

3681 ± 1019 4178 ± 1034 5114 ± 1469 5276 ± 1467

31 ± 14 41 ± 15 58 ± 16 60 ± 13

Transgenic lineages

Heterozygous G4

Silk: Materials, Processes, and Applications

Reproduced with permission through Creative Commons Attribution 4.0 International License.

Regenerated silk fibers103 W(k)

The strengthening glass slide

L

Oxygen plasma treatment

Wo Wt

Oxygen plasma treatment

x Single-stage exponential function

(C)

(D)

The strengthening glass slide

Elongation part RSF aqueous solution

Shearing part The inlet 2mm

PDMS layer with channel pattern RSF fiber roller

The strengthened PDMS inlet

85µm

(A)

PDMS cover Side view direction

(B)

100/250µm The outlet PDMS with channel pattern

PDMS cover

Fig. 4.12  Schematic depiction of the experimental set up for microfluidic spinning (A), crosssection of the channel in the device (B), mathematical representation of the elongation in the microfluidic channel (C) and side view of the microfluidic with reinforcing glass slides (D) (Luo et al., 2014). Reproduced with permission from Elsevier.

were obtained. Dissolution of fibroin in LiBr and regenerating the fibers using alcohol solutions has been reported to reduce the molecular weight from 300,000 kDa to about 100,000 kDa. Consequently, the crystallinity, heat of degradation and tensile strength were lower than raw silk fibers (Zuo et al., 2006). Tensile strength of the fibers was 0.82 cN/dtex and orientation degree was 77% compared to 2.56 cN/dtex and 87.1% for native silk fibers. However, the lower orientation and weaker properties were considered to increase the biodegradability of silk and hence useful for certain applications. In a recent study, silk nanofibers with structure of raw fibers were developed using low intensity ultrasound assisted acid hydrolysis (Hu et al., 2019). Degummed B. mori silk fibers were added into various concentrations of sulfuric acid and maintained for 0.5 to 5 h at temperatures between 30 and 90 °C. Hydrolyzed fibers were subject to homogenization at 500 W and 20 kHz for 15 min to separate the silk nanofibers. Up to 90% yield of silk nanofibers could be obtained depending on the dissolution conditions. Nanofibers formed had diameters between 4 and 18 nm and length of 306 nm. FTIR and X-ray diffraction studies did not show any major difference in crystal structure between the raw and regenerated fibers. However, reduction in the amorphous regions during hydrolysis increased the % crystallinity of the fibers from 38% to 44% (Hu et al., 2019).

104

Table 4.16  Comparison of the properties of regenerated silk fibers obtained at different humidities and fibroin concentrations (Luo et al., 2014). Fibroin, %

Humidity, %

Fiber diameter, μm

Modulus, GPa

Strength, MPa

Strain, %

Breaking energy, kJ/kg

β-sheet content, %

Crystallinity, %

44 44 47 47

40 50 40 50

9.0 ± 1.3 7.5 ± 0.8 6.5 ± 0.3 5.4 ± 0.5

9.4 ± 1.2 9.3 ± 2.4 9.1 ± 0.7 10.5 ± 0.7

541 ± 26 481 ± 31 484 ± 34 493 ± 45

19 ± 4.8 17 ± 4.7 14.8 ± 2.2 12.7 ± 2.3

57 ± 17 43 ± 16 38 ± 9 33 ± 7

38.9 26.7 47.1 34.7

36.5 28.1 29.8 26.6 Silk: Materials, Processes, and Applications

Reproduced with permission from Elsevier.

Sample

Diameter, μm

Stress, MPa

Strain, %

Modulus, GPa

Breaking energy, kJ/kg

As-spun 90% Methanol 80% Ethanol 90% Isopropanol Saturated (NH4)2 SO4 Degummed silk

16.2 ± 1.8 9.1 ± 0.7 8.5 ± 1.5 8.2 ± 1.7 15.3 ± 1.4 10.2 ± 0.8

29.9 ± 13.5 162.8 ± 13.7 199.2 ± 51.9 188.5 ± 35.9 51.1 ± 23.0 314.2 ± 63.6

3.6 ± 1.9 14.6 ± 12.3 55.4 ± 21.3 4.4 ± 1.0 2.6 ± 0.8 16.4 ± 5.1

1.1 ± 0.5 5.3 ± 0.8 6.8 ± 1.2 4.8 ± 1.8 3.0 ± 0.3 4.0 ± 2.9

0.5 ± 0.4 19.6 ± 9.3 58.3 ± 32.1 3.3 ± 0.4 0.5 ± 0.2 30.4 ± 11.3

Reproduced with permission from Elsevier.

Regenerated silk fibers105

Table 4.17  Changes in the properties of the regenerated silk fibers due to various post treatments in comparison to as-spun and degummed silk fibroin fibers (Wei et al., 2012).

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References Cao, K., Liu, Y., Ramakrishna, S., 2017. Recent developments in regenerated silk fibers. J. Nanosci. Nanotechnol. 17, 8667–8682. Chen, X., Knight, D.P., Shao, Z., Vollrath, F., 2001. Regenerated Bombyx silk solutions studied with rheometry and FTIR. Polymer 42 (25), 09969–09974. Cho, H.J., Yoo, Y.J., Kim, J.W., Park, Y.H., Bae, D.G., Um, I.C., 2012. Effect of molecular weight and storage time on the wet-and electro-spinning of regenerated silk fibroin. Polym. Degrad. Stab. 97 (6), 1060–1066. Chung, D.E., Kim, H.H., Kim, M.K., Ki, H.L., Park, Y.H., In, C.U., 2015. Effects of different Bombyx mori silkworm varieties on the structural characteristics and properties of silk. Int. J. Biol. Macromol. 79, 943–951. Corsini, P., Perez‐Rigueiro, J., Guinea, G.V., Plaza, G.R., Elices, M., Marsano, E., Carnasciali, M.M., Freddi, G., 2007. Influence of the draw ratio on the tensile and fracture behavior of NMMO regenerated silk fibers. J Polym Sci B 45 (18), 2568–2579. Fang, G., Huang, Y., Tang, Y., Qi, Z., Yao, J., Shao, Z., Chen, X., 2016. Insights into silk formation process: correlation of mechanical properties and structural evolution during artificial spinning of silk fibers. ACS Biomater Sci. Eng. 2 (11), 1992–2000. Goujon, N., Wang, X., Rajkowa, R., Byrne, N., 2012. Regenerated silk fibroin using protic ionic liquids solvents: Towards an all-ionic-liquid process for producing silk with tunable properties. Chem. Commun. 48 (9), 1278–1280. Ha, S.-W., Park, Y.H., Hudson, S.M., 2003. Dissolution of Bombyx mori silk fibroin in the calcium nitrate Tetrahydrate—methanol system and aspects of wet spinning of fibroin solution. Biomacromolecules 4 (3), 488–496. Hu, Y., Yu, J., Liu, L., Fan, Y., 2019. Preparation of natural amphoteric silk nanofibers by acid hydrolysis. J. Mater. Chem. B https://doi.org/10.1039/C8TB03005G. Jin, Y., Zhang, Y., Hang, Y., Shao, H., Hu, X., 2013. A simple process for dry spinning of regenerated silk fibroin aqueous solution. J. Mater. Res. 28 (20), 2897–2902. Kim, H.J., Um, I.C., 2014. Effect of degumming ratio on wet spinning and post drawing performance of regenerated silk. Int. J. Biol. Macromol. 67, 387–393. Koeppel, A., Holland, C., 2017. Progress and trends in artificial silk spinning: a systematic review. ACS Biomater Sci. Eng. 3 (3), 226–237. Koh, L., Cheng, Y., Teng, C., Kin, Y., Loh, X., et al., 2015. Structures, mechanical properties and applications of silk fibroin materials. Prog. Polym. Sci. 46, 86–110. Luo, J., Zhang, L., Peng, Q., Sun, M., Zhang, Y., Shao, H., Hu, X., 2014. Tough silk fibers prepared in air using a biomimetic microfluidic chip. Int. J. Biol. Macromol. 66, 319–324. Magaz, A., Roberts, A.D., Faraji, S., Nascimento, T.R.L., Medeiros, E.S., Zhang, W., Greenhalgh, R.D., Mautner, A., Li, X., Blaker, J.J., 2018. Porous, aligned, and biomimetic fibers of regenerated silk fibroin produced by solution blow spinning. Biomacromolecules 19 (12), 4542–4553. Marsano, E., Corsini, P., Arosio, C., Boschi, A., Mormino, M., Freddi, G., 2005. Wet spinning of Bombyx mori silk fibroin dissolved in N-methyl morpholine N-oxide and properties of regenerated fibres. Int. J. Biol. Macromol. 37 (4), 179–188. Peng, Q., Shao, H., Hu, X., Zhang, Y., 2015. Role of humidity on the structures and properties of regenerated silk fibers. Progr. Nat. Sci. Mater. Int. 25 (5), 430–436. Peng, Q., Shao, H., Xuechao, H., Zhang, Y., 2017. Microfluidic dry-spinning and characterization of regenerated silk fibroin fibers. JoVE (J. Visual. Exp.) 127, e56271. Perez-Rigueiro, J., Biancotto, L., Corsini, P., Marsano, E., Elices, M., Plaza, G.R., Guinea, G.V., 2009. Supramolecular organization of regenerated silkworm silk fibers. Int. J. Biol. Macromol. 44 (2), 195–202.

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Phillips, D.M., Drummy, L.F., Naik, R.R., Hugh, C., Fox, D.M., Trulove, P.C., Mantz, R.A., 2005. Regenerated silk fiber wet spinning from an ionic liquid solution. J. Mater. Chem. 15 (39), 4206–4208. Plaza, G.R., Corsini, P., Pérez‐Rigueiro, J., Marsano, E., Guinea, G.V., Elices, M., 2008. Effect of water on Bombyx mori regenerated silk fibers and its application in modifying their mechanical properties. J. Appl. Polym. Sci. 109 (3), 1793–1801. Xia, X.-X., Qian, Z.-G., Ki, C.S., Park, Y.H., Kaplan, D.L., Lee, S.Y., 2010. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber. Proc. Natl. Acad. Sci. 107 (32), 14059–14063. Seidel, A., Liivak, O., Calve, S., Adaska, J., Ji, G., Yang, Z., Grubb, D., Zax, D.B., Jelinski, L.W., 2000. Regenerated spider silk: Processing, properties, and structure. Macromolecules 33 (3), 775–780. Sun, M., Zhang, Y., Zhao, Y., Shao, H., Hu, X., 2012. The structure–property relationships of artificial silk fabricated by dry-spinning process. J. Mater. Chem. 22 (35), 18372–18379. Um, I.C., Kweon, H.Y., Park, Y.H., Hudson, S., 2001. Structural characteristics and properties of the regenerated silk fibroin prepared from formic acid. Int. J. Biol. Macromol. 29 (2), 91–97. Um, I.C., Kweon, H.Y., Lee, K.G., Ihm, D.W., Lee, J.-H., Park, Y.H., 2004a. Wet spinning of silk polymer: I. Effect of coagulation conditions on the morphological feature of filament. Int. J. Biol. Macromol. 34 (1–2), 89–105. Um, I.C., Ki, C.S., Kweon, H.Y., Lee, K.G., Ihm, D.W., Park, Y.H., 2004b. Wet spinning of silk polymer: II. Effect of drawing on the structural characteristics and properties of filament. Int. J. Biol. Macromol. 34 (1–2), 107–119. Weatherbee-Martin, N., Xu, L., Hupe, A., Kreplak, L., Fudge, D.S., Liu, X.-Q., Rainey, J.K., 2016. Identification of wet-spinning and post-spin stretching methods amenable to recombinant spider aciniform silk. Biomacromolecules 17 (8), 2737–2746. Wei, W., Zhang, Y., Zhao, Y., Shao, H., Xuechao, H., 2012. Studies on the post-treatment of the dry-spun fibers from regenerated silk fibroin solution: Post-treatment agent and method. Mater. Des. 36, 816–822. (1980–2015). Wei, W., Zhang, Y., Shao, H., Hu, X., 2011a. Posttreatment of the dry-spun fibers obtained from regenerated silk fibroin aqueous solution in ethanol aqueous solution. J. Mater. Res. 26 (9), 1100–1106. Wei, W., Zhang, Y., Zhao, Y., Luo, J., Shao, H., Hu, X., 2011b. Bio-inspired capillary dry spinning of regenerated silk fibroin aqueous solution. Mater. Sci. Eng. C 31 (7), 1602–1608. Xu, Y., Zhang, Y., Shao, H., Hu, X., 2005. Solubility and rheological behavior of silk fibroin (Bombyx mori) in N-methyl morpholine N-oxide. Int. J. Biol. Macromol. 35 (3–4), 155–161. Yan, J., Zhou, G., Knight, D.P., Shao, Z., Chen, X., 2009. Wet-spinning of regenerated silk fiber from aqueous silk fibroin solution: Discussion of spinning parameters. Biomacromolecules 11 (1), 1–5. Yao, J., Masuda, H., Zhao, C., Asakura, T., 2002. Artificial spinning and characterization of silk Fiber from Bombyx mori silk fibroin in Hexafluoroacetone hydrate. Macromolecules 35 (1), 6–9. Yoshioka, T., Tashiro, K., Ohta, N., 2016. Molecular orientation enhancement of silk by the hot-stretching-induced transition from α-helix-HFIP complex to β-sheet. Biomacromolecules 17 (4), 1437–1448. You, Z., Ye, X., Ye, L., Qian, Q., Wu, M., Song, J., Che, J., Zhong, B., 2018. Extraordinary mechanical properties of composite silk through hereditable transgenic silkworm expressing recombinant major Ampullate Spidroin. Sci. Rep. 8, 1–14.

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Zhang, F., Lu, Q., Ming, J., Dou, H., Liu, Z., Zuo, B., Qin, M., Li, F., Kaplan, D.L., Zhang, X., 2014. Silk dissolution and regeneration at the nanofibril scale. J. Mater. Chem. B 2 (24), 3879–3885. Zhang, F., Lu, Q., Yue, X., Zuo, B., Qin, M., Li, F., Kaplan, D.L., Zhang, X., 2015. Regeneration of high-quality silk fibroin fiber by wet spinning from CaCl2–formic acid solvent. Acta Biomater. 12, 139–145. Zhou, G., Shao, Z., Knight, D.P., Yan, J., Chen, X., 2009. Silk fibers extruded artificially from aqueous solutions of regenerated Bombyx mori silk fibroin are tougher than their natural counterparts. Adv. Mater. 21 (3), 366–370. Zhu, Z., Kikuchi, Y., Kojima, K., Tamura, T., Kuwabara, N., Nakamura, T., Asakura, T., 2010. Mechanical properties of regenerated Bombyx mori silk fibers and recombinant silk fibers produced by transgenic silkworms. J. Biomater. Sci. Polym. Ed. 21 (3), 395–411. Zuo, B., Dai, L., Wu, Z., 2006. Analysis of structure and properties of biodegradable regenerated silk fibroin fibers. J. Mater. Sci. 41 (11), 3357–3361.

Electrospun silk fibers

5

5.1 Electrospun fibers from B. mori silk fibroin 5.1.1 Electrospinning using chemical solvent systems Electrospun fibers were obtained from silk fibroin retaining the nanofibrillar structure seen in natural silk (Zhang et  al., 2014). To form the electrospun fibers, degummed silk was directly dissolved using LiBr-FA as solvents for 3 h at room temperature. A 12 kV potential was applied for electrospinning with the distance between needle and syringe being 10 cm and flow rate was 1 mL/h. After electrospinning the fibers were immersed in 75% ethanol for 1 h. Up to 35% fibroin could be dissolved depending on the concentration of CaCl2 and type of silk used. SEM images showed that the dissolution process formed nanofibrils and micelles with diameters ranging from 20 to 200 nm. Electrospinning solutions (1–10%) resulted in formation of fibers under all concentrations. However, diameter of the fibers increased from 10 nm up to 1200 nm with increasing solution concentration. Finer fibers could also be obtained even after removing LiBr. Before treating with ethanol, strong absorption peaks were noticed at 1648 cm−1 (amide I), 1548 cm−1 (amide II) and 1232 cm−1 (amide III) suggesting that the fibroin in the fibers was amorphous in nature. However, conformational transformation to silk II occurs when the fibers are dipped in 75% ethanol solution as evident from the absorption peaks at 1625 cm−1, 1517 cm−1, and 1260 cm−1 from the amide I, amide II and amide III regions, respectively. Under stress, the nanofibers were seen to become thin and orient themselves towards the fiber axis which provides good tensile properties. Electrospun fibers obtained had tensile stress ranging from 0.9 to 9.4 MPa, elongation from 2.8% to 183% and modulus from 9.2 to 352 MPa depending on the draw ratio and type of fibroin used (Zhang et  al., 2014). In another study, B. mori cocoons were degummed, dissolved using CaCl2 and dialyzed to obtain fibroin. The fibroin was redissolved in formic acid and electrospun into fibers. Several electrospinning conditions were studied to ensure that the fibers with desired properties were obtained. It was reported that fibers with diameters less than 100 nm were produced using a solution concentration of 12–15% and electric field of 3–4 kV (Sukigara et al., 2003). Continuing their study, the researchers also observed that the concentration of the solution played a critical part in the formation of fibers with 9% fibroin concentration producing fibers in the range of 8–223 nm and 15% fibroin providing fibers in the range of 12–397 nm (Ayutsede et al., 2005). Similarly, distance between the tip of the needle to the collecting drum was 5, 7, or 10 cm which produced fibers having average diameters of 70, 61 and 55 nm. Crystallinity ratio of the nanofibers was lower than that in the native silk whereas the β-sheet content was higher. The fiber mats obtained has strength of 7.3 MPa, modulus of 515 MPa and elongation of 3.2% (Ayutsede et al., 2005). Post extrusion modifications such as drawing have also shown to substantially improve the mechanical properties of electrospun fibers. B. mori silk was degummed, Silk: Materials, Processes, and Applications. https://doi.org/10.1016/B978-0-12-818495-0.00005-3 © 2020 Elsevier Ltd. All rights reserved.

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dissolved in 9 M LiBr and proteins separated using a molecular weight cut off of 14,000 ± 2000 kDa. Electrospun fibers were obtained using this protein made into a 33% concentration in LiBr. After electrospinning, the fiber mats were subject to several stages of drawing and immersion in 90% ethanol solution for 30 min. Various draw rates (0.1–0.9 mm/s) and draw ratios (1.1–1.4×) were used to modify the fibers (Fan et al., 2013). Increasing draw ratio increased the β-sheet content thus reducing the unordered regions and also leading to the increase in % crystallinity. The as-spun fibers had a breaking strength of 1.8 MPa and elongation of 8.7% which increased to 8.6 MPa. Mats obtained were considered to be suitable for use as sutures since the suture retention strength of the 1.4× drawn fibers was 1.3 N, similar to that of natural tissue (Fan et al., 2013). Unique fibroin tubular scaffolds were developed for filtration, medical and other applications (Zhou et al., 2009). B. mori silk was degummed using 0.5% Na2CO3 and later dissolved using CaCl2/H2O/C2H5OH and a fibroin solution of 37–41 wt% was used for electrospinning. To form the tubular scaffolds, the solution was electrospun onto a rotating metal rod. Diameters of the tube could be controlled by varying the size of the rod and flow rate between 0.15 and 0.6 mL/min. Scaffolds obtained were immersed in methanol for 40 min for crystallization to occur. Fig. 5.1 shows the images of the fibers and the digital picture of the tubular scaffold formed (Zhou et al., 2009). Treating with methanol increased the strength of the scaffolds from 0.4 MPa to 3.6 MPa but decreased elongation from 26% to 12%. The strength and elongation of the scaffolds was considered to be suitable for use as vascular grafts. A detailed investigation was done to understand the effect of electrospinning conditions on the properties of the fibers obtained (Zhang et al., 2012). Silk fibroin obtained from Bombyx mori was dissolved using LiBr and dialyzed to obtain a 7% solution. This solution was diluted and made into films and dried using slow or fast drying which led to the formation of films containing nanofilaments or nanospheres. The films obtained were redissolved in 98% formic acid solution for 3 h and electrospun into fibers. Nanofibers formed were treated with 75% ethanol for 1 h to improve the mechanical properties and water stability. Fibers with diameters between 25 and 47 nm were obtained using the nanosphere solution compared to 54–334 nm for the fibers obtained using the nanofilaments. Diameter of the fibers was dependent on the concentration of the fibroin solution used (Fig. 5.2). Treating with 75% ethanol resulted in transformation

Fig. 5.1  SEM image of electrospun silk fibroin fibers (A) and digital pictures of the tubular scaffold (B) and the scaffold immersed in 75% methanol (C) (Zhou et al., 2009). Reproduced with permission from Elsevier.

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Fig. 5.2  SEM images of the morphology of silk fibroin nanofibers obtained using various concentrations of the nanosphere solutions (Zhang et al., 2012). Reproduced with permission from American Chemical Society.

to silk and the fibers displayed the characteristic amide peaks at 1630 cm−1 (amide I) and 1525 cm−1 (amide II) wavelengths. In terms of mechanical properties, the fibers obtained using the filament approach had breaking strength of 17.7 MPa and elongation of 25%, considerably higher than the properties of fibroin nanofibers reported earlier (Zhang et al., 2012). Electrospun tubular grafts suitable for vascular tissue engineering were fabricated using a blend of recombinant spider silk proteins pNSR32, poly(caprolactone) and chitosan (Zhao et  al., 2013) in 98% formic acid. Scaffolds obtained were composed of nanofibers in tubular form with internal diameter of 3 mm and 1.2 mm. Inclusion of the spider silk proteins considerably enhanced the adhesion and proliferation and also the ability of chitosan to form fibers. When implanted in mice, the scaffolds were able to maintain their structural integrity for at least 8 weeks and hence were suggested to be suitable as vascular grafts (Zhao et al., 2013). B. mori fibroin was degummed to obtain a sericin content of 0–16% as shown in Table 5.1. An 18% fibroin solution containing different levels of sericin was electrospun using a ternary solvent system. Considerable variations were observed in the morphology, structure and properties of the fibers depending on the extent of sericin in them. A sericin content of less than 1% (degumming ratio between 25% and 28%) was found to be necessary to obtain good electrospinnability and fiber properties (Ko et al., 2013).

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Table 5.1  Extent of degumming achieved at different degumming conditions (Ko et al., 2013). Degumming condition Sodium oleate, w/v

Sodium carbonate, % w/v

Degumming time, hours

Degumming ratio, %

Residual sericin content, %

0.6 0.10 0.024 0

0.4 0.067 0.016 0

2 1 1 1

28.5 25.6 19.5 12

0 0.6 8.2 16.0

Reproduced with permission from Elsevier.

Similarly, the electric field during electrospinning was also observed to have a profound influence on the properties of the fibers obtained (Park and Um, 2017). The rate of electrospinning was controlled by changing the concentration of the solution (7–11%) and the electric field (1.0–2.5 kV/cm). Under these conditions, a highest production rate of 3 mL/h was achieved. Electric field did not show a major influence on the fiber diameters at low solution concentrations but changed the diameters from about 100 nm up to 4 μm at high solution concentration and electric fields (Park and Um, 2017). Kim et al. also used a ternary solvent system (CaCl2/CH3CH2OH/H2O) (1/2/8 in mole ratio) at 70°C for 6 h to produce electrospun fibers from B. mori fibroin (Kim et al., 2003). Regenerated fibroin was redissolved in formic acid and electrospun into fibers using solution concentrations between 6 and 15 wt%. Fibers obtained had diameters in the range of 30 to 120 nm with average diameter of 80 nm. Fibrous mats formed were highly porous with a porosity of 76% and pore volume of 2 mL/g (Kim et al., 2003). It was reported that fibroin in the as spun fibers converted in to β-sheet form within 10 min after extrusion and hence provided good properties. In another process of all aqueous electrospinning of fibroin, two degumming processes, one with and one without sodium carbonate was used for degumming. It was found that boiling in plain water without any chemicals was sufficient to obtain desired level of degumming (30%). However, prolonged boiling caused damage to the fibroin if the boiling time was higher than 120 min. Proteins with higher molecular weight (180–350 kDa) could be obtained by the new degumming approach. However, these proteins could not be electrospun into fibers. Changing the pH of the spinning dope was considered to improve the electrospinnability (Table 5.2). Considerable variation was observed in the electrospinnability of the solution (Kishimoto et al., 2017). Solutions with pH between 10.5 and 11.5 were able to form stable fibers when the concentration of the solution was 5%. A viscosity range between 14 and 100 MPa s was found to give satisfactory electrospinnability. It was inferred that the concentration of the solution was more critical than the molecular weight of the proteins. Using pH 10.5, fibers with average strength of about 0.83 MPa and elongation of 12% was obtained. However, the diameter of the fibers obtained was not reported.

Concentration of SF aqueous solution (wt%) Sample code

pH

4

5

6

7

8

9

10

12

14

16

18

20

22

SF0–120

7 10.5 11 11.5 7 10.5 7 10.5

× Δ Δ Δ – – – –

× ○ Δ Δ – – – –

× ○ Δ Δ – – – –

× ○ Δ Δ – – – –

× ○ ○ Δ × Δ × –

× ○ ○ ○ × Δ × –

× ○ ○ ○ × ○ × –

– – – – × ○ × –

– – – – × ○ × Δ

– – – – × ○ × ○

– – – – × ○ × ○

– – – – × ○ × ○

– – – – × ○ × ○

SF0.5–30 SF2.5–30

○: Fibers, Δ: fibers with beads, ×: no spinning, –: not experiment. Reproduced with permission from Elsevier.

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Table 5.2  Influence of pH and concentration of silk on the electrospinnability (Kishimoto et al., 2017).

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5.1.2 Aqueous system for production of electrospun silk fibroin solutions Due to the economics and complexities involved in using expensive solvents or copolymers, attempts have been made to develop all aqueous B. mori spinning solutions using a different degumming and dissolution process (Chen et al., 2006; Kishimoto et  al., 2017). In one approach, degummed silk was dissolved in a ternary solvent system consisting of CaCl2/H2O/EtOH in 1/8/2M ratio for 40 min at 80 °C. Obtained solution was concentrated to 28–37% by slow stirring at 50–60 °C (Chen et  al., 2006). Electrospinning was done using a voltage between 12 and 20 kV and a tip to collector distance of 18 cm. Mats of fibers (500 nm to 10 µm) obtained were immersed in a 90/10 methanol/water solution for 10 min. Both XRD and FTIR studies indicated that treatment with methanol increased the β-sheet content due to crystallization. Similarly, the mechanical properties of the mats increased from 0.8 MPa to 1.49 MPa and elongation increased to 1.6% from 0.8% after treating with methanol. Using the same ternary solvent system, fibroin was extracted and redissolved using1,1,1,3,3,3-hexafluoro-2-isopropyl alcohol (HFIP) solution (Jeong et al., 2006). Post treatment was done by immersing the fibers in water, ethanol, methanol or proponol to induce crystallization of the fibroin. Fibers electrospun had diameters of about 380 nm but were soluble in water. Treating the fibers with alcohols changed the fibroin structure from α to β-form i.e. silk I to silk II but the extent of change was dependent on the type of alcohol and duration of treatment. Water vapor was also found to induce the conformational change and make the fibers insoluble (Jeong et al., 2006). Up to 3 times increase in elastic modulus was also observed due to the treatment. However, the electrospinnability and properties of the fibers obtained were also found to be dependent on the extent of degumming (Ko et  al., 2013). A new method of producing electrospun fibers using aqueous solutions and no chemicals was considered to be suitable for large scale applications (Kishimoto et al., 2017). pH of the silk fibroin solution in the posterior gland of silkworm from which the fibers are extruded is about 6.9. It was hypothesized that similar conditions could be used to produce electrospun fibers. To mimic the conditions in the silkworm gland, degummed silk was dissolved in LiBr and later dialyzed and concentrated into a 20% aqueous solution. This solution was adjusted to pH 6.9 by adding 0.1 M citric acid‑sodium hydroxide-hydrochloric acid buffer. A solution concentration of 30%, voltage of 40 kV and distance of 20 cm were reported to produce the finest fibers (1700 nm) (Zhu et al., 2007). X-ray diffractogram showed peaks at 9.1, 20.6 and 24.6° characteristic of the silk II (β-sheet) structure in the degummed silk whereas the electrospun mats had weak peaks at 27.6 suggesting the presence of mostly amorphous silk I structure. However, using a similar electrospinning technique, fibers with diameters ranging from 400 to 1000 nm were obtained by Wang et al. (2005). Electrospun fibers had a diffraction peak at 17.6° which shifted to 18.6° with increase in electrospinning voltage. It was suggested that the silk fibroin molecules were able to undergo conformational change from Silk I to Silk II during electrospinning. However, the concentration of the spinning solution and other electrospinning parameters determined the extent of change in the silk II conformation with the presence of both α-helix and

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β-sheets (Wang et al., 2005). In yet another study on electrospinning using aqueous solutions, silk fibroin (80–90 kDa) after dissolution in LiBr and dialysis was electrospun into fibers (Wang et al., 2006a,b). A solution concentration of 28% (viscosity of 250 mPa s) produced fibers with diameters in the range of 400–800 nm, considerably smaller than the diameters of natural silk fibers. Ribbons of fibers were obtained when the concentration of the fibroin solution was 38% due to high viscosity. Partial transformation of the silk structure from α to β-form occurred during electrospinning (Wang et al., 2006a,b). To improve the properties of electrospun fibers produced using aqueous solutions, a new process of electrospinning was developed. In this approach, the spinning solution was subject to shear under dehumidified and forced air. Shearing action increased the viscosity and simultaneously decreased the surface tension between 40 and 38 mN/m. Considerable increase in β-sheet content from 17% to 34% occurred assisting fiber formation. Fibers with average diameter of 183 nm were obtained when the solution concentration was 17%. Mats obtained with strength of 17 MPa and elongation of 12% suitable for cell adhesion and proliferation were obtained (Singh et al., 2016). Another alternative approach has also been considered to produce electrospun fibers using aqueous solutions. In this approach, several additives including LiBr, NaCl or Na2HOPO4 were added into aqueous solution to increase the electrical conductivity of the solution and obtain better fibers. Electrospinning was done using a spinneret to winder distance of about 10–20 cm and voltage between 0.9 and 1.35 KV. Fibers formed were immersed in pure ethanol for 10 min to make them insoluble in water. Optimum conditions for the electrospinning was with a solution concentration between 11% and 17% resulting in the thickness of mat being 0.21 ± 0.03 μm to 0.49 ± 0.06 μm (Cao et al., 2009). Substantial decrease were observed in the diameter of the fibers due to the addition of the salts which was suggested to be due to the higher net charge density that increases the electrical force and leads to the formation of thinner fibers. Raman spectroscopy indicated that the amide I band around 1667 cm−1 decreased after the ethanol treatment and that the amide III band shifted from 1249 cm−1 to 1104 cm−1. These changes indicate that the β-sheet content increases due to the conversion of silk I structure to silk II structure. Similar changes were also observed in the x-ray diffraction pattern suggesting corresponding increase in the crystallinity (Cao et al., 2009). An attempt was made to produce electrospun silk fibers by using a spinning dope similar to that found in the native silkworm glands. To do this, B. mori silk was dissolved in LiBr to obtain a 20% solution. This solution was adjusted to various pHs and electrospun into fibers using a flow rate of 2 mL/h (and solution concentration from 20% to 38% (Zhu et al., 2008). Concentration of the proteins and pH played a critical role in fiber formation. Reducing pH resulted in finer fibers and the morphology of the fibers changed from being belt-like to becoming a uniform cylinder. Conformational change from random coil (silk I) to silk II form also occurred with reduction in pH. However, no major effect was observed on the crystallization of the fibers with change in pH. A biomimic approach was also used to produce electrospun fibers with diameters as low as 50 nm (He et al., 2008).

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5.2 Silk fiber blends Since pure fibroin is not easily electrospinnable, a blend of silk fibroin and PEO was used for electrospinning (Jin et al., 2002). Fibroin was extracted using LiBr and the solution formed was dialyzed using a MWCO of 2000–3500. PEO (900,000 g/mol) was added into the fibroin solution to obtain a silk/fibroin ratio of 80/20 and a 7.1% solution. This solution was electrospun into fibers and the fibers were immersed into a 90/10 methanol/water solution for 10 min at room temperature and later washed in water for 48 h at 37 °C to remove the PEO from the mats (Wang et al., 2004). Blend fibers with average diameter of 800 ± 50 nm were obtained but the diameter was highly dependent on the ratio of silk/PEO and spinning conditions used (Jin et al., 2002; Wang et al., 2004). Although the as spun fibers did not show any crystalline peaks after treating with methanol and washing with water, the PEO was removed which led to the formation of broad peaks at 2θ of 19.80° corresponding to a crystalline spacing of 4.43 Ȧ a characteristic of the β-sheet structure. Similar changes were also observed from the FTIR spectrum conforming the transformation of the silk from α-coils to β-sheets. Morphologically, the fibers were observed to have a hard shell and softer core due to the gradual crystallization of the fibers in the methanol solution. Based on the AFM measurements, the as spun fibers had modulus of 0.75 GPa which increased to 1.28 GPa and 8 GPa after methanol and water treatments, respectively (Wang et al., 2004). In another study, silk/PEO blend (80/20) was electrospun into fibers having average diameter of 700 nm and the fibers obtained were studied for potential use as tissue engineering scaffolds (Jin et al., 2004). At a higher magnification, the fibers were seen to be composed of nanofibrils of about 110 nm in diameter. Tensile strength and elongation of the electrospun mats after methanol treatment were 13.6 MPa and 4% respectively. Properties of the fibroin/PEO blend fibers were also found to be dependent on the humidity and speed of the collecting drum (Meinel et al., 2009). B. mori silk fibroin was degummed and dissolved using 9 M LiBr at 55 °C and dialyzed to obtain a 12.5% solution. PEO was added into the fibroin solution and the blend was electrospun using a voltage between 12 and 15 kV and rotating drum speed between 1000 and 4000 rpm. Methanol treatment followed by washing with water was done to change the structure and remove PEO. Fibers obtained were bead free when electrospun at 30% RH with diameters of 530 ± 100 nm. Morphology of the fibers, particularly orientation changed considerably with increase in rotating speed of the collection drum (Fig. 5.3). The fibers obtained were suggested to be suitable for tissue engineering and other medical applications. Multiwell carbon nanotubes having length of 1 μm and diameters of 15–20 nm were combined (0.5–2%) with fibroin in formic acid solution and electropsun. Fibers with average diameter of 85 nm were obtained. CNTs showed uniform distribution and alignment along the length of the fibers. In addition to the characteristic β-sheet peaks seen at 20.2° and 23.3°, another peak was observed at 25.8° which increased in intensity as the amount of CNTs were increased. This suggests that some molecular bonding occurred between the fibroin proteins and CNTs. Due these interactions, substantial increase in tensile properties were achieved (Zuo et al., 2017). Adding 1% CNTs into the silk increased modulus from 32 MPa to 107 MPa and strength increased

Electrospun silk fibers117

Fig. 5.3  Scanning electron microscope images of the electrospun SF/PEO fibers at two different RHs (A and B) and three different rates of collecting the fibers (Meinel et al., 2009). Reproduced with permission from Elsevier.

from 6 to 10 MPa. Since MWTs increased orientation (alignment) and restricted the movement of the molecules, elongation of the fibers decreased to 9% from the initial level of 19%. Further increase in the MWT content decreased all the tensile parameters considerably (Table 5.3). Contrary to the tensile properties, electrical conductiivty increased continually with the increase in the CNT content (Zuo et al., 2017). Table 5.3  Changes in the properties of the silk fibers containing various levels of CNTs (Zuo et al., 2017).

CNT, %

Tensile strength, MPa

Modulus, MPa

Elongation, %

Electrical conductivity, s/cm

0 0.5 1.0 1.5 2.0

6.3 ± 0.8 7.2 ± 0.6 9.9 ± 1.2 6.1 ± 0.9 4.3 ± 0.4

32.5 ± 4.3 58.5 ± 2.0 107.5 ± 9.2 91.8 ± 10.6 85.9 ± 15.5

19.3 ± 1.2 12.3 ± 0.8 9.3 ± 1.5 6.7 ± 0.6 5.0 ± 0.9

5.04 ± 10−14 8.66 ± 10−6 1.2 ± 10−4 8.61 ± 10−8 7.15 ± 10−9

Open access publication.

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Complex nanofibrous mats consisting of silk fibroin, polyacrylonitrile (PAN) and TiO2 nanoparticles was developed for potential use as dye absorbent (Aziz et  al., 2017). Silk fibroin solution was obtained by dissolving in 12% regenerated fibroin sponges in 98% formic acid for 3 h. PAN was dissolved (12%) using DMF. These two solutions were combined and electrospinning was done using voltage between 16 and 22 kV and maintaining a flow rate of 1 mL/h. Based on FTIR spectra, it was suggested that PAN was able to form hydrogen bonds with the peptide linkages in the silk fibroin. Diameters of the fibers increased from about 58 nm up to 117 nm with increase in PAN content. Compared to neat PAN, the hybrid fibers had considerably higher strength and modulus. Extent of dye removal from the fibers was dependent on the conditions. At pH 3, an high removal efficiency of 92% was obtained for anonic dyes due to the high electrostatic attraction between the anionic dyes and the positively charged surfaces of the electrospun mats (Aziz et al., 2017).

5.3 New systems of electrospinning Using a new system called stable jet electrospinning, nanofibers with excellent mechanical properties were developed from a blend of B. mori silk fibroin and PEO. B. mori fibers were dissolved in a ternary solvent system consisting of CaCl2/CH3CH2OH/ H2O to obtain fibroin. The fibroin obtained along with various ratios of PEO was dissolved using formic acid (Yi et al., 2018). Blend solution was electrospun into fibers using the new stable jet spinning (without jet whipping and fiber spraying) and also the conventional method. Fibers obtained were treated with methanol/water (90/10) solution for 10 min to convert the silk fibroin to β-sheet configuration and make it insoluble in water. Later, the fibers were immersed in distilled water to remove the water soluble PEO. Fig. 5.4 shows the process used to obtain the stable jet electrospun

Fig. 5.4  Schematic representation of the new stable jet electrospinning (A), Changes in viscosity (B) and jet length (C) with varying SF: PEO ratio and SEM images (D) showing the morphology of the fibers at different polymer ratios. (Yi et al., 2018). Reproduced with permission from Royal Society of Chemistry.

Electrospun silk fibers119

Table 5.4  Changes in the properties of the electrospun fibers with increase in concentration of silk in the solution (Yi et al., 2018). Conc. of silk, %

PEO/silk ratio

Total conc.

Conductivity, μS

Applied field strength, kV/cm

Fiber diameter, nm

7.2 7.2 7.2 6.3 6.0 5.3 4.1 3.0 PEO

0/100 1/3 1/4 1/4 1/3 1/3 1/2 2/3 100/0

7.2 8.8 8.3 7.3 7.4 6.6 5.8 4.8 4.0

240 217 192 185 209 182 175 154 61

0.50 0.60 0.60 0.53 0.55 0.55 0.55 0.55 0.60

– 840 ± 80 740 ± 150 700 ± 100 730 ± 50 720 ± 100 850 ± 50 880 ± 50 410 ± 90

Reproduced with permission from Royal Society of Chemistry.

fibers, properties of the protein solution used for electrospinning and morphology of the blend fibers obtained. Average diameter of the fibers obtained varied from 1.8 to 2.4 μm depending on the ratio of fibroin to PEO. Tensile properties of the electrospun mats obtained using the new spinning system were considerably higher than those obtained using the conventional electrospinning (Table 5.4). The new spinning system assisted in achieving high molecular orientation and also better conversion from α-­helix to the β-sheet form which provides substantially better mechanical properties even under wet conditions (Table 5.5). A new system of electrospinning was used to reduce the surface tension of the electrospinning solution (Fig. 5.5). When compressed air was passed through the solution, numerous small size bubbles were formed on the surface of the solution. These bubbles gets charged under an electric field and eventually force themselves into a upward jet which helps in the formation of the nanofibers. It was suggested that fibers with Table 5.5  Comparison of silk fibroin and fibroin/PEO blend membranes with and without various treatments obtained using the stable jet electrospinning and conventional electrospinning (ES) approaches (Yi et al., 2018).

Sample

Condition

Tensile Strength, MPa

Fibroin/PEO Methanol Without PEO Without PEO Regular ES Methanol ES Methanol ES

Dry Dry Dry Wet Dry Dry Wet

50.9 ± 1.1 73.9 ± 5.2 41.5 ± 7.8 14.3 ± 1.6 2.3 ± 0.1 5.2 ± 0.7 1.1 ± 0.1

Elongation, %

Modulus, MPa

Energy at break, kJ/m2

29.2 ± 1.1 2.4 ± 0.3 1.3 ± 0.1 125.1 ± 10.4 24.5 ± 1.9 4.6 ± 0.3 137 ± 10.5

1186 ± 165 2426 ± 87 1824 ± 615 160 ± 7.7 24 ± 3.7 186 ± 23.2 2.4 ± 0.3

364 ± 26.2 30.6 ± 2.4 11.0 ± 0.1 337 ± 37 13.7 ± 0.3 2.4 ± 0.3 22.1 ± 2.5

Reproduced with permission from Royal Society of Chemistry.

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e

nd

ou

Gr

o dc

r

cto

lle

–

Jets Bubbles

Solution reservoir

Metal electrode

+ Gas tube

DC high voltage generator

Gas pump

N2

Fig. 5.5  A new system of electrospinning that reduces surface tension and enables production of electrospun fibers (He et al., 2008). Reproduced with permission from Elsevier.

diameters less than that of spider silk (2.5–4 μm) could be obtained at high rates since there were no needles or other conditions that restrict the efficiency of electrospinning (He et al., 2008). A unique two-fluid electrospinning system was adopted to form submicron fibers from fibroin. A 8% silk fibroin solution was added into a 2% PEO solution to obtain a silk: PEO ratio of 16:1. Silk and PEO in two separate storage tanks were extruded concurrently to form a core-shell fiber with PEO as shell and fibroin as the core (Fig. 5.6). Once the fibers were formed, the mats were stored under high humidity (90% RH) at 25 °C for over 72 h. Later, the mats were washed with water for 5 days at room temperature. A ratio of outer and inner flow rates had to be maintained in the range of 6:1 to 10:1 to obtain uniform and continuous core-shell fibers. Storing the spun fibers

Two-fluid electrospinning

PEO

Silk

Post Treatment

Core/Shell Fiber

Water Extraction

Crystallized silk component

Small diameter Crystallized silk fiber

Fig. 5.6  Schematic representation of the process of developing crystallized silk fiber using a two polymer system (Wang et al., 2006a, b). Reproduced with permission from American Chemical Society.

Electrospun silk fibers121

at high humidity lead to crystallization and further washing in water made the fibers insoluble. It was suggested that storing at high humidity disrupts the hydrogen bonds and facilitates orientation of the crystals and hence reduced solubility. It was reported that fibers with diameters as low as 170 nm could be obtained with this approach (Wang et al., 2006a,b).

5.4 Electrospun fibers from wild silks In addition to B. mori or spider silk, other wild silks have also been used to develop electrospun fibers. B. mori and Samia cynthia ricini which is one of the common wild silks were degummed and later dissolved using HFA. The solutions were electrospun into fibers using a voltage between 15 and 30 kV and distance of 10–15 cm. Fibers developed were treated with methanol to remove HFA (Ohgo et al., 2003). The two silks produced fibers with diameters ranging from 100 to 1000 nm but under different optimum conditions. NMR spectroscopy results showed that the Ala residues transformed into silk II structure in the B. mori silk whereas such transformation did not occur in the S. ricini silk (Table 5.6). Due to the lower level of β-sheets, the S. ricini silk fibers had maximum strength of 20 MPa and elongation of 20% whereas the B. mori silk had a strength of 15 MPa and elongation of 40%. Further, recombinant hybrid silk proteins were developed to mimic the structure of B. mori and S. ricini. A primary structure of [GGAGSGYGGGYGHGYGSDGG (GAGAGS)3]6 was developed in recombinant protein mats containing fibers of about 100 nm (Ohgo et al., 2003). Genetically engineered dragline spider silk proteins were used to understand the electrospinnability and changes in secondary structure after electrospinning (Stephens et al., 2005). The recombinant spider silk proteins were obtained using E. coli and dissolved in HFIP. Based on the frequencies and assignments of various polypetides and their position before and after the electrospinning, it was reported that the regenerated proteins and electrospun fibers were initially composed of α-helixes, redissolving in HFIP converts the α-helices into β-form (Stephens et al., 2005).

Table 5.6  Frequencies and assignment of Polypeptides in Fourier Transform Raman Spectroscopy (Stephens et al., 2005). Assignments

Frequency, cm−1

Relative intensity

1650–1667 1666–180 1664–1666

Moderate/strong Strong Moderate

1270 1230 1240

Weak Strong moderate

Amide I

Α-helix β-sheets disordered

Amide II

Α-helix β-sheets disordered

Reproduced with permission from American Chemical Society.

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Table 5.7  Properties of electrospun fibers obtained from Eri and Tassar silks before and after treatment with ethanol (Andiappan et al., 2016). Eri silk

Tassar silk

Property

Untreated

Ethanol treated

Untreated

Ethanol treated

Average pore diameter, μm % Crystallinity Crystallite size (Ȧ) Tensile stress, MPa Tensile Strain, % Water uptake, %

2.0 40.9 26.6 0.86 4.9 75.3

0.4 46.7 33.3 1.4 2.4 59.5

2.85 41.1 26.0 0.5 3.0 73.5

1.4 42.7 38.5 1.4 3.7 57.2

Reproduced with permission through creative common license.

Fibroin extracted from the silk glands of fifth instar larvae of the wild silk, Antheraea assama was used to develop electrospun silk fibers. The silk proteins were washed in water and dissolved using 1% sodium dodecyl sulfate solution and then dissolved solution was dialyzed to obtain the protein concentrate suitable for electrospinning. The protein solution was combined with PVA (13%) at 1:4 fibroin to PVA ratio and electrospun at a voltage of 25 kV and flow rate of 0.8 ± 0.1 mL/h and rotating drum speed of 500 rpm. Obtained fibers were immersed in 70% ethanol for 2 h for the β-sheet formation to occur. Further, the membranes were coated with recombinant spider silk proteins to increase cell adhesion and proliferation (Chouhan et al., 2018). Although the morphology or the mechanical properties of the membranes were not reported, it was found that the fibers were able to support the growth of cells and could be ideal for tissue engineering applications. A comparison of the properties of nanofiber membranes obtained from silk fibroin from two different wild silks (Eri and Tasar) was done by Andiappan et al. (Andiappan et al., 2016). The two silks were degummed and fibroin extracted was dissolved using trifluoroacetic acid to form a 15% protein solution which was electrospun into fibers at a voltage of 20 kV. Post treatment with ethanol was also done to improve properties and stability in aqueous media. Fibers produced from eri silk had diameters between 400 and 500 nm compared to 800–1000 nm for tasar silk. The variation in diameter was suggested to be due to the molecular weight differences in the fibroin obtained from the two silks. Considerable differences were also observed in the pore size, pore diameter and water uptake % of the two fibers (Table 5.7). It was suggested that either eri or tassar silk could be chosen to obtain electrospun fibers with distinct properties depending on the end use requirements.

5.5 Electrospun fibers from spider silk proteins Dragline spider silk (N. clavipes) were dissolved in HFIP at room temperature. Spiders silk (0.23–1.25%) dissolved in the HFIP solution within 20 min whereas B. mori silk takes 5 months at room temperature to dissolve completely. Both the ­solutions were

Electrospun silk fibers123

electrospun resulting in fibers of 8–200 nm for the spider silk and 6.5 nm to 100 nm for B. mori silk. Diameters of the fibers obtained for both the silks were considerably smaller than the natural silks (2–5 μm). In fact, the smallest electrospun fibers obtained had diameters that correspond to 200 molecules in the cross-section of N. clavipes and 150 nm for the B. mori silk (Zarkoob et al., 2004). B. mori silk fibers were thermally stable up to 245 °C and the spider silk was stable up to 280 °C. This approach was proposed to be unique and provided fibers with sub-nano diameters, not possible with other approaches (Zarkoob et  al., 2004). Similar to HFIP, hexafluoro acetone (HFA) hydrates were also able to dissolve silk and suggested to provide better properties than HFIP (Yao et al., 2002). It took 2 h for regenerated silk fibroin film to dissolve in HFA compared to 2 days in HFIP. Comparatively, silk fibroin fibers and S. ricini silk could dissolve in 2 months and 5 days, respectively but were undissolvable in HFIP. Electrospun fibers were coagulated using methanol and further subject to steam annealing to improve crystallization and orientation. It was suggested that the regenerated fibers obtained using HFA were more similar structurally to the native fibers compared to those obtained using other approaches (Yao et al., 2002). Dragline silk obtained from the spider Araneus ventricosus was dissolved using HFIP and electrospun into a coagulation bath made of various organic solvents. Post treatment of the fibers was done by treating with 90% aqueous ethanol or methanol solution for 30 min and later sterilized using UV disinfection. Fibers obtained by coagulation in acetone had diameters between 600 nm and 7 μm and had considerable beads. Comparatively, fibers spun into methanol solution had average diameters of 700 nm and were relatively bead free (Yu et al., 2014). In addition to the size of the fibers, the type of coagulation bath used affected the conversion into β-sheets, crystallinity and mechanical properties. Membranes developed had good biocompatibility for PC 12 cells and had slow degradation profile required for cell culture applications (Yu et al., 2014). Silk fibroin blends were also prepared by combining with poly (D-lactic acid) PLA and electrospining using an emulsion system. Spider silk was obtained from Agelema labyrinthica and dissolved using formic acid at 70 °C for 7 days and PLA synthesized was dissolved using acetone (Zhou et al., 2008). Ratios of the fibroin and PLA were varied from 5%, 10%, 15%, 20%, 25%, and 30% and fibers were obtained with diameters in the range of 200 nm to 1.2 μm (Table 5.8). Considerable changes were observed in the primary and secondary structure of the fibroin with addition of PLA (Table 5.9). These changes were also reflected in the tensile properties. Pure PLA fibers had strength and elongation of 1.8 MPa and 20%, respectively compared to 3.7 MPa and 62% for the blend containing 30% silk (Zhou et al., 2008). Analysis of the electrospun fibers showed an increase in the β-sheet content due to the addition of fibroin which leads to increase in strength. In a similar study, spider silk was collected from Araneus ventricosus and dissolved in HFIP. PLA having molecular weight of 100,000 was also dissolved in HFIP and the two solutions were combined in different ratios. Electrospinning was done using 12 kV and extrusion rate of 1 mL/h and fibers formed were collected on a rotating mandrel at 1300 rpm (Yu and Sun, 2015). Three-dimensional membranes with different surface features and morphologies were obtained after treating with acetone (Fig. 5.7). The structure and properties of the 3D formation was dependent on the thickness, ratio of

124

Table 5.8  Properties of 3D membranes developed from spider silk fibroin and PLLA in dry and wet condition (Yu and Sun, 2015). Strength, MPa

Elongation, %

Initial Modulus, MPa

Longitudinal

Transverse

Longitudinal

Transverse

Longitudinal

Transverse

1:1 2:3 1:2 1:2 wet

17.9 ± 2.7 15.9 ± 2.6 13.7 ± 2.6 9.3 ± 2.7

6.9 ± 1.2 4.7 ± 0.5 2.3 ± 0.1 2.2 ± 0.1

10.2 ± 2.5 6.9 ± 1.9 10.3 ± 3.4 7.2 ± 2.5

4.9 ± 0.8 6.6 ± 1.3 5.3 ± 0.6 6.8 ± 1.2

4.1 ± 0.2 0.4 ± 0.00 0.3 ± 0.1 0.1 ± 0.08

3.9 ± 0.2 0.4 ± 0.03 0.33 ± 0.09 0.18 ± 0.08

Reproduced with permission from John Wiley and Sons.

Silk: Materials, Processes, and Applications

Weigth Ratio (Protein/PLLA)

Electrospun silk fibers125

Table 5.9  Changes in the diameters of the fibers with increasing ratio of fibroin (Zhou et al., 2008). Fiber diameter

Secondary structure proportion (%)

Samples

Mean ± SD

Max

Min

β-sheets

Random

α-helix

turns

Pure silk Regenerated fibroin PDLLA 5% 10% 15% 20% 25% 30%

– –

– –

– –

39 60

14 1

22 18

25 21

236 ± 54 455 ± 115 411 ± 164 575 ± 217 231 ± 57 216 ± 54 494 ± 168

341 573 794 1050 364 302 893

162 142 125 237 161 89 217

– – – 40 3 46 40

– – – 17 16 10 15

– – – 32 37 27 29

– – – 11 14 17 16

Reproduced with permission from American Chemical Society.

Fig. 5.7  SEM images of the blend fibers after treating with acetone. (A) is made from 1:2 ratio of protein: PLLA and had thickness of 0.5 mm; (B) is 2:3 and 0.53 mm; (C) is 1:1 and 0.53 mm and (D) is 1:2 and 0.12 mm thick scaffold (Yu and Sun, 2015). Reproduced with permission from John Wiley and Sons.

protein: PLA and extent of ­treatment with acetone. Similar observations were also made for the mechanical properties. Interestingly, the wet tensile properties of the scaffolds were also high and hence considered to be suitable for tissue engineering applications (Yu and Sun, 2015). To improve the mechanical properties and suitability for cell culture, proteins from the major ampullate spidroins 1 and 2 from the dragline spider silk were used to

126

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Fig. 5.8  Comparison of the mechanical properties of spider silk-CNT electrospun membranes with collagen-CNT membranes. SEM and AFM images reveal that higher CNT content leads to less oriented fibers (Chi and Wang, 2018). Reproduced with permission from Elsevier.

d­ evelop electrospun fibers with the addition of 0.05%, 0.25% or 0.5% carbon nanotubes. Recombinant MaSp1 and MaSp2 were obtained from goat milk and combined in the ratio of 4:1 and dissolved using 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). CNTs were added into the solution and electrospun into fibers followed by treatment with 70% ethanol and UV irradiation (Chi and Wang, 2018). Advantages of using the spider silk proteins was compared with collagen. Mechanical properties of the silk-CNT membranes was higher than that of the collagen-CNTs. However, the properties were considerably influenced by the % CNTs (Fig. 5.8). Lower levels of CNTs provided higher orientation. Silk-CNTs membranes were highly conductive and increased proliferation of fibroblasts by nearly 20 times compared to collagen-CNTs. In a similar approach, recombinant spider silk proteins were made into nanofibrous membranes for mechanical vibration and humidity sensors (Shehata et  al., 2018). Fibers with diameters as low as 70 nm and elastic modulus of 4.3 MPa and maximum strain of 41% were obtained. Mechanical properties (Table 5.10) of the membranes varied considerably with humidity and vibration. A piezoelectric co-efficient of 3.62 pC/N and electrical resistance detection with sensitivity of 0.15 Giga Ohm per 1% change in RH was possible.

Electrospun silk fibers127

Table 5.10  Mechanical properties of electrospun recombinant spider silk protein membranes at different humidities (Shehata et al., 2018). Relative humidity, % Property

Dry

45

75

85

99

Elastic modulus, MPa Tensile strength, MPa Strain, % Strength at break MPa Energy at break, kJ/m2

1.89 ± 0.3

4.3 ± 0.8

1.2 ± 0.3

1.0 ± 0.2

1.5 ± 0.2

3.57 ± 0.5

4.5 ± 0.6

1.5 ± 0.3

1.3 ± 0.2

1.3 ± 0.2

16.1 ± 4.2 11.1 ± 1.9

35.9 ± 7.1 12.6 ± 2.1

35.7 ± 3.6 13.9 ± 3.6

40.9 ± 6.9 14.1 ± 3.1

37.7 ± 3.5 15.5 ± 1.4

24.7 ± 7.9

72.5 ± 9.7

59.8 ± 9.1

61.9 ± 8.3

66.1 ± 9.7

Reproduced with permission through Creative Commons Attribution License.

A recombinant spider silk protein containing the cell binding RGD peptides were synthesized and labeled as pNSR16. The protein was dissolved in 98% formic acid alongwith PVA in 50/50 ratio to obtain a 15% solution concentration. Electrospinning was done with varying voltage, extrusion speed and distance at a temperature of 45 °C (Zhang et  al., 2014). Average diameter of the fibers was between 325 and 450 nm. Treating with alcohol lead to transformation from random coil to β-sheets and removal of PVA lead to highly porous fibers (84% porosity). Membranes obtained were considered to be suitable for promoting wound healing and expressing fibroblast growth factors. The pNSR16 peptides were also used to develop a bilayered tubular scaffold by blending with gelatin and polyurethane for potential use as vascular grafts (Fig.  5.9). Polyurethane was dissolved in DMF/AEC and combined with pNSR16/ gelatin solution in formic acid and the mixture was electrospun at a voltage of 15 kV and flow rate of 1 mL/h (Zhang et al., 2016). A smooth double layered scaffold consisting of two distinct layers was obtained. The pNSR/gelatin formed separate layers with nanofibrils having diameters of 377 nm whereas the PU formed the outside layer with average fiber diameter of 653 nm. Scaffold formed had average length of 6 cm and 3 mm outer diameter. Mechanical properties of the scaffolds were considerably higher than the synthetic polymer based arterties available on the market (Table 5.11).

Fig. 5.9  Digital image of the pNSR/gelatin-PU tubular scaffold (A), SEM image shows clear separation of the two layers (B), morphology of the fibers in the inner (C) and outer (D) layer (Zhang et al., 2016). Reproduced with permission from John Wiley and Sons.

128

Table 5.11  Comparison of the properties of silk based scaffold with commercially available scaffolds (Zhang et al., 2016). Substrate

Burst pressure, KPa

Tensile stress, MPa

Strain at break, %

Suture strength, N

Water uptake, %

Porosity, %

13.1 ± 0.9 7.3 ± 1.1 6.8 ± 0.2

263 ± 9.8 25 ± 3.5 276 ± 7.1

26.1 ± 6.8 7.6 ± 0.2 24.6 ± 3.6

155 ± 5.3 59 ± 3.6 145 ± 3.8

4.8 ± 0.6 0.5 ± 0.3 4.9 ± 0.8

7.3 ± 1.1 80.2 ± 1.4 27.1 ± 3.8

91.3 ± 1.6 86.6 ± 1.4 88.7 ± 1.5

–

–

1–2

63–76

–

–

–

– –

– –

1.4–11.1 6–5

45–99 20–30

– –

– –

– –

Reproduced with permission from John Wiley and Sons.

Silk: Materials, Processes, and Applications

PU pNSR16/Gt pNSR 16/ Gt-PU Human femoral artery Coronary artery ePTFE

Water leakage (ml/min/cm2)

Electrospun silk fibers129

In addition to the structure and mechanical properties, biocompatibility and biodegradation are one of the key requirements for using protein based scaffolds for medical applications. Recombinant spider silk proteins eADF4 were obtained through e.coli and made into electropsun fibers. Proteins were dissolved in HFIP and electrospun at a voltage of 25 kV. Obtained fibers were studied for their ability to be degraded by various enzymes. The non-woven membranes made from the spider silk proteins had distinct solubility in various proteolytic enzymes. Only two of the five proteases were able to digest the proteins. Scaffolds were more readily dissolved in PXIV compared to CHC. Extent of degradation by the enzymes was also dependent on the morphology of the fibers (Müller-Herrmann and Scheibel, 2015).

References Andiappan, M., Kumari, T., Sundaramoorthy, S., Meiyazhagan, G., Manoharan, P., Venkataraman, G., 2016. Comparison of eri and tasar silk fibroin scaffolds for biomedical applications. Progr. Biomater. 5 (2), 81–91. Ayutsede, J., Gandhi, M., Sukigara, S., Micklus, M., Chen, H.-E., Ko, F., 2005. Regeneration of Bombyx mori silk by electrospinning. Part 3: Characterization of electrospun nonwoven mat. Polymer 46 (5), 1625–1634. Aziz, S., Sabzi, M., Fattahi, A., Arkan, E., 2017. Electrospun silk fibroin/PAN double-layer nanofibrous membranes containing polyaniline/TiO2 nanoparticles for anionic dye removal. J. Polym. Res. 24 (9), 140. Cao, H., Chen, X., Huang, L., Shao, Z., 2009. Electrospinning of reconstituted silk fiber from aqueous silk fibroin solution. Mater. Sci. Eng. C 29 (7), 2270–2274. Chen, C., Chuanbao, C., Ma, X., Yin, T., Hesun, Z., 2006. Preparation of non-woven mats from all-aqueous silk fibroin solution with electrospinning method. Polymer 47 (18), 6322–6327. Chi, N., Wang, R., 2018. Electrospun protein-CNT composite fibers and the application in ­fibroblast stimulation. Biochem. Biophys. Res. Commun. 504 (1), 211–217. Chouhan, D., Thatikonda, N., Nilebäck, L., Widhe, M., My, H., Mandal, B.B., 2018. Recombinant spider silk functionalized silkworm silk matrices as potential bioactive wound dressings and skin grafts. ACS Appl. Mater. Interfaces 10 (28), 23560–23572. Fan, S., Zhang, Y., Shao, H., Hu, X., 2013. Electrospun regenerated silk fibroin mats with enhanced mechanical properties. Int. J. Biol. Macromol. 56, 83–88. He, J.-H., Liu, Y., Xu, L., Yu, J.-Y., Sun, G., 2008. Biomimic fabrication of electrospun nanofibers with high-throughput. Chaos, Solitons Fractals 37 (3), 643–651. Jeong, L., Lee, K.Y., Liu, J.W., Park, W.H., 2006. Time-resolved structural investigation of regenerated silk fibroin nanofibers treated with solvent vapor. Int. J. Biol. Macromol. 38 (2), 140–144. Jin, H.-J., Fridrikh, S.V., Rutledge, G.C., Kaplan, D.L., 2002. Electrospinning Bombyx mori silk with poly (ethylene oxide). Biomacromolecules 3 (6), 1233–1239. Jin, H.-J., Chen, J., Karageorgiou, V., Altman, G.H., Kaplan, D.L., 2004. Human bone marrow stromal cell responses on electrospun silk fibroin mats. Biomaterials 25 (6), 1039–1047. Kim, S.H., Nam, Y.S., Lee, T.S., Park, W.H., 2003. Silk fibroin nanofiber. Electrospinning, properties, and structure. Polym. J. 35 (2), 185. Kishimoto, Y., Morikawa, H., Yamanaka, S., Tamada, Y., 2017. Electrospinning of silk fibroin from all aqueous solution at low concentration. Mater. Sci. Eng. C 73, 498–506.

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Ko, J.S., Yoon, K., Ki, C.S., Kim, H.J., Bae, D.G., Lee, K.H., Park, Y.H., Um, I.C., 2013. Effect of degumming condition on the solution properties and electrospinnablity of regenerated silk solution. Int. J. Biol. Macromol. 55, 161–168. Meinel, A.J., Kubow, K.E., Klotzsch, E., Garcia-Fuentes, M., Smith, M.L., Vogel, V., Merkle, H.P., Meinel, L., 2009. Optimization strategies for electrospun silk fibroin tissue engineering scaffolds. Biomaterials 30 (17), 3058–3067. Müller-Herrmann, S., Scheibel, T., 2015. Enzymatic degradation of films, particles, and nonwoven meshes made of a recombinant spider silk protein. ACS Biomater Sci. Eng. 1 (4), 247–259. Ohgo, K., Zhao, C., Kobayashi, M., Asakura, T., 2003. Preparation of non-woven nanofibers of Bombyx mori silk, Samia cynthia ricini silk and recombinant hybrid silk with electrospinning method. Polymer 44 (3), 841–846. Park, B.K., Um, I.C., 2017. Effects of electric field on the maximum electro-spinning rate of silk fibroin solutions. Int. J. Biol. Macromol. 95, 8–13. Shehata, N., Kandas, I., Hassounah, I., Sobolčiak, P., Krupa, I., Mrlik, M., Popelka, A., Steadman, J., Lewis, R., 2018. Piezoresponse, mechanical, and electrical characteristics of synthetic spider silk nanofibers. Nanomaterials 8 (8), 585. Singh, B.N., Panda, N.N., Pramanik, K., 2016. A novel electrospinning approach to fabricate high strength aqueous silk fibroin nanofibers. Int. J. Biol. Macromol. 87, 201–207. Stephens, J.S., Fahnestock, S.R., Farmer, R.S., Kiick, K.L., Chase, D.B., Rabolt, J.F., 2005. Effects of electrospinning and solution casting protocols on the secondary structure of a genetically engineered dragline spider silk analogue investigated via Fourier transform Raman spectroscopy. Biomacromolecules 6 (3), 1405–1413. Sukigara, S., Gandhi, M., Ayutsede, J., Micklus, M., Ko, F., 2003. Regeneration of Bombyx mori silk by electrospinning—Part  1: Processing parameters and geometric properties. Polymer 44 (19), 5721–5727. Wang, M., Jin, H.-J., Kaplan, D.L., Rutledge, G.C., 2004. Mechanical properties of electrospun silk fibers. Macromolecules 37 (18), 6856–6864. Wang, H., Zhang, Y., Shao, H., Hu, X., 2005. Electrospun ultra-fine silk fibroin fibers from aqueous solutions. J. Mater. Sci. 40 (20), 5359–5363. Wang, H., Shao, H., Hu, X., 2006a. Structure of silk fibroin fibers made by an electrospinning process from a silk fibroin aqueous solution. J. Appl. Polym. Sci. 101 (2), 961–968. Wang, M., Yu, J.H., Kaplan, D.L., Rutledge, G.C., 2006b. Production of submicron diameter silk fibers under benign processing conditions by two-fluid electrospinning. Macromolecules 39 (3), 1102–1107. Yao, J., Masuda, H., Zhao, C., Asakura, T., 2002. Artificial spinning and characterization of silk Fiber from Bombyx mori silk fibroin in Hexafluoroacetone hydrate. Macromolecules 35 (1), 6–9. Yi, B., Zhang, H., Yu, Z., Yuan, H., Wang, X., Zhang, Y., 2018. Fabrication of high performance silk fibroin fibers via stable jet electrospinning for potential use in anisotropic tissue regeneration. J. Mater. Chem. B 6 (23), 3934–3945. Yu, Q., Sun, C., 2015. A three‐dimensional multiporous fibrous scaffold fabricated with regenerated spider silk protein/poly (l‐lactic acid) for tissue engineering. J. Biomed. Mater. Res. A 103 (2), 721–729. Yu, Q., Xu, S., Zhang, H., Gu, L., Xu, Y., Ko, F., 2014. Structure–property relationship of regenerated spider silk protein nano/microfibrous scaffold fabricated by electrospinning. J. Biomed. Mater. Res. A 102 (11), 3828–3837. Zarkoob, S., Eby, R.K., Reneker, D.H., Hudson, S.D., Ertley, D., Adams, W.W., 2004. Structure and morphology of electrospun silk nanofibers. Polymer 45 (11), 3973–3977.

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Zhang, F., Zuo, B., Fan, Z., Xie, Z., Lu, Q., Zhang, X., Kaplan, D.L., 2012. Mechanisms and control of silk-based electrospinning. Biomacromolecules 13 (3), 798–804. Zhang, F., Lu, Q., Ming, J., Dou, H., Liu, Z., Zuo, B., Qin, M., Li, F., Kaplan, D.L., Zhang, X., 2014. Silk dissolution and regeneration at the nanofibril scale. J. Mater. Chem. B 2 (24), 3879–3885. Zhang, C.‐y., Zhang, D.‐c., Chen, D.‐l., Li, M., 2016. A bilayered scaffold based on RGD recombinant spider silk proteins for small diameter tissue engineering. Polym. Compos. 37 (2), 523–531. Zhao, J., Qiu, H., Chen, D.-l., Zhang, W.-x., Zhang, D.-c., Li, M., 2013. Development of nanofibrous scaffolds for vascular tissue engineering. Int. J. Biol. Macromol. 56, 106–113. Zhou, S., Peng, H., Yu, X., Zheng, X., Cui, W., Zhang, Z., Li, X., et al., 2008. Preparation and characterization of a novel electrospun spider silk fibroin/poly (D, L-lactide) composite fiber. J. Phys. Chem. B 112 (36), 11209–11216. Zhou, J., Cao, C., Ma, X., 2009. A novel three-dimensional tubular scaffold prepared from silk fibroin by electrospinning. Int. J. Biol. Macromol. 45 (5), 504–510. Zhu, J., Shao, H., Hu, X., 2007. Morphology and structure of electrospun mats from regenerated silk fibroin aqueous solutions with adjusting pH. Int. J. Biol. Macromol. 41 (4), 469–474. Zhu, J., Zhang, Y., Shao, H., Hu, X., 2008. Electrospinning and rheology of regenerated Bombyx mori silk fibroin aqueous solutions: The effects of pH and concentration. Polymer 49 (12), 2880–2885. Zuo, L., Zhang, F., Gao, B., Zuo, B., 2017. Fabrication of electrical conductivity and reinforced electrospun silk Nanofibers with MWNTs. Fibres Text. Eastern Eur.

Further reading Zhao, L., Chen, D., Yao, Q., Li, M., 2017. Studies on the use of recombinant spider silk protein/ polyvinyl alcohol electrospinning membrane as wound dressing. Int. J. Nanomedicine 12, 8103–8112.

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Applications of silk

6

6.1 Medical applications of silk fibroin 6.1.1 Biomaterials from B. mori silk Due to the large scale availability and presence of cell growth promoting peptides, B. mori silk has been commonly used to develop fibers, films, hydrogels, 2D and 3D structures for medical applications. Many approaches have been used to develop silk based scaffolds for cartilage tissue engineering. For instance, anterior cruciate ligament (ACL) is one of the most mechanically demanding materials in the body. It has been difficult to develop artificial ACLs with properties similar to that of native ACL. A silk fiber matrix was developed to study the possibility of using it as a scaffold to develop ACL. B. mori silk fibers were degummed and treated with detergent to remove sericin. Degummed fibers were twisted into bundles of 5 or 10 parallel fibers until they had mechanical properties similar to that of natural ACL (Table 6.1). Fibers retained their strength even after being in the cell culture medium for 21 days. Cells seeded (human BMSCs) were found to be well adhered on the matrix and formed a uniform extracellular matrix after 14 days of culture. Collagen type II and bone sialoprotein which were markers specific to cartilage and bone were detected on the scaffolds suggesting that the fibers were ideal substrates for regeneration of ACL (Altman et al., 2002). In addition to demonstrating the suitability of silk for generation of ACL under laboratory conditions, Fan et al. generated ACL in an pig model using mesenchymal stem cells on silk filaments. In their approach, microporous silk mesh was fabricated by adding silk sponge into knitted silk mesh and later by fabricating the silk into a cord with tightly wound shaft (Fig. 6.1). Scaffold developed had a maximum load bearing capacity of 399 N and stiffness of 59 N/mm compared to 770 N and 94 N/mm for native ACL, respectively. MSCs were found to be able to proliferate completely and generate collagen, I, III and tenascin-C genes. The scaffold with the seeded cells was implanted in a pig to generate ACL within 24 weeks. Required level of ACL formation had occurred and the scaffold had retained most of its strength even after 24 weeks of implantation suggesting that the silk scaffold were suitable for clinical applications (Fan et al., 2009). Other studies have shown that adding collagen into silk fibers provided better mechanical properties, generated higher collagen, good interface healing and a possibility for functional ligament repair (Chen et al., 2008). A cartilage like tissue was engineered from human mesenchymal stem cells using silk scaffolds. B. mori cocoons were degummed and dissolved using lithium bromide (LiBr). To improve cell adhesion onto silk, RGD sequences were also attached by coupling with a GRGDS peptide (Meinel et al., 2004). Scaffolds were developed from both modified and unmodified proteins by lyophilization and later immersion in methanol for the protein conformation to take place. Chondrogenesis of hMSCs could be induced on the silk scaffolds and continuous formation of cartilage was observed, particularly on Silk: Materials, Processes, and Applications. https://doi.org/10.1016/B978-0-12-818495-0.00006-5 © 2020 Elsevier Ltd. All rights reserved.

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Table 6.1  Comparison of the properties of silk fiber scaffold compared to natural ACL (Altman et al., 2002).

Silk matrix Parallel silk matrix Human ACL

Tensile strength (N)

Stiffness, N/ mm

Yield (N)

Elongation, %

2337 ± 72 2214

354 ± 26 1740

1262 ± 36 1274

38.6 ± 2.4 26.5

2160 ± 157

242 ± 28

1200

33

Reproduced with permission from Elsevier.

Fig. 6.1  Digital images of the braided silk cord (A), knitted silk mesh (B), silk mesh rolled around a silk cord (C), and cross-sectional morphology of the silk scaffold (D) (Fan et al., 2009). Reproduced with permission from Elsevier.

the RGD modified scaffolds. These scaffolds were suggested to be ideal for tissue engineering and bone repair (Meinel et al., 2004). Another study also showed that hMSCs seeded on silk scaffolds were able to generate three times higher glycosaminoglucan content compared to collagen scaffolds. Cartilage like tissue successfully grew on the scaffold with homogenous distribution throughout. Interestingly, mechanical properties of the scaffolds after generation of the cartilage were 2 fold higher than the unseeded scaffolds suggesting that the scaffolds were suitable as autologous cartilage tissue engineering substrates and durable implants (Hofmann et al., 2006). Fibroin from B. mori cocoons was immersed in 9 M lithium bromide and the protein obtained was redissolved in HFIP and made into disc shaped scaffolds of 5 mm in diameter and 2 mm in thickness (Hofmann et al., 2006). Scaffolds were also treated with methanol for 30 min

Applications of silk135

to reduce solubility and improve mechanical properties. Thickness and modulus of the scaffolds used for culturing the cells was considerably high compared to the raw silk scaffold (Hofmann et al., 2006). Amount of DNA on the membranes and deposition of chondroitin sulfate per scaffolds was also higher after methanol treatment. A single silk scaffold but with different pore sizes on each side could be developed from silk fibroin for cartilage tissue engineering (Hofmann et  al., 2007). Scaffolds were formed over a period of two days with porosity higher than 90%. Cells (hMSCs) were seeded using both static and dynamic approach. Pore sizes ranging from 112 to 500 μm were obtained. However, it was also possible to develop scaffolds with smaller diameters on one side and larger diameters on the other. When the size of the pores was smaller, the porosity was 93.5% whereas it was 95.6% for larger pores. After 3 weeks of cell culture, no mineralization was found in the control medium but considerably interconnected and calcified structures were observed in the scaffolds. However, large variations in ability to support cell growth were observed between different types of scaffolds and also on the type of seeding method used (Fig. 6.2). Ratio of bone surface to volume ratio also changed with pore structures and ranged from 77 to 66 mm. This approach of having different pore diameters in a single scaffold was considered to be suitable for musculoskeletal tissue engineering (Hofmann et al., 2007). Instead of using a single silk fiber, a biocomposite scaffold was fabricated using a blend of mulberry and non-mulberry silk. Fig. 6.3 shows one of the common approaches used to fabricate the blend scaffold (Singh et  al., 2017). Porcine primary chondrocytes were seeded on the scaffolds and their ability to support the development of adult cartilage was studied. Composite scaffolds had pore size ranging from 20 to 80 μm and showed considerably lower swelling in pH 7.4 solution. Reinforcing the silk solution with silk fibers increased compressive modulus and stiffness nearly 8 times compared to using only pure silk solution (Singh et al., 2017). Considerable deposition of glycosaminoglycan and collagen were observed in the non-mulberry silk solution reinforced with silk fibers. These scaffolds were considered to be viable alternatives as scaffolds for cartilage tissue engineering. In a similar approach, silk fibroin solution was reinforced with silk fibers and ­poly(caprolactone) to form a synthetic periosteum. Considerable increase in elongation and decrease in the rate of degradation of the pure silk membranes was observed due to the addition of PCL. Higher levels of osteogenic differentiators, alkaline phosphatase and osteopontin were observed in the silk fibroin based scaffold compared to PCL or plastic tissue culture plates. Composites of silk fibroin and PCL showed higher levels of osteocalcin expression indicating that the scaffolds are suitable for treating fractures (Cheng et al., 2018). B. mori silk fibroin was extracted, dissolved in a ternary solvent system and combined with PEO to form electrospun fibers for anisotropic tissue engineering (Yi et al., 2018). Blended scaffolds were also washed with methanol to improve mechanical properties and stability in the cell culture media. Murine iPS-MSCs were seeded on the scaffolds and the affinity and proliferation was studied. Formation of spindle shaped cells was observed around the scaffolds suggesting good affinity for the substrates. However, presence of PEO was found to adversely affect cell proliferation. In addition, the scaffolds were able to guide cell growth and tissue formation along the fiber direction as seen from the long and thin shaped cells (Fig. 6.4).

mixed pores

small pores

large pores

(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

(I)

(J)

(K)

(L)

dynamic

static

dynamic

static

Fig. 6.2  Confocal laser scanning and histology section images of silk scaffolds of various pore sizes seeded with hMSCs after 24 h of seeding (Hofmann et al., 2007). Reproduced with permission from Elsevier.

A. assamensis Silk Solution (AS)

1% SDS Dissolution

Silk Fibers of A. Assamensis (AF) Silk Fibers of B. mori (BF)

9.3 M LiBr Dissolution

Porcine Primary Chondrocytes Silk Fiber-reinforced Composite

Silk Gland of A. assamensis

ASAF

• Lyophilization • Ethanol Treatment • Media Conditioning BSBF

Cocoons of B. mori

Cartilage Construct

B. Mori Silk Solution (BS)

Fig. 6.3  Schematic representation of the process used to develop 3D scaffolds from mulberry silk, non-mulberry silk solutions reinforced with silk fibers (Singh et al., 2017). Reproduced with permission from IOP publishing.

Applications of silk137

Fig. 6.4  Confocal images and cell proliferation values for the iPS-MSCs seeded on the fibroin based scaffolds compared to TCP (Yi et al., 2018). Proliferation of the cells along the direction of the fibers is indicated by the arrows. Reproduced with permission from Royal Society of Chemistry.

Porous silk scaffolds were also found to be suitable for developing micro-­organoid liver tissue for potential treatment of hepatitis (Wei et al., 2018). B. mori fibroin was dissolved using sodium carbonate and LiBr to which NaCl particles were added. A salt leaching approach was followed to remove the salt and form pores with diameters between 425 and 500 μm. A co-culture of primary hepatocytes and stellate cells was seeded on to the scaffold and incubated to form the tissue. Good viability and proliferation of the cells was observed on the scaffold. Appropriate formation of liver like tissue and adequate response to metabolic treatment was also found when co-­ culture was done on the scaffold (Wei et al., 2018). To create an environment similar to that found in the liver, silk scaffolds were developed using a blend of B. mori and Antreraea assamensis which is rich in cell binding amino acids (RGD peptides). 3D porous scaffolds were developed from the blend of the two silks. Pure B. mori scaffolds had porosity of 93% and compressive strength of 12.8 kPa compared to 89% and 23 kPa for the wild silk. Blend scaffolds has porosity of 89% but a much lower compressive strength of 8 kPa. Human hepatocarcinoma cells and primary neonatal rat hepatocytes were seeded on the scaffolds. Blend scaffolds supported formation of high density hepatocyte clusters and cell–cell interactions. Synthesis of liver specific compounds such as albumin, urea and cytochrome P450 enzyme were observed over a period of 21 days. No macrophage growth was observed when the scaffolds were subcutaneously implanted in Swiss mice suggesting that the scaffolds are ideally suited for growing functional liver cells (Janani and Samit, 2017).

6.1.2 Hydrogels from silk fibroin Several reports are available on the development and application of hydrogels from silk fibroin. A novel approach of vortex induced gelation was used to form h­ ydrogels

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Silk: Materials, Processes, and Applications

in which the rate of β-sheet formation and gelation kinetics could be controlled (Yucel et al., 2009). Silk cocoons were degummed and later dissolved using LiBr to obtain a solution concentration of approximately 5.2%. This solution was vortexed at 3200 rpm for various time periods to induce silk self-assembly and hydrogelation. Physiochemical characterization of the hydrogel showed that the gels had properties suitable for cell encapsulation and controlled delivery. Similar to vortexing, ultrasonication was also found to influence β-sheet formation and gelation (Wang et al., 2008). Properties of the gels could be controlled by varying the fibroin concentration, gelation time or sonication conditions which allowed the incorporation of cells into the scaffolds. Human bone marrow derived mesenchymal stem cells were successfully incorporated into the fibroin solutions and rapidly formed into hydrogels. Cells inside the hydrogel were able to grow and proliferate up to 21 days indicating their suitability for cell encapsulation and delivery (Wang et al., 2008). Another approach of in situ formation of fibroin hydrogels was by using the anionic surfactant sodium dodecyl sulfate (SDS). In the presence of SDS, the fibroin chains had high hydrophobic interactions or electrostatic repulsion leading to varying gelation times (Wu et  al., 2012) (Fig.  6.5). A shortest gelation time of 15–18 min was obtained when

Hydrophobic interaction

Unstable

(A)

Stable

Ionic electrostatic effect Unfolding

Self-assembling into b-sheets

Repetitive GAGAGS sequences

(B)

Hydrophobic microdomain

(C)

Mixed micellar particles

(D) Clusters

Random chain of SF

b-sheet structure

DS Group Fibroin-Surfactant Hybrid Particles Negatively charged micelle formed by DS Groups

Fig. 6.5  Schematic representation of the formation of hydrogels from silk fibroin. Mechanism of the effect of SDS on the conformation on the proteins (A); nucleation of the micelles (B); clusters of micelles due to the presence of SDS (C); negatively charged micelles accumulating and formation of clusters (D) (Wu et al., 2012). Reproduced with permission from Elsevier.

Applications of silk139

SDS ­concentration was ­between 8 and 12 mM. SDS in the gel was gradually released with the degradation of the hydrogel suggesting the suitability for controlled release applications. A two-step approach was used to obtain fibrous fibroin hydrogels suitable for tissue engineering. In the first step, silk fibers were dissolved with formic acid/ calcium chloride resulting in formation of nanofibrillar solution. In the second step, the solution was added into 9.3 M LiBr at 70 °C for complete dissolution. During the two step process, fibroin in the form of nanofibers transforms into microfibers and finally into hydrogels with fibrous networks when fibroin transitions from random α-coils to β-sheets within 30 min (Ming et al., 2016). Compressive strength of the hydrogels ranged from 22 to 32 kPa depending on the conditions during gelation. BMSCs showed good attachment and proliferation indicating that the hydrogels are suitable for tissue engineering applications (Ming et al., 2016). Similar fibrous hydrogels were also obtained from a combination of silk fibroin/sodium alginate. These hydrogels were able to support controlled crystallization and growth of hydroxyapatite into nanorods and rectangular nanorod columns depending on the conditions during gelation. Ability to control the morphology of the biomineralization was suggested to be ideal for bone repairs (Ming et al., 2015). Biomimetic mineralization was shown to be possible in 3D fibroin hydrogels by inducing Ca2+ into the scaffolds by ion diffusion. Presence of calcium ions provided nucleation sites for the hydroxyapatite crystals and also regulated their growth (Jin et al., 2015). Scaffolds had higher compressive strength after mineralization due to the presence of both organic and inorganic materials. MG-63 cells seeded on the scaffold showed good viability, proliferation and differentiation. Similar to formation of HA crystals, it was discovered that calcium carbonate biomineralization can also occur on fibroin hydrogels (Ma et al., 2013). Various sizes and morphologies of calcium carbonate crystals (calcites) were formed depending on the calcium concentration and processing conditions (Fig. 6.6). Since hydrogels typically have poor mechanical properties, a new approach of controlling the β-sheet transformation was used to form 3D hydrogels with considerably high modulus (Zhu et al., 2018). In this approach, B. mori silk was dissolved using HFIP and freeze dried to form a powder. Later, the powder was dissolved (15%) in HFIP and the solution was aged for several days for gelation to occur. Fig.  6.7 shows the schematic of the process used to develop the hydrogels. The structure and properties of the hydrogels were highly dependent on the fibroin concentration and the temperature. It was suggested that the new approach of using water and HFIP solution allowed formation of large number of intraprotein hydrogen bonds by about 40% which subsequently leads to higher β-sheet content. Mechanical properties of the hydrogels varied from 0.08 to 0.7 MPa (Stress), modulus from 1.0 to 6.5 MPa and toughness from 0.003 to 0.60 MJ/m3 depending on the conditions during hydrogel formation (Zhu et al., 2018) (Table 6.2). Modulus of the hydrogels were considerably higher compared to conventional hydrogels which have a modulus between 0.01 and 0.1 MPa. The new method of hydrogel formation was considered to be suitable for biomedicine, engineering and soft robotics applications.

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Silk: Materials, Processes, and Applications

Fig. 6.6  Scanning electron microscopy image of pure silk fibroin (A); calcium carbonate crystals (B); silk gels containing various concentrations of CaCO3 crystals (C-5%, D-10%, E-15% and F-25%) (Ma et al., 2013). Reproduced with permission from Elsevier.

6.1.3 Microneedles from silk fibroin Microneedles based transdermal delivery of vaccines and macromolecules have been preferred to avoid degradation of the drugs in the gastrointestinal track. Silk fibroin has been found to assist in transdermal delivery of vaccines and other biomolecules when made into micro needles. Using a conventional approach of micromolding, needle structures of 500 μm height and tip radii of 10 μm were built using aluminum. The aluminum master molds were used to make negative microneedles of polydimethylsiloxane (PDMS) by soft lithography. Later, fibroin solution was cast on the PDMS

Applications of silk141

Freeze drying

SF aqueous solution

Dissolution

SF powder or sponge

Incubation

SF/HFIP mixture (15 wt%)

SF/HFIP solution DI water

Washing

SF hydrogel

SF/HFIP/water hydrogel

Incubation

SF/HFIP/water solution

Mixing

SF/HFIP/water solution

Fig. 6.7  Schematic representation of the formation of the silk fibroin hydrogels with very high modulus (Zhu et al., 2018). Reproduced with permission from John Wiley and Sons.

mold, allowed to dry and needles scaffolds were separated. Post treatment of the needles was done by exposing to high humidity, methanol, temperature etc. (Tsioris et al., 2012). Fabricated needles were inserted into mouse skin and their ability to release horse radish peroxidase was measured. Release of the enzyme was dependent on the treatment condition and a maximum of 54 g of HRP per needle was released after 48 h. Further, the microneedles were also able to deliver antibiotics and prevent the growth of bacteria. Adopting the PDMS process, microneedles were fabricated with height of 700 μm, tip diameter of 15 μm and base diameter of 360 μm with total needle array size of 1.5 cm×1.5 cm. Fibroin solution (6–8%) was cast onto the mold and made into needles after drying for 2–3 days. Antigens to treat three pathogens influenza, C. difficile and Shigella were loaded onto the needles and administered onto mice using the transdermal approach. It was suggested that the microneedles were ideally suited for transdermal delivery to vaccines (Stinson et al., 2017). A new thermal drawing method of fabricating high aspect ratio microneedles was developed using silk fibroin as the polymer. Microneedles were first drawn using PLGA and PDMS (Fig. 6.8). The fabricated microneedles were dipped into silk fibroin solution which formed a layer on the substrate. Methanol treatment was done to improve the mechanical properties of the needles and the substrate was removed once the needles were stable (Lee et al., 2015). Needles with different shape and size could be obtained with height/width between 1.43 and 1.26 µm, tip angles between 13° and 99° and radii of curvature from 177 to 190 μm. Mechanical properties of the needles were also dependent on the shape and size with the bullet shaped needles which had a force of 54 g/needle compared to 24 g/needle for the slender needle. A drug RB was combined with silk fibroin which was made into the needles followed by ethanol treatment for 1–60 min. Amount of rhodamine B loaded onto the needles was between 7.5 and 8.2 μg. Up to 90% of the loaded drugs could be released and the release profile was dependent on the extent of methanol treatment with longer treatment leading to

142

Table 6.2  Variations in the properties of the fibroin hydrogels with changes in fabrication conditions (Zhu et al., 2018). Dosage of SF and solvent

% of each component

Structure and properties

HFIP, ml

SF (g)

Water, %

HFIP, %

SF, %

Gelation Time, days

SF/water content of hydrogel (%)

Β-Sheet content, %

Transmittance at 800 nm

0.6 0.9 1.2 1.5 1.8 2.1 2.4 2.7 3.3

3 3 3 3 3 3 3 3 3

0.45 0.45 0.45 0.45 0.45 0.45 0.45 0.45 0.45

10 15 19 22 26 29 31 34 39

82 78 74 71 68 65 63 60 56

8 7 7 7 6 6 6 6 5

>10 >5 3 2 2 1 1 1 1

– 14.0 ± 0.4/86.0 ± 0.4 11.0 ± 0.3/89.0 ± 0.3 10.6 ± 0.1±/89.4 ± 0.1 10.0 ± 0.1/90.0 ± 0.1 10.7 ± 0.3/89.3 ± 0.3 9.2 ± 0.3/90.8 ± 0.3 8.7 ± 0.2/91.3 ± 0.2 8.3 ± 0.3/91.7 ± 0.3

– 35 ± 1 35 ± 1 37 ± 1 36 ± 2 35 ± 4 35 ± 1 35 ± 1 35 ± 1

– 90 88 89 76 73 67 55 47

Reproduced with permission from John Wiley and Sons.

Silk: Materials, Processes, and Applications

Water, ml

Applications of silk143

micropillar 130°C

(A)

PLGA film 200°C

PDMS

(D)

160°C

(B)

160°C

200°C

(C)

PDMS mold

(E)

60°C

Aqueous SF Solution

(F)

Dried SF MeOH

(G)

(H)

(I)

Fig. 6.8  Schematic representation of the formation of the silk fibroin microneedles. Heating and drawing the PLGA films (A–C), casting the needles onto the micromold (D and E) and coating (F), drying (G) and methanol treatment of the needles (H). Developed silk fibroin microneedles (I) (Lee et al., 2015). Reproduced with permission from Elsevier.

slower release (Fig. 6.9). The needles could be inserted into porcine skin (Fig. 6.10) and able to deliver the drugs efficiently suggesting suitability for transdermal drug delivery (Lee et al., 2015). Silk based microneedles for vaccine delivery have also been developed using poly(acrylic) acid as the base (DeMuth et  al., 2014). Fibroin was extracted from B. mori silk worms and dissolved using 9 M lithium bromide at 60 °C for 4 h. Microneedles were fabricated from the fibroin using poly(dimethyl siloxane) (PDMS) as the mold. The needles were made using fibroin as the tip and PAA as the base. An array of microneedles was developed as shown in Fig.  6.11. The microneedles produced had a height of 550 μm and width of 250 μm and tip radius of 4.7 ± 0.6 μm. Vaccines (AF555-OVA) loaded on the needles were found to be concentrated at the tip of the silk portion. The needles were able to disintegrate when inserted into the skin with burst release from the PAA part but with a sustained release from the fibroin section. It was suggested that the needles would be suitable to develop programmable delivery of vaccines to the skin for immune therapy and other applications

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Fig. 6.9  Amount of the drug RB released from the silk microneedles treated with methanol for different time (A) and the release rate of the drug from the needles (B) (Lee et al., 2015). Reproduced with permission from Elsevier.

Fig. 6.10  Digital images of dip coating the drug on the surface of the needles (i); inserting the microneedles into the procine skin (ii); procine skin during insertion (iii) and after insertion (iv) (Lee et al., 2015). Reproduced with permission from Elsevier.

Applications of silk145

(1) PDMS Mold

(B) (D)

(3) Silk Drying ± MeOH AF647 – OVA in Silk (4) PAA Addition AF555 – OVA in PAA (5) PAA Drying/Removal Overlay

(A)

(C)

Normalized OVA Release

(2) Silk Addition

1.0 MN-PAA MN-Silk MN-MeOH-Silk

0.5

0.0

(E)

0

2

4 6 8 10 12 Days Post Delivery

14

16

Fig. 6.11  Schematic depiction of the process of preparing the silk fibroin/PAA microneedles (A); Optical image of the needle containing the vaccine (blue color) (B); Confocal images of the needles shows the vaccine (AF647-OVA) in blue in the silk tips and AF 555-OVA in red at the pedestal section (C); scanning electron microscope image of a separated needle (D) and release profile of the vaccine from the different silk scaffolds (DeMuth et al., 2014). Reproduced with permission from John Wiley and Sons.

(DeMuth et al., 2014). Silk fibroin based needles that can swell and also are insoluble were developed by treating with urea, N-dimethylformamide, glycine and 2-­ethoxyethanol. Modified fibroin was cast on the PDMS molds resulting in microarray of needles of 15 × 15 of 300–700 μm depth and intervals of 150–1000 μm. Modified needles were insoluble and had about 200% lower swelling capacity before insertion compared to unmodified needles (Yin et al., 2018). However, the needles were able to swell and become a gel after insertion and transform into a porous network of 500–700 nm. The needles were able to penetrate into the skin to a depth of 200 μm and deliver drugs at a much controlled rate compared to the soluble fibroin microneedles.

6.1.4 Silk micro and nanoparticles for drug delivery Several researchers have developed micro and nanospheres from silk and its blends and studied their potential for biomedical applications. Fibroin from B. mori silkworms was degummed and dissolved in LiBr solution (Wang et al., 2010). Similarly, PVA solution was also prepared and combined with the silk solution and stirred at 150 rpm for 2 h at room temperature. The blend solution was poured onto petri dishes and allowed to form films by evaporation at room temperature. To form the nano and microspheres, the blend films were dissolved in ultrapure water and subject to intense ultrasonication using a sonicator at 10% amplitude to form the particles. Average particle size varied depending on the ratio of silk/PVA and was between 308 and 578 nm. PVA was removed from the silk by washing and centrifugation resulting in

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Mixing & freezing

Particle Nucleation

(A)

(PVA) (Ethanol)

(B) Fig. 6.12  Schematic representation of the formation of regular silk particles with PVA (A) and unpredictable particles without PVA (B) (Shi and Goh, 2012). Reproduced with permission from Elsevier.

only fibroin particles. These particles showed high preference for loading of bovine serum albumin (BSA), dextran and Rhodamine B (Wang et al., 2010). About 95% of the drugs could be loaded onto the particles and efficiency of release was about 80%. PVA was also used with ethanol to develop silk nanoparticles with tunable size and appearance using a self-assembly approach (Shi and Goh, 2012). A schematic of the approach used to prepare the nanoparticles is shown below. Fibroin was dissolved using a ternary solvent system to obtain a 2% solution which was combined with 2% PVA solution and ethanol and mixed vigorously. The solution was frozen for 48 h and later centrifuged and lyophilized to form powder. Particles obtained had diameter between 900 nm to 1.5 μm. However, the shape of the particles was dependent on the amount of PVA used. It was suggested that PVA would form a hydrogel during freezing and restrict the nucleation and aggregation resulting in uniform particle size (Fig. 6.12). However, the amount of PVA on the particles or methods to remove PVA and obtain pure silk nanoparticles was not reported. Silk nanoparticles were found to be ideal to transport the antioxidant quercetin. B. mori was dissolved in a mixture of CaCl2/ethanol and water and left at 70 °C for three hours and later dialyzed to obtain a 2% solution in water (Lozano-Pérez et al., 2017). The fibroin solution was slowly added into methanol to form the particles which were lyophilized and collected. Various loading and unloading conditions were studied to understand the potential of the particles to deliver the antioxidant. Particles obtained had a diameter of 139 nm but increased to about 171 nm after sorption of the antioxidant. Similarly, the zeta potential of the particles was −27 mV in the presence of the drug compared to −17 mV without any drug which was suggested to be due to the “protein corona effect”. An initial burst followed by a sustained release was observed with about 40% of the drug in the GI tract. Additionally, excellent free radical scavenging activity was also observed due to the antioxidant

Applications of silk147

Table 6.3  BMP loading and encapsulation efficiencies (Bessa et al., 2010a).

Growth Factor

Concentration of BMP/mg fibroin (μg)

Encapsulation efficiency (%)

Loading capacity (μg/BMP/mg particle)

BMP-2 BMP-9 BMP-14 BMP-2 BMP-9 BMP-14

0.5 0.5 0.5 5.0 5.0 5.0

97.7 ± 2.0 90.2 ± 5.9 85.8 ± 6.0 76.8 ± 3.5 72.4 ± 4.4 67.9 ± 6.1

0.69 ± 0.06 0.63 ± 0.17 0.60 ± 0.17 5.4 ± 0.5 5.1 ± 0.5 4.8 ± 0.6

Reproduced with permission from John Wiley and Sons.

properties of quercetin. Using the same fibroin dissolution and particle formation approach, another biomolecule bone morphogenic protein (BMP) were loaded onto silk nanoparticles in an attempt to promote new bone formation (Bessa et al., 2010a,b). Particles initially had a mean diameter of 580 nm when 1:2 ratio of ethanol to silk was used and increased up to 1.2 μm when the ratio was 1:4. Up to 97.7% of the BMP was loaded onto the microspheres with loading capacity ranging from 0.60 to 5.0 μg/g of particle depending on the type of BMP used (BMP-2,9 or 14) (Table 6.3). An initial burst and later a sustained release was observed with release rates between 80% and 90% corresponding to 15.7 and 8 ng/day after 14 days for the three types of BMP. It was proposed that the silk nanoparticles had slow degradation and required release rates suitable for bone regeneration. Further, studies showed that when incorporated into cells, BMP-2 retained most of its activity. Higher mineralization was observed in the BMP-2 containing fibroin particles compared to the particles added into the media. In vivo studies also showed bone formation and increase in bone density (Bessa et al., 2010b). The particles provided sustained release of BMP-2 in the liver suggesting that they are suitable for both drug delivery and bone tissue engineering (Bessa et al., 2010b). Silk nanoparticles with controllable features were obtained by a phase separation approach. Here, silk solution obtained using LiBr was mixed with potassium phosphate at different proportions, pH and ionic strength. Later, the phosphate was thoroughly washed and removed by salting out resulting in silk particles. To study the influence of various post-treatments, the particles were incubated in ethanol or methanol for 24 h which changed the primary and secondary structure of silk (Lammel et al., 2010). Particles were further sonicated at 20% amplitude for 5–60 s. Average size of the particles varied from 486 nm to 4 μm depending on the conditions, particularly, concentration of phosphate. Small drug molecules such as crystal violet, alcian blue and Rhodamine blue showed loading efficiency of up to 95% with a 6% matrix loading and these molecules were also able to diffuse into the matrix. High burst release was seen for Rhodamine B (83%) followed by crystal violet (17%) and alcian blue (3%). Size, structure and loading and release conditions could be easily controlled using this approach and the all aqueous preparation method was considered suitable for developing the silk particles for medical applications (Lammel et al., 2010).

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Table 6.4  Size of the fibroin microparticles obtained at different conditions (Baimark et al., 2010). Fibroin concentration, %

Fibroin solution, mL

Organic solvent

Microparticle yield, %

Average particle, μm

4.0 4.0 4.0 4.0 4.0 4.0 4.0 2.0 1.0

0.2 0.4 0.8 0.2 0.4 0.2 0.05 0.2 0.2

Ethyl acetate Ethyl acetate Ethyl acetate Diethyl ether Diethyl ether Dichloromethane Chloroform Ethyl acetate Ethyl acetate

85 80 76 89 85 92 95 88 90

134 ± 65 141 ± 58 148 ± 69 125 ± 72 131 ± 81 91 ± 35 48 ± 18 105 ± 46 101 ± 48

Reproduced with permission from John Wiley and Sons.

A water-in-oil-emulsion-diffusion method was developed to generate hollow fibroin microparticles (Baimark et al., 2010). Fibroin having molecular weight between 6 and 8 kDa was made into concentrations of 1%, 2%, and 4%. This solution was slowly added into three different types of organic solvents (Table 6.4). Particles with diameters ranging from 48 to 150 μm were obtained depending on the concentration of the fibroin solution and type of organic solvent used. Most of the particles existed in the random configuration but had varying size of holes. These porous particles were suggested to be suitable for both medical and non-medical applications (Baimark et al., 2010). Using the same approach, fibroin particles with diameters between 20 and 26 μm were prepared. Ethyl acetate was used as the solvent and span 80 was added as the surfactant to prevent agglomeration. Fibroin solution was also crosslinked with genipin and later used to form the particles. Uniform and spherical particles were obtained with the fibroin being transformed to the β-sheet form after crosslinking (Imsombut et al., 2010). Lipid vesicles were used as substrates to prepare silk fibroin microspheres for controlled drug delivery applications (Wang et al., 2007). 1,2-Dioleoyl-sn-glycero-3phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein) (DOPE) were used as the lipid substrates. The DOPC was dissolved in chloroform and added into the silk fibroin solution. The mixture was later frozen in liquid nitrogen for 15 min and then thawed at 37 °C for 15 min. Samples were subject to the freeze–thaw cycle three times and the solution was pipetted into water under fast stirring for the fibroin to be converted into microspheres. Instead of water, methanol was also used to form the microspheres but without the freeze–thaw cycle. Lyophilization was done for 3 days at 4 °C and pure methanol or saturated sodium chloride was added to remove the lipid templates and obtain self-assembled sheets. Microspheres were formed when the silk material was suspended in MeOH and later centrifuged and sonicated aggressively at 30% amplitude resulting in the particles. The lyophilized material was treated with 2 mL of NaCl solution and incubated for up

Applications of silk149

solvent

MeOH-based microspheres

Separated Silk/Drug

water or buffer lipid silk/drug

Lipid Removal

silk drug

MeOH

Centrifuge

Pellet Resuspend

tion

hiliza

Lyop

OH

Fre ez e

Me

N2

-th

Lipid in Chloroform

Lipid Film

Lipid-Silk Mixture

aw Ly

NaCl-based microspheres

op

hil

iza

tio

Lipid Removal

n

NaCl

Centrifuge

Solution

Pellet Resuspend

Mixed silk/drug

Fig. 6.13  Schematic of the process used for formation of silk microspheres (Wang et al., 2007). Reproduced with permission from Elsevier.

to 15 h. Later, the pellets formed were washed and sonicated to form the microspheres. A schematic representation of the process is shown in Fig. 6.13. The prepared microspheres were used to load horseradish peroxidase (HRP) and the efficiency of loading and release were studied. Yield of microspheres was higher for the MeOH treated samples (30–60%) compared to the NaCl approach (8–25%) (Wang et al., 2007). Particle sizes also varied from 1.7 to 2.7 μm depending on the preparation conditions. HRP loading efficiency was also between 7% and 20%. These microspheres were suggested to be suitable for controlled release of enzymes and drugs. Fibroin was extracted from B. mori cocoons and made into an aqueous solution in 3% and 9% concentrations. Microspheres were prepared from the solution using an Inotech-40-encapsulator with nozzle diameters of 80 or 200 μm. A process called jet break-up was used by applying an oscillation frequency of 1300 Hz at a voltage of 1700 V for the solution (Fig. 6.14). Droplets obtained were freeze dried and if necessary treated with 90% methanol solution and later again exposed to methanol at room temperature for 30 min. Alternatively, the spheres were exposed to 96% relative humidity in the presence of saturated Na2SO4 solution at room temperature for 24 h. Two drugs (salicylic acid and propranolol hydrochloride) were loaded onto the microspheres and the loading and release efficiency was investigated (Wenk et al., 2008). Diameter of the microspheres obtained ranged from 80 to 200 μm with methanol treated fibroin producing smaller size microspheres compared to those obtained using salt based approach. Encapsulation efficiencies for the drugs was about 11–25% for salicylic acid compared to 22–59% for hydrochloride. About 43% of the drug was released after 7 days indicating high level of affinity between drugs and fibroin. No major inhibition of cells was observed due to the presence of the microspheres or the drugs (Wenk et al., 2008).

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Fig. 6.14  Images of silk fibroin spheres prepared using different concentrations of solution and magnified to different levels (Wenk et al., 2008). Reproduced with permission from Elsevier.

In another unique approach, silk microspheres were prepared using the microfluidics approach (Breslauer et al., 2010). A 8% silk fibroin solution was made using LiBr as the solvent. This solution was made into a dispersed phase using 75% oleic acid and 25% methanol. Solutions were passed through specially made microfluidic machine using a flow rate of 1 mL/h for the dispersed phase and 6 to 14 mL/h for the continuous phase. The microspheres formed were passed into a methanol, ethanol or isopropanol bath for 24 h for the crystallization to occur (Breslauer et  al., 2010). Based on the diameter of the microfluidic device, the size of the particles obtained was between 145 and 200 μm. Measurements using atomic force microscopy (AFM) indicated a Young’s modulus of 1.5 kPa for the particles which was much lower than the modulus reported for silk fibers. These microparticles were suggested to be suitable for use in emulsions and cosmetics (Breslauer et al., 2010).

Applications of silk151

Instead of using raw fibroin, the potential of developing nanoparticles from regenerated fibroin was also studied (Cao et al., 2007). To obtain the regenerated solution, the B. mori silk was dissolved using LiBr and then dialyzed to obtain proteins with molecular weight of 12–14 kDa. Silk fibroin concentrations of 1–10% were added into ethanol and frozen at −40C for 24 h. Later, the solution was lyophilized to form microspheres with size between 0.2 and 1.5 μm. Zeta potential of the particles was about −27 mV between pH 7 to 11. Both silk I and silk II structures were observed in the particles depending on the concentration of ethanol used. It was suggested that the silk could be formed into microspheres due to the presence of hydrophilic and hydrophobic amino groups. The polar side groups in the amino acids had strong affinity to water and hence remained in the soluble state.

6.1.5 Blends of fibroin and other biopolymers Several studies have also been conducted to develop scaffolds by blending fibroin with other biopolymers. In one such attempt, collagen from decellularized extracellular matrix and fibroin were made into nano and microporous scaffolds using a low temperature 3D printing process. Solutions of the three polymers were extruded using a computerized 3D printer at a temperature of −40 °C. Later, the scaffolds were freeze dried for 1 day and crosslinked with 100 mM of EDC solution in 95% ethanol for 1 h. Subsequent treatment with methanol was done to induce β-sheet formation (Li et al., 2018). Mouse preosteoblast cells were seeded on the scaffolds and cell attachment and proliferation studies were conducted. Diameter of the macropores was 600, 614, and 604 μm for the collagen matrix and blend scaffolds, respectively. Similarly, the scaffolds had a compressive modulus of about 0.03 MPa which increased to 0.3 MPa after treating with methanol due to the formation of β-sheets (Li et  al., 2018). Excellent cell growth and proliferation was possible and combined with the good compressive strength, the collagen-silk scaffolds were suggested to be suitable for bone tissue engineering. Electrospun fibers developed from blends of B. mori silk fibroin with PEO were studied for their potential to support growth and proliferation of human bone marrow stromal cells (BMSCs) with and without the presence of PEO (Jin et al., 2004). The fibers in the electrospun membranes had diameter of 700 nm and tensile strength, modulus and elongation of 13.6 MPa, 625 MPa and 4%, respectively. Extensive growth of BMSCs were observed on the electrospun silk mats after 14 days of incubation. Silk fibroin was also used to coat commercially available scaffolds and improve the cell adhesion and proliferation (Cassinelli et al., 2006). Flat sheets of PP and porous polyamide membranes were coated with fibroin solution by inhibition or deposition. Later, the coated sheets were treated with methanol to induce β-sheet formation. SF coating increased the strength substantially but decreased the elongation by >50–100%. The coating was non-toxic and promoted the adhesion and proliferation of mouse fibroblast cells (Cassinelli et al., 2006). Novel 3D scaffolds for skin tissue regeneration could be developed by combing silk fibroin with functionalized, laminated and oxidized citrus pectin (Türkkan et al., 2018). Scaffolds with high porosity (83%) and interconnected pores were obtained with pore size of about 120 μm. These scaffolds were further crosslinked using b­ orax

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and calcium chloride. Mechanical property studies showed that the scaffold had a compressive strength of 860 kPa, strain of 62% and Young’s modulus of 1.9 kPa. Scaffolds had good stability and absorbed large amounts of water (800%). High biocompatibility with good adhesion, proliferation and penetration of the fibroblasts into the pores was also observed. The scaffolds were considered to be ideal to develop skin dermal substitutes (Türkkan et al., 2018). A completely biodegradable scaffold suitable for medical applications was developed by combining PLA with silk fibers obtained from tussah silk worms (Cheng et al., 2018). Silk fibers were cut into 3–6 mm and combined with PLA in 1–7 wt%. The silk fibers and PLA mix was passed through a Hakke minilab twin-screw microextuder at a temperature of 183 °C. Composite coming out of the extruder was again passed through an injection molding machine at 200 °C to obtain the desired shape of the samples. Addition of 5% of fibroin into PLA decreased the peak stress from 65 to 62 MPa but increased the modulus from 1.8 to 2.5 GPa. Similarly, the elongation of the composite samples was considerably higher at 10% compared to 6.7% for the pure PLA (Cheng et al., 2018). Although no cell culture or biocompatibility studies were done, it was suggested that the composite would be suitable for medical applications (Cheung et al., 2008). Further, studies showed that the silk/PLA composites had similar weight loss but could biodegrade at a faster rate up to 16 weeks in PBS solution (Cheung et al., 2010). Biodegradation rates of the composite could be controlled by varying the amount of PLA/silk fibroin. Three different methods were used to develop porous 3D scaffolds from regenerated fibroin. Schematic of the approach is shown in Fig.  6.15. Considerable variations were found in the properties of the scaffold depending on the type of processing and post-treatment used (Table  6.5). Salt leaching approach produced scaffold with highest pore size (202 μm) compared to 15 μm for the scaffold frozen at −80 °C. In another study, gas forming was considered to be the best approach to obtain 3D scaffolds with the requirements for bone tissue engineering specifically having highly open and interconnected porous structure (Nazarov et al., 2004). Fibroin has also been made into hydrogels for potential delivery of stem cells by blending with pullulan and crosslinking with enzymes (Li et al., 2018). Silk cocoons were degummed and later dissolved using LiBr. Carboxymethylated pullulan was added into the silk solutions in various concentrations in the presence of horse radish peroxidase and hydrogen peroxide and formed into hydrogels by freeze drying. Hydrogels made had swelling ratio ranging from 79.6% to 84.8% but no significant degradation was observed up to 28 days. Compressive strength of fibroin scaffolds increased substantially from 7 to 71 kPa when amount of pullulan was increased to 6%. Similar changes were also observed in the porosity and pore size with pore sizes ranging from 10 to 180 μm. Up to 90% cell proliferation was observed on the blend scaffolds indicating their suitability for musculoskeletal tissue engineering (Li et al., 2018). The wild silkworm Antheraea mylitta and B. mori were used to extract fibroin and develop nanoparticles for drug delivery and other applications (Kundu et  al., 2010). The process of desolvation in which DMSO was used as the desolvating agent was employed to generate the nanoparticles. In this process, 10 mL of DMSO was put into

Applications of silk153

Silk Processing Extraction of sericin

Cocoon silk

(B)

0.02 M Na2CO3 boiling water

Dissolving silk fibroin Filtering & Dialysis

Air dried fibroin

Freeze drying Silk foam

5~15% in 9.3 M LiBr

2~5.8% Aqueous silk solution

Silk Scaffold Fabrication

Freeze Drying Method Aqueous silk solution (5.8 w/v%) in Teflon mold Add Methanol

Freeze drying

Viscocus silk solution (17%) in Teflon mold

Salt Leaching Method NaCl

Silk hydrogel

Dissolving silk foam in HFIP (17 w/v%)

(A)

Gas Foaming Method

Add porogens

NH4HCO3

HFIP evaporation Methanol Leaching salt in water at room temp., 24 hrs

Sublimation of NH4HCO3 in hot water, 10 min

Porous silk matrix

Fig. 6.15  Schematic representation of the processing of silk (A) and development of the scaffolds (B) (Nazarov et al., 2004). Reproduced with permission from American Chemical Society.

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Table 6.5  Comparison of the compressive strength and modulus of porous silk fiber scaffolds obtained using various fabrication approaches (Nazarov et al., 2004). Method

Sample

Treatment

Compressive strength, kPa

Compressive modulus, kPa

NH4HCO3/silk wt% 10:1

Methanol

280 ± 4

900 ± 94

1-Butanol 2-Propanol Methanol 1-Butanol 2-Propanol

230 ± 9 250 ± 28 250 ± 21 150 ± 8 100 ± 11

500 ± 37 800 ± 44 1000 ± 75 300 ± 40 200 ± 30

Methanol 1-Butanol 2-Propanol Methanol 1-Butanol 2-Propanol None

30 ± 10 150 ± 14 100 ± 20 175 ± 3 250 ± 4 200 ± 3 80 ± 1

100 ± 2 400 ± 50 400 ± 58 450 ± 94 490 ± 94 790 ± 3 170 ± 7

15% Methanol 25% Methanol 15% 2-Proponol 25% 2-Proponol None 15% Methanol 25% Methanol 15% 2-Proponol 25% 2-Proponol

10 ± 2 10 ± 3 10 ± 2

20 ± 1 10 ± 3 40 ± 4

10 ± 3

50 ± 8

20 ± 2 20 ± 3 5 ± 4 30 ± 2

220 ± 7 90 ± 21 90 ± 40 100 ± 1

20 ± 1

130 ± 1

Gas foaming

20:1 Salt leaching NaCl/silk wt% 10:1

20:1 Freeze drying

Temperature −20 °C

−80 °C

Reproduced with permission from American Chemical Society.

10 mL of regenerated silk fibroin solution and left at room temperature during which the silk fibroin forms nanoaggregates and precipitates at the bottom. These nanoaggregates were later sonicated and extensively washed to remove DMSO and later freeze dried to obtain the nanoparticles (Kundu et al., 2010). Nanoparticles formed had average diameters between 150 and 170 nm with the wild silk providing smaller particles than B. mori silk. No cytotoxicity was observed in cells due to the nanoparticles and bioimaging showed that the particles were able to enter the cytoplasm of the cells. VEGF loaded on the nanoparticles showed sustained release for up to 3 weeks. Fibroin extracted from A. mylitta was also used to combine with functionalized carbon nanotubes to develop scaffolds suitable for in vitro and in vivo bone generation (Naskar et al., 2017). Prior

Applications of silk155

to combining with silk, the CNTs were functionalized using non-covalent surfactant Triton X. Fibroin and CNT solutions were blended in various proportions and cast into 96 well plates. These plates were frozen in −20 °C for 24 h and later freeze dried to form sponges. Scaffolds were seeded with fibroblasts and their ability to load and release two growth factors was investigated (TGFβ-1 and BMP-2). Diameter of the pores in the scaffolds was between 49 and 78 μm and porosity was between 87% and 76%. Compressive strength and Young’s modulus was highly dependent on the amount of CNTs and ranged from 2.5 to 35 MPa. Scaffolds with 0.5% and 1.0% carbon nanofibers did not show any cytotoxicity. Formation of ECM was also evident in these scaffolds whereas higher CNT content affected cell proliferation and formation of ECM (Naskar et al., 2017). Desired level of sustained release was also obtainable from the scaffolds containing lower levels of CNTs. Osteoblastic differentiation and gene expression data demonstrated that the scaffolds were suitable for bone regeneration.

6.2 Medical applications of spider silk proteins Spider silk has also been extensively studied for tissue engineering and other medical applications. Regenerated spider silk was combined with poly(lactic acid) and developed into porous fiber scaffolds for culturing PC 12 cell lines (Yu and Sun, 2015). Spider silk and PPLA were dissolved in HFIP and electrospun into fibrous scaffolds. These scaffolds were treated with acetone and washed with water and later dried to form the 3D multi-porous scaffolds. In addition to being biodegradable, the scaffolds were able to direct cell growth and the cell showed elliptical shape and elongation along the axis of the fibers. Hence, it was suggested that the scaffolds could be suitable for growth of axons and tendons (Yu and Sun, 2015). A blend of recombinantly produced spider silks (pNSR32) with poly(caprolactone) and/or chitosan was made into tubular scaffolds for vascular tissue engineering (Zhao et al., 2013). Considerably higher proliferation of cells (Sprague–Dawley rat aortic endothelial cells, SDRAECs) (Fig. 6.16) was observed on the blend scaffolds compared to the individual scaffolds. In addition, the vascular grafts were able to maintain their structural integrity for 8 weeks and able to heal an abdominal aortic defect in SD rats suggesting that the scaffolds could be used for vascular tissue engineering (Fig. 6.17). Silk fibers have been reported to be ideally suited for developing conduits for nerve regeneration. Among the different silks, spider silk has been found to be particularly suitable for nerve regeneration. When implanted in vivo in both rodents and sheep, spider silk conduits showed good nerve regeneration without any toxicity or immunological reactions (Radtke et al., 2011). Similarly, axonal regeneration was achieved using spider silk based constructs. It was found that Schwann cells migrated into the nerve constructs and a large number of axons grew through the constructs (Radtke et  al., 2011). Spider silk was directly extracted from Nephila spiders in lengths of about 150 m. These silk fibers were made into constructs by passing through acellularized venules (Radtke et al., 2011). Fig. 6.18 shows the regeneration of axons and migration of the schwann cells into the constructs. Hydrogels incorporating various biological molecules have been developed using recombinant spider silk proteins. Recombinant proteins obtained from eADF4(C16)

156

Silk: Materials, Processes, and Applications

Cell Viability (OD570nm-630nm)

2.0

1.5

PCL pNSR32/PCL pNSR32/PCL/CS Coverslip

*#D *#D

*#

*# *

1.0

*#D

*

*# 0.5

*# *#

*

0.0 1

5

3

7

Time (d)

Fig. 6.16  Comparison of the viability of the SDRAECs on individual and blend scaffolds after incubation for 1–7 days (Zhao et al., 2013). Reproduced with permission from Elsevier.

Fig. 6.17  Images of the natural blood vessel (A) compared to the spider silk/PCl/CS blend before transplantation (B) and after 2, 4, and 8 weeks after implantation (C, D and E, respectively) (Zhao et al., 2013). Reproduced with permission from Elsevier.

Applications of silk157

Fig. 6.18  Images show the regeneration and migration of the axons and nerve cells through the spider silk conduits. Neurofilaments are shown in green and Schwann cells in red (Radtke et al., 2011). Reproduced with permission through open access licensing.

were dissolved in 6 M guanidinium thiocyanate at 5 mg/mL and dialyzed to obtain proteins between 6000 and 8000 Da which were redissolved to obtain solution of 3–5% concentration (Kumari et al., 2018). Three biomolecules bovine serum albumin (BSA), horse radish peroxidase (HRP) and lysozyme (LYZ) were loaded directly or through the diffusion process and made into hydrogels by incubation at 37 °C. Further, spider silk was made into particles (320 to 540 nm) and combined with fluorescently labeled LYZ and BSA to determine the loading and release efficiencies. Amount of the biomolecule released from the hydrogels was dependent on the media, type of biomolecule and other conditions (Fig. 6.19). Protein solutions containing the silk particles could be made into 3D printed hydrogel scaffolds for potential drug delivery applications.

6.3 Clinical uses of silk Silk fibroin based scaffolds are being commercially used for medical applications. Table 6.6 lists some of the reports on the use of silk based (SERI) scaffolds for various end-uses. A company Sofregen Medical Inc. (https://www.sofregen.com/silk-­proteinplatform) is manufacturing and selling the SERI scaffolds.

158

gelation at 37°C b sheets biologicals

(B)

60 40 10 mM Tris/HCl, pH 7.5 25 mM PB, pH 7.5 25 mM PBS, pH 7.5

20 0

0

2

4 6 Time (h)

8

10

100

(C)

80 60 40 10 mM Tris/HCl, pH 7.5 25 mM PB, pH 7.5 25 mM PBS, pH 7.5

20 0

0

2

4 6 Time (h)

8

10

Accumulated release of LYS (%)

80

eADF4(C16) hydrogels with encapsulated biologicals

50 40 30 20 10

(D)

10 mM Tris/HCl, pH 7.5 25 mM PB, pH 7.5 25 mM PBS, pH 7.5

0 0

2

4 6 Time (h)

8

10

Fig. 6.19  Process of preparing the recombinant silk protein hydrogel using simple diffusion (A); release profile of BSA (B), HRP (C) and LYS (D) from the hydrogels (Kumari et al., 2018). Reproduced with permission from American Chemical Society.

Silk: Materials, Processes, and Applications

Accumulated release of BSA (%)

100

Accumulated release of HRP (%)

eADF4(C16) solution biologicals (4% w/v) (BSA, HRP and LYS)

(A)

Article type

Study sponsor

Retrospective case report

Allergan

Retrospective study Multi center

Allergan

Prospective study Multi center

Allergan

Retrospective case report

Allergan

Retrospective case report

Allergan

Retrospective study

Not disclosed

Intervention

Clinical follow up (months)

Reported outcome

Abdominoplasty and use of scaffold to provide soft-tissue support to the abdominal fascia in patient with massive weight loss Revision of breast augmentation (n = 40); revision of breast reconstruction (n = 24); mastopexy augmentation (n = 20); mastopexy augmentation-revision (n = 16); hernia repair (n = 11); other (n = 30) 2-Stage implant-based breast reconstruction

24

Contour and flatness of the anterior abdominal wall was maintained

0–12

Abdominoplasty and lower body lift of in patient with massive weight loss. Scaffold implantation on left lower body only Brachioplasty

7

Adverse side effect reporting voluntary Surgeons rated the ease of use a mean of 2.86 (scale 0–3) Surgeons rated their satisfaction a mean of 9.31 (scale 0–10) 75 subjects undergone stage 2, subject satisfaction score 4.3 ± 0.91 (5 best). Investigator satisfaction score was 9.4 ± 0.84 (10 best). Adverse effects in 214 breasts: tissue necrosis (6.1%), seroma (6.1%), hematoma (2.8%), breast infection (1.9%), cellulitis (1.9%), implant loss (1.9%), capsular contracture (0%) No complications reported, improve patient satisfaction

77 Patients (71 women, 6 men) underwent

18.4 ± 7.5

6

6

No complications reported, perceived faster maturation process and a better-quality scar The overall complication rate (N77) was 6.5%, consisting of 2 wound Continued

Applications of silk159

Table 6.6  Details of the applications and outcome of the use of SERI scaffolds under practical conditions (Holland et al., 2019)

Table 6.6  Details of the applications and outcome of the use of SERI scaffolds under practical conditions (Holland et al., 2019)—cont'd Study sponsor

Multi center

Retrospective study Correspondence Retrospective study Prospective study

No No

Intervention Abdominal wall fascial repair or reinforcement. The remaining 95 patients not reported on Direct-to-implant after skin-sparing mastectomy Unilateral skin-sparing mastectomy and immediate reconstruction

Clinical follow up (months)

6–13 Not reported

71 patients undergoing 2-stage breast reconstruction

12

No

Breast reconstruction

12

Allergan No

2-stage implant-based breast reconstruction Direct-to-implant reconstruction with surgical scaffold after skin-sparing mastectomy

24 24–37

Multi center

Correspondence Retrospective study Single center Prospective study Prospective study Single center

Reported outcome dehiscences, 1 with device exposure, 1 seroma, 1 infection with explantation, and a perioperative bulge requiring reoperation Capsular contraction (35%); loss of scaffold due to necrosis (n = 1); Late infection (6 weeks and 3.5 months postsurgery) in 2 breasts leading to scaffold and implant removal. In 2 patients successful completion Investigator satisfaction score was 9.4 ± 0.91 (10 best) and patient scores was 4.5 ± 0.82 (5 best). Complication rates in 105 breasts were tissue necrosis (6.7%), seroma (5.7%), hematoma (4.8%), implant loss (3.8%), capsular contracture (1.9%), breast infection (1.0%) Late infection with Ps. aeruginosa in 2 patients at 5 months resulting in implant replacement. Lack of mesh integration (or degradation) in all 4 patients Investigator satisfaction high No intraoperative complications. Adverse effects in 22 breasts: Postoperative bleeding, that required reoperation occurred in 5% breast, postoperative seroma in 45% and surgical site infection in 9%. Scaffold-related

Silk: Materials, Processes, and Applications

Allergan

160

Article type

Applications of silk161

6.4 Biotechnological applications of silk Genetically modified silk having fluorescent properties were made into a solution (Fig.  6.20) and used for detection of cancer and also for bio-imaging (Kim et  al., 2015). Further, the solution obtained was made into scaffolds and electrospun fibers for implanting into mice and detection of antibodies. Localization of antibodies and specific binding was observed in HeLa cells suggesting the suitability for detection of tumors and metastatic nodes. Fluorescent nanoparticles prepared from the silk fibers were ingested into mice and the particles were found to attach to the epithelial surface of intestine (Fig. 6.21). Since the particles were resistant to acid, it was suggested that they could be used for detection of Esophageal perforations that are difficult to be found using normal techniques (Kim et al., 2015). Biomimetic layers for water purification were developed using silk fibroin nanofibrils and hydroaxapatite (HAP) (Ling et al., 2017). In this approach, silk solution was made into self-assembled nanofibrils and was used for biomineralization. It was reported that the silk/HAP membranes could be formulated rapidly in 1000 times higher than commercial filtration membranes. A comparison of the separation performance of the SNF/HAP membranes for various model compounds is given in Table 6.7. Hybrid membranes of

Fluorescence silk cocoon

Cut cocoons and dispose of worm

Cocoons for 1day in 0.04 M NaHCO3 & Alcalase 1.5cc/L

Rinse fiber several times

Squeeze out excess water and allow to dry overnight

Dialysis 1mM DTT solution Fluorescence silk solution Dialysis membrane

Silk fiber put in 9.5M LiBr solution and add of 1mM DTT and incubate at 45°C for 4h

Fig. 6.20  Schematic representation of preparing the fluorescent silk solution from the cocoons (Kim et al., 2015). Reproduced with permission from Elsevier.

162

Silk: Materials, Processes, and Applications CON

EGFP-SF

Fig. 6.21  Confocal images of the gastric mucous membranes of rat fed with silk fibroin nanoparticles (left) and those fed with fluorescently labeled silk nanoparticles (right) (Kim et al., 2015). Reproduced with permission from Elsevier.

poly(lactide-co-glycolic acid)/silk were developed for purification of human adipose derived stem cells (hADSCs) (Chen et al., 2014). The membranes were able to preferentially differentiate osteogenic cells depending on the alkali phosphatase activity which were difficult to be separated using conventional techniques. Hybrid membranes were able to separate the cells within 30 min compared to 5–12 days for the conventional approach (Chen et al., 2014).

6.5 Cosmetic applications Numerous studies have been done to evaluate the benefits of using silk fibroin and other silk based materials for cosmetic applications. Some of the most common types of using silk based proteins in cosmetics include fibroin, sericin, hydrolyzed fibroin and sericin, silk extract, silk powder and silkworm cocoon extract (Table 6.8). Due to the high value and large market, many companies have reported the possibility of using silk fibroin in cosmetics and many patents have been filed related to the use of silk in various cosmetic products (WO 2019030661 A1 20,190,214; WO 2019040850 A1 20,190,228; WO 2019091682 A1 20,190,516; US 20150079012 A1 20,150,319,). Silk fibroin was used to develop hydrogels for incorporating hair growth promoting agents such as FGF-2. The growth factor was initially encapsulated into lyposomes and later added into silk fibroin (FGF-2-LIP-SF). Growth factors having particle size in the range of 85 nm to 120 nm were applied on to the skin and found to penetrate the dermis. It was found that mice treated with the growth factor containing fibroin hydrogel showed rapid growth of hair follicles and expression of inflammatory TNF-α and suppression of IL-6 cytokines (Xu et al., 2018). Similar results were also obtained when silk sericin was coated onto hair. Silk fibroin was blended with either chitosan or partially hydrolyzed polyacrylamide and made into films for cosmetic applications (Sionkowska et al., 2014). Pure fibroin film had surface roughness of 22.9 nm (Rms) but decreased to 13.4 after blending with chitosan but the surface had increased hydrophilicity. These properties were

Compound

Concentration

Flux, liter/h/m2/ bar

Green fluorescent protein Cytochrome C Bovine serum albumin Gold nanoparticles Eosin B Orange G Alcian Blue 8GX Brilliant Blue G Brilliant Blue R-250 Alizarin Red S Rhodamine B Congo Red Direct Red 81 Fluorescent Brightener 28

0.05 mg/mL

1079

91.1

92.6

–

2.0 mg/mL 2.0 mg/mL

1254 636

97.8 70.2

61.8 33.3

– 10.7

5.5 × 1013 units/ml 251 μm 306 μm 80 μm 139 μm 44 μm 434 μm 171 μm 153 μm 240 μm 85 μm

1185 1288 1015 881 928 687 819 1107 967 1020 1145

100 64.9 76.7 100 100 100 99.3 100 100 99.8 100

100 57.0 32.1 100 100 99.8 91.1 49.3 100.0 88.3 93.7

– 13.5 18.7 100 100 99.8 88.8 40.8 90.9 56.4 92.2

Reproduced with permission through Open Access Publishing.

Rejection at 10 min, %

Rejection at 2 h, %

Rejection at 24 h, %

Applications of silk163

Table 6.7  Ability of the SNF/HAP membranes to separate various model compounds (Ling et al., 2017).

164

Silk: Materials, Processes, and Applications

Table 6.8  Form of silk and method of manufacture for cosmetic applications. Form of silk

Method of manufacture

Hydrolyzed silk

A solution of silk obtained by hydrolyzing silk using acid, alkali or enzymes Hydrolyzed silk of specific molecular weight in ethanol solution Trimethyl quaternary ammonium derivative of hydrolyzed silk Particles of silk with average size of about 5.85 μm Pigment with silk fibroin coating on the surface.

Hydrolyzed silk ether ester Quaternary silk polypeptide Silk powder Silk coated pigment

suggested to be suitable for cosmetic applications such as hair cream. In a study, novel vitamin E was loaded onto electrospun silk fibroin nanofibrous mats for potential use for skin treatment and regeneration (Sheng et al., 2013). Up to 8% vitamin could be loaded onto the mats and a sustained release of up to 80% was observed over a period of 80 h. Substantially higher growth and proliferation of mouse fibroblasts was possible on the vitamin containing fibroin mats and hence considered to be suitable for personal skin care and also tissue regeneration (Sheng et al., 2013). In a similar study, l-ascorbic acid 2-phosphate (VC-2-p)- was loaded onto electrospun nanofibroin (360 to 510 nm diameter) mats for skin care applications (Fan et al., 2012). Up to 3% of the bioactive agent could be loaded onto the fibers and a release of up to 80% was possible within 250 h. The growth factor containing silk mats showed excellent fibroblast viability and also higher expression of functional genes indicating suitability of the scaffolds for anti-aging skin care and other applications (Sheng et al., 2013). Silk fibroin obtained in a form soluble in water was found to improve the moisturizing efficiency of skin. It was suggested that the higher hydroxyproline content in fibroin was responsible for the moisturizing effect as indicated by the decreased impedance values on the skin (Daithankar et al., 2005). A considerably smoother skin texture and higher flexibility was reported for the fibroin containing films. Micro and nanoparticles of silk have been used as additives in various cosmetics. For instance, powder from silk glands was used as baby cosmetic powder. It was observed that addition of fibroin lead to 50% increase in moisture sorption and with a considerably low irritation index of 0.5. Several companies are selling silk powder based cosmetics (https://www.amsilk.com/news/#c236). Hydrolyzed silk in various forms and shapes is suggested to have antistatic and humectant properties and hence suitable for tissue engineering and skin care applications. A few researchers have also explored the possibility of using wild silk for cosmetic applications. Antheraea pernyi silk was hydrolyzed to separate alanine rich and tyrosine rich fractions (Lee et al., 2011). No arsenic or mercury was detected but trace elements lead, cadmium etc. were detected in the fractions and hence it was suggested that the A. pernyi silk was suitable for cosmetic applications. In addition to fibroin form silks, the byproduct of silk processing sericin has also been studied as a moisturizer for skin care applications (Padamwar et  al., 2005).

Applications of silk165 1 Time (h) 0

TEWL (mg/cm2/h)

–1 –2

0

1

2

3

4

5

6 Control skin SS 1.5

–3 –4 –5

SS 2 PL 10 PL 5 CB 1.5 CB 2

–6 –7

Fig. 6.22  Changes in the transepidermal water loss (TEWL) of sericin hydrogels containing various amount of gel stabilizers (Padamwar et al., 2005). Reproduced with permission from John Wiley and Sons.

Sericin gels were prepared by dissolving 1.5% or 2% protein in water and with the addition of pluoronic and carbopol, heating the solution to 45–50 °C and aging the solution overnight at room temperature. The gel was applied onto the skin (forearms) and measurements were done to determine the impedance and transepidermal water loss (Fig. 6.22) and other properties. Sericin reduced skin impedance and water evaporation due to the moisturizing effect particularly due to the presence of hydroxyproline. Sericin is reported to have UV and oxidation resistance and provides wrinkle resistance, highly desirable for cosmetic applications. It has been shown that gels and sponges with desired mechanical properties can be prepared from sericin by varying the conditions during fabrication (Jang and Um, 2017). Sericin sponges were made using repeated freezing and thawing cycles and later treating with ethanol. Sponges with porosity up to 91% and swelling of 3600% were formed. Gels developed had strength up to 6000 Pa depending on sericin concentration gelation and storage time. Comparatively, the sericin sponge had bending strength of 1100 Pa and strain of 15% in the wet state (Jang and Um, 2017). Although the amount of literature on silk cosmetics is relatively less, the interest in using silk based materials for cosmetics is increasing rapidly. This is evident by the number of patents filed on silk related cosmetics. Major companies manufacturing cosmetic products such as proctor and gamble and L’oreal have claimed many inventions on using silk for cosmetic applications. In addition to the silk fibroin, silkworm itself has been considered to develop moisturizers, stain reduction and age spot relief creams. Similarly, native and modified spider silk proteins have also been used for cosmetic applications (KR1020087007719A). Although extensively used, concerns have been expressed on the safety of using silk based proteins for cosmetics applications. For instance, the personal care products council suggests that using fibroin,

166

Silk: Materials, Processes, and Applications

MEA-hydrolyzed silk and even silkworm cocoon extract for cosmetics is not advisable. Further studies particularly using in  vivo models are in progress to ascertain the benefits and disadvantages of using several silk based products for skin and other personal care applications.

6.6 Miscellaneous applications 6.6.1 Removal of metal ions Potential of using modified silk fibroin membranes for removal of metal contaminants in waste water was investigated (Gao et al., 2017). B. mori silk fibroin was degummed and dissolved using a ternary solvent system. The solution obtained was combined with methacryloxypropyltrimethoxysilane (MTPS) and cast into membranes. These membranes were used for sorption of six different metal ions. Among the different metals studied, highest removal (80%) was observed for Pd (II) and lowest for Ni (II) and Cr (III) (Fig. 6.23). Absorption of the metal ions onto the silk membrane followed second order kinetics and considered to be suitable for waste water treatment. Instead of using pure silk fibroin, a blend of wool keratose and silk fibroin was electrospun into nanofibers and evaluated for removal of Cu2+ metal ions (Baek et al., 2007). The morphology and mechanical properties of the membranes varied with the relative percentage of the two polymers. Similarly, the extent of removal of Cu2+ depended on the pH during sorption and the blend ratio. Amount of metal ions sorbed varied from 7 to 20 mg/g with membranes containing 70% wool providing the highest sorption (Fig.  6.24). Blending with fibroin was found to improve the sorption of the metal

Ion removal percentage (%)

80

60

40

20

0

Co(II)

Ni(II)

Cu(II)

Cr(III)

Cd(II)

Pd(II)

Metal Ions

Fig. 6.23  Comparison of the extent of metal ion removal by the modified silk fibroin membranes (Gao et al., 2017). Reproduced with permission from Elsevier.

Applications of silk167 70 20

50

15 qe (mg/g)

Removal of Cu2+ (%)

60

40 30 20

10

5

10 0

0 4.5

7 pH

WK/SF

8.5

WK/ WK/ W W W (100/0 SF (70/30)K/SF (50/50 K/SF (30/70 SF (0/100)ool sliver (con ) ) ) trol)

Adsorbent

2+

Fig. 6.24  Changes in the sorption of Cu ions with variations in pH (left) and ratio of wool and silk fibroin (right) (Baek et al., 2007). Reproduced with permission from Springer Nature.

ions by wool fibers. In a similar approach, blends of wool and silk fibroin were electrospun into fibers having diameters between 200 and 400 nm and used for sorption of Cu2+ ions (Ki et al., 2007). Blend membranes prepared had a specific surface area of 1.429 × 107/m and porosity of 63%, much higher than pure wool or filter paper. Consequently, the blend membranes had a higher sorption capacity of 2.9 μg/g but lower than that reported in another study for the same metal ion.

6.6.2 Development of supercapacitors Extensive studies have been made to understand the potential of using silk for various electronic applications (Fig. 6.25) (Zhu et al., 2016). Although developing supercapacitors using silk based materials is predominant, studies have shown that transparent electronics and devices can also be fabricated using silk. Unique hierarchical porous carbon nanosheets were developed from silk for potential supercapacitor applications. The silk based carbon has very high specific surface area of 2494 m2/g and the pore content was 2.28 cm3/g, ideally suited for preparation of carbon anode materials for lithium batteries. The battery developed had storage capacity of 186 mA h/g, capacitance of 242 F/g and energy density of 102 W h/kg. Even after 10,000 cycles, there was only a 9% loss in capacitance. Ability to fabricate the electrode in simple one-step process and high performance were considered favorable for various energy applications (Hou et al., 2015). Instead of using raw silk, fibroin was dissolved in 9.3 M LiBr to obtain a 20% solution and later concentrated to 7–8%. Further, the fibroin obtained was cast into films and later treated with KOH and carbonized at 800 °C for 3 h. A specific capacitance of 264 F/g at current density of 6.2 A/g compared to 120F/g at 52.5 A/g in acid electrolyte was obtained which was considered to be unprecedented. Also, an energy density of 133 Wh/kg and power density of 217 KW/Kg were considered to be several orders higher than that of lithium ions batteries. The supercapacitors developed also had excellent charge/discharge functions with only 6.8% decrease in initial capacitance even after 10,000 cycles (Yun et al., 2013).

168

Silk: Materials, Processes, and Applications

Fig. 6.25  Potential applications of silk in developing electronics devices and components (Zhu et al., 2016). Reproduced with permission from John Wiley and Sons.

Antheraea mylitta silk cocoons were carbonized by heating up to 400 °C under argon atmosphere (Sahu et al., 2015) to obtain silk carbon. The carbon obtained was treated with HNO3: H2O solution for 24 h at room temperature to improve performance. Such treatment resulted in the carbon being highly doped with nitrogen (15%), beneficial for energy applications. CV studies showed that the capacitance of the modified carbon was 348 F/g at 5 mV/s and increased to as high as 631 F/g. Instead of using chemical modifications, highly nitrogen doped B. mori silk fibroin was developed by using heat treatment under inert conditions. In this approach, silk carbon formed at 500 and 700 °C were activated using saturated steam at 850 °C. Some of the properties of the carbon obtained using different conditions are shown in Table  6.9. The silk carbon had lower volumetric capacitance but substantially higher specific capacitance compared to phenol based carbon. It was suggested that steam activation was a greener and economical approach to develop silk based supercapacitors for energy storage applications (Kim et al., 2007). Nitrogen doping was also done to obtain porous carbon nanosheets from silk cocoons for potential application in lithium ion batteries. In this method (Fig. 6.26), cocoons were cut into pieces and combined with ZnCl2 and FeCl3 solution and heated up to 900 °C for 1 h under nitrogen atmosphere for simultaneous activation and carbonization (Xiang et al., 2017). The electrode carbon was further treated with different sulfur loadings to improve electrical conductivity. The carbon developed had a unique interconnected sheet like morphology (20 to 50 nm thick nanosheets) with a specific surface area of 1540 m2/g and large pore volume

Applications of silk169

Table 6.9  Conditions used and properties of carbon obtained from silk fibroin (Sahu et al., 2015).

Temperature, °C

N, %

Conductivity, S/m

400 600 800

15.2 9.8 4.1

38 × 10−5 9.5 × 10−5 16 × 10−6

Specific capacitance, F/g

Charge transfer resistance, Ω

Equivalent series resistance

Specific capacitance (F/g)

348 83 70

1.3 1.57 3.5

3.8 6.0 8.0

220.5 37.6 18.0

Reproduced with permission from Elsevier.

ZnCl2 Cutting

Stirring and evaporation

Dissolution

Drying

Silkworm cocoon Recovery

Fe and Zn

S8 NPCN/S

Melting infusion

NPCN

NPCN

FeCl3 solution Water-bath heater (1) Grinding (2) HCl washing (3) H2O washing (4) Drying

Activation and carbonization

N2 Tube furnace

Fig. 6.26  Schematic representation of the process used to develop the carbon from silk cocoons (Xiang et al., 2017). Reproduced with permission from Elsevier.

of 1.85 cm3/g. With pore sizes between 1.03 and 108 nm and specific surface area of 1540 m2/g and pore volume of 1.85 cm3/g, the carbon material before sulfur treatment was considered to be ideal for transport of electrons and Li+ ions during discharge/ recharge cycles. However, the sulfur treated carbon had better charge/discharge capabilities of up to 1303 mAh/g and retained the performance even after 100 cycles when used as cathode (Xiang et al., 2017). Overall, it was suggested that properties of silk and the easy processing would provide ideal electrodes for lithium sulfur batteries.

6.6.3 Electronic devices Electrochemical transistors were developed by doping PEDOT onto woven silk fibers (Müller et al., 2011). Silk monofilaments were arranged in a cross-junction pattern to form the transistors with the two ends of the fibers forming the source and the drain terminals. Treating the silk fibers with PEDOT decreased the mechanical properties but provided good electrochemical properties such as ON/OFF ratio and VOFF voltage. In another study, flexible electrodes were made using silk fibroin (Hu et al., 2018). To obtain such a flexible electrode, silk fabrics were carbonized and cut into rectangular strips. Later, the silk carbon was treated with Ti3C2Tx Mxene powder for a period of 20 days resulting in

170

Silk: Materials, Processes, and Applications

Fig. 6.27  Ended and twisted silk carbon fabric flexible electrodes treated with Ti3C2Tx (Hu et al., 2018). Reproduced with permission from Elsevier.

the formation of a coating on the fiber surface. Optical images of the bended and twisted flexible electrodes are shown in Fig. 6.27. The electrode developed had a specific capacitance of 362 mF/cm2 at 2 mV/s, much higher than many other materials used as electrodes (Hu et al., 2018). The electrodes had high stability with capacitance retention ratio higher than 90% even after 1000 charge/discharge cycles (Zhu et al., 2016). A dual mode electronic skin (e-skin) was developed for simultaneous detection of pressure and temperature using carbon fiber membranes derived from silk fibers (Wang et al., 2017). B. mori silk was dissolved in formic acid and electrospun using a flow rate of 0.6 mL/h onto copper foils. Later, the fibers on the foil were thermally treated at 1050 °C under argon and hydrogen atmosphere. Carbon obtained was coated onto polyethylene terephthalate films for the temperature sensors and p­ olydimethysiloxane (PDMS) to form the strain sensors (Fig. 6.28). The sensors developed had remarkable performance for sensing with sensitivity of 0.81% resistance change per °C. However,

Electrospinning

Silk Cocoons

Heat Treatment

Silk Nanofibers

Patterning & Encapsulation Pre-stretching, Patterning & Encapsulation

Stamp Transfer

Silk-derived Carbon Fiber SilkCFM@Polymer Film Membrane (SilkCFM)

Lamination Temperature Sensor

Pressure Sensor

Combo E-Skin Sensor

Fig. 6.28  Schematic representation of the process to develop the dual sensor “e-skin” from electrospun silk nanofiber carbon (Wang et al., 2017). Reproduced with permission from American Chemical Society.

Applications of silk171

Fig. 6.29  Process of developing the ultra-flexible silk fabric supercapacitor. Raw silk fabric (A); after screen printing silver (B), after printing carbon (C), two active layers (D), removal of PDMS support (E) and finally the single textile supported supercapacitor (Zhang et al., 2016). Reproduced with permission from American Chemical Society.

the sensitivity was found to be dependent on the temperature during measurement. Similarly, the strain sensor showed high sensitivity at 50% strain with excellent stability and durability and the readings were not influenced by the external temperature stimulus. Hence, the sensors could be used for monitoring activities such as exhaling, finger pressing and distribution of external stimuli (Wang et al., 2017). An ultra-flexible supercapacitor was developed using silk fabric and screen printing and transfer printing, (Fig. 6.29). The capacitor had a high specific capacitance of 19.23 MF/cm2 at a current density of 1 mA/cm2 and good capacitance retention of 84% after 2000 charge–discharge cycles (Zhang et al., 2016). Unlike the conventional two substrate devices, the silk based device had excellent mechanical properties even in the bent state and was able to tolerate up to 100 bending and twisting cycles without any deterioration in performance. This electrode was suggested to be suitable for use in smart textiles and wearable electronics. In a similar approach, silk fabrics were carbonized and used as flexible strain sensor that was able to be twisted and even knotted (Fig. 6.30) (Wang et al., 2016). The sensors had strain of about 500% and fast response of 10,000 cycles and unusually high electrical ­conductivity of 140 Ω/ sq. Such high performance was reported to be due to the unique hierarchical structure of the silk fabrics after carbonization. These sensors were ­suitable for monitoring motions during vigorous jumping, jogging, respiration and other activities through wearable

172

Silk: Materials, Processes, and Applications

Fig. 6.30  Developing highly flexible and robust strain sensors from carbonized silk fabric. Process of developing the sensor (A); optical images of the raw (B and D) and carbonized (C and E) silk fabrics. The sensor was able to twist and turn even after connecting to an LED (F–H) (Wang et al., 2016). Reproduced with permission from John Wiley and Sons.

electronics. In another study, flexible supercapacitors were developed by combining reduced graphene oxide and silk fibroin and converting the blends into films (Rath et al., 2017). Initially, graphene oxide was exfoliated using a microbial strain and later mixed with silk fibroin solution and the resulting composite was made into an electrode for a supercapacitor device. The device had a specific capacitance of 104 F/g at a current density of 0.5 A/g in an ionic liquid electrolyte. High stability was observed for the electrode with a discharge rate of 1A/g and capacitance retention ratio of 89% after 10,000 cycles. Also, a high energy density of 28.3 Wh/kg at a power density of 78.3 kW/kg in ionic liquid electrolyte and maximum energy storage capacity and maximum available power at 28.3 Wh/kg and 78.2 kW/kg were considered to be suitable for supercapacitor applications (Rath et al., 2017).

References Altman, G.H., Horan, R.L., Lu, H.H., Moreau, J., Martin, I., Richmond, J.C., Kaplan, D.L., 2002. Silk matrix for tissue engineered anterior cruciate ligaments. Biomaterials 23 (20), 4131–4141.

Applications of silk173

Baek, D.H., Ki, C.S., Um, I.C., Park, Y.H., 2007. Metal ion adsorbability of electrospun wool keratose/silk fibroin blend nanofiber mats. Fibers and Polymers 8 (3), 271–277. Baimark, Y., Srihanam, P., Srisuwan, Y., Phinyocheep, P., 2010. Preparation of porous silk fibroin microparticles by a water‐in‐oil emulsification‐diffusion method. J. Appl. Polym. Sci. 118 (2), 1127–1133. Bessa, P.C., Balmayor, E.R., Azevedo, H.S., Nürnberger, S., Casal, M., Van Griensven, M., Reis, R.L., Redl, H., 2010a. Silk fibroin microparticles as carriers for delivery of human recombinant BMPs. Physical characterization and drug release. J. Tissue Eng. Regen. Med. 4 (5), 349–355. Bessa, P.C., Balmayor, E.R., Hartinger, J., Zanoni, G., Dopler, D., Meinl, A., Banerjee, A., et  al., 2010b. Silk fibroin microparticles as carriers for delivery of human recombinant bone morphogenetic protein-2: In vitro and in vivo bioactivity. Tissue Engineering Part C: Methods 16 (5), 937–945. Breslauer, D.N., Muller, S.J., Lee, L.P., 2010. Generation of monodisperse silk microspheres prepared with microfluidics. Biomacromolecules 11 (3), 643–647. Cao, Z., Chen, X., Yao, J., Huang, L., Shao, Z., 2007. The preparation of regenerated silk fibroin microspheres. Soft Matter 3 (7), 910–915. Cassinelli, C., Cascardo, G., Morra, M., Draghi, L., Motta, A., Catapano, G., 2006. Physicalchemical and biological characterization of silk fibroin-coated porous membranes for medical applications. Int. J. Artif. Organs 29 (9), 881. Chen, X., Qi, Y.-Y., Wang, L.-L., Yin, Z., Yin, G.-L., Zou, X.-H., Ouyang, H.-W., 2008. Ligament regeneration using a knitted silk scaffold combined with collagen matrix. Biomaterials 29 (27), 3683–3692. Chen, D.-C., Chen, L.-Y., Ling, Q.-D., Wu, M.-H., Wang, C.-T., Suresh Kumar, S., Chang, Y., et al., 2014. Purification of human adipose-derived stem cells from fat tissues using PLGA/ silk screen hybrid membranes. Biomaterials 35 (14), 4278–4287. Cheng, G., Chen, J., Wang, Q., Yang, X., Cheng, Y., Li, Z., Tu, H., Deng, H., Li, Z., 2018. Promoting osteogenic differentiation in pre-osteoblasts and reducing tibial fracture healing time using functional nanofibers. Nano Res. 11 (7), 3658–3677. Cheung, H.-Y., Lau, K.-T., Tao, X.-M., Hui, D., 2008. A potential material for tissue engineering: Silkworm silk/PLA biocomposite. Compos. Part B 39 (6), 1026–1033. Cheung, H.-Y., Lau, K.-T., Pow, Y.-F., Zhao, Y.-Q., Hui, D., 2010. Biodegradation of a silkworm silk/PLA composite. Compos. Part B 41 (3), 223–228. Daithankar, A.V., Padamwar, M.N., Pisal, S.S., Paradkar, A.R., Mahadik, K.R., 2005. Moisturizing efficiency of silk protein hydrolysate: silk fibroin. Indian J. Biotechnol. 4 (1), 115–121. DeMuth, P.C., Min, Y., Irvine, D.J., Hammond, P.T., 2014. Implantable silk composite microneedles for programmable vaccine release kinetics and enhanced immunogenicity in transcutaneous immunization. Advanced Healthcare Materials 3 (1), 47–58. Fan, H., Liu, H., Toh, S.L., Goh, J.C.H., 2009. Anterior cruciate ligament regeneration using mesenchymal stem cells and silk scaffold in large animal model. Biomaterials 30 (28), 4967–4977. Fan, L., Wang, H., Zhang, K., Cai, Z., He, C., Sheng, X., Mo, X., 2012. Vitamin C-reinforcing silk fibroin nanofibrous matrices for skin care application. RSC Adv. 2 (10), 4110–4119. Gao, A., Xie, K., Song, X., Zhang, K., Hou, A., 2017. Removal of the heavy metal ions from aqueous solution using modified natural biomaterial membrane based on silk fibroin. Ecol. Eng. 99, 343–348. Hofmann, S., Knecht, S., Langer, R., Kaplan, D.L., Vunjak-Novakovic, G., Merkle, H.P., Meinel, L., 2006. Cartilage-like tissue engineering using silk scaffolds and mesenchymal stem cells. Tissue Eng. 12 (10), 2729–2738.

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Hofmann, S., Hagenmüller, H., Koch, A.M., Müller, R., Vunjak-Novakovic, G., Kaplan, D.L., Merkle, H.P., Meinel, L., 2007. Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds. Biomaterials 28 (6), 1152–1162. Holland, C., Numata, K., Rnjak‐Kovacina, J., Seib, F.P., 2019. The biomedical use of silk: Past, present, future. Advanced Healthcare Materials 8 (1). Hou, J., Cao, C., Idrees, F., Ma, X., 2015. Hierarchical porous nitrogen-doped carbon nanosheets derived from silk for ultrahigh-capacity battery anodes and supercapacitors. ACS Nano 9 (3), 2556–2564. Hu, M., Hu, T., Cheng, R., Yang, J., Cui, C., Zhang, C., Wang, X., 2018. MXene-coated silk-­ derived carbon cloth toward flexible electrode for supercapacitor application. Journal of Energy Chemistry 27 (1), 161–166. Imsombut, T., Srisuwan, Y., Srihanam, P., Baimark, Y., 2010. Genipin-cross-linked silk fibroin microspheres prepared by the simple water-in-oil emulsion solvent diffusion method. Powder Technol. 203 (3), 603–608. Janani, G., Samit, K., 2017. Nandi, and Biman B. Mandal. Functional hepatocyte clusters on bioactive blend silk matrices towards generating bioartificial liver constructs. Acta Biomater. Jang, M.J., Um, I.C., 2017. Effect of sericin concentration and ethanol content on gelation behavior, rheological properties, and sponge characteristics of silk sericin. Eur. Polym. J. 93, 761–774. Jin, H.-J., Chen, J., Karageorgiou, V., Altman, G.H., Kaplan, D.L., 2004. Human bone marrow stromal cell responses on electrospun silk fibroin mats. Biomaterials 25 (6), 1039–1047. Jin, Y., Kundu, B., Cai, Y., Kundu, S.C., Yao, J., 2015. Bio-inspired mineralization of hydroxyapatite in 3D silk fibroin hydrogel for bone tissue engineering. Colloids Surf. B: Biointerfaces 134, 339–345. Ki, C.S., Gang, E.H., In, C.U., Park, Y.H., 2007. Nanofibrous membrane of wool keratose/silk fibroin blend for heavy metal ion adsorption. J. Membr. Sci. 302 (1–2), 20–26. Kim, Y.J., Abe, Y., Yanagiura, T., Park, K.C., Shimizu, M., Iwazaki, T., Nakagawa, S., Endo, M., Dresselhaus, M.S., 2007. Easy preparation of nitrogen-enriched carbon materials from peptides of silk fibroins and their use to produce a high volumetric energy density in supercapacitors. Carbon 45 (10), 2116–2125. Kim, D.W., Lee, O.J., Kim, S.W., Ki, C.S., Chao, J.R., Yoo, H., Yoon, S.I., Lee, J.E., Park, Y.R., Kweon, H., Lee, K.G., 2015. Novel fabrication of fluorescent silk utilized in biotechnological and medical applications. Biomaterials 70, 48–56. Kumari, S., Bargel, H., Anby, M.U., Lafargue, D., Scheibel, T., 2018. Recombinant spider silk hydrogels for sustained release of biologicals. ACS Biomaterials Science & Engineering 4 (5), 1750–1759. Kundu, J., Chung, Y.-I., Kim, Y.H., Tae, G., Kundu, S.C., 2010. Silk fibroin nanoparticles for cellular uptake and control release. Int. J. Pharm. 388 (1–2), 242–250. Lammel, A.S., Hu, X., Park, S.-H., Kaplan, D.L., Scheibel, T.R., 2010. Controlling silk fibroin particle features for drug delivery. Biomaterials 31 (16), 4583–4591. Lee, K.-g., Kweon, H.Y., Yeo, J.-h., Woo, S.O., Han, S.M., Kim, J.-H., 2011. Characterization of tyrosine-rich Antheraea pernyi silk fibroin hydrolysate. Int. J. Biol. Macromol. 48 (1), 223–226. Lee, J.Y., Park, S.H., Seo, I.H., Lee, K.J., Ryu, W.H., 2015. Rapid and repeatable fabrication of high A/R silk fibroin microneedles using thermally-drawn micromolds. Eur. J. Pharm. Biopharm. 94, 11–19. Li, T., Song, X., Weng, C., Wang, X., Wu, J., Sun, L., Gong, X., Zeng, W.-N., Yang, L., Chen, C., 2018. Enzymatically crosslinked and mechanically tunable silk fibroin/pullulan hydrogels for mesenchymal stem cells delivery. Int. J. Biol. Macromol. 115, 300–307.

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Ling, S., Qin, Z., Huang, W., Cao, S., Kaplan, D.L., Buehler, M.J., 2017. Design and function of biomimetic multilayer water purification membranes. Sci. Adv. 3 (4), e1601939. Lozano-Pérez, A.A., Rivero, H.C., Pérez Hernández, M.d.C., Pagán, A., Montalbán, M.G., Víllora, G., Cénis, J.L., 2017. Silk fibroin nanoparticles: Efficient vehicles for the natural antioxidant quercetin. Int. J. Pharm. 518 (1–2), 11–19. Ma, Y., Feng, Q., Bourrat, X., 2013. A novel growth process of calcium carbonate crystals in silk fibroin hydrogel system. Mater. Sci. Eng. C 33 (4), 2413–2420. Meinel, L., Hofmann, S., Karageorgiou, V., Zichner, L., Langer, R., Kaplan, D., Vunjak‐ Novakovic, G., 2004. Engineering cartilage‐like tissue using human mesenchymal stem cells and silk protein scaffolds. Biotechnol. Bioeng. 88 (3), 379–391. Ming, J., Jiang, Z., Wang, P., Bie, S., Zuo, B., 2015. Silk fibroin/sodium alginate fibrous hydrogels regulated hydroxyapatite crystal growth. Mater. Sci. Eng. C 51, 287–293. Ming, J., Li, M., Han, Y., Chen, Y., Li, H., Zuo, B., Pan, F., 2016. Novel two-step method to form silk fibroin fibrous hydrogel. Mater. Sci. Eng. C 59, 185–192. Müller, C., Hamedi, M., Karlsson, R., Jansson, R., Marcilla, R., Hedhammar, M., Inganäs, O., 2011. Woven electrochemical transistors on silk fibers. Adv. Mater. 23 (7), 898–901. Naskar, D., Ghosh, A.K., Mandal, M., Das, P., Nandi, S.K., Kundu, S.C., 2017. Dual growth factor loaded nonmulberry silk fibroin/carbon nanofiber composite 3D scaffolds for in vitro and in vivo bone regeneration. Biomaterials 136, 67–85. Nazarov, R., Jin, H.-J., Kaplan, D.L., 2004. Porous 3-D scaffolds from regenerated silk fibroin. Biomacromolecules 5 (3), 718–726. Padamwar, M.N., Pawar, A.P., Daithankar, A.V., Mahadik, K.R., 2005. Silk sericin as a moisturizer: an in vivo study. J. Cosmet. Dermatol. 4 (4), 250–257. Radtke, C., Allmeling, C., Waldmann, K.-H., Reimers, K., Thies, K., Schenk, H.C., Hillmer, A., Guggenheim, M., Brandes, G., Vogt, P.M., 2011. Spider silk constructs enhance axonal regeneration and remyelination in long nerve defects in sheep. PLoS One 6 (2), e16990. Rath, T., Pramanik, N., Kumar, S., 2017. High electrochemical performance flexible solid-state supercapacitor based on co-doped reduced graphene oxide and silk fibroin composites. Energy 141, 1982–1988. Sahu, V., Grover, S., Tulachan, B., Sharma, M., Srivastava, G., Roy, M., Saxena, M., et  al., 2015. Heavily nitrogen doped, graphene supercapacitor from silk cocoon. Electrochim. Acta 160, 244–253. Sheng, X., Fan, L., He, C., Zhang, K., Mo, X., Wang, H., 2013. Vitamin E-loaded silk fibroin nanofibrous mats fabricated by green process for skin care application. Int. J. Biol. Macromol. 56, 49–56. Shi, P., Goh, J.C.H., 2012. Self-assembled silk fibroin particles: Tunable size and appearance. Powder Technol. 215, 85–90. Singh, Y.P., Adhikary, M., Bhardwaj, N., Bhunia, B.K., Mandal, B.B., 2017. Silk fiber reinforcement modulates in vitro chondrogenesis in 3D composite scaffolds. Biomed. Mater. 12 (4), 045012. Sionkowska, A., Lewandowska, K., Planecka, A., Szarszewska, P., Krasinska, K., Kaczmarek, B., Kozlowska, J., 2014. Biopolymer blends as potential biomaterials and cosmetic materials. In: Key Engineering Materials. 583. Trans Tech Publications, pp. 95–100. Stinson, J.A., Raja, W.K., Lee, S., Kim, H.B., Diwan, I., Tutunjian, S., Panilaitis, B., Omenetto, F.G., Tzipori, S., Kaplan, D.L., 2017. Silk fibroin microneedles for transdermal vaccine delivery. ACS Biomaterials Science & Engineering 3 (3), 360–369. Tsioris, K., Raja, W.K., Pritchard, E.M., Panilaitis, B., Kaplan, D.L., Omenetto, F.G., 2012. Fabrication of silk microneedles for controlled‐release drug delivery. Adv. Funct. Mater. 22 (2), 330–335.

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Türkkan, S., Atila, D., Akdağ, A., Tezcaner, A., 2018. Fabrication of functionalized citrus pectin/silk fibroin scaffolds for skin tissue engineering. J. Biomed. Mater. Res. B Appl. Biomater. 106 (7), 2625–2635. Wang, X., Wenk, E., Matsumoto, A., Meinel, L., Li, C., Kaplan, D.L., 2007. Silk microspheres for encapsulation and controlled release. J. Control. Release 117 (3), 360–370. Wang, X., Kluge, J.A., Leisk, G.G., Kaplan, D.L., 2008. Sonication-induced gelation of silk fibroin for cell encapsulation. Biomaterials 29 (8), 1054–1064. Wang, X., Yucel, T., Lu, Q., Hu, X., Kaplan, D.L., 2010. Silk nanospheres and microspheres from silk/pva blend films for drug delivery. Biomaterials 31 (6), 1025–1035. Wang, C., Li, X., Gao, E., Jian, M., Xia, K., Wang, Q., Xu, Z., Ren, T., Zhang, Y., 2016. Carbonized silk fabric for ultrastretchable, highly sensitive, and wearable strain sensors. Adv. Mater. 28 (31), 6640–6648. Wang, C., Xia, K., Zhang, M., Jian, M., Zhang, Y., 2017. An all-silk-derived dual-mode E-skin for simultaneous temperature–pressure detection. ACS Appl. Mater. Interfaces 9 (45), 39484–39492. Wei, G., Wang, J., Lv, Q., Liu, M., Xu, H., Zhang, H., Jin, L., Yu, J., Wang, X., 2018. Three‐dimensional co‐culture of primary hepatocytes and stellate cells in silk scaffold improves hepatic morphology and functionality in vitro. J. Biomed. Mater. Res. A 106 (8), 2171–2180. Wenk, E., Wandrey, A.J., Merkle, H.P., Meinel, L., 2008. Silk fibroin spheres as a platform for controlled drug delivery. J. Control. Release 132 (1), 26–34. Wu, X., Hou, J., Li, M., Wang, J., Kaplan, D.L., Lu, S., 2012. Sodium dodecyl sulfate-induced rapid gelation of silk fibroin. Acta Biomater. 8 (6), 2185–2192. Xiang, M., Wang, Y., Wu, J., Guo, Y., Wu, H., Zhang, Y., Liu, H., 2017. Natural silk cocoon derived nitrogen-doped porous carbon nanosheets for high performance lithium-sulfur batteries. Electrochim. Acta 227, 7–16. Xu, H.-L., Chen, P.-P., Wang, L.-f., Xue, W., Fu, T.-L., 2018. Hair regenerative effect of silk fibroin hydrogel with incorporation of FGF-2-liposome and its potential mechanism in mice with testosterone-induced alopecia areata. Journal of Drug Delivery Science and Technology 48, 128–136. Yi, B., Zhang, H., Yu, Z., Yuan, H., Wang, X., Zhang, Y., 2018. Fabrication of high performance silk fibroin fibers via stable jet electrospinning for potential use in anisotropic tissue regeneration. J. Mater. Chem. B 6 (23), 3934–3945. Yin, Z., Kuang, D., Wang, S., Zheng, Z., Yadavalli, V.K., Lu, S., 2018. Swellable silk fibroin microneedles for transdermal drug delivery. Int. J. Biol. Macromol. 106, 48–56. Yu, Q., Sun, C., 2015. A three‐dimensional multiporous fibrous scaffold fabricated with regenerated spider silk protein/poly (l‐lactic acid) for tissue engineering. J. Biomed. Mater. Res. A 103 (2), 721–729. Yucel, T., Cebe, P., Kaplan, D.L., 2009. Vortex-induced injectable silk fibroin hydrogels. Biophys. J. 97 (7), 2044–2050. Yun, Y.S., Se, Y.C., Shim, J., Kim, B.H., Chang, S.‐.J., Baek, S.J., Huh, Y.S., et  al., 2013. Microporous carbon nanoplates from regenerated silk proteins for supercapacitors. Adv. Mater. 25 (14), 1993–1998. Zhang, H., Qiao, Y., Lu, Z., 2016. Fully printed ultraflexible supercapacitor supported by a ­single-textile substrate. ACS Appl. Mater. Interfaces 8 (47), 32317–32323. Zhao, J., Qiu, H., Chen, D.-l., Zhang, W.-x., Zhang, D.-c., Li, M., 2013. Development of nanofibrous scaffolds for vascular tissue engineering. Int. J. Biol. Macromol. 56, 106–113. Zhu, B., Wang, H., Leow, W.R., Cai, Y., Loh, X.J., Han, M.‐.Y., Chen, X., 2016. Silk fibroin for flexible electronic devices. Adv. Mater. 28 (22), 4250–4265.

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Zhu, Z., Ling, S., Yeo, J., Zhao, S., Tozzi, L., Buehler, M.J., Omenetto, F., Li, C., Kaplan, D.L., 2018. High‐strength, durable all‐silk fibroin hydrogels with versatile Processability toward multifunctional applications. Adv. Funct. Mater. 28 (10), 1704757.

Further reading Lee, H., Yang, G.H., Kim, M., Lee, J.Y., Huh, J.T., Kim, G.H., 2018. Fabrication of micro/ nanoporous collagen/dECM/silk-fibroin biocomposite scaffolds using a low temperature 3D printing process for bone tissue regeneration. Mater. Sci. Eng. C 84, 140–147. Rockwood, D.N., Preda, R.C., Yücel, T., Wang, X., Lovett, M.L., Kaplan, D.L., 2011. Materials fabrication from Bombyx mori silk fibroin. Nat. Protoc. 6 (10), 1612. Zhang, Y.-Q., Shen, W.-D., Xiang, R.-L., Zhuge, L.-J., Gao, W.-J., Wang, W.-B., 2007. Formation of silk fibroin nanoparticles in water-miscible organic solvent and their characterization. J. Nanopart. Res. 9 (5), 885–900.

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3D printing silk

7

7.1 Scaffolds developed from silk fibroin and blends with other biopolymers The structure, properties and applications of the 3D printed silk structures depends on the form of silk, the printing method used and the printing conditions (Table 7.1). Bombyx mori silk fibroin was used as ink to develop 3D scaffolds using an approach called direct-write-assembly (DWA) (Ghosh et al., 2008). Fibroin dissolved in LiBr solution at 60 °C for 4 h was concentrated to 8% after dialysis using 3500 MW membranes and further concentrated up to 28–30% using PEG membranes of 8000 g/mol with the solution having a viscosity of about 2.9 Pa, appropriate for printing. For the 3D printing, the silk solution in a syringe was extruded using a nozzle having outer and inner diameters of 1.0 and 0.58 mm, respectively. Extrusion was done at a speed of 2 mm/s and pressure of 20–70 kPa into an 86% methanol bath where the proteins coagulated into filaments. Scaffolds were built layer by layer to obtain a size of 2 mm × 2 mm with 2–6 layers (Fig. 7.1). It took about 1–3 min to build the scaffolds and after drying and crystallization, the fibers had a diameter of about 4.5 μm. Ability of the scaffolds to support the growth and differentiation of hMSCs was studied in terms of expression of TGF-β1 and GAG content. The three layer scaffold built had periodic structure and a modulus of 5.6 GPa. Cells seeded on the scaffolds showed continued growth and proliferation and after 21 days, there was higher GAG content on the protein scaffolds compared to control (Ghosh et al., 2008). DWA was also used to develop 3D scaffolds from blends of silk fibroin and hydroxyapatite (HA) (Sun et al., 2012). Scaffolds with pore sizes ranging from 200 to 750 μm and filaments of 200 μm in diameter and elastic modulus of 223 MPa, considerably higher than similar scaffolds developed by electrospining were obtained. Silk/ HA scaffolds were used to co-culture hMSCs and hMMECs which formed networks of extracellular matrices. Cells followed the morphology of the scaffolds and it was suggested that the desired 3D structure could be developed by varying the pore spacings (Sun et al., 2012). A combination of silk fibroin nanoparticles with size between 500 and 800 nm and rod shaped hydroxyapatite nanoparticles of size 30–60 nm were mixed in various ratios with poly(lactic acid) and extruded on a 3D printer to form bone clips with the mechanical properties and biocompatibility suitable for in  vivo applications (Yeon et al., 2018). A mixture of 94% PLA, 3% HA and 3% silk fibroin nanoparticles were made into a filament and used for printing the 3D clips (Fig. 7.2). Ability of the clips to support cell growth and their biocompatibility when implanted in mice to heal femoral bone fractures were studied. Clips made from the blends of the three polymers had similar (2.4 MPa) compressive strength but higher cell growth compared to the neat PLA. The composite scaffolds showed excellent alignment to the bones and were able to repair the fractured segments (Fig. 7.3). After 4 weeks of Silk: Materials, Processes, and Applications. https://doi.org/10.1016/B978-0-12-818495-0.00007-7 © 2020 Elsevier Ltd. All rights reserved.

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Table 7.1  Some of the parameters that are adopted and influence the properties of 3D printed scaffolds from silk (DeBari et al., 2018). Parameter

Inkjet printing

Extrusion printing

Printing speed Printing pressure Piezoelectric voltage Ultrasound frequency Nozzle diameter Cell density Viscosity Resolution Cell viability

100,000 drops/s N/A 70–450 V 60–300 Hz 20–120 μm  90%

15 mm/s–10 μm/s 1–99 psi NA NA 150–610 μm 1 × 107 cells/ml 30–600 × 106 mPas 100–1000 μm 68–86%

Fig. 7.1  Scaffolds of various shapes (A–C) produced with the 3D direct ink writing approach and SEM image of a single silk fiber in the scaffold (D) (Ghosh et al., 2008). Reproduced with permission from John Wiley and Sons.

3D printing silk181

(A)

Silk solution

Dry casting at 30°C for 12h

Silk membrane

Grind

Seramic ball SF particle Silk fibroin nano-particle

(B)

PLA HA

Ball milling

SF particle

Load materials to filament maker.

PLA filament including HA and Silk.

Printing clips using 3D printer.

Fig. 7.2  Schematic representation of the process of developing 3D scaffolds (clips) from the blend of PLA, HA and silk fibroin nanoparticles (Yeon et al., 2018). Reproduced with permission from Taylor and Francis.

Fig. 7.3  Digital images of the femur in mice (A), cut femur (B) and the PLA/HA/Silk 3D clip supporting the broken femur (C) (Yeon et al., 2018). Reproduced with permission from Taylor and Francis.

implantation, there was formation of osteoids and osteoblast deposition suggesting complete biocompatibility of the clips. In another study, silk fibroin was combined with hydroxyapatite and made into nanocomposite scaffolds using 3D printing. Fibroin powder was combined with ternary solution of CaCl2, C2H5OH and water and later with an aqueous solution of (NH4)2HPO4 and the pH was adjusted to 10. The resulting SF/HA powder was

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l­ yophilized and used for 3D printing. The mineralized SF/HA was made into particles of