Bioactive Materials for Bone Regeneration [1 ed.] 0128135034, 9780128135037

Table of contents :
Bioactive Materials for Bone Regeneration
Copyright
Preface
1 - Material characteristics, surface/interface, and biological effects on the osteogenesis of bioactive materials
1.1. Fabrication methods of bioactive materials for bone regeneration
1.1.1 Material characteristics of bioactive materials for bone regeneration
1.1.1.1 Chemical composition
1.1.1.2 Porous structure
1.1.1.3 Surface micro- and nanostructure
1.1.2 Design of porous bioactive materials
1.1.2.1 Synthesis of initial nanopowder and precursor
1.1.2.2 Molding of porous structure
1.1.2.3 Sintering technologies
1.1.2.4 Surface modification methods
1.1.3 Main challenges and prospects
1.1.3.1 Main challenges of bioactive materials
1.1.3.2 Enhancing bioactivity and mechanical property methods
1.1.3.2.1 Bonelike apatite formation
1.1.3.2.2 Nanoscale topography
1.1.3.2.3 Whisker reinforcement
1.1.3.2.4 Trace ion doping
References
1.2. Surface micro-/nanostructure regulation of bioactive materials for osteogenesis
1.2.1 Surface morphology of bioactive materials for osteogenesis
1.2.1.1 Orderly micropatterned surface morphology of calcium phosphate–based bioceramics
1.2.1.2 Randomly structured surface morphology of calcium phosphate–based bioceramics
1.2.1.2.1 Hydrothermal treatment of randomly structured surface morphology
1.2.1.2.2 Simulated body fluid immersion and inducing of random calcium phosphate surface morphology
1.2.1.2.3 Other fabrication methods of randomly structured surface morphology
1.2.2 Porosity of bioactive porous materials for osteogenesis
1.2.3 Grain size of bioactive materials for osteogenesis
1.2.3.1 Microscale and submicroscale grain sizes
1.2.3.2 Nanoscale grain size
1.2.4 Summary
References
1.3. Protein adsorption on bioactive materials and its effect on osteogenesis
1.3.1 Current methods for studying protein adsorption
1.3.1.1 Experimental methods
1.3.1.2 Computing methods
1.3.2 Material factors influencing protein adsorption
1.3.2.1 Material factors
1.3.2.1.1 Topography
1.3.2.1.2 Chemical properties
1.3.2.1.3 Hydrophobicity
1.3.2.2 Interactions between proteins and bioactive materials
1.3.3 The effect of protein adsorption on the osteogenesis of bioactive materials
1.3.3.1 Extracellular protein adsorption
1.3.3.2 Adsorption of specific proteins (bone morphogenetic proteins and transcription growth factor beta)
1.3.3.3 Other growth factor adsorption
1.3.3.4 Cytokine adsorption
1.3.4 Summary
References
1.4. Osteogenesis induced by bioactive porous materials and the related molecular mechanism
1.4.1 Angiogenesis of bioactive materials and the involved molecular mechanism
1.4.2 Osteogenesis of bioactive materials and material-mediated mesenchymal stem cell function
1.4.2.1 Osteogenic ionic environment created in the porous structure
1.4.2.1.1 Ca2+ gradient
1.4.2.1.2 PO43− internalization
1.4.2.2 Cells of origin and cellular events in material-induced osteogenesis
1.4.2.2.1 Cells of origin
1.4.2.2.2 Events at cellular level
1.4.2.3 Osteogenic mechanism of bioactive porous titanium
1.4.3 Role of immunoresponse in the osteogenesis of bioactive materials
1.4.3.1 Autocrine effect of mesenchymal stem cells
1.4.3.2 Paracrine effect from immune cells
1.4.4 Summary
References
2 - Biomaterial-induced microenvironment and host reaction in bone regeneration
2.1 Bioactive inorganic ions for the manipulation ofosteoimmunomodulation to improve bone regeneration
2.1.1 Introduction
2.1.2 Application of bioactive ions in developing bone biomaterials and their possible application in manipulating osteoimmunomod ...
2.1.2.1 Strontium
2.1.2.2 Zinc
2.1.2.3 Magnesium
2.1.2.4 Calcium
2.1.2.5 Silicon
2.1.2.6 Cobalt
2.1.2.7 Copper
2.1.2.8 Europium
2.1.2.9 Fluorine
2.1.3 Combining bioactive elements to develop novel bone biomaterials with osteoimmunomodulatory properties as well as promote os ...
2.1.4 Summaries and future prospects
References
2.2 Silicate-based bone cements for hardtissue regeneration
2.2.1 Preparation of silicate-based bone cement
2.2.2 Self-setting properties and drug delivery performance of silicate-based bone cement
2.2.2.1 Setting time and mechanical strength
2.2.2.2 Injectability and washout resistance
2.2.2.3 Drug loading and release properties of silicate-based bone cements
2.2.3 In vitro and in vivo bioactivity and osteoinductivity of silicate-based bone cement
References
2.3 Trace elemente based biomaterials for osteochondral regeneration
2.3.1 Introduction
2.3.2 Biomaterials for osteochondral regeneration
2.3.2.1 The clinical need for osteochondral regeneration
2.3.2.2 The anatomy and properties of osteochondral tissue
2.3.2.3 Current strategies for osteochondral regeneration
2.3.2.4 Biomaterials for cartilage and bone regeneration
2.3.2.4.1 Biomaterials for cartilage regeneration
2.3.2.4.2 Biomaterials for bone regeneration
2.3.3 Trace element–based biomaterials for osteochondral regeneration
2.3.3.1 Manganese-doped biomaterials for osteochondral regeneration
2.3.3.2 Molybdenum-doped biomaterials for osteochondral regeneration
2.3.3.3 Lithium-based biomaterials for osteochondral regeneration
2.3.3.4 Strontium-based biomaterials for osteochondral regeneration
2.3.3.5 Other nutrient elements for osteochondral regeneration
2.3.4 Conclusions and perspectives
References
2.4 Bioactive ions for bone tissue engineering design
2.4.1 Introduction
2.4.2 Effects of bioactive ions on cell proliferation and stemness maintenance
2.4.3 Effects of bioactive ions on osteogenesis and osteoclastogenesis
2.4.4 Effects of bioactive ions on angiogenesis
2.4.5 Design of bioactive ion composite biomaterials for bone tissue engineering
2.4.6 Conclusions and perspectives
References
3 - A bone regeneration concept based on immune microenvironment regulation
3.1. Characteristics of the immune microenvironment in biomaterial-based regeneration
3.1.1 Immune response after biomaterial implantation
3.1.2 Macrophage in bone-relevant physiological and pathological processes
3.1.2.1 Initiating factors of macrophages: injury, protein absorption, danger signals, and polymorphonuclear leukocyte activation
3.1.2.2 Introduction of macrophages and foreign body giant cells
3.1.3 T cells and B cells in bone-relevant physiological and pathological processes
3.1.3.1 T lymphocyte activation and features of immunological function
3.1.3.2 B-lymphocyte response to antigens
3.1.4 Dendritic and natural killer cells in bone-relevant physiological and pathological processes
References
3.2. Biomaterials and their degradation products in the immune microenvironment and regeneration
3.2.1 Metallic implants, the immune microenvironment, and regeneration
3.2.1.1 Immune response to titanium implants
3.2.1.1.1 Macrophage role in immune response to titanium implants
3.2.1.1.2 T cell and dendritic cell roles in immune response to titanium implants
3.2.1.1.3 Influence of implant surface characteristics on immune response to titanium implants
3.2.1.1.4 Aseptic loosening in titanium implants
3.2.1.1.5 Immune response to cobalt–chromium alloy implants
3.2.1.1.6 Aseptic loosening in cobalt-chromium alloy implants
3.2.2 Inorganic materials implants, immune microenvironment, and regeneration
3.2.2.1 General immune response to bioceramic implants
3.2.2.2 Calcium phosphate ceramics in bone regeneration
3.2.2.2.1 Calcium ion immune microenvironment regulation through inflammation adjustment
3.2.2.2.2 Positive effects of phosphate ions on bone regeneration
3.2.2.3 Other ions that regulate the immune microenvironment
3.2.2.3.1 Silicon regulation of the immune microenvironment through macrophage inhibition
3.2.2.3.2 Positive effect of silicon on bone regeneration
3.2.2.3.3 Zinc regulation of the immune microenvironment through changes in tumor necrosis factor alpha and interleukin-1 beta levels
3.2.2.3.4 Magnesium regulation of the immune microenvironment by inhibition of the toll-like receptor pathway
3.2.2.3.5 Different immune responses to hydroxyapatite and strontium-doped calcium phosphate
3.2.2.4 Appropriate pore and particle sizes for influencing the immune microenvironment to strengthen bone regeneration
3.2.3 Organic materials implants, immune microenvironment, and regeneration
3.2.3.1 Immune response to ultra-high-molecular-weight polyethylene
3.2.3.2 Ultra-high-molecular-weight polyethylene particle activation of the inflammasome
3.2.3.3 Polylactic acid regulation of the immune microenvironment by macrophage
3.2.3.4 Polylactic acid degradation product influences on the microenvironment
3.2.3.5 Surface property regulation of the immune microenvironment
3.2.3.6 Topography property regulation of macrophage
3.2.3.7 Geometry property regulation of inflammation response
3.2.3.8 Delivering bioactive factors through polymers
References
3.3. Biomaterial research and development aim to produce a “good” bone immune microenvironment by regulating immune cell respons ...
3.3.1 Surface property regulation of immune-mediated osteogenesis
3.3.1.1 Topography
3.3.1.2 Charge
3.3.1.3 Hydrophilicity and hydrophobicity
3.3.2 Particle size regulation of immune-mediated osteogenesis
3.3.3 Microporosity regulation of immune-mediated osteogenesis
3.3.4 Ion regulation of immune-mediated osteogenesis
3.3.5 Summary and expectations
References
Index
A
B
C
D
E
F
G
H
I
L
M
N
O
P
Q
S
T
U
V
Z

Citation preview

Bioactive Materials for Bone Regeneration

Jiang Chang Biomaterials and Tissue Engineering Research Center Shanghai Institute of Ceramics Chinese Academy of Sciences Shanghai, Shanghai, China

Xingdong Zhang National Engineering Research Center for Biomaterials Sichuan University Chengdu, Sichuan, China

Kerong Dai Shanghai Ninth People’s Hospital Shanghai Jiaotong University School of Medicine Shanghai, Shanghai, China

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2020 Higher Education Press. Published by 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: 978-0-12-813503-7 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Matthew Deans Acquisitions Editor: Glyn Jones Editorial Project Manager: Naomi Robertson Production Project Manager: Sojan P. Pazhayattil Cover Designer: Christian J. Bilbow Typeset by TNQ Technologies

Preface Several thousand years ago people began using materials to fix damaged tissue such as bones and teeth, and nowadays different materials including metals, ceramics, and polymers are widely used for orthopedic and dental applications. In most of these clinical applications, we mainly utilize physical properties of materials such as mechanical support, physical coverage, and mechanical fixation to support bone regeneration. However, with increased economic development and an aging population, regenerative medicine is facing new challenges and questions that need to be answered including how to enhance chronic wound healing, heal aging people or patients with osteoporosis, and reduce bone healing time (reduction in treatment time and costs). One of the fundamental questions is whether, instead of physical support for bone regeneration, biomaterials have biological activities that can actively stimulate the bone-healing process. In recent years, many studies have shown that specific structural and chemical material signals such as the surface micro-/nanostructure of bone graft materials and ions released from bioceramics and bioactive glasses indeed have activity to stimulate bone regeneration through the regulation of cell proliferation, stem cell differentiation, cellecell interaction, and macrophage polarization. However, how these material signals activate the biological system and their related mechanisms are still unclear. Elucidating the mechanisms of biomaterials in stimulating cellular activity and bone regeneration will provide important information for designing optimal materials for bone regeneration. With the support of the Natural Science Foundation of China, we conducted a five-year project to investigate bioactive bone-regeneration materials with an emphasis on the aforementioned scientific questions, and these studies have resulted in the establishment of several research teams with extended research collaboration nationally and internationally. These studies have also further extended our knowledge about the interaction between biomaterials and biological systems and our understanding of the bioactivity of bone-regeneration biomaterials. We believe that the concept of bioactive materials with biological activity derived from pure materials may significantly contribute to the development of new-generation biomaterials for regenerative medicine. Therefore, with the help of project team members and their collaborators, we decided

ix

x Preface

to edit this book, which summarizes related studies in the field of bone biomaterials and gives an overview of updated research progress on bioactive materials for bone regeneration. We hope this book may be interesting for scientists, engineers, and graduate students in biomedical engineering and provide useful information for the development of new-generation biomaterials for regenerative medicine. Jiang Chang Xingdong Zhang Kerong Dai

Chapter 1

Material characteristics, surface/interface, and biological effects on the osteogenesis of bioactive materials Chapter outline 1.1 Fabrication methods of bioactive materials for bone regeneration 1.1.1 Material characteristics of bioactive materials for bone regeneration 1.1.1.1 Chemical composition 1.1.1.2 Porous structure 1.1.1.3 Surface micro- and nanostructure 1.1.2 Design of porous bioactive materials 1.1.2.1 Synthesis of initial nanopowder and precursor 1.1.2.2 Molding of porous structure 1.1.2.3 Sintering technologies 1.1.2.4 Surface modification methods 1.1.3 Main challenges and prospects 1.1.3.1 Main challenges of bioactive materials 1.1.3.2 Enhancing bioactivity and mechanical property methods

3

4 4 4 5 6

6 7 9 11 12 12

1.1.3.2.1 Bonelike apatite formation 1.1.3.2.2 Nanoscale topography 1.1.3.2.3 Whisker reinforcement 1.1.3.2.4 Trace ion doping References 1.2 Surface micro-/nanostructure regulation of bioactive materials for osteogenesis 1.2.1 Surface morphology of bioactive materials for osteogenesis 1.2.1.1 Orderly micropatterned surface morphology of calcium phosphatee based bioceramics 1.2.1.2 Randomly structured surface morphology of calcium phosphatee based bioceramics

14 14

15 16 16

26

26

26

29

14

Bioactive Materials for Bone Regeneration. https://doi.org/10.1016/B978-0-12-813503-7.00001-7 Copyright © 2020 Higher Education Press. Published by Elsevier Ltd. All rights reserved.

1

2 Bioactive Materials for Bone Regeneration 1.2.1.2.1 Hydrothermal treatment of randomly structured surface morphology 1.2.1.2.2 Simulated body fluid immersion and inducing of random calcium phosphate surface morphology 1.2.1.2.3 Other fabrication methods of randomly structured surface morphology 1.2.2 Porosity of bioactive porous materials for osteogenesis 1.2.3 Grain size of bioactive materials for osteogenesis 1.2.3.1 Microscale and submicroscale grain sizes 1.2.3.2 Nanoscale grain size 1.2.4 Summary References 1.3 Protein adsorption on bioactive materials and its effect on osteogenesis 1.3.1 Current methods for studying protein adsorption 1.3.1.1 Experimental methods 1.3.1.2 Computing methods 1.3.2 Material factors influencing protein adsorption 1.3.2.1 Material factors 1.3.2.1.1 Topography 1.3.2.1.2 Chemical properties 1.3.2.1.3 Hydrophobicity

29

31

33 35 36

36 40 44 44

53 53 53 54 56 56 56 58 60

1.3.2.2 Interactions between proteins and bioactive materials 1.3.3 The effect of protein adsorption on the osteogenesis of bioactive materials 1.3.3.1 Extracellular protein adsorption 1.3.3.2 Adsorption of specific proteins (bone morphogenetic proteins and transcription growth factor beta) 1.3.3.3 Other growth factor adsorption 1.3.3.4 Cytokine adsorption 1.3.4 Summary References 1.4 Osteogenesis induced by bioactive porous materials and the related molecular mechanism 1.4.1 Angiogenesis of bioactive materials and the involved molecular mechanism 1.4.2 Osteogenesis of bioactive materials and materialmediated mesenchymal stem cell function 1.4.2.1 Osteogenic ionic environment created in the porous structure 1.4.2.1.1 Ca2þ gradient 1.4.2.1.2 PO3 4 internalization 1.4.2.2 Cells of origin and cellular events in material-induced osteogenesis 1.4.2.2.1 Cells of origin 1.4.2.2.2 Events at cellular level 1.4.2.3 Osteogenic mechanism of bioactive porous titanium 1.4.3 Role of immunoresponse in the osteogenesis of bioactive materials

61

62 63

65 67 67 68 69

79

79

82

82 82 84

84 84 86

88

90

Material characteristics, surface/interface Chapter | 1 1.4.3.1 Autocrine effect of mesenchymal stem cells

90

1.4.3.2 Paracrine effect from immune cells 1.4.4 Summary References

3

92 95 96

Bioactive materials play an increasingly important role in regenerative medicine and tissue engineering for bone. Many reports have shown that the biological properties of bioactive materials depend greatly on material characteristics and surface/interface properties. Therefore, this chapter firstly focuses on different fabrication methods for the preparation of bone regenerative biomaterials with an emphasis on accurate control of material characteristics such as chemical composition, macro-/microstructure, and mechanical properties. Methods for surface modification of bone regenerative biomaterials and evaluation of physicochemical properties of prepared materials are also introduced. Protein adsorption is the initial event after implantation of a biomaterial and directly influences subsequent cell behavior and implant fate. This chapter introduces the interactions between bone regenerative biomaterials and various bone-related proteins and discusses the key contributions of adsorbed functional proteins in biomaterials to material-induced bone regeneration. The cell is the fundamental unit of the human body, and the behavior of cells under the influence of biomaterials determines the progress of bone regeneration and repair. Finally, this chapter elucidates the interactions of bone regenerative biomaterials with cells and tissues and the specific effects of material characteristics on osteogenesis and the involved molecular mechanism.

Chapter 1.1

Fabrication methods of bioactive materials for bone regeneration Inspired by the concept of regenerative medicine, the design of biomaterials with tissue-inducing abilities is a new direction for bioactive materials. Bioactive materials should be bioactive not only to bond with the tissue interface, but also to induce tissue regeneration, thus permanently healing damaged or missing tissues and organs. This discovery of material osteoinductivity indicates that materials might be endowed with the biofunction of inducing tissue regeneration, thus making them hopeful solutions for establishing tissue function through optimized design of the material itself without adding any living cells or growth factors. That is to say, fabrication methods are quite important for bioactive materials, as they are critical to biological performance in bone regeneration.

4 Bioactive Materials for Bone Regeneration

1.1.1 Material characteristics of bioactive materials for bone regeneration 1.1.1.1 Chemical composition Among the current bone substitute materials, calcium phosphate (Ca-P) ceramics undoubtedly have the most potential owing to their similar composition to that of the bone mineral as well as their confirmed biocompatibility, osteoconductivity, and osteoinductivity. The most studied Ca-P ceramics are hydroxyapatite (HA), b-TCP, and BCP (HA/b-TCP) [1e4]. Among them, HA is the most stable and occasionally achieves osteoinductivity due to its low dissolution rate. The solubility of b-TCP is much higher than that of HA, but the fast dissolution rate makes it difficult to retain the basic mechanical support for the desired duration [5]. Therefore, biphasic calcium phosphate (BCP) with different b-TCP/HA ratios can achieve optimum solubility and good osteoinductivity [1,2,4e7]. Our previous work compared the osteoinductivity of BCP ceramics with different b-TCP/HA ratios, the results demonstrating that BCP with a b-TCP/HA ratio of 3/7 could promote BMP-2 expression and owned a higher osteoinductivity than those of BCP of 7/3, pure b-TCP, and HA ceramics [2]. Our present work introduced a novel alginate gelatinizing technology to stabilize Ca-deficient hydroxyapatite (CDHA) in BCP ceramics; the obtained BCP ceramics with a high CDHA phase content showed excellent bioactivity and osteoinductivity because the the composition of CDHA was closer to that of bony mineral [8]. Furthermore, the osteoinductivity of nonceramic Ca-P materials (i.e., Ca-P cements, Ca-P composites) was usually weaker than that of Ca-P ceramics, partly due to the lack of a 3D porous structure and high solubility. Silicon (Si) is one of the indispensable trace elements in the human body, found in extracellular matrix compounds and bone [9,10]. It was reported that Si is mainly distributed in the active calcification sites of bone and directly involved in the bony mineralization process [9,11]. Up to now, many kinds of Si-based bioactive materials have been developed and widely applied. Research emphases include ceramic preparation methods, mechanical strength, apatite mineralization, dissolution, bioactive properties, and corresponding mechanisms [12e14]. Due to their variable chemical compositions, the physical, chemical, and biological properties could be well optimized to satisfy the varied requirements of tissue regeneration [9]. One of the most popular silicate ceramics is bioglass, which has been approved by the FDA and employed for orthopedic applications in clinic under the name NovaBone@ [15e17].

1.1.1.2 Porous structure 3D porous structures also play a critical role in determining the osteoinductivity of materials. Osteoinductivity is generally observed in porous Ca-P

Material characteristics, surface/interface Chapter | 1

5

ceramics, while dense Ca-P ceramics cannot induce bone formation [18,19]. The porous structure mainly facilitates the exchange of oxygen and nutrition and allows tissue, blood, and cells to migrate the scaffold interior [1,4,18e21]. It is well known that pore structure parameters (i.e., porosity, shape, size, and connectivity) have a great influence on the biological performance of scaffolds. Generally, high porosity is beneficial to osteogenesis, but the scaffold strength with overly high porosity is too low to provide stable support during the implantation process [1]. It is generally believed that a porosity ranging from 40% to 80% is suitable for bone repair. Moreover, pore connectivity is related to osteogenesis, and the connected pores allow nutrients, cells, and tissue to grow into the inner part of the scaffolds [1,4,22,23]. Yuan HP et al. observed that new bone was mainly generated in the interior of the peripheral channels (close to the openings) of DCPA cement bulk in goat intramuscular implantation [24]. Much previous research has also certified that the suitable pore diameter for bone-repairing scaffolds is about 200e600 mm, and a connected pore size within a range of 50e200 mm is relatively optimal [1,4]. Moreover, micropores (20 nm) could adsorb more fibrinogen and insulin than particles with low porosity [25]. We further certified that the distribution of micropores on the walls of macropores favored the adsorption of lowmolecular-weight proteins [26]. These studies strongly indicate that high levels of micropores in Ca-P ceramics favor protein adsorption that in turn induces osteogenesis.

1.1.1.3 Surface micro- and nanostructure Surface micro- and nanostructure also are an important factor for inducing the bioactivity of biomaterials. Many studies have investigated the effects of surface topography on cellular behaviors (i.e., cell adhesion, proliferation, and differentiation [27e33]. Dalby MJ et al. [34,35] fabricated several kinds of surface topographies with nanostructure and observed that the responses of mesenchymal stem or stromal cells (MSCs) were greatly influenced by surface topography. A kind of nanodisplaced topography could significantly promote osteospecific differentiation; further study found that the disordered nanopit pattern could induce osteogenic differentiation, while symmetric and random nanopit arrays could not. For Ca-P ceramics, surface topography can be tailored by adjusting their grain sizes. Osteoinductivity of BCP ceramics

6 Bioactive Materials for Bone Regeneration

FIGURE 1.1 Material characteristics of bioactive porous materials for bone regeneration.

increased with decreasing crystal size, and the different surface microstructure was regarded as an important factor affecting osteoinductivity [4,36]. The surface pore structure is generally regarded as another important surface topography that has an important influence on cell response, biofunctions, and even osteogenic processes. Our previous work fabricated several kinds of pore structures on HA-dense ceramic discs [37], and the results revealed that macropore structures favored cell proliferation, while micropore structures upregulated early osteoblastic differentiation. Another of our works fabricated that HA ceramics with orderly micropatterned surfaces varied in groove width [38] and found that cell response also changed with the micropatterns. Based on the above analysis, it is inferred that surface topography plays a crucial role in material osteoinductivity by modulating cell behaviors. Overall, chemical composition, porous structure, and the surface microand nanostructure of bioactive materials were regarded as playing important roles in new bone regeneration. The relationships and interactions between them are shown in Fig. 1.1.

1.1.2 Design of porous bioactive materials 1.1.2.1 Synthesis of initial nanopowder and precursor Up to now, many methods have been developed for the synthesis of Ca-P nanopowder and precursor: liquid precipitation [39e43], sol-gel processing [44e48], emulsion technique [49e51], hydrothermal process [52e55], ultrasonic technique [56e58], mechanochemical method [59e61], template method [62e66], microwave processing [67,68], and so on. Various morphologies and structures of Ca-P nanopowder and precursor have been synthesized by means of these methods including spherical, needlelike, fibrous, nanorods, layer nanostructures, hollow nanospheres, and flowers. However, each method for the synthesis of Ca-P nanopowder and precursor

Material characteristics, surface/interface Chapter | 1

7

has advantages and disadvantages. For example, Ca-P nanocrystals with homogenous morphologies can be easily synthesized by the sol-gel process, but the process needs a high sintering temperature to decompose the organic content. Similarly, biomimetic, needlelike/spherical nanocrystals, or nanorods can be prepared using a simple liquid precipitation process, but the preparation process is difficult to control, and the obtained particles easily aggregate. As mentioned above, Ca-P powder or precursor with nanostructure can be fabricated using many methods, and few products by means of corresponding methods can be further considered for the fabrication of Ca-P bioceramics. On the one hand, the yield of most methods is too low to supply fabricating Ca-P bioceramics. For example, the hydrothermal process can synthesize high crystallinity, small size, and good-shaped nanocrystals, but the yield of Ca-P nanocrystals is quite low. In addition, the structures and morphologies of Ca-P nanocrystals are important factors for determining the property of the obtained ceramics. Those with plate, flower, and fiber morphologies are undesirable for the fabrication of porous Ca-P ceramics. Among them, liquid precipitation seems to be most available to fabricate porous Ca-P bioceramics. The well-dispersed, needlelike Ca-P nanoparticles have been synthesized by liquid precipitation with the aid of dispersants (i.e., citric acid, polyethylene glycol), which have been well employed in assembling porous Ca-P bioceramics in our laboratory [36,39,69].

1.1.2.2 Molding of porous structure To endow ceramics with bioactivity, the design of the scaffold porous structure is vital. Porosity, pore size and shape, and interconnectivity are typical parameters for determining the biological and mechanical properties of the bone scaffold. It is generally believed that porosity is necessary for cell migration, attachment, and proliferation as wel as for neovascularization processes [20]. It is well known that natural bone has a multilevel pore structure (from nanometer to micrometer thus to satisfy the growth of different tissue growth [70e72]. Generally, a macropores range from 100 to 1000 mm facilitates the ingrowth of bone tissue and blood vessels, and interconnected pores ranging from 10 to 100 mm are beneficial for nutrient transport. Moreover, micropores less than 10 mm favor protein adsorption and cell attachment [73e75]. Up to now, various methods have been developed to construct porous structure, involving microsphere-sintering, gas-foaming, freeze-drying, organic foam impregnation, and electrospinning (Table 1.1). Among them, microsphere-sintering seems optimal because pore size and porosity can be easily controlled via this method, but it is difficult to produce abundant micropores, so ceramics by this approach do not possess good bioactivity including osteoinductivity [4]. One promising approach is an H2O2 gasfoaming method that is favorable for producing ceramics with abundant micropores besides interconnecting macropores (shown in Fig. 1.2), and the

8 Bioactive Materials for Bone Regeneration

TABLE 1.1 Fabrication methods for three-dimensional porous ceramic scaffolds.

Methods

Pore diameter (mm)

Advantages

Disadvantages

Microspheresintering [76,77]

10e1000

High mechanical properties; controlled pore size and porosity

Lack of micropores; use of template

Gas-foaming [4,78e80]

100 e800; 80%) and pore sizes ranging from 200 to 600 mm are optimal for new bone tissue regeneration [82e84]. Some researchers have pointed out that limiting bone cells to growth in pores does not reflect the size of the aperture but rather the degree of connectivity and size of channels. However, a systematic understanding of the effects of porosity and pore size on the osteogenic outcome of different bioactive porous materials is still needed [85,86]. On the other hand, the mechanical properties of bioactive porous materials are closely related to porosity. In natural bone tissues, the tangent elastic modulus generally decreases as their porosity increases [87,88]. These rules also work in synthetic biomaterials. For example, in a porous tantalum scaffold, the tangent elastic modulus was reduced from 2.2 GPa to 373 MPa, and the yield stress changed from 4 to 12.7 MPa, with an adjustment of the porosity from 66% to 88%. In another example, porous titanium scaffolds with 21%e48% porosity were achieved by metallurgical powder method, and Young’s modulus for these scaffolds was about 6e11 GPa [89], which is similar to that of wet compact human bone [90]. Similarly, Ti6Al4V scaffolds with porosity of 34%e54% showed about 40%e42% yield strength for human cortical bone in Guden’s study [91]. In composite scaffolds, material composition and porosity work together to regulate mechanical properties. For example, Xu et al. [92] incorporated water-soluble mannitol crystals into calcium phosphate cement (CPC) to obtain macroporous scaffolds. By changing the ratio of mannitol in the mixtures, the porosity of CPC scaffolds could be changed within a range of 45%e70%. Both flexural modulus and flexural strength were significantly decreased with increased porosity [92].

1.2.3 Grain size of bioactive materials for osteogenesis 1.2.3.1 Microscale and submicroscale grain sizes Numerous studies have demonstrated that surface microstructure properties (e.g., grain size, surface structural dimensions) of bioactive porous materials can significantly affect the osteoblastic differentiation of progenitor cells in vitro and influence osteoinductivity when implanted heterotopically in vitro. By now, a plenty of Ca-P-based bioceramics with grains in nano- (&100 nm), submicro- (100e1000 nm), and microscale (S1 mm) have been successfully fabricated and their biological properties have been extensively investigated. Researchers have readily fabricated Ca-P-based bioceramics with similar chemical composition but different surface microstructures (i.e., grain size in micron to submicron scale) by varying their sintering temperatures, as changes in sintering temperature are considered to show little effects on the chemistry and macroporosity of bioceramics. Generally speaking, crystal grain size of ceramics gradually increases with the increment of sintering temperature, leading to a significant decrease in specific surface area (Table 1.3). For instance, our group produced porous HA/tricalcium phosphate (HA/TCP) ceramics (HT11 and HT12) by using H2O2 foaming and sintering at 1100 C and 1200 C, respectively [93]. Both ceramics exhibited almost the same phase

TABLE 1.3 Calcium phosphate ceramics with grain size in micron to submicron scale. Refs

Implanted intramuscularly into dogs

BCP1100 > BCP1200

[93]

No

Implanted intramuscularly into goats

HA1150 > HA1250 (no indication), BCP1100 z BCP1150 > BCP1200

[94]

BCP1050 < BCP1125 < BCP1200

No

Filled inside PTFE cylinders and implanted in goat femoral condyle

BCP1125 > BCP1050 > BCP1200 (no bone)

[95]

Different sintering temperatures

BCP1150 < BCP1300

hMSCs

Implanted subcutaneously into immunodeficient mice; implanted intramuscularly into dogs and sheeps; repair posterolateral spinal fusion

BCP1150 > BCP1300

[96]

Different sintering temperatures

BCP1150 ( BCP1300 (no bone)

[97]

Materials (grain size)

In vitro

In vivo

Different sintering temperatures

BCP1100 < BCP1200

No

Different sintering temperatures

HA1150 < HA1250 BCP1100 < BCP1150 < BCP1200

Different sintering temperatures

37

Continued

Material characteristics, surface/interface Chapter | 1

Capacity of osteoinduction or osteoconduction

Methods

Capacity of osteoinduction or osteoconduction

Refs

Implanted intramuscularly into goats; repair iliac wing defect in goats

BCP1150 > BCP1300

[98]

No

Implanted intramuscularly and subcutaneously into goats

BCP1150 > BCP1300

[99]

TCPs ( TCPb (no bone)

[100]

Different powders and sintering process

TCPs ( TCPb (no bone)

[101]

Different powders and sintering process

TCPs ( TCPb (no bone)

[102]

Methods

Materials (grain size)

In vitro

In vivo

Different sintering temperatures

BCP1150 < BCP1300

No

Different sintering temperatures

BCP1150 < BCP1300

Different powders and sintering process

38 Bioactive Materials for Bone Regeneration

TABLE 1.3 Calcium phosphate ceramics with grain size in micron to submicron scale.dcont’d

Material characteristics, surface/interface Chapter | 1

39

composition (HA:TCP ¼ 60:40) and similar macroporous structures; however, HT11 had smaller grain size and higher microporosity than HT12. After implantation in the dorsal muscles of dogs, HT11 ceramics induced earlier bone formation than HT12, as new bones were observed in almost all HT11 but in only 50% of HT12 at 6 weeks. The area of newly formed bone tissue in HT11 was obviously larger than that in HT12. Habibovic et al. [94] fabricated porous HA ceramics by sintering at 1150 C and 1250 C (abbr. HA1150 and HA1250) and biphasic calcium phosphate (BCP) ceramics by sintering at 1100, 1150 and 1200 C (abbr. BCP1100, BCP1150, and BCP1200). With increasing sintering temperatures, grain sizes of either HA or BCP ceramics increased, but their osteoinductivity decreased with the orders as HA1150 > HA1250 (no indication at all) and BCP1100 z BCP1150 > BCP1200 based on a 6- and 12-week goat intramuscular implantation model. Fellah et al. [95] also compared three BCP ceramic granules (HA:TCP ¼ 60:40) sintered at different temperatures (e.g., 1050 C, 1120 C, 1200 C). After filled inside polytetrafluoroethylene (PTFE) cylinders and implanted orthotopically, histological analysis showed that only BCP granules sintered at lower temperature (BCP1050 and BCP1120) with smaller grain sizes promoted a substantial amount of new bone formation. Yuan et al. [96] and Davison et al. [97] also fabricated BCP (HAp:TCP ¼ 80:20) ceramic scaffolds with different surface structural dimensions by varying sintering temperatures, including BCP1150 with submicrosized grains (700 nm), including compressive strength of 2.4e2.6 MPa, porosity of w75%, and macropore size of 230e250 mm for HA and 320e350 mm for BCP [139,141]. The decrease of Ca-P grain size into nanoscale can mimic the inorganic components of natural

Material characteristics, surface/interface Chapter | 1

43

bone, and thus it is speculated that Ca-P nanoceramics will display the superior biological properties [141]. Our studies found that compared with conventional ones (cHA and cBCP), Ca-P nanoceramics (nHA and nBCP) with nanoscale grains could adsorb higher amounts of both large-molecular bovine serum albumin (BSA) and small-molecular lysozyme (LSZ) [141]. We also investigated the effects of CaP nanoceramics on the cellular behaviors of osteoblasts. Our results suggested that Ca-P nanoceramics could promote cell adhesion, spreading, and proliferation, as osteoblasts spread out abundant filapodia to grasp nanograins of CaP nanoceramics, while few filapodia were seen on the surface of the conventional ones. These findings were consistent with the reports from other groups [142,143]. Furthermore, our studies demonstrated that compared with conventional ones with Ca-P grains in micron scale, porous Ca-P nanoceramics with nanosize grains could promote osteoblastic differentiation in vitro more effectively by upregulating expression of specific osteogenic genes like BMP-2 and Cbfa1/Runx2 and increasing activity of ALP [139,141]. In vivo, Ca-P ceramics with grain size at nano- and microscale were implanted into the doral muscles of dogs, respectively. After 45 days postsurgery, formation of ectopic bone was only observed in BCP nanoceramics (nBCP). After 90 day-implantation, abundant bone tissue with medullary cavities formed in nBCP ceramics, while a significantly lower amount of new bone formed in the conventional BCP ceramics (cBCP) [141]. To sum up, in comparison with conventional Ca-P ceramics composed of grains in the micron range, Ca-P nanoceramics with a grain size below 100 nm can be endowed with evidently higher protein adsorption capacity and superior osteoinductivity, which may be attributed to higher specific surface area, more surface defects, more abundant micropores, and smaller surface topography in nanoceramics than in conventional ones. By now the fabrication of porous Ca-P nanoceramics is still challenging due to difficulty in creating large-size bulks, the requirement of expensive devices, and low yield. In our work, we used a dip-coating technology to create a biomimetic nano-HA coating layer onto the surface of highly porous BCP scaffolds with trabecular structure and microsize grains, which were prepared by a polymeric sponge replication method [144]. The results showed that compared with BCP scaffolds with micrograins (0.5e2 mm), nHA-coated BCP scaffolds with nanosize HA grains (40e60 nm) showed superior biocompatibility and bioactivity as evidenced by higher amount of protein adsorption, more complex bonelike apatite formation, and increased adhesion and proliferation of BMSCs. Moreover, nanostructured Ca-P coatings can also be created on the surface of metallic or other implants via plasma spraying [145,146], sputter deposition [147], electrochemical deposition [148], and so on. The surface modification of implants in nanoscale is expected to improve their biomimicry, bioactivity, and osteoconductivity [116,149].

44 Bioactive Materials for Bone Regeneration

1.2.4 Summary These findings have deepened our knowledge about the role of material surface structural cues in material-induced osteogenesis. It has demonstrated that surface micro-/nanostructured features of bioactive materials should provide structural cues to regulate the adhesion, proliferation, and differentiation of osteoprogenitors/osteoblasts, which subsequently promotes new bone formation. Interconnective pore structure with suitable porosity and pore size is a prerequisite for good osteogenesis. In general, nanoscale structural features show superior bioactivity and osteoinductivity to those of microscale ones; bioceramics with orderly micropatterns close to cell size (20e50 mm) favor osteoblastic differentiation of MSCs; and biomaterials with a hierarchical micro/nano hybrid surface stimulate a prominent cell response. Therefore, bioactive scaffolds with a well-designed surface micro-/nanostructure are expected to direct MSC differentiation into osteoblastic lineages in order to achieve desired osteogenic effects.

References [1] D. Sari, S. Bang, L. Nguyen, Y. Cho, K. Park, S. Lee, Micro/nano surface topography and 3D bioprinting of biomaterials in tissue engineering, J. Nanosci. Nanotechnol. 16 (2016) 8909e8922. [2] Z. Tang, X. Li, Y. Tan, H. Fan, X. Zhang, The material and biological characteristics of osteoinductive calcium phosphate ceramics, Regen. Biomater. 5 (2018) 43e59. [3] E.K.F. Yim, K.W. Leong, Significance of synthetic nanostructures in dictating cellular response, Nanomed. Nanotechnol. Biol. Med. 1 (2005) 10e21. [4] M.S. Lord, M. Foss, F. Besenbacher, Influence of nanoscale surface topography on protein adsorption and cellular response, Nano Today 5 (2010) 66e78. [5] S. Tawfick, M. De Volder, D. Copic, S.J. Park, C.R. Oliver, E.S. Polsen, M.J. Roberts, A.J. Hart, Engineering of micro- and nanostructured surfaces with anisotropic geometries and properties, Adv. Mater. 24 (2012) 1628e1674. [6] T. Ozdemir, A.M. Higgins, J.L. Brown, Osteoinductive biomaterial geometries for bone regenerative engineering, Curr. Pharmaceut. Des. 19 (2013) 3446e3455. [7] U. Ripamonti, Functionalized surface geometries induce: “Bone: formation by autoinduction, Front. Physiol. 8 (2018) 1e18. [8] S.A. Skoog, G. Kumar, R.J. Narayan, P.L. Goering, Biological responses to immobilized microscale and nanoscale surface topographies, Pharmacol. Ther. 182 (2018) 33e55. [9] M.J. Dalby, N. Gadegaard, R. Tare, A. Andar, M.O. Riehle, P. Herzyk, C.D.W. Wilkinson, R.O.C. Oreffo, The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder, Nat. Mater. 6 (2007) 997e1003. [10] A.M. Ross, J. Lahann, Surface engineering the cellular microenvironment via patterning and gradients, J. Polym. Sci., Part B: Polym. Phys. 51 (2013) 775e794. [11] C. Scotchford, C. Gilmore, E. Cooper, G. Leggett, S. Downes, Protein adsorption and human osteoblast-like cell attachment and growth on alkylthiol on gold self-assembled monolayers, J. Biomed. Mater. Res. 59 (2002) 84e99.

Material characteristics, surface/interface Chapter | 1

45

[12] S. Bose, S.F. Robertson, A. Bandyopadhyay, Surface modification of biomaterials and biomedical devices using additive manufacturing, Acta Biomater. 66 (2018) 6e22. [13] H. Liu, T.J. Webster, Enhanced biological and mechanical properties of well-dispersed nanophase ceramics in polymer composites: from 2D to 3D printed structures, Mater. Sci. Eng. C 31 (2011) 77e89. [14] X. Chen, X. Fu, J. Shi, H. Wang, Regulation of the osteogenesis of pre-osteoblasts by spatial arrangement of electrospun nanofibers in two- and three-dimensional environments, Nanomedicine 9 (2013) 1283e1292. [15] G. Yang, X. Li, Y. He, J. Ma, G. Ni, S. Zhou, From nano to micro to macro: electrospun hierarchically structured polymeric fibers for biomedical applications, Prog. Polym. Sci. 81 (2018) 80e113. [16] X. Chen, X. Zhu, H. Fan, X. Zhang, Rat bone marrow cell responses on the surface of hydroxyapatite with different topography, Key Eng. Mater. 361e363 (2008) 1107e1110. [17] X. Chen, X. Chen, X. Zhu, B. Cai, H. Fan, X. Zhang, Effect of surface topography of hydroxyapatite on Human osteosarcoma MG-63 cell, J. Inorg. Mater. 28 (2013) 901e906. [18] Z. Wang, Z. Xiao, H. Fan, X. Zhang, Fabrication of micro-grooved patterns on hydroxyapatite ceramics and observation of earlier response of osteoblasts to the patterns, J. Inorg. Mater. 28 (2013) 51e57, https://doi.org/10.3724/SP.J.1077.2013.12093. [19] B. Fang, J. Chang, K. Lin, Designing ordered micropatterned hydroxyapatite bioceramics to promote the growth and osteogenic differentiation of bone marrow stromal cells, J. Mater. Chem. B Mater. Biol. Med. 3 (2015) 968e976. [20] R. Wang, Y.X. Hu, Patterning hydroxyapatite biocoating by electrophoretic deposition, J. Biomed. Mater. Res. A 67 (2003) 270e275. [21] K. Teshima, S. Lee, K. Yubuta, S. Mori, T. Shishido, S. Oishi, Selective growth of highly crystalline hydroxyapatite in a micro-reaction cell of agar gel, CrystEngComm 13 (2011) 827e830. [22] Y.H. Tseng, M.E. Birkbak, H. Birkedal, Spatial organization of hydroxyapatite nanorods on a substrate via a biomimetic approach, Cryst. Growth Des. 13 (2013) 4213e4219. [23] W. Suchanek, M. Yashima, M. Kakihana, M. Yoshimura, Processing and mechanical properties of hydroxyapatite reinforced with hydroxyapatite whiskers, Biomaterials 17 (1996) 1715e1723. [24] F. Mu¨ller, U. Gbureck, T. Kasuga, Y. Mizutani, J.E. Barralet, U. Lohbauer, Whiskerreinforced calcium phosphate cements, J. Am. Ceram. Soc. 90 (2007) 3694e3697. [25] X. Ye, C. Zhou, Z. Xiao, Y. Fan, X. Zhu, Y. Sun, X. Zhang, Fabrication and characterization of porous 3D whisker-covered calcium phosphate scaffolds, Mater. Lett. 128 (2014) 179e182. [26] Y. Zhu, K. Zhang, R. Zhao, X. Ye, X. Chen, Z. Xiao, X. Yang, X. Zhu, K. Zhang, Y. Fan, X. Zhang, Bone regeneration with micro/nano hybrid-structured biphasic calcium phosphate bioceramics at segmental bone defect and the induced immunoregulation of MSCs, Biomaterials 147 (2017) 133e144. [27] K. Lin, L. Xia, J. Gan, Z. Zhang, H. Chen, X. Jiang, J. Chang, Tailoring the nanostructured surfaces of hydroxyapatite bioceramics to promote protein adsorption, osteoblast growth, and osteogenic differentiation, ACS Appl. Mater. Interfaces 5 (2013) 8008e8017. [28] L. Xia, K. Lin, X. Jiang, Y. Xu, M. Zhang, J. Chang, Z. Zhang, Enhanced osteogenesis through nano-structured surface design of macroporous hydroxyapatite bioceramic scaffolds via activation of ERK and p38 MAPK signaling pathways, J. Mater. Chem. B. 1 (2013) 5403.

46 Bioactive Materials for Bone Regeneration [29] L. Xia, K. Lin, X. Jiang, B. Fang, Y. Xu, J. Liu, D. Zeng, M. Zhang, X. Zhang, J. Chang, Z. Zhang, Effect of nano-structured bioceramic surface on osteogenic differentiation of adipose derived stem cells, Biomaterials 35 (2014) 8514e8527. [30] L. Mao, J. Liu, J. Zhao, J. Chang, L. Xia, X. Wang, K. Lin, B. Fang, Effect of micro-nanohybrid structured hydroxyapatite bioceramics on osteogenic and cementogenic differentiation of human periodontal ligament stem cell via Wnt signaling pathway, Int. J. Nanomed. 12 (2015) 7031e7044. [31] L. Xia, N. Zhang, X. Wang, Y. Zhou, L. Mao, J. Liu, X. Jiang, Z. Zhang, J. Chang, K. Lin, B. Fang, The synergetic effect of nano-structures and silicon-substitution on the properties of hydroxyapatite scaffolds for bone regeneration, J. Mater. Chem. B. 4 (2016) 3313e3323. [32] M. Nagano, T. Kitsugi, T. Nakamura, T. Kokubo, M. Tanahashi, Bone bonding ability of an apatite-coated polymer produced using a biomimetic method: a mechanical and histological study in vivo, J. Biomed. Mater. Res. 31 (1996) 487e494. [33] T. Kokubo, H. Kim, M. Kawashita, Novel bioactive materials with different mechanical properties, Biomaterials 24 (2003) 2161e2175. [34] D. Vasudev, J. Ricci, C. Sabatino, P. Li, J. Parsons, In vivo evaluation of a biomimetic apatite coating grown on titanium surfaces, J. Biomed. Mater. Res. A 69 (2004) 629e636. [35] F. Barre`re, C. van der Valk, R. Dalmeijer, C. van Blitterswijk, K. de Groot, P. Layrolle, In vitro and in vivo degradation of biomimetic octacalcium phosphate and carbonate apatite coatings on titanium implants, J. Biomed. Mater. Res. A 64 (2003) 378e387. [36] P. Li, Biomimetic nano-apatite coating capable of promoting bone ingrowth, J. Biomed. Mater. Res. A 66 (2003) 79e85. [37] F. Barre`re, van der Valk, G. CM, M.,R. Dalmeijer, K. de Groot, P. Layrolle, Osteointegration of biomimetic apatite coating applied onto dense and porous metal implants in femurs of goats, J. Biomed. Mater. Res. B Appl. Biomater. 67 (2003) 655e665. [38] J. de Bruijn, H. Yuan, R. Dekker, K. Layrolle, K. de Groot, C. van Blitterswijk, Osteoinductive Biomimetic Calcium-Phosphate Coatings and Their Potential Use as Tissue-Engineering Scaffolds, em squared incorporated, Toronto, 2000. [39] M. Tanahashi, T. Yao, T. Kokubo, M. Minoda, T. Miyamoto, T. Nakamura, T. Yamamuro, Apatite coated on organic polymers by biomimetic process: improvement in its adhesion to substrate by NaOH treatment, J. Appl. Biomater. 5 (1994) 339e347. [40] M. Tanahashi, T. Yao, T. Kokubo, M. Minoda, T. Miyamoto, T. Nakamura, T. Yamamuro, Apatite coated on organic polymers by biomimetic process: improvement in its adhesion to substrate by glow-discharge treatment, J. Biomed. Mater. Res. 29 (1995) 349e357. [41] W. Yan, T. Nakamura, K. Kawanabe, S. Nishigochi, M. Oka, T. Kokubo, Apatite layercoated titanium for use as bone bonding implants, Biomaterials 18 (1997) 1185e1190. [42] N. Koju, P. Sikder, Y. Ren, H. Zhou, S.B. Bhaduri, Biomimetic coating technology for orthopedic implants, Curr. Opin. Chem. Eng. 15 (2017) 49e55. [43] Y.F. Tan, S.F. Hong, X.L. Wang, J. Lu, H. Wang, X.D. Zhang, Regulation of bone-related genes expression by bone-like apatite in MC3T3-E1 cells, J. Mater. Sci. Mater. Med. 18 (2007) 2237e2241. [44] H. Ohgushi, A. Caplan, Stem cell technology and bioceramics: from cell to gene engineering, J. Biomed. Mater. Res. 48 (1999) 913e927. [45] Y. Chou, J. Dunn, B. Wu, In vitro response of MC3T3-E1 pre-osteoblasts within threedimensional apatite-coated PLGA scaffolds, J. Biomed. Mater. Res. B Appl. Biomater. 75 (2005) 81e90. [46] J. Chen, Y. Duan, X. Zhang, Effect of microstructure on osteoinductivity of biomaterials, Key Eng. Mater. 284e286 (2005) 289e292.

Material characteristics, surface/interface Chapter | 1

47

[47] C. Deng, J. Chen, H. Fan, X. Zuang, Effect of flowing speed on bone-like apatite formation in porous calcium phosphate in dynamic RSBF, J. Mater. Sci. 40 (2005) 1809e1812. [48] F. Barrere, C.A. Van Blitterswijk, K. De Groot, P. Layrolle, Influence of ionic strength and carbonate on the Ca-P coating formation from SBF5 solution, Biomaterials 23 (2002) 1921e1930. [49] F. Barrere, C.A. Van Blitterswijk, K. De Groot, P. Layrolle, Nucleation of biomimetic Ca-P coatings on Ti6Al4V from a SBF  5 solution: influence of magnesium, Biomaterials 23 (2002) 2211e2220. [50] Y.F. Chou, W. Huang, J.C.Y. Dunn, T.A. Miller, B.M. Wu, The effect of biomimetic apatite structure on osteoblast viability, proliferation, and gene expression, Biomaterials 26 (2005) 285e295. [51] X. Wei, C. Fu, K. Savino, M.Z. Yates, Carbonated hydroxyapatite coatings with aligned crystal domains, Cryst. Growth Des. 12 (2012) 3474e3480. [52] W. Xue, F. Cong, S. Keith, Y. Matthew Z, Fully dense yttrium-substituted hydroxyapatite coatings with aligned crystal domains, Cryst. Growth Des. 12 (2012) 217e223. [53] D. Liu, K. Savino, M.Z. Yates, Microstructural engineering of Hydroxyapatite membranes to enhance proton conductivity, Adv. Funct. Mater. 19 (2009) 3941e3947. [54] X. Wang, J. Zhuang, Q. Peng, Y. Li, Liquid-solid-solution synthesis of biomedical hydroxyapatite nanorods, Adv. Mater. 18 (2006) 2031e2034. [55] F. Nudelman, K. Pieterse, A. George, P. Bommans, H. Friedrich, L. Brylka, P. Hilbers, G. de With, N. Sommerdijk, The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors, Nat. Mater. 9 (2010) 1004e1009. [56] L. Li, C. Mao, J. Wang, X. Xu, H. Pan, Y. Peng, X. Gu, R. Tang, Bio-inspired enamel repair via glu-directed assembly of apatite nanoparticles an approach to biomaterials with optimal characteristics, Adv. Mater. 23 (2011) 4695e4701. [57] X. Liu, K. Lin, R. Qian, L. Chen, S. Zhuo, J. Chang, Growth of highly oriented hydroxyapatite arrays tuned by quercetin, Chemistry 18 (2012) 5519e5523. [58] K. Lin, C. Wu, J. Chang, Advances in synthesis of calcium phosphate crystals with controlled size and shape, Acta Biomater. 10 (2014) 4071e4102. [59] X. Liu, K. Lin, C. Wu, Y. Wang, Z. Zou, J. Chang, Multilevel hierarchically ordered artificial biomineral, Small 10 (2014) 152e159. [60] J. Ryu, S.H. Ku, M. Lee, C.B. Park, Bone-like peptide/hydroxyapatite nanocomposites assembled with multi-level hierarchical structures, Soft Matter 7 (2011) 7201e7206. [61] J. Ryu, S.H. Ku, H. Lee, C.B. Park, Mussel-inspired polydopamine coating as a universal route to hydroxyapatite crystallization, Adv. Funct. Mater. 20 (2010) 2132e2139. [62] K.C.R. Kolan, M.C. Leu, G.E. Hilmas, M. Velez, Effect of material, process parameters, and simulated body fluids on mechanical properties of 13-93 bioactive glass porous constructs made by selective laser sintering, J. Mech. Behav. Biomed. Mater. 13 (2012) 14e24. [63] S. Fujibayashi, M. Takemoto, M. Neo, T. Matsushita, T. Kokubo, K. Doi, T. Ito, A. Shimizu, T. Nakamura, A novel synthetic material for spinal fusion: a prospective clinical trial of porous bioactive titanium metal for lumbar interbody fusion, Eur. Spine J. 20 (2011) 1486e1495. [64] A. Fedotov, V. Komlev, V. Smirnov, A. Fomin, I. Fadeeva, N. Sergeeva, I. Sviridova, V. Kirsanova, S. Barinov, Porous composite materials chitosan - bioactive calcium compound particulate for bone tissue engineering, Tissue Eng. A 15 (2009) 727. [65] A.R. Walpole, Z. Xia, C.W. Wilson, J.T. Triffitt, P.R. Wilshaw, A novel nano-porous alumina biomaterial with potential for loading with bioactive materials, J. Biomed. Mater. Res. A 90 (2009) 46e54.

48 Bioactive Materials for Bone Regeneration [66] C. Wu, Y. Ramaswamy, H. Zreiqat, Porous diopside (CaMgSi2O6) scaffold: a promising bioactive material for bone tissue engineering, Acta Biomater. 6 (2010) 2237e2245. [67] D. Jagadeesan, C. Deepak, K. Siva, M.S. Inamdar, M. Eswaramoorthy, Carbon spheres assisted synthesis of porous bioactive glass containing hydroxycarbonate apatite nanocrystals: a material with high in vitro bioactivity, J. Phys. Chem. C 112 (2008) 7379e7384. [68] H. Yuan, J. De Bruijn, Y. Li, J. Feng, Z. Yang, K. De Groot, X. Zhang, Bone formation induced by calcium phosphate ceramics in soft tissue of dogs: a comparative study between porous alpha-TCP and beta-TCP, J. Mater. Sci. Mater. Med. 12 (2001) 7e13. [69] A. Lecomte, H. Gautier, J. Bouler, A. Gouyette, Y. Pegon, G. Daculsi, C. Merle, Biphasic calcium phosphate: a comparative study of interconnected porosity in two ceramics, J. Biomed. Mater. Res. 84B (2008) 1e6. [70] D. Tancred, B. McCormack, A. Carr, A synthetic bone implant macroscopically identical to cancellous bone, Biomaterials 19 (1998) 2303e2311. [71] J. Zhang, W. Liu, O. Gauthier, S. Sourice, P. Pilet, G. Rethore, K. Khairoun, J. Bouler, F. Tancret, P. Weiss, A simple and effective approach to prepare injectable macroporous calcium phosphate cement for bone repair: syringe-foaming using a viscous hydrophilic polymeric solution, Acta Biomater. 31 (2016) 326e338. [72] K. Hing, B. Annaz, S. Saeed, P. Revell, T. Buckland, Microporosity enhances bioactivity of synthetic bone graft substitutes, J. Mater. Sci. Mater. Med. 16 (2005) 467e475. [73] Z. Wang, Z. Tang, F. Qing, Y. Hong, Applications of calcium phosphate nanoparticles in porous hard tissue, Nanobr. Reports Rev. 7 (2012) 1e18. [74] A.M.C. Barradas, H. Yuan, C.A. van Blitterswijk, P. Habibovic, Osteoinductive biomaterials: current knowledge of properties, experimental models and biological mechanisms, Eur. Cells Mater. 21 (2011) 407e429. [75] H. Yuan, Z. Yang, Y. Li, X. Zhang, J. De Bruijn, K. De Groot, Osteoinduction by calcium phosphate biomaterials, J. Mater. Sci. Mater. Med. 9 (1998) 723e726. [76] S. Samavedi, A.R. Whittington, A.S. Goldstein, Calcium phosphate ceramics in bone tissue engineering: a review of properties and their influence on cell behavior, Acta Biomater. 9 (2013) 8037e8045. [77] X. Zhang, A Calcium Phosphate, Bioceramics with Osteoinduction, The 4th World Biomaterials Congress., 1992. [78] X. Zhu, H. Zhang, H. Fan, W. Li, X. Zhang, Effect of phase composition and microstructure of calcium phosphate ceramic particles on protein adsorption, Acta Biomater. 6 (4) (April 2010) 1536e1541. Acta Biomater. [79] X.D. Zhu, H.S. Fan, Y.M. Xiao, D.X. Li, H.J. Zhang, T. Luxbacher, X.D. Zhang, Effect of surface structure on protein adsorption to biphasic calcium-phosphate ceramics in vitro and in vivo, Acta Biomater. 5 (2009) 1311e1318. [80] M. Schumacher, U. Deisinger, G. Ziegler, R. Detsch, Indirect rapid prototyping of biphasic calcium phosphate scaffolds as bone substitutes: influence of phase composition, macroporosity and pore geometry on mechanical properties, J. Mater. Sci. Mater. Med. 21 (2010) 3119e3127. [81] P. Daugela, M. Pranskunas, G. Juodzbalys, J. Liesiene, O. Baniukaitiene, A. Afonso, P. Sousa Gomes, Novel cellulose/hydroxyapatite scaffolds for bone tissue regeneration: in vitro and in vivo study, J. Tissue Eng. Regenerat. Med. 12 (2018) 1195e1208. [82] F. Zhao, M.J. Mc Garrigle, T.J. Vaughan, L.M. McNamara, In silico study of bone tissue regeneration in an idealised porous hydrogel scaffold using a mechano-regulation algorithm, Biomechanics Model. Mechanobiol. 17 (2018) 5e18.

Material characteristics, surface/interface Chapter | 1

49

[83] A. Raafat, W. Abd-Allah, In vitro apatite forming ability and ketoprofen release of radiation synthesized (gelatin-polyvinyl alcohol)/bioglass composite scaffolds for bone tissue regeneration, Polym. Compos. 39 (2018) 606e615. [84] K. Shim, S. Kim, Y. Yun, D. Jeon, H. Kim, K. Park, H. Song, Surface immobilization of biphasic calcium phosphate nanoparticles on 3D printed poly(caprolactone) scaffolds enhances osteogenesis and bone tissue regeneration, J. Ind. Eng. Chem. 55 (2017) 101e109. [85] H. Lee, G.H. Yang, M. Kim, J.Y. Lee, J.T. Huh, G.H. Kim, 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 (2017) 140e147. [86] C. Xue, H. Ren, H. Zhu, X. Gu, Q. Guo, Y. Zhou, J. Huang, S. Wang, G. Zha, J. Gu, Y. Yang, Y. Gu, X. Gu, Bone marrow mesenchymal stem cell-derived acellular matrixcoated chitosan/silk scaffolds for neural tissue regeneration, J. Mater. Chem. B. 5 (2017) 1246e1257. [87] H. Shao, X. Ke, A. Liu, M. Sun, Y. He, X. Yang, J. Fu, Y. Liu, L. Zhang, G. Yang, S. Xu, Z. Gou, Bone regeneration in 3D printing bioactive ceramic scaffolds with improved tissue/material interface pore architecture in thin-wall bone defect, Biofabrication 9 (2017) 025003. [88] S. Maheshwari, K. Govindan, M. Raja, A. Raja, M. Pravin, S. Kumar, Synthesis and characterization of calcium phosphate ceramic/(Poly(vinyl alcohol)-Polycaprolactone) bilayer nanocomposites-A bone tissue regeneration scaffold, J. Nanosci. Nanotechnol. 18 (2018) 1548e1556. [89] S. Faghihi, F. Azari, H. Li, M.R. Bateni, J.A. Szpunar, H. Vali, M. Tabrizian, The significance of crystallographic texture of titanium alloy substrates on pre-osteoblast responses, Biomaterials 27 (2006) 3532e3539. [90] S.W. Tsai, F.Y. Hsu, P.L. Chen, Beads of collagen-nanohydroxyapatite composites prepared by a biomimetic process and the effects of their surface texture on cellular behavior in MG63 osteoblast-like cells, Acta Biomater. 4 (2008) 1332e1341. [91] D. Chappard, B. Guillaume, R. Mallet, F. Pascaretti-Grizon, M.F. Basle´, H. Libouban, Sinus lift augmentation and b-TCP: a microCT and histologic analysis on human bone biopsies, Micron 41 (2010) 321e326. [92] M. Schuler, G.R. Owen, D.W. Hamilton, M. de Wild, M. Textor, D.M. Brunette, S.G.P. Tosatti, Biomimetic modification of titanium dental implant model surfaces using the RGDSP-peptide sequence: a cell morphology study, Biomaterials 27 (2006) 4003e4015. [93] H. Fan, T. Ikoma, C. Bao, H. Wang, L. Zhang, J. Tanaka, X. Zhang, Surface characteristics and osteoinductivity of biphasic calcium phosphate ceramics with different sintering temperature, Key Eng. Mater. 309e311 (2006) 1299e1302. [94] P. Habibovic, H. Yuan, C.M. Van Der Valk, G. Meijer, C.A. Van Blitterswijk, K. De Groot, 3D microenvironment as essential element for osteoinduction by biomaterials, Biomaterials 26 (2005) 3565e3575. [95] B.H. Fellah, O. Gauthier, P. Weiss, D. Chappard, P. Layrolle, Osteogenicity of biphasic calcium phosphate ceramics and bone autograft in a goat model, Biomaterials 29 (2008) 1177e1188, https://doi.org/10.1016/j.biomaterials.2007.11.034. [96] H. Yuan, H. Fernandes, P. Habibovic, J. De Boer, A.M.C. Barradas, A. De Ruiter, Osteoinductive ceramics as a synthetic alternative to autologous bone grafting, Proc. Natl. Acad. Sci. 107 (2010) 10e15. [97] N.L. Davison, J. Su, H. Yuan, J.J.J.P. van den Beucken, J.D. de Bruijn, F.B. de Groot, Influence of surface microstructure and chemistry on osteoinduction and osteoclastogenesis by biphasic calcium phosphate discs, Eur. Cells Mater. 29 (2015) 314e329.

50 Bioactive Materials for Bone Regeneration [98] P. Habibovic, H. Yuan, M. van den Doel, T. Sees, C. van Blitterswijk, K. de Groot, Relevance of osteoinductive biomaterials in critical-sized orthotopic defect, J. Orthop. Res. 24 (2006) 867e876. [99] P. Habibovic, T.M. Sees, M.A. Van Den Doel, C.A. Van Blitterswijk, K. De Groot, Osteoinduction by biomaterials d physicochemical and structural influences, J. Biomed. Mater. Res. A 77 (2006) 747e762. [100] J. Zhang, X. Luo, D. Barbieri, A.M.C. Barradas, J.D. De Bruijn, C.A. Van Blitterswijk, H. Yuan, The size of surface microstructures as an osteogenic factor in calcium phosphate ceramics, Acta Biomater. 10 (2014) 3254e3263. [101] N. Davison, X. Luo, T. Schoenmaker, V. Everts, H. Yuan, F. Barre`re-de Groot, J. de Bruijn, Submicron-scale surface architecture of tricalcium phosphate directs osteogenesis in vitro and in vivo, Eur. Cells Mater. 27 (2014) 281e297. [102] N.L. Davison, A.L. Gamblin, P. Layrolle, H. Yuan, J.D. de Bruijn, F. Barre`re-de Groot, Liposomal clodronate inhibition of osteoclastogenesis and osteoinduction by submicrostructured beta-tricalcium phosphate, Biomaterials 35 (2014) 5088e5097. [103] J. Huh, J. Lee, W. Kim, M. Yeo, G. Kim, Preparation and characterization of gelatin/alphaTCP/SF biocomposite scaffold for bone tissue regeneration, Int. J. Biol. Macromol. 110 (2018) 488e496. [104] X. Wang, H.C. Schro¨der, W.E.G. Mu¨ller, Amorphous polyphosphate, a smart bioinspired nano-/bio-material for bone and cartilage regeneration: towards a new paradigm in tissue engineering, J. Mater. Chem. B. 6 (2018) 2385e2412. [105] M. Meskinfam, S. Bertoldi, N. Albanese, A. Cerri, M.C. Tanzi, R. Imani, N. Baheiraei, M. Farokhi, S. Fare`, Polyurethane foam/nano hydroxyapatite composite as a suitable scaffold for bone tissue regeneration, Mater. Sci. Eng. C 82 (2018) 130e140. [106] D.N. Heo, N.J. Castro, S.-J. Lee, H. Noh, W. Zhu, L.G. Zhang, Enhanced bone tissue regeneration using a 3D printed microstructure incorporated with a hybrid nano hydrogel, Nanoscale 9 (2017) 5055e5062. [107] M. Hadavi, S. Hasannia, S. Faghihi, F. Mashayekhi, H.H. Zadeh, S.B. Mostofi, Novel calcified gum Arabic porous nano-composite scaffold for bone tissue regeneration, Biochem. Biophys. Res. Commun. 488 (2017) 671e678. [108] A. Samadikuchaksaraei, M. Gholipourmalekabadi, E. Erfani Ezadyar, M. Azami, M. Mozafari, B. Johari, S. Kargozar, S.B. Jameie, A. Korourian, A.M. Seifalian, Fabrication and in vivo evaluation of an osteoblast-conditioned nano-hydroxyapatite/gelatin composite scaffold for bone tissue regeneration, J. Biomed. Mater. Res. A 104 (2016) 2001e2010. [109] H. Hosseinkhani, M. Hosseinkhani, A. Khademhosseini, H. Kobayashi, Bone regeneration through controlled release of bone morphogenetic protein-2 from 3-D tissue engineered nano-scaffold, J. Control. Release 117 (2007) 380e386. [110] A. Tae Young, J.H. Kang, D.J. Kang, J. Venkatesan, H.K. Chang, I. Bhatnagar, K.Y. Chang, J.H. Hwang, Z. Salameh, S.K. Kim, H.T. Kim, D.G. Kim, Interaction of stem cells with nano hydroxyapatite-fucoidan bionanocomposites for bone tissue regeneration, Int. J. Biol. Macromol. 93 (2016) 1488e1491. [111] R. Emadi, S.I. Roohani Esfahani, F. Tavangarian, A novel, low temperature method for the preparation of ß-TCP/HAP biphasic nanostructured ceramic scaffold from natural cancellous bone, Mater. Lett. 64 (2010) 993e996. [112] T.J. Webster, C. Ergun, R.H. Doremus, R.W. Siegel, R. Bizios, Enhanced functions of osteoblasts on nanophase ceramics, Biomaterials 21 (2000) 1803e1810.

Material characteristics, surface/interface Chapter | 1

51

[113] Y. Yang, J.L. Ong, J. Tian, Rapid sintering of hydroxyapatite by microwave processing, J. Mater. Sci. Lett. 21 (2002) 67e69. [114] X. Guo, J. Gough, P. Xiao, J. Liu, Z. Shen, Fabrication of nanostructured hydroxyapatite and analysis of human osteoblastic cellular response, J. Biomed. Mater. Res. A 82 (2007) 1022e1032. [115] Y. Zhao, Y. Zhang, F. Ning, D. Guo, Z. Xu, Synthesis and cellular biocompatibility of two kinds of HAP with different nanocrystal morphology, J. Biomed. Mater. Res. B Appl. Biomater. 83 (2007) 121e126. [116] P. Wang, L. Zhao, J. Liu, M.D. Weir, X. Zhou, H.H.K. Xu, Bone tissue engineering via nanostructured calcium phosphate biomaterials and stem cells, Bone Res 2 (2015) 14017. [117] X. Li, L. Wang, Y. Fan, Q. Feng, F.Z. Cui, F. Watari, Nanostructured scaffolds for bone tissue engineering, J. Biomed. Mater. Res. A 101 A (2013) 2424e2435. [118] B. Snoddy, A.C. Jayasuriya, The use of nanomaterials to treat bone infections, Mater. Sci. Eng. C 67 (2016) 822e833. [119] C. Zhou, Y. Hong, X. Zhang, Applications of nanostructured calcium phosphate in tissue engineering, Biomater. Sci. 1 (2013) 1012e1028. [120] S. Sai Nievethitha, N. Subhapradha, D. Saravanan, N. Selvamurugan, W.B. Tsai, N. Srinivasan, R. Murugesan, A. Moorthi, Nanoceramics on osteoblast proliferation and differentiation in bone tissue engineering, Int. J. Biol. Macromol. 98 (2017) 67e74. [121] X. Chen, H. Fan, X. Deng, L. Wu, T. Yi, L. Gu, C. Zhou, Y. Fan, X. Zhang, Scaffold structural microenvironmental cues to guide tissue regeneration in bone tissue applications, Nanomaterials 8 (2018) 960e975. [122] B. Zhang, P. Zhang, Z. Wang, Z. Lyu, H. Wu, Tissue-engineered composite scaffold of poly(lactide-co-glycolide) and hydroxyapatite nanoparticles seeded with autologous mesenchymal stem cells for bone regeneration, J. Zhejiang Univ. - Sci. B. 18 (2017) 963e976. [123] N. Siddiqui, K. Pramanik, Development of fibrin conjugated chitosan/nano b-TCP composite scaffolds with improved cell supportive property for bone tissue regeneration, J. Appl. Polym. Sci. 132 (2015) 1e10. [124] L. Li, M. Zhao, J. Li, Y. Zuo, Q. Zou, Y. Li, Preparation and cell infiltration of lotus-type porous nano-hydroxyapatite/polyurethane scaffold for bone tissue regeneration, Mater. Lett. 149 (2015) 25e28. [125] J.S. Lee, S.D. Baek, J. Venkatesan, I. Bhatnagar, H.K. Chang, H.T. Kim, S.K. Kim, In vivo study of chitosan-natural nano hydroxyapatite scaffolds for bone tissue regeneration, Int. J. Biol. Macromol. 67 (2014) 360e366. [126] O.Y. Alothman, F.N. Almajhdi, H. Fouad, Effect of gamma radiation and accelerated aging on the mechanical and thermal behavior of HDPE/HA nano-composites for bone tissue regeneration, Biomed. Eng. Online 12 (2013) 95. [127] Y. Hao, H. Yan, X. Wang, B. Zhu, C. Ning, S. Ge, Evaluation of osteoinduction and proliferation on nano-Sr-HAP: a novel orthopedic biomaterial for bone tissue regeneration, J. Nanosci. Nanotechnol. 12 (2012) 207e212. [128] K. Pielichowska, S. Blazewicz, Bioactive polymer/hydroxyapatite (Nano)composites for bone tissue regeneration, Adv. Polym. Sci. 232 (2010) 97e207. [129] X. Liao, S. Lu, Y. Zhuo, C. Winter, W. Xu, B. Li, Y. Wang, Bone physiology, biomaterial and the effect of mechanical/physical microenvironment on MSC osteogenesis: a tribute to Shu Chien’s 80th Birthday, Cell. Mol. Bioeng. 4 (2011) 579e590. [130] R. LeGeros, Biodegradation and bioresorption of calcium phosphate ceramics, Clin. Mater. 14 (1993) 65e88. [131] K. Lin, J. Chang, J. Lu, W. Wu, Y. Zeng, Properties of b-Ca3(PO4)2 bioceramics prepared using nano-sized powders, Ceram. Int. 33 (2007) 979e985.

52 Bioactive Materials for Bone Regeneration [132] J. Wang, L. Shaw, Morphology-enhanced low-temperature sintering of nanocrystalline hydroxyapatite, Adv. Mater. 19 (2007) 2364e2369. [133] R. Webster, T.J., C. Ergun, R.H. Doremus, R.W. Seigel, Bizios, Enhanced osteoclast - like functions on nanophase ceramics, Biomaterials 22 (2001) 1327e1333. [134] H.W. Kim, H.E. Kim, V. Salih, Stimulation of osteoblast responses to biomimetic nanocomposites of gelatin-hydroxyapatite for tissue engineering scaffolds, Biomaterials 26 (2005) 5221e5230. [135] M. Sato, M.A. Sambito, A. Aslani, N.M. Kalkhoran, E.B. Slamovich, T.J. Webster, Increased osteoblast functions on undoped and yttrium-doped nanocrystalline hydroxyapatite coatings on titanium, Biomaterials 27 (2006) 2358e2369. [136] E.S. Thian, J. Huang, S.M. Best, Z.H. Barber, R.A. Brooks, N. Rushton, W. Bonfield, The response of osteoblasts to nanocrystalline silicon-substituted hydroxyapatite thin films, Biomaterials 27 (2006) 2692e2698. [137] W. Sun, C. Chu, J. Wang, H. Zhao, Comparison of periodontal ligament cells responses to dense and nanophase hydroxyapatite, J. Mater. Sci. Mater. Med. 18 (2007) 677e683. [138] X. Wang, H. Fan, Y. Xiao, X. Zhang, Fabrication and characterization of porous hydroxyapatite/b-tricalcium phosphate ceramics by microwave sintering, Mater. Lett. 60 (2006) 455e458. [139] B. Li, X. Chen, B. Guo, X. Wang, H. Fan, X. Zhang, Fabrication and cellular biocompatibility of porous carbonated biphasic calcium phosphate ceramics with a nanostructure, Acta Biomater. 5 (2009) 134e143. [140] W. Sutton, Microwave processing of ceramic materials, Am. Ceram. Soc. Bull. 68 (1989) 376e386. [141] Y. Hong, H. Fan, B. Li, B. Guo, M. Liu, X. Zhang, Fabrication, biological effects, and medical applications of calcium phosphate nanoceramics, Mater. Sci. Eng. R Rep. 70 (2010) 225e242. [142] J.S. Suwandi, R.E.M. Toes, T. Nikolic, B.O. Roep, Inducing tissue specific tolerance in autoimmune disease with tolerogenic dendritic cells, Clin. Exp. Rheumatol. 33 (2015) 97e103. [143] Y.-C. Ho, F.-M. Huang, Y.-C. Chang, Cytotoxicity of formaldehyde on human osteoblastic cells is related to intracellular glutathione levels, J. Biomed. Mater. Res. B Appl. Biomater. 83 (2007) 340e344. [144] J. Wang, Y. Zhu, M. Wang, D. Liu, X. Chen, X. Zhu, X. Yang, K. Zhang, Y. Fan, X. Zhang, Fabrication and preliminary biological evaluation of a highly porous biphasic calcium phosphate scaffold with nano-hydroxyapatite surface coating, Ceram. Int. 44 (2018) 1304e1311. [145] Y. Huang, Y. Qu, B. Yang, W. Li, B. Zhang, X. Zhang, In vivo biological responses of plasma sprayed hydroxyapatite coatings with an electric polarized treatment in alkaline solution, Mater. Sci. Eng. C 29 (2009) 2411e2416. [146] R.A. Surmenev, A review of plasma-assisted methods for calcium phosphate-based coatings fabrication, Surf. Coat. Technol. 206 (2012) 2035e2056. [147] M. McCafferty, G. Burke, B. Meenan, Mesenchymal stem cell response to conformal sputter deposited calcium phosphate thin films on nanostructured titanium surfaces, J. Biomed. Mater. Res. A 102 (2014) 3585e3597. [148] J. Hernandez-Montelongo, D. Gallach, N. Naveas, V. Torres-Costa, A. Climent-Font, J.P. Garcı´a-Ruiz, M. Manso-Silvan, Calcium phosphate/porous silicon biocomposites prepared by cyclic deposition methods: spin coating vs electrochemical activation, Mater. Sci. Eng. C 34 (2014) 245e251. [149] A. Tomsia, J. Lee, U. Wegst, E. Saiz, Nanotechnology for dental implants, Int. J. Oral Maxillofac. Implant. 28 (2013) e535ee546.

Material characteristics, surface/interface Chapter | 1

53

Chapter 1.3

Protein adsorption on bioactive materials and its effect on osteogenesis Protein adsorption is important for bioactive materials [1,2]. When bioactive materials are implanted into a living body, proteins from the surrounding body fluids will be spontaneously adsorbed onto their surfaces or interfaces, and then cellular attachment, proliferation, and migration occur [3e5]. Thus, protein adsorption behavior plays a vital role during the osteogenesis process [6,7]. Many studies of protein adsorption behaviors have been done in order to better understand the mechanism of protein adsorption and the reasons that bioactive materials have excellent biological properties. The results and conclusions greatly improved our knowledge in the biomaterial field. Many facets of protein adsorption have attracted much attention. For example, the effects of different material properties including chemical components and surface properties on adsorption behavior were further studied; the effects of different protein properties and environments (such as the pH value of protein solution, the acidity/basicity or electric charge of proteins, and the conformation change of protein upon adsorption onto the surface) on adsorption behavior were also investigated. Basically, protein adsorption behaviors on bioactive materials are also affected by these issues. Selective adsorption of bone-relative proteins, like BMP2, VEGF, and serum album, is the key property of bioactive porous bone repair materials. In this chapter, we will simply review the research/ characterization methods of protein adsorption and discuss the effects of kinds of properties on protein adsorption, the interaction mechanism between protein and bioactive materials, and its effect on osteogenesis.

1.3.1 Current methods for studying protein adsorption 1.3.1.1 Experimental methods In order to fully understand the mechanism of protein adsorption, a quantitative description is necessary. This description includes adsorption energy, adsorption and desorption kinetics, conformation changes of adsorbed proteins, adsorption sites in contact with material surfaces, and even selective/ competitive adsorption on bioactive materials. Some research protocols [8] and a set of characterization techniques were developed to further study the

54 Bioactive Materials for Bone Regeneration

protein adsorption mechanism. Protein characterization is mainly dependent on biochemistry technologies, like polyacrylamide gel electrophoresis, and includes certain equipment: nuclear magnetic resonance, secondary ion mass spectrometry, and atomic force microscopy for the analysis of protein structure. On the other hand, in general, solution depletion, optical, and spectroscopic techniques are most popular in protein adsorption research [8]. Ellipsometry, an optical technique, is a surface-sensitive method for the investigation of various aspects of protein adsorption mainly at reflecting metal surfaces and ceramic surfaces, which has been applied in the research of protein adsorption on bioactive materials [9]. Fourier transform infrared attenuated total internal reflection techniques can be used for the study of material surfaces and events at material surfaces such as protein adsorption. It can provide the adsorbed amount of plasma protein onto surfaces of different materials and studies of secondary structure alterations in adsorbed proteins after being subjected to different sorbents and protein aggregation [8,10]. Recently, biological and physiochemical methodologies and their combination are widely adopted in the research of protein adsorption on materials. For instance, Prof. Zhang and Prof. Zhu’s group adopted advanced protein analysis technology (ITRAQ, ELISA, IHC, etc.) to qualitatively and quantitatively analyze the properties of protein adsorption on the bioactive Ca-P materials. Adsorption characteristics showed that porous Ca-P ceramics exhibit certain selective adsorption properties for different proteins, especially for the cell adhesion proteins, angiogenesis, and osteogenic factors. The surface micro-/ nanostructure can significantly enhance the adsorption/enrichment ability of protein, especially low-molecular-weight BMPs. Although experimental methods for studying protein adsorption have greatly enhanced our understanding of the mechanism of protein adsorption, detailed information at microscale is still inadequate.

1.3.1.2 Computing methods With the rapid development of computer technology, a simulation method has been increasingly applied in this area that has provided a significant amount of adsorption information at the atomic level. The molecular dynamics (MD) simulation method is most employed and has been widely applied in chemistry and biology. MD simulation is a technique based on classical mechanics and calculates the time-dependent behaviors of a molecular system. In this computational methodology, atoms are described as soft bodies. Combining the suitable force field parameters for proteins, the MD simulation has been widely applied in the research of protein adsorption on solid substrate [11e13]. It also began to play an increasingly prominent role in the research of biomaterials. For example, oligopeptide, Arg-Gly-Asp (RGD) tripeptides, fibronectin, and HSA adsorption behavior on the rutile (110) surface have been studied by MD based on the Amber force field [14e17] and the Charmm force

Material characteristics, surface/interface Chapter | 1

55

field [18]. The conformational change of HSA in the process of adsorption on carbon nanotube surface also is simulated by MD method based on Charmm force field [19]. The interactive behaviors between different subdomain of HSA and graphite surface with or without water [20,21], and adsorption behavior of certain oligopeptide on quartz surface, have been evaluated by MD based on CVFF force field [22]. Meanwhile, many MD works of interactions between protein and Ca-P have been done. The Charmm force field-based simulations of Fibronectin type III with different orientations and BMP-7 adsorption on HA (001) surface indicated that electrostatic energy plays a dominant role in the interaction, and charged eCOO- and eNHþ 3 are the strongest groups that interact with the HA surface [23,24]. CVFF force field-based simulations of polyacrylic acid adsorption on HA surface indicated that potential sites for chelation and hydrogen bond formation between HA and polyacrylic acid exist, which depend on the exposed surface of HA. The COO group strongly attached to calcium atoms and is a more prominent site for HA mineralization than the COOH group [25,26]. The Amber force field-based simulation of interaction between the Hyp-Pro-Gly tripeptide in collagen protein and the HA surfaces indicated that this tripeptide interacted primarily with HA (110), rather than HA (001) plane according to the results of adsorption energy, which is in agreement with experiment that in natural bone the (110) surface grows preferentially from a collagen matrix [27e29]. Adsorption of Gly and Glu amino acids on HA surface is investigated by MD based on a mixed BHM and Lennard_Jones force field. Its results indicated that the amino acids adsorbed on the HA (001) and (100) surfaces with their positive amino groups occupied vacant calcium sites, and their negative carboxylate groups occupied vacant P or OH sites precisely and formed an ordered adsorption layer; Glupreferred to adsorb strongly onto the HA (001) surface, which resulted in the formation of plate like HA. However, Gly did not show any significantly preferential adsorption between these two HA surfaces [30]. Although MD simulation has been used in many research fields, we should note the limitation of MD calculations when applying the results in experiments. The simulation scales of time and space cannot match the real environments because of the limitations of computer power. Moreover, the differences between the parameters of force fields that were calculated based on quantum mechanics and practical results also existed. Thus, there is still a degree of discrepancy between computational results and experimental data. Nevertheless, the general trend and detailed information at atomic level are useful in predicting and explaining the adsorption phenomena and is sufficient to narrow the experimental tests or reduce the analysis cost, even though the simulation results may not be exact results.

56 Bioactive Materials for Bone Regeneration

1.3.2 Material factors influencing protein adsorption 1.3.2.1 Material factors 1.3.2.1.1 Topography The physiochemical feature of material surface is one of the decisive factors of protein adsorption. Surface topography, like roughness, porosity, pore size, and particle size determines the size of surface area which interacts with the protein molecules. More surface area exposed can provide more interaction sites for adsorbing proteins. These sites bond protein molecules through different ways, such as electrostatic force, hydrophobicity and so on. It is generally accepted that higher surface area/specific surface area (SSA), higher amount of protein adsorption based on lots of experimental results. Essentially, modulating the roughness, porosity, pore size, and particle size of material is to get more surface area which could benefit protein adsorption. Note that increasing surface area is valid for improving protein adsorption on all bioceramics including Ca-P materials. Higher roughness could lead to higher surface area, but it may be variable in nano scale. EA. Dos Santos et al. [31] found that the albumin and fibronectin adsorption on the HA (with Au coated or not) with lower nanoroughness (32  6 nm) was higher than that observed on the b-TCP (with Au coated or not) along time. Cai et al. [32] also showed that the nanoscale roughness on titanium surfaces had little effect on the structure and amount of adsorbed albumin and fibrinogen. However, the adsorption of larger molecules like collagen can be influenced by different roughness degrees of polymer surfaces [33]. It was observed not only differences on the amount of proteins but also on their structure. The reason may be that when the roughness scale increases from nanometers to micrometers, topography may appear smooth to the protein, considering the protein size, lead to little effects on adsorption process [34,35]. Presently, it is known that nanoscale roughness of Ca-P can affect the protein adsorption process, but more studies need to be done so as to understand the influencing trend, especially for different proteins adsorption on different Ca-P surfaces. Porosity, pore size/distribution, and particle size also impact the protein adsorption through regulating the surface area. The presence of porosity enlarges greatly the surface area of materials and improves the protein adsorption. XD. Zhu et al. [36] reported that the amount of the adsorbed total proteins on porous biphasic Ca-P (BCP (HA/TCP ¼ 7:3)) was far beyond that on dense BCP. Many pores with a size distribution from 100 to 500 mm in diameter presented on porous BCP, and many micropores distributed on the wall of the macropores. The increased surface area of materials mainly attributed to the presence of macropores and micropores. Higher porosity leads to higher surface area. The porosity could further increase the protein adsorption and the subsequent cell attachment [37]. Many other studies also proved the effect of

Material characteristics, surface/interface Chapter | 1

57

porosity on protein adsorption on Ca-P [38e40]. Most proteins would be suffered the structural or conformational rearrangement after adsorption on the substrate surface [41]. The behavior of proteins adsorbed on porous Ca-P is a multilayer adsorption and that on dense Ca-P is a monolayer one. It could be attributed to the holdback effect of porous structure [36]. Furthermore, the effect of porous structure on protein adsorption has been considered as an interpretation of the osteoinductive potential of Ca-P bioceramics after implanted to the ectopic sites [42,43]. Ca-P bioceramics can adsorb and enrich the native bone morphogenetic proteins (BMPs) from the body fluid, which induced bone formation in a dose-dependent manner [44]. If the threshold of BMP local concentration for triggering osteoinduction satisfied, the Ca-P bioceramics could have potential to be osteoinductive. The porous structure of Ca-P enlarges the SSA, which lead Ca-P bonds more BMPs, and plays a vital role for the osteoinductivity of Ca-P bioceramics. On the other hand, pore size is another factor that controls protein adsorption. It should correlate to the protein size and cell size. If the size of the nano-/micropore is smaller than that of protein, the protein could not be adsorbed in the pores and thus the efficient surface area for this protein adsorption must have been decreased. Contrarily, the protein is easily trapped in mesopores and improves adsorption. Many experimental results have validated this phenomenon. E. Fujii et al. [45] reported that zinc-substituted HA (ZnHA) nanocrystals with certain content of Zn was more appropriate for b2-microglobulin (b2-MG) adsorption than for BSA adsorption, which is attributed to that the pore size presented on ZnHA was suitable for b2-MG adsorption. The same story also happened on carbonate HA and other Ca-P bioceramics [40,46,47]. Meanwhile, cell size is the key benchmark for macropore size selection. The size of most bone-related cells is about 400 mm and thus 100e500 mm was generally employed in the Ca-P bioceramics for benefiting the cell adhesion [42,43]. As for the particle size, it mainly correlated to the SSA. Smaller particle size leads to higher SSA, which enhances the protein adsorption [47,48]. M. Rouahi et al. [38] reported that HA powder with 100 nm particles led to higher adsorption of proteins than that on HA powder with 1 mm particles. This was attributed to the higher SSA of nano scale HA powder compared with the microscale HA. Thus, the quantity of proteins adsorbed on powders was positively correlated to their SSA. Considering the above factors, it can be concluded that the higher SSA of Ca-P particles, the more protein adsorption; and the lower microporosity of ceramics, the less protein adsorption and initial cell attachment as shown in Fig. 1.12. It should be noted that increasing the SSA does not mean that the size of particle should be as small as possible, as there are other impacts when the size reaches the nanometer level [49]. Certain specific properties of nanomaterials may affect protein adsorption. Unfortunately, studies on the effect of nanoeffect on protein adsorption on bioactive materials are still lacking.

58 Bioactive Materials for Bone Regeneration

FIGURE 1.12 Schematic representation of the inverse correlation existing between SSA, protein adsorption capacity of HA powder, and protein adsorption and cell attachment and growth on sintered HA ceramics [38].

1.3.2.1.2 Chemical properties It is well known that protein adsorption behaviors can be controlled by the substrate surface parameters [50e53]. The chemical properties of material surface play an important role in determining the rates of protein adsorption and the amount of protein adsorbed by interaction between the functional groups on substratum and proteins, even the adsorbed proteins conformation. The chemical nature of the surface can induce greater protein-surface interactions through either electrostatic or hydrophobic interactions [54]. It is generally accepted that electrostatic force played a vital role in the protein adsorption process and has been proved by lots of experimental and computer simulation studies [23,24,48,55]. The charged ions or groups on the substrate surface can bond the functional groups, including amino group, carbonyl group, carboxyl group and aromatic group etc, on the protein molecules to dominate the protein adsorption. The bonded ions or groups on substrate surface or proteins generally called adsorption sites. There are many studies focused on the effect of chemical component or solubility/degradation rate and zeta potential of material on the protein adsorption. Essentially, these effects are just to regulate the charge density, charge distribution, or adsorption site distribution on the substrate surface so as to benefit the protein binding. Ca2þ and PO3 4 are believed to be the protein binding sites on Ca-P surfaces and provide the major driving force for protein adsorption [55e57]. Different component substrate materials have different structure which can lead to different charge/adsorption sites distribution on the surfaces. For instance, the distribution of charged ions or groups (mainly include Ca2þ and PO3 4 ) on HA is far different from that on OCP (Fig. 1.13). The same story also happened on other Ca-P. This difference could induce the discrepancy of net charge on the substrate surface. It is known that proteins can be divided into two types generally, one is acidic protein whose isoelectric point (pI) < 7 and the other is basic protein whose pI > 7. When the pH value is at 7.4, which is equal to that of body fluid, acidic protein and basic protein carry a negative charge and positive charge respectively. The interaction between surface and proteins through electrostatic force could be affected by the surface charge and protein net charge in different solution. XD Zhu et al. [48] reported that HA, BCP and TCP had negative surface charge, and preferred to adsorb more basic protein

Material characteristics, surface/interface Chapter | 1

59

FIGURE 1.13 The top view of charged ions or group distributions on HA (001) and OCP (100) planes. Ca, green (gray in printed version); P, violet (light gray in printed version); O, red (dark gray in printed version).

lysozyme (LSZ) than acidic protein BSA in the pH 7.4 phosphate buffered saline (PBS) solution. HA with higher surface net charge and thus higher value of zeta potential exhibited higher LSZ adsorption due to the stronger electrostatic attraction between them. Another basic protein transforming growth factor-b1 (TGF-b1), which can promote the proliferation and differentiation of bone forming cell [58], also preferred to adsorb on the porous BCP with higher zeta potential than that on dense BCP in the rat serum and in vivo [36]. K. Ohta et al. [59] reported that positive charge Ca2þsites adsorbed acidic proteins and negative PO3 4 sites bonded basic proteins. The order of the ratios of Ca to P sites was estimated to be DCPD > OCP > HA >> DCPA >> b-TCP, which is in agreement with the order of the surface zeta potentials. And the amount of the adsorbed proteins is proportional to the surface charge. The type of adsorbed proteins is dependent on the distributions of Ca, P sites. The effect of distribution of charged ions or groups on protein adsorption is relative obvious on the amorphous Ca-P. There is no significant difference in protein adsorption found between amorphous HA and fluorapatite due to the surface structure of  them is not highly ordered with respect to the position of Ca2þ, PO3 4 , OH  and F ions. Thus the difference in the number of binding sites and the adsorption strength which also depends on the proteins is minimized [60]. The vacancies and defects in the materials crystal surface also impact the protein adsorption [56]. Webster et al. [61,62] reported that the amount of adsorbed BSA was decreased by the Zn2þ substituted into the HA crystal. E. Fujii et al. [45] found that the SSA of Zn-HA increased with increasing Zn content and the amounts of BSA adsorbed on Zn-HA decreased with increasing Zn content in spite of the increase in SSA. It could be attributed to the special arrangement of ions or groups on the substituted HA, which was in cooperation with the effect of surface topography on adsorption and led ZnHA had a highly selective adsorption of b2-MG. Elangovan et al. [63] reported that less proline-rich acidic salivary protein (PRP1) was adsorbed onto carbonated HA (CHA) than onto HA, and a smaller degree of BSA adsorption on CHA than HA with increasing the carbonate content, citing changes in

60 Bioactive Materials for Bone Regeneration

crystal morphology and texture as a possible cause [64]. However, Takemoto et al. [47] found that higher carbonate content of CHA adsorbed more b2-MG than lower that of CHA and HA. S.J. Segvich et al. [39] also reported the different adsorption behaviors of three artificial peptides on the CHA and HA. Accordingly, these differences in adsorption caused by material defect/substitution are attributed to the difference in bonding sites distribution or surface charge density/distribution on the substrate surfaces. Considering the variation of charge groups on proteins, the selective adsorption of proteins on different component/structure Ca-P is easy to understand. The incubation environment is also an important factor that impacts protein adsorption. Kinds of ions in the solution are redistributed under the influence of substrate surface charges, which leads the properties of solution around the surface to change. The counter ions in the solution are attracted to the surface of substrate and make the water molecules on the surface more ordered. These differences in the surface charge distribution caused by incubation solution have potential to improve or inhibit the protein adsorption on the material surfaces. Therefore, different solutions could induce different protein adsorption behaviors. However, for Ca-P bioceramics, the incubation solutions have similar properties according to the Ca-P application field. PBS, Hank’s balanced salt solution (HBSS), serum and in vivo are most employed for protein adsorption on Ca-P investigation. But the protein adsorption differences in different solution environments are rarely reported. The pH value is an important factor that affects the electrical properties of the incubation solution. Studies showed that a decrease in pH led to an increase in acidic protein adsorption and binding affinity [65]. Meanwhile, the solubility of Ca-P is an important parameter for influencing the properties of solution. A representative example happened on protein adsorption on BCP and HA. It is known that b-TCP has a higher solubility than HA [66] and the dissolution of Ca2þ, PO3 4 and other ions from b-TCP would lead to an increasing ionic strength of the solution. Higher ionic strength in solution could induce protein to exposing more polar ionized residues to the solvent [65,67]. Thus, the amount of the protein adsorbed on BCP could be increased by the stronger interaction between protein and the surface binding sites of BCP, which always has a higher ability to adsorb proteins than HA, considering the effect of topography at the same time [40]. This also could be the reason that BCP has better osteoinductivity than HA. For the bioceramics, the sintering temperature has great effect on the solubility of Ca-P in the incubation solution according to the differences in their thermodynamic properties [38]. Higher temperature leads to higher crystallinity, therefore lower solubility [68e72].

1.3.2.1.3 Hydrophobicity Besides the electrostatic force, the hydrophobic interaction is another important way to induce greater protein-surface affinity. It is generally true that a

Material characteristics, surface/interface Chapter | 1

61

hydrophobic surface will adsorb proteins more strongly than a neutrally charged hydrophilic surface and thus adsorb a greater amount of proteins [73e75]. The proteins will tend to adsorb on to the hydrophobic surface by kinds of hydrophobic patches of residues present on the protein’s amphiphilic surface. Protein would unfold and spread its hydrophobic core over the surface due to the thermodynamic driving force to reduce the net hydrophobic surface area of the system exposed to the solvent [76,77]. The charged and polar functional groups of proteins will tend to interact with the hydrophilic surface. For Ca-P bioceramics, BCP has a higher hydrophobicity than that of HA, but lower than that of b-TCP according to the contact angle measuring results [78,79]. This could be another reason that BCP has a higher ability to adsorb proteins than HA. All the variation of parameters of substrate materials, such as component, zeta potential, defect of crystalline and solubility, etc. are just to regulate the chemical properties or hydrophobicity of materials so as to get suitable surface charge, binding sites and polar sites distribution to improve or inhibit protein adsorption. Based on above review, it is clear that bioactive materials also obey the regulation of protein adsorption on substrate materials. In summary, the higher porosity and SSA, relative smaller particle size, suitable surface charge distribution, binding and hydrophobic/polar sites distribution for different kinds of proteins and incubation environments are beneficial to protein adsorption on bioactive materials, which are showed in Table 1.4.

1.3.2.2 Interactions between proteins and bioactive materials All the protein adsorption behaviors on materials are the results of the interaction between proteins and materials surface. Different interaction behaviors at the organic-inorganic interface can lead to different properties of protein

TABLE 1.4 Material factors that affect protein adsorption. Feature

Essential factors

Effect

Structural roughness; topography; porosity; pore size/distribution; particle size; pore connectivity.

Surface area/ specific surface area

More surface area/specific surface area, more protein adsorption

Chemical composition; solubility/degradation rate; zeta potential; vacancy; defects; pH value; hydrophobicity.

Surface charge density/ distribution and polar group arrangement

Protein adsorbs on surface mainly through electrostatic force and polar force. Suitable combination of chemical features of materials can lead to adsorb target proteins.

62 Bioactive Materials for Bone Regeneration

adsorption and structure, morphology, size, orientation, nucleation and growth of Ca-P precipitates. Investigations of interaction between proteins and materials, combining the theoretical analysis method, are helpful to better understand the mechanism of protein adsorption, even biomineralization. Interaction between proteins and materials surface mainly depends on the electrostatic force, sometimes hydrogen bond, which was proved by experimental and computer simulation results [23,24,80]. Proteins adsorbed on the surfaces of Ca-P mainly through positive Ca sites binding negative carboxylate groups and negative P/OH sites binding positive amino groups in protein, while other groups like charged guanidino group, neutral amino, and hydroxyl groups have relative weak interactions with surfaces. Different proteins have different arrangements of charged groups which leads to different adsorption behaviors. As the Ca-P crystals have different structures on their planes, considering the pattern recognition between crystal surfaces and molecules [81], proteins could have the property of selective adsorption on Ca-P surface planes, which should be the reason of protein effects on morphology, size, and orientation of Ca-P crystals. For instance, acidic proteins preferentially adsorbed on the (100) face of HA and OCP crystals [82,83]. While, the strength of interaction between amelogenin and the crystal faces of OCP was in the order of (010) > (001) > (100), which indicated that amelogenin adsorption on OCP should block the growth of (010) face [84]. Different arrangements of carboxylate group and amino group compose different types of residues in protein, called acidic residue and basic residue which are negative and positive, respectively. Thus, based on the electrostatic attraction, the acidic proteins should be preferably adsorbed on the Ca sites based surfaces, basic proteins preferentially adsorbed on the P/OH sites based surfaces; acidic residues preferably bonded to the Ca sites, basic residues preferentially bonded to the P/OH sites. Overall, many of the mineralization-related proteins are acidic and phosphorylated, which are believed to play a key role in biomineralization [85,86]. The acidic phosphoproteins are obviously rich in acidic residues, Aspartic acid (Asp) and glutamic acid (Glu), but also contain basic residues, Arginine (Arg) and Lysine (Lys). Glu preferred to adsorb strongly onto the HA (001) face, which resulted in formation of platelike HA, glycine (Gly) did not show any significantly preferential adsorption on HA surfaces, which resulted rodlike HA [24,30,59].

1.3.3 The effect of protein adsorption on the osteogenesis of bioactive materials A lot of studies have demonstrated that biomaterials have a strong affinity for serum proteins, extracellular matrix (ECM) proteins, growth factors and other cytokines, especially for Ca-P porous ceramic due to its high specific surface area and positive charged binding site provided by Ca2þ available for adsorption. Protein adsorption was reported as the first event after implantation, which

Material characteristics, surface/interface Chapter | 1

63

means it occurred prior to cell arrival to implants around [87]. Therefore, the adsorbed proteins from the blood or other body fluids play a key role in affecting the subsequent cell behavior and other biological process. Based on above effects, protein adsorption on biomaterials is always considered as an important factor that contributes to osteogenesis, including direct effects on cell function.

1.3.3.1 Extracellular protein adsorption ECM proteins, especially the adhesive proteins among them, were confirmed to be adsorbed strongly on Ca-P ceramic and other biomaterials. Generally, assessment of a biomaterial’s osteoinductivity can be carried out by in vitro evaluating its ability to induce the osteogenic differentiation of undifferentiated mesenchymal stem cells (MSCs) [88]. Therefore, the effect of adsorbed proteins on osteogenesis can be evaluated by investigating the regulation on cell behavior and functions. Considering ECM is a main microenvironment after material implantation or the main factor influencing implant’s properties, it always plays a crucial role in mediating the interaction between cells and biomaterials (e.g., cell adhesion and subsequent tissue formation on biomaterial surface) and contributing to the enhancement of the interactions between the implants and host tissues, which ultimately strengthens the implant-tissue interface [89e91]. In vitro, it was reported to regulate the osteogenic differentiation of bone marrow derived MSCs [92]. Human umbilical vein endothelial cell (HUVEC)-derived ECM coating on b-TCP can increase the osteogenic differentiation of MSCs by activating the signaling pathway of MAPK/ERK [90]. Cell behavior and functions are mediated via integrin or other receptors binding to ECM proteins, which depends on the density, conformation, culture condition, and type of adsorbed proteins and cells [93e96]. Integrins, a kind of transmembrane protein, are key receptors in charge of ECM-cell interaction and stem cell adhesion, spreading and homing. It has a large family of subunits that connect the extracellular environment to the intracellular cytoskeleton, thus mediating cell proliferation, migration, and differentiation [97]. The integrin expression on cell depends on the culture conditions and contacting surface, thus difference in the integrin binding and subsequent signal transduction is responsible for the difference in cell behavior and function [98]. Major integrin-ECM protein combinations and interaction as summarized by Humphries. JD et al. are shown in Fig. 1.14. Collagen (Col) is an important component of ECM proteins that could be adsorbed on biomaterials easily. Col I was found to mediating the adhesion of MSCs on polymer scaffolds by increasing the activation of MAPK and PI3K signaling pathways [100,101] and promoting the proliferation and osteogenesis of human MSCs via ERK and AKT pathways [102]. It was reported to be combined with avb3 by the RGD domain to induce MSCs calcification and

64 Bioactive Materials for Bone Regeneration

FIGURE 1.14 The combination and interaction of multiple integrin receptors with ECM ligands and cell surface adhesive proteins [99].

differentiation [103,104]. Col IV was also reported as an important mediator for tissue regeneration [105]. Another important class of ECM proteins is the noncollagenous glycoprotein, such as the adhesive proteins of fibronectin (FN), vitronectin (VN) and laminin (LN). Many in vitro experiments have confirmed that FN plays an essential role in regulating cell attachment and expansion [106]. It always regulates cell behavior by being combined with its receptor, which is also substrate-dependent and cell-niche dependent [107]. During tissue repair process, FN is synthesized and deposited at where early recruitment and commitment of MSCs [108]. Cho et al. [109] reported that a recombinant FNderived oligopeptide F20 coating on titanium discs stimulate cellular proliferation as well as osteoblasts differentiation through the extracellular signal regulated kinase (ERK) signaling pathway, and it acted as a suitable biomolecule for surface modification of dental implants for improving osseointegration. In research of Xing et al. [110], FN can be functioned together with cadherin to strengthen the MSC-recruiting capacity and provide a favorable microenvironment for the proliferation and osteogenic differentiation of MSCs, thus to improve the bone repair eventually. Besides, as Torres et al. reported [111], the presence of FN is not bone ECM-specific, but its distinctive role in the osteogenic commitment of MSCs is density-dependent.

Material characteristics, surface/interface Chapter | 1

65

For example, a lower FN density at position 10 mm in the gradient (corresponding to 48 ng/cm2) on PCL could elicited stronger osteogenic expression, higher cytoskeleton spreading and specific markers of the stem cell commitment to the osteoblast lineage. VN was found to mediating the adhesion of MSCs on polymer scaffolds by increased the activation of FAK and paxillin signaling pathways [101]. Felgueiras et al. reported that VN-coated surface enhanced the levels of MC3T3E1 adhesion, spreading and the formation of intracellular actin cytoskeleton and focal contacts [112]. Previous studies have showed that VN-coated plate could enhance bone matrix synthesis and calcium deposition mainly through the integrin-mediated signal transduction of ERK/MAPK pathway by interaction with integrin avb3 and avb5 [92,113]. In our previous study, VN was preferably adsorbed on BCP ceramic in serum proteins. It also revealed that upregulated adsorption of VN was in favor of MSCs attachment, spreading and osteogenic differentiation by providing a binding domain to avb3 and avb5 receptors, thus activated the signal transduction pathway from outside-in to enhance RUNX2 gene expression. Yamaguchi reported that LN5 promotes the adhesion, migration and moderate proliferation of human corneal endothelial cells by interaction with integrin a3b1 [114]. In Robert et al. research, LN can also promote MSCs adhesion and enhance the gene expression of osteoblasts markers through an ERK-dependent pathway [115]. Yeo et al. found the LN-derived bioactive core peptides are involved in adhesion and spreading of osteoblast-like cells [116]. Our previous work revealed that the elevated gene expressions of a6b1 and a6b4 in MSCs on BCP ceramic could be related with LN-participated cellular events. The dissimilarities in the research above might be ascribed to the different cell system, substrate and culture condition. Generally speaking, ECM adsorbed on biomaterials was required for cell attachment and spreading, which could contribute to the osteogenesis at different time through diverse integrin-mediated signal transduction pathways. These integrins recognize a number of ECM proteins typically by an RGD peptide domain.

1.3.3.2 Adsorption of specific proteins (bone morphogenetic proteins and transcription growth factor beta) Besides ECM proteins, many in vitro experiments showed that growth factors also had a strong adsorption capacity on Ca-P ceramics and other biomaterials [117,118]. It was also confirmed that BMPs, TGF-b and other growth factors made a huge contribution to cell’s migration, proliferation and differentiation [119,120]. BMPs, an important member of TGF-b superfamily [121], play a critical role in regulating growth, differentiation and apoptosis of various cell types, including MSCs, osteoblasts and chondroblasts. Besides, it was also confirmed to stimulate the synthesis and secretion of other bone-related growth

66 Bioactive Materials for Bone Regeneration

factors [122,123]. BMPs always bind to the serine/threonine kinase receptors to initiate the Smad intracellular signaling cascade and then translate into the nucleus to regulating the target genes transcription [124,125]. BMP-2 might be the most potent to induce osteogenic differentiation among them [126,127]. Many in vitro and in vivo studies have confirmed that Ca-P ceramics had a strong affinity for BMP-2. The endogenous BMP-2 might be the specific promoter of bone formation induced by Ca-P ceramics [128e130]. In early study, BMP-2 was recognized as the earliest gene to be induced in vivo and appeared the second elevation during osteogenesis, which had a specific response in vivo of recruitment of MSCs [131]. Therefore, the adsorbed BMP2 on implanted biomaterials could induce MSCs aggregation to material surface and provide a suitable microenvironment for osteogenesis. The expression of BMP-2 in vivo was usually consistent with the ectopic bone formation ability in biomaterials [132]. In Liu et al.’s research, Ca-P-coated titanium with BMP-2 adsorption enhanced the volume of bone deposition within the immediate vicinity of the implant surface [133]. Other research also revealed that the sustained release of BMP-2 adsorbed on bone biomaterials had a stronger effect on osteoblasts and MSCs [118,134]. Besides, BMP-6 and BMP-9 were also considered as the most potent to induce osteoblasts lineage-specific differentiation of MSCs [126]. Mbalaviele G et al. research revealed that BMP-2 could also functioned with b-catenin synergistically to promote bone formation in vivo, as the result showed in Fig. 1.15. TGF-b is one of the most important growth factors with a strong adsorption on biomaterials. Most evidences were collected by cell experiments in vitro that it is a potent chemotactic for bone forming cells and macrophage at the

FIGURE 1.15 New bone formation in vivo induced by the synergistic effect of BMP-2 and the active b-catenin. A solution containing LacZ or DN151 (a mutant for b-catenin with constitutive transcriptional activity) was injected subcutaneously over the parietal bone of the calvariae in the absence or presence of BMP-2 for 3 weeks [135].

Material characteristics, surface/interface Chapter | 1

67

early stage of osteogenesis [131,136,137]. In physiological condition, TGF-b was produced by osteoblasts, degranulating platelets, inflammatory cells, endothelium, chondrocytes, and ECM [138]. When biomaterials were implanted, an inflammation response appeared immediately and more TGF-b was produced. A part of them would be adsorbed on the surface and some would be dispersed in the microenvironment around the substrate. TGF-b was also found to initiate the signaling for BMP synthesis as well as extracellular proteins such as collagen, ALP, OCN, and OPN [139,140]. In vivo, autocrine and paracrine processes stimulated by TGF-b play an important role in MSC and osteoblasts maintenance and expansion [141]. The deficiency of TGF-b usually leads to a lower mineralization and bone formation [142]. Recently, TGF-b was suggested as an indispensable molecules in RANKL-induced osteoclast differentiation that it can advance the matrix production and osteoblasts differentiation when it reduced the RANKL secretion [143]. It was well established that TGF-b signaling in bone was Smad-dependent and the RSmads respond to TGF-b receptors are Smad2 and 3 [144].

1.3.3.3 Other growth factor adsorption Besides BMPs and TGF-b, it has been proved that other growth factors also have a strong adsorption capacity on bone materials, such as VEGF, PDGF, IGF, and FGF. The source of these molecules in vivo is either inflammatory cells or bone-related cells [145]. PDGF was released at the very early stage of bone repair or wound healing and served as a mitogenic and chemotactic agent for MSCs, macrophages, and osteoblasts. An animal study also confirmed that PDGF could significantly increase the number of osteoblasts and the thickness of callus in bone defects [146]. IGF always expressed throughout the bone formation process and was responsible for cell proliferation and protein synthesis. FGF, which was involved in cell proliferation, usually expressed from the early stage to osteoblast formation. It also plays a critical role in angiogenesis and MSC mitogenesis [147]. VEGF always expressed during bone and endochondral formation and made a contribution to osteogenesis by stimulating endothelial cell proliferation and vascularization. It is also a key regulator of physiological angiogenesis during embryogenesis, skeletal growth, and reproductive functions [148]. In addition, all these factors, but not limited to them, impact MSC proliferation in in vitro culture [149].

1.3.3.4 Cytokine adsorption Cytokines are another important factor that can be adsorbed easily by implants in vivo. The majority of supporting evidence was listed by in vitro experiments [150e152]. As we known, the inflammatory reactions initiating fracture healing largely depend on the secreted cytokines from macrophage and other inflammatory cells [153]. Recent reports have indicated that chemotactic

68 Bioactive Materials for Bone Regeneration

factor is a kind of promoter for inflammatory cells and other bone-related cells migration, including macrophage chemotactic proteins (MCP), tumor necrosis factor-a (TNF-a) and interleukin (IL-1, IL-6 et al.). During the process of osteogenesis, MSC-derived osteoblasts are another critical source beyond the endogenous ones. Therefore, the migrated MSCs as well as distant osteoblasts could accelerate bone formation. In addition, they could stimulate the synthesis of ECM that is helpful for healing. Absence of TNF-a would result in delayed resorption of mineralized cartilage and suppression of new bone formation [154]; however, TNF-a can mediate RANKL stimulation of osteoclast differentiation through an autocrine mechanism [155]. The role of IL-6 in bone research is controversial so far. Some reports showed that IL-6 exhibited a positive effect on osteoblast differentiation [156,157]; others found IL-6 could promote osteoclast differentiation that will lead to bone resorption [158]. In fact, the osteogenesis is the result of a complex interaction between cytokines, growth factors, and other supporting molecules. For example, IL-6 was also found to participate in IL-1- and TNF-a induced osteoclast formation. Our previous study showed a significant influence of BCP-mediated inflammatory reaction on its osteoinductivity, which was improved from the interaction between an MSC and soluble factor mixture composed of cytokines and growth factors [159]. Traditionally, the consensus was basically reached that M1 macrophages (secrete proinflammatory factors: IL-1, IL-6 and TNF-a et al.) were responsible for promoting the osteoclast differentiation and M2 macrophage (secrete antiinflammatory factors: L-10, TGF-b et al.) played a more important role during osteogenesis [160]. However, some recent studies present the idea of that osteogenesis was enhanced in the response of M1 macrophages, rather than M2 [161]. Whatever the case is, macrophages whose phenotype occupies a transition between M1 and M2 was hard to be identified. Therefore, both phenotypes are indispensable during bone formation process and the phenotype switch pattern might determines osteogenesis rather than a specific phenotype. In addition, the adsorption of these cytokines on biomaterial implants plays a decisive role in osteogenesis by affecting different molecular and cellular events. In conclusion, different protein adsorption on biomaterials could produce diverse effects on cell behavior and function, including cell adhesion, proliferation, migration, and differentiation during the osteoinduction process. However, further specific experiments should be designed to get a more comprehensive understanding of the effecting process and thus could help to reveal the mechanism of osteogenesis.

1.3.4 Summary Protein adsorption is an important way for bioactive materials to achieve their biological properties.Various material surfaces with different structures and properties can be predicted, designed and prepared by theoretical calculation

Material characteristics, surface/interface Chapter | 1

69

simulations and kinds of advanced experimental methods so as to improve the adsorption capacity of the target protein and even achieve the selective adsorption of a certain protein. Finally, the bioactive materials can achieve their biological properties related to the adsorbed proteins. The future development direction of protein adsorption on bioactive materials lies in the development of advanced technology to more sophisticatedly regulate the material factors affecting protein adsorption as well as various combinations of these material factors to achieve specific protein or protein group adsorption, so as to achieve specific biological properties.

References [1] [2] [3]

[4] [5] [6] [7]

[8] [9] [10] [11] [12] [13]

[14] [15]

P. Ducheyne, Q. Qiu, Bioactive ceramics: the effect of surface reactivity on bone formation and bone cell function, Biomaterials 20 (24) (1999) 2287e2303. W.Q. Yan, M. Oka, T. Nakamura, Bone bonding in bioactive glass ceramics combined with bone matrix gelatin, J. Biomed. Mater. Res. 42 (2) (2015) 258e265. A. Łaczka-Osyczka, M. Łaczka, S. Kasugai, K. Ohya, Behavior of Bone Marrow Cells Cultured on Three Different Coatings of Gel-Derived Bioactive GlasseCeramics at Early Stages of Cell Differentiation, 1998. S.B. Low, Bioactive ceramics in implant, grafting, and periodontal treatment, Dent. Implant. Update 9 (9) (1998) 65. D.A. Puleo, A. Nanci, Understanding and controlling the boneeimplant interface, Biomaterials 20 (23e24) (1999) 2311e2321. S. Radin, P. Ducheyne, Effect of serum proteins on solution-induced surface transformations of bioactive ceramics, J. Biomed. Mater. Res. A 30 (3) (1996) 273e279. A.A. Sawyer, K.M. Hennessy, S.L. Bellis, Regulation of mesenchymal stem cell attachment and spreading on hydroxyapatite by RGD peptides and adsorbed serum proteins, Biomaterials 26 (13) (2005) 1467e1475. V. Hlady, J. Buijs, H.P. Jennissen, Methods for studying protein adsorption, Methods Enzymol. 309 (99) (1999) 402e429. H. Elwing, Protein absorption and ellipsometry in biomaterial research, Biomaterials 19 (4e5) (1998) 397e406. K.K. Chittur, FTIR/ATR for protein adsorption to biomaterial surfaces, Biomaterials 19 (4e5) (1998) 357e369. B.R. Brooks, R.E. Bruccoleri, B.D. Olafson, CHARMM: a program for macromolecular energy, minimization, and dynamics calculations, J. Comput. Chem. 4 (2) (1983) 187e217. A.D. MacKerell Jr., B. Brooks, C.L. Brooks III, L. Nilsson, B. Roux, Y. Won, M. Karplus, CHARMM: The Energy Function and its Parameterization, 1998. B.R. Brooks, C. Brooks III, A. Mackerell Jr., L. Nilsson, R. Petrella, B. Roux, Y. Won, G. Archontis, C. Bartels, S. Boresch, CHARMM: the biomolecular simulation program, J. Comput. Chem. 30 (10) (2009) 1545e1614. V. Carravetta, S. Monti, Peptide-TiO2 surface interaction in solution by ab initio and molecular dynamics simulations, J. Phys. Chem. B 110 (12) (2006) 6160e6169. Y. Kang, X. Li, Y. Tu, Q. Wang, H. Agren, On the mechanism of protein adsorption onto hydroxylated and nonhydroxylated TiO2 surfaces, J. Phys. Chem. C 114 (2010) 14496e14502.

70 Bioactive Materials for Bone Regeneration [16] C. Wu, M. Chen, C. Guo, X. Zhao, C. Yuan, Peptide- TiO2 interaction in aqueous solution: conformational dynamics of RGD using different water models, J. Phys. Chem. B 114 (13) (2010) 4692e4701. [17] C. Wu, M. Chen, C. Xing, Molecular understanding of conformational dynamics of a fibronectin module on rutile (110) surface, Langmuir (2010). [18] A.A. Skelton, T. Liang, T.R. Walsh, Interplay of sequence, conformation, and binding at the peptide- titania interface as mediated by water, ACS Appl. Mater. Interfaces 1 (7) (2009) 1482e1491. [19] J.-W. Shen, T. Wu, Q. Wang, Y. Kang, Induced stepwise conformational change of human serum albumin on carbon nanotube surfaces, Biomaterials 29 (28) (2008) 3847e3855. [20] G. Raffaini, F. Ganazzoli, Simulation study of the interaction of some albumin subdomains with a flat graphite surface, Langmuir 19 (8) (2003) 3403e3412. [21] F. Ganazzoli, G. Raffaini, Computer simulation of polypeptide adsorption on model biomaterials, Phys. Chem. Chem. Phys. 7 (21) (2005) 3651e3663. [22] G. Forte, A. Grassi, G. Marletta, Molecular modeling of oligopeptide adsorption onto functionalized quartz surfaces, J. Phys. Chem. B 111 (38) (2007) 11237. [23] H. Zhou, T. Wu, X. Dong, Q. Wang, J. Shen, Adsorption mechanism of BMP-7 on hydroxyapatite (001) surfaces, Biochem. Biophys. Res. Commun. 361 (1) (2007) 91e96. [24] J. Shen, T. Wu, Q. Wang, H. Pan, Molecular simulation of protein adsorption and desorption on hydroxyapatite surfaces, Biomaterials 29 (5) (2008) 513e532. [25] R. Bhowmik, K. Katti, D. Katti, Molecular dynamics simulation of hydroxyapatitee polyacrylic acid interfaces, Polymer 48 (2) (2007) 664e674. [26] R. Bhowmik, K.S. Katti, D. Verma, D.R. Katti, Probing molecular interactions in bone biomaterials: through molecular dynamics and Fourier transform infrared spectroscopy, Mater. Sci. Eng. C 27 (3) (2007) 352e371. [27] N. Almora-Barrios, K.F. Austen, N.H. de Leeuw, Density functional theory study of the ˆ 0) binding of Glycine, proline, and hydroxyproline to the hydroxyapatite (0001) and (011¡A surfaces, Langmuir 25 (9) (2009) 5018e5025. [28] N. Almora-Barrios, N.H. de Leeuw, Modelling the interaction of a Hyp-Pro-Gly peptide with hydroxyapatite surfaces in aqueous environment, CrystEngComm (2009) 960e967. [29] N. Almora-Barrios, N.H. de Leeuw, A density functional theory study of the interaction of collagen peptides with hydroxyapatite surfaces, Langmuir (2010). [30] H. Pan, J. Tao, X. Xu, R. Tang, Adsorption processes of Gly and Glu amino acids on hydroxyapatite surfaces at the atomic level, Langmuir 23 (17) (2007) 8972e8981. [31] E. Dos Santos, M. Farina, G. Soares, K. Anselme, Surface energy of hydroxyapatite and b-tricalcium phosphate ceramics driving serum protein adsorption and osteoblast adhesion, J. Mater. Sci. Mater. Med. 19 (6) (2008) 2307e2316. [32] K. Cai, M. Frant, J. Bossert, G. Hildebrand, K. Liefeith, K.D. Jandt, Surface functionalized titanium thin films: zeta-potential, protein adsorption and cell proliferation, Colloids Surfaces B Biointerfaces 50 (1) (2006) 1e8. [33] Y.F. Dufreˆne, T.G. Marchal, P.G. Rouxhet, Influence of substratum surface properties on the organization of adsorbed collagen films: in situ characterization by atomic force microscopy, Langmuir 15 (8) (1999) 2871e2878. [34] M. Han, A. Sethuraman, R.S. Kane, G. Belfort, Nanometer-scale roughness having little effect on the amount or structure of adsorbed protein, Langmuir 19 (23) (2003) 9868e9872. [35] F. Yap, Y. Zhang, Protein and cell micropatterning and its integration with micro/nanoparticles assembly, Biosens. Bioelectron. 22 (6) (2007) 775e788.

Material characteristics, surface/interface Chapter | 1

71

[36] X. Zhu, H. Fan, Y. Xiao, D. Li, H. Zhang, T. Luxbacher, X. Zhang, Effect of surface structure on protein adsorption to biphasic calcium-phosphate ceramics in vitro and in vivo, Acta Biomaterialia 5 (4) (2009) 1311e1318. [37] M. Rouahi, O. Gallet, E. Champion, J. Dentzer, P. Hardouin, K. Anselme, Influence of hydroxyapatite microstructure on human bone cell response, J. Biomed. Mater. Res. A 78 (2) (2006) 222e235. [38] M. Rouahi, E. Champion, O. Gallet, A. Jada, K. Anselme, Physico-chemical characteristics and protein adsorption potential of hydroxyapatite particles: influence on in vitro biocompatibility of ceramics after sintering, Colloids Surfaces B Biointerfaces 47 (1) (2006) 10e19. [39] S.J. Segvich, H.C. Smith, D.H. Kohn, The adsorption of preferential binding peptides to apatite-based materials, Biomaterials 30 (7) (2009) 1287e1298. [40] X. Zhu, H. Zhang, H. Fan, W. Li, X. Zhang, Effect of phase composition and microstructure of calcium phosphate ceramic particles on protein adsorption, Acta Biomaterialia 6 (4) (2010) 1536e1541. [41] K.C. Dee, D.A. Puleo, R. Bizios, Tissue-Biomaterial Interactions, Jonh Willey and Sons Inc., New Jersey, 2002. [42] H. Yuan, K. Kurashina, J.D. de Bruijn, Y. Li, K. De Groot, X. Zhang, A preliminary study on osteoinduction of two kinds of calcium phosphate ceramics, Biomaterials 20 (19) (1999) 1799e1806. [43] H. Yuan, Z. Yang, Y. Li, X. Zhang, J. De Bruijn, K. De Groot, Osteoinduction by calcium phosphate biomaterials, J. Mater. Sci. Mater. Med. 9 (12) (1998) 723e726. [44] H. Yuan, P. Zou, Z. Yang, X. Zhang, J. De Bruijn, K. De Groot, Bone morphogenetic protein and ceramic-induced osteogenesis, J. Mater. Sci. Mater. Med. 9 (12) (1998) 717e721. [45] E. Fujii, M. Ohkubo, K. Tsuru, S. Hayakawa, A. Osaka, K. Kawabata, C. Bonhomme, F. Babonneau, Selective protein adsorption property and characterization of nanocrystalline zinc-containing hydroxyapatite, Acta Biomaterialia 2 (1) (2006) 69e74. [46] S. Takashima, Y. Kusudo, S. Takemoto, K. Tsuru, S. Hayakawa, A. Osaka, Synthesis of carbonate-hydroxy apatite and selective adsorption activity against specific pathogenic substances, Key Eng. Mater. 218 (2001) 175e178. [47] S. Takemoto, Y. Kusudo, K. Tsuru, S. Hayakawa, A. Osaka, S. Takashima, Selective protein adsorption and blood compatibility of hydroxy-carbonate apatites, J. Biomed. Mater. Res. A 69 (3) (2004) 544e551. [48] X. Zhu, H. Fan, C. Zhao, J. Lu, T. Ikoma, J. Tanaka, X. Zhang, Competitive adsorption of bovine serum albumin and lysozyme on characterized calcium phosphates by polyacrylamide gel electrophoresis method, J. Mater. Sci. Mater. Med. 18 (11) (2007) 2243e2249. [49] G. Cao, Nanostructures & Nanomaterials: Synthesis, Properties & Applications, Imperial College, 2004. [50] G.B. Sigal, M. Mrksich, G.M. Whitesides, Effect of surface wettability on the adsorption of proteins and detergents, J. Am. Chem. Soc. 120 (14) (1998) 3464e3473. [51] S. Srivastava, A. Verma, B.L. Frankamp, V.M. Rotello, Controlled assembly of proteine nanoparticle composites through protein surface recognition, Adv. Mater. 17 (5) (2005) 617e621. [52] T.C. Ta, M.T. McDermott, Mapping interfacial chemistry induced variations in protein adsorption with scanning force microscopy, Anal. Chem. 72 (11) (2000) 2627e2634.

72 Bioactive Materials for Bone Regeneration [53] G. Iwamoto, L. Winterton, R. Stoker, R. Van Wagenen, J. Andrade, D. Mosher, Fibronectin adsorption detected by interfacial fluorescence, J. Colloid Interface Sci. 106 (2) (1985) 459e464. [54] P. Roach, D. Eglin, K. Rohde, C.C. Perry, Modern biomaterials: a reviewdbulk properties and implications of surface modifications, J. Mater. Sci. Mater. Med. 18 (7) (2007) 1263e1277. [55] B. Feng, J. Chen, X. Zhang, Interaction of calcium and phosphate in apatite coating on titanium with serum albumin, Biomaterials 23 (12) (2002) 2499e2507. [56] D. Hay, E. Moreno, Differential adsorption and chemical affinities of proteins for apatitic surfaces, J. Dent. Res. 58 (2 Suppl. l) (1979) 930e942. [57] Y. Liu, P. Layrolle, J. de Bruijn, C. van Blitterswijk, K. de Groot, Biomimetic coprecipitation of calcium phosphate and bovine serum albumin on titanium alloy, Mater. Res. 57 (2001) 327e335. [58] T. Boontheekul, D.J. Mooney, Protein-based signaling systems in tissue engineering, Curr. Opin. Biotechnol. 14 (5) (2003) 559e565. [59] K. Ohta, H. Monma, S. Takahashi, Adsorption characteristics of proteins on calcium phosphates using liquid chromatography, J. Biomed. Mater. Res. 55 (3) (2001) 409e414. [60] H. Zeng, K.K. Chittur, W.R. Lacefield, Analysis of bovine serum albumin adsorption on calcium phosphate and titanium surfaces, Biomaterials 20 (4) (1999) 377e384. [61] C. Ergun, T.J. Webster, R. Bizios, R.H. Doremus, Hydroxylapatite with substituted magnesium, zinc, cadmium, and yttrium. I. Structure and microstructure, J. Biomed. Mater. Res. 59 (2) (2002) 305e311. [62] T.J. Webster, C. Ergun, R.H. Doremus, R. Bizios, Hydroxylapatite with substituted magnesium, zinc, cadmium, and yttrium. II. Mechanisms of osteoblast adhesion, J. Biomed. Mater. Res. 59 (2) (2002) 312e317. [63] S. Elangovan, H.C. Margolis, F.G. Oppenheim, E. Beniash, Conformational changes in salivary proline-rich protein 1 upon adsorption to calcium phosphate crystals, Langmuir 23 (22) (2007) 11200e11205. [64] K. Kandori, M. Saito, T. Takebe, A. Yasukawa, T. Ishikawa, Adsorption of bovine serum albumin on synthetic carbonate calcium hydroxyapatite, J. Colloid Interface Sci. 174 (1) (1995) 124e129. [65] D.T.H. Wassell, R.C. Hall, G. Embery, Adsorption of bovine serum albumin onto hydroxyapatite, Biomaterials 16 (9) (1995) 697e702. [66] G. Daculsi, Biphasic calcium phosphate concept applied to artificial bone, implant coating and injectable bone substitute, Biomaterials 19 (16) (1998) 1473e1478. [67] C. Yongli, Z. Xiufang, G. Yandao, Z. Nanming, Z. Tingying, S. Xinqi, Conformational changes of fibrinogen adsorption onto hydroxyapatite and titanium oxide nanoparticles, J. Colloid Interface Sci. 214 (1) (1999) 38e45. [68] N. Kivrak, A.C. Tas¸, Synthesis of calcium hydroxyapatite-tricalcium phosphate (HA-TCP) composite bioceramic powders and their sintering behavior, J. Am. Ceram. Soc. 81 (9) (1998) 2245e2252. [69] R. LeGeros, S. Lin, R. Rohanizadeh, D. Mijares, J. LeGeros, Biphasic calcium phosphate bioceramics: preparation, properties and applications, J. Mater. Sci. Mater. Med. 14 (3) (2003) 201e209. [70] J. Osborn, H. Newesely, The material science of calcium phosphate ceramics, Biomaterials 1 (2) (1980) 108e111.

Material characteristics, surface/interface Chapter | 1

73

[71] D. Bernache-Assollant, A. Ababou, E. Champion, M. Heughebaert, Sintering of calcium phosphate hydroxyapatite Ca10(PO4)6(OH)2 I. Calcination and particle growth, J. Eur. Ceram. Soc. 23 (2) (2003) 229e241. [72] H.Y. Juang, M.H. Hon, Effect of calcination on sintering of hydroxyapatite, Biomaterials 17 (21) (1996) 2059e2064. [73] M. Malmsten, Formation of adsorbed protein layers, J. Colloid Interface Sci. 207 (2) (1998) 186e199. [74] C. Grunwald, W. Eck, N. Opitz, J. Kuhlmann, C. Wo¨ll, Fabrication of 2D-protein arrays using biotinylated thiols: results from fluorescence microscopy and atomic force microscopy, Phys. Chem. Chem. Phys. 6 (17) (2004) 4358e4362. [75] A.A. Vertegel, R.W. Siegel, J.S. Dordick, Silica nanoparticle size influences the structure and enzymatic activity of adsorbed lysozyme, Langmuir 20 (16) (2004) 6800e6807. [76] C.F. Wertz, M.M. Santore, Effect of surface hydrophobicity on adsorption and relaxation kinetics of albumin and fibrinogen: single-species and competitive behavior, Langmuir 17 (10) (2001) 3006e3016. [77] R. Latour, Biomaterials: protein-surface interactions, Encyclopedia of biomaterials and biomedical engineering 1 (2005) 270e278. [78] X. Zhu, Surface Structure and Properties of Calcium Phosphate Bioceramics and the Relation with the Special Adsorption of Proteins, National research center for biomaterials, Sichuan university, China, 2006. [79] M. Lopes, F. Monteiro, J. Santos, A. Serro, B. Saramago, Hydrophobicity, surface tension, and zeta potential measurements of glass-reinforced hydroxyapatite composites, J. Biomed. Mater. Res. 45 (4) (1999) 370e375. [80] L. Addadi, S. Weiner, M. Geva, On how proteins interact with crystals and their effect on crystal formation, Z. Kardiol. 90 (3) (2001) 92e98. [81] R. Fujisawa, Y. Kuboki, Preferential adsorption of dentin and bone acidic proteins on the (100) face of hydroxyapatite crystals, Biochim. Biophys. Acta 1075 (1) (1991) 56. [82] L. Addadi, J. Moradian-Oldak, H. Furedimilhofer, S. Weiner, A. Veis, H. Slavkin, P. Price, Chemistry and Biology of Mineralized Tissues, Elsevier, Amsterdam, 1992. [83] M. Iijima, J. Moradian-Oldak, Control of octacalcium phosphate and apatite crystal growth by amelogenin matrices, J. Mater. Chem. 14 (14) (2004) 2189e2199. [84] L.C. Palmer, C.J. Newcomb, S.R. Kaltz, E.D. Spoerke, S.I. Stupp, Biomimetic systems for hydroxyapatite mineralization inspired by bone and enamel, Chem. Rev. 108 (11) (2008) 4754e4783. [85] A. George, B. Sabsay, P. Simonian, A. Veis, Characterization of a novel dentin matrix acidic phosphoprotein. Implications for induction of biomineralization, J. Biol. Chem. 268 (17) (1993) 12624. [86] C.J. Wilson, R.E. Clegg, D.I. Leavesley, M.J. Pearcy, Mediation of biomaterial-cell interactions by adsorbed proteins: a review, Tissue Eng. 11 (2) (2005) 1e18. [87] P. Sibilla, A. Sereni, A.G., Effects of a hydroxyapatite-based biomaterial on gene expression in osteoblast-like cells, J. Dent. Res. 85 (2006) 354e358. [88] O.F. Zouani, C. Chollet, B. Guillotin, M.C. Durrieu, Differentiation of pre-osteoblast cells on poly(ethylene terephthalate) grafted with RGD and/or BMPs mimetic peptides, Biomaterials 31 (32) (2010) 8245e8253. [89] Y. Kang, S. Kim, J. Bishop, A. Khademhosseini, Y. Yang, The osteogenic differentiation of human bone marrow MSCs on HUVEC-derived ECM and beta-TCP scaffold, Biomaterials 33 (29) (2012) 6998e7007.

74 Bioactive Materials for Bone Regeneration [90] C.J. Flaim, S. Chien, S.N. Bhatia, An extracellular matrix microarray for probing cellular differentiation, Nat. Methods 2 (2) (2005) 119e125. [91] S. Mathews, R. Bhonde, P.K. Gupta, S. Totey, Extracellular matrix protein mediated regulation of the osteoblast differentiation of bone marrow derived human mesenchymal stem cells, Differentiation, Research in Biological Diversity 84 (2) (2012) 185e192. [92] B.G. Keselowsky, D.M. Collard, A.s.J. Garcı´a, Surface chemistry modulates fibronectin conformation and directs integrin binding and specificity to control cell adhesion, J. Biomed. Mater. Res. A 66 (5) (2002) 247e259. [93] A. Avigdor, P. Goichberg, S. Shivtiel, A. Dar, A. Peled, S. Samira, O. Kollet, R. Hershkoviz, R. Alon, I. Hardan, H. Ben-Hur, D. Naor, A. Nagler, T. Lapidot, CD44 and hyaluronic acid cooperate with SDF-1 in the trafficking of human CD34þ stem/progenitor cells to bone marrow, Blood 103 (8) (2004) 2981e2989. [94] F. Gattazzo, A. Urciuolo, P. Bonaldo, Extracellular matrix: a dynamic microenvironment for stem cell niche, Biochim. Biophys. Acta 1840 (8) (2014) 2506e2519. [95] N.R. Gandavarapu, P.D. Mariner, M.P. Schwartz, K.S. Anseth, Extracellular matrix protein adsorption to phosphate-functionalized gels from serum promotes osteogenic differentiation of human mesenchymal stem cells, Acta Biomaterialia 9 (1) (2013) 4525. [96] K.R. Legate, S.A. Wickstrom, R. Fassler, Genetic and cell biological analysis of integrin outside-in signaling, Genes Dev. 23 (4) (2009) 397e418. [97] R.K. Sinha, R.S. Tuan, Regulation of human osteoblast integrin expression by orthopedic implant materials, Bone 18 (5) (1996) 451e457. [98] J.D Humphries, A Byron, M.J Humphries, INTEGRIN LIGANDS, J Cell Sci. 119 (2006) 3901e3903, https://doi.org/10.1242/jcs.03098. [99] S.R. Chastain, A.K. Kundu, S. Dhar, J.W. Calvert, A.J. Putnam, Adhesion of mesenchymal stem cells to polymer scaffolds occurs via distinct ECM ligands and controls their osteogenic differentiation, J. Biomed. Mater. Res. A 78 (1) (2006) 73e85. [100] A.K. Kundu, A.J. Putnam, Vitronectin and collagen I differentially regulate osteogenesis in mesenchymal stem cells, Biochem. Biophys. Res. Commun. 347 (1) (2006) 347e357. [101] K.S. Tsai, S.Y. Kao, C.Y. Wang, Y.J. Wang, J.P. Wang, S.C. Hung, Type I collagen promotes proliferation and osteogenesis of human mesenchymal stem cells via activation of ERK and Akt pathways, J. Biomed. Mater. Res. A 94 (3) (2010) 673e682. [102] G.E. Davis, Affinity of integrins for damaged extracellular matrix: alpha v beta 3 binds to denatured collagen type I through RGD sites, Biochem. Biophys. Res. Commun. 182 (3) (1992) 1025e1031. [103] M. Mizuno, Y. Kuboki, Osteoblast-related gene expression of bone marrow cells during the osteoblastic differentiation induced by type I collagen, J. Biochem. 129 (2001) 133e138. [104] A. Urciuolo, M. Quarta, V. Morbidoni, F. Gattazzo, S. Molon, P. Grumati, F. Montemurro, F.S. Tedesco, B. Blaauw, G. Cossu, Collagen VI regulates satellite cell self-renewal and muscle regeneration, Nat. Commun. 4 (3) (2013) 1964. [105] C.F. Bentzinger, Y.X. Wang, J. von Maltzahn, V.D. Soleimani, H. Yin, M.A. Rudnicki, Fibronectin regulates Wnt7a signaling and satellite cell expansion, Cell stem cell 12 (1) (2013) 75e87. [106] D. Missirlis, T. Haraszti, H. Kessler, J.P. Spatz, Fibronectin promotes directional persistence in fibroblast migration through interactions with both its cell-binding and heparinbinding domains, Sci. Rep. 7 (1) (2017) 3711. [107] W.S. To, K.S. Midwood, Plasma and cellular fibronectin: distinct and independent functions during tissue repair, Fibrogenesis Tissue Repair 4 (2011) 21.

Material characteristics, surface/interface Chapter | 1

75

[108] Y.D. Cho, S.J. Kim, H.S. Bae, W.J. Yoon, K.H. Kim, H.M. Ryoo, Y.J. Seol, Y.M. Lee, I.C. Rhyu, Y. Ku, Biomimetic approach to stimulate osteogenesis on titanium implant surfaces using fibronectin derived oligopeptide, Curr. Pharmaceut. Des. 22 (30) (2016) 4729e4735. [109] J. Xing, T. Mei, K. Luo, Z. Li, A. Yang, Z. Li, Z. Xie, Z. Zhang, S. Dong, T. Hou, J. Xu, F. Luo, A nano-scaled and multi-layered recombinant fibronectin/cadherin chimera composite selectively concentrates osteogenesis-related cells and factors to aid bone repair, Acta Biomaterialia 53 (2017) 470e482. [110] A.B. Faia-Torres, T. Goren, T.O. Ihalainen, S. Guimond-Lischer, M. Charnley, M. Rottmar, K. Maniura-Weber, N.D. Spencer, R.L. Reis, M. Textor, N.M. Neves, Regulation of human mesenchymal stem cell osteogenesis by specific surface density of fibronectin: a gradient study, ACS Appl. Mater. Interfaces 7 (4) (2015) 2367e2375. [111] H.P. Felgueiras, M.D.M. Evans, V. Migonney, Contribution of fibronectin and vitronectin to the adhesion and morphology of MC3T3-E1 osteoblastic cells to poly(NaSS) grafted Ti6Al4V, Acta Biomaterialia 28 (2015) 225e233. [112] T.I. Kim, J.H. Jang, H.W. Kim, J.C. Knowles, Y. Ku, Biomimetic approach to dental implants, Curr. Pharmaceut. Des. 14 (22) (2008) 2201e2211. [113] M. Yamaguchi, N. Ebihara, N. Shima, M. Kimoto, T. Funaki, S. Yokoo, A. Murakami, S. Yamagami, Adhesion, migration, and proliferation of cultured human corneal endothelial cells by laminin-5, Investig. Ophthalmol. Vis. Sci. 52 (2) (2011) 679e684. [114] R.F. Klees, R.M. Salasznyk, K. Kingsley, W.A. Williams, A. Boskey, G.E. Plopper, Laminin-5 induces osteogenic gene expression in human mesenchymal stem cells through an ERK-dependent pathway, Mol. Biol. Cell 16 (2005) 881e890. [115] I.S. Yeo, S.K. Min, H. Ki Kang, T.K. Kwon, S. Youn Jung, B.M. Min, Adhesion and spreading of osteoblast-like cells on surfaces coated with laminin-derived bioactive core peptides, Data in brief 5 (2015) 411e415. [116] M. Lin, S. Overgaard, H. Glerup, K. Søballe, C. Bu¨nger, Transforming growth factor-beta1 adsorbed to tricalciumphosphate coated implants increases peri-implant bone remodeling, Biomaterials 22 (3) (2001) 189e193. [117] F.G. Draenert, A.L. Nonnenmacher, P.W. Ka¨mmerer, J. Goldschmitt, W. Wagner, BMP-2 and bFGF release and in vitro effect on human osteoblasts after adsorption to bone grafts and biomaterials, Clin. Oral Implant. Res. 24 (7) (2013) 750e757. [118] E.Y. Yang, H.L. Moses, Transforming growth factor b1-induced changes in cell migration, proliferation, and angiogenesis in the chicken chorioallantoic membrane, JCB (J. Cell Biol.) 111 (2) (1990) 731e741. [119] K. Inai, R.A. Norris, S. Hoffman, R.R. Markwald, Y. Sugi, BMP-2 induces cell migration and periostin expression during atrioventricular valvulogenesis, Dev. Biol. 315 (2) (2008) 383e396. [120] D.P. Ten, J. Fu, P. Schaap, B.A. Roelen, Signal transduction of bone morphogenetic proteins in osteoblast differentiation, J. Bone Joint Surg. Am 85-A (Suppl. 3) (2003) 34. [121] H. Peng, J. Cummins, J. Huard, Synergistic enhancement of bone formation and healing by stem celleexpressed VEGF and bone morphogenetic protein-4, J. Clin. Investig. 110 (6) (2002) 751e759. [122] G. Valdimarsdottir, M.J. Goumans, A. Rosendahl, M. Brugman, S. Itoh, F. Lebrin, P. Sideras, P.T. Dijke, Stimulation of Id1 expression by bone morphogenetic protein is sufficient and necessary for bone morphogenetic proteineinduced activation of endothelial cells, Circulation 106 (17) (2002) 2263.

76 Bioactive Materials for Bone Regeneration [123] L. Wang, X. Zhang, Y. Guo, X. Chen, R. Li, L. Liu, C. Shi, C. Guo, Y. Zhang, Involvement of BMPs/Smad signaling pathway in mechanical response in osteoblasts, Cell. Physiol. Biochem. 26 (6) (2010) 1093e1102. [124] T. Katagiri, S. Tsukamoto, The unique activity of bone morphogenetic proteins in bone: a critical role of the Smad signaling pathway, Biol. Chem. 394 (6) (2013) 703e714. [125] H. Cheng, W. Jiang, F.M. Phillips, R.C. Haydon, Y. Peng, L. Zhou, H.H. Luu, N. An, B. Breyer, P. Vanichakarn, Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs), Urol. Oncol. Semi. Orig. Invest. 22 (1) (2003) 1544e1552. [126] D.I. Israel, K.M. Nove JKerns, R.J. Kaufman, V. Rosen, K.A. Cox, J.M. Wozney, Heterodimeric bone morphogenetic proteins show enhanced activity in vitro and in vivo, Growth Factors 13 (3e4) (1996) 291e300. [127] J. Wang, H. Zhang, X. Zhu, H. Fan, Y. Fan, X. Zhang, Dynamic competitive adsorption of bone-related proteins on calcium phosphate ceramic particles with different phase composition and microstructure, J. Biomed. Mater. Res. B Appl. Biomater. 101 (6) (2013) 1069e1077. [128] H. Rao, Z. Lu, W. Liu, Y. Wang, H. Ge, P. Zou, H. He, The adsorption of bone-related proteins on calcium phosphate ceramic particles with different phase composition and its adsorption kinetics, Surf. Interface Anal. 48 (10) (2016) 1048e1055. [129] Z. Yang, H. Yuan, W. Tong, P. Zou, W. Chen, X. Zhang, Osteogenesis in extraskeletally implanted porous calcium phosphate ceramics: variability among different kinds of animals, Biomaterials 17 (22) (1996) 2131e2137. [130] T.J. Cho, L.C. Gerstenfeld, T.A. Einhorn, Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing, J. Bone Miner. Res. 17 (3) (2002) 513e520. [131] J. Wang, Y. Chen, X. Zhu, T. Yuan, Y. Tan, Y. Fan, X. Zhang, Effect of phase composition on protein adsorption and osteoinduction of porous calcium phosphate ceramics in mice, J. Biomed. Mater. Res. A 102 (12) (2014) 4234e4243. [132] Y. Liu, L. Enggist, A.F. Kuffer, D. Buser, E.B. Hunziker, The influence of BMP-2 and its mode of delivery on the osteoconductivity of implant surfaces during the early phase of osseointegration, Biomaterials 28 (16) (2007) 2677e2686. [133] E.R. Balmayor, J.P. Geiger, C. Koch, M.K. Aneja, G.M. Van, C. Rudolph, C. Plank, Modified mRNA for BMP 2 in combination with biomaterials serves as a transcript activated matrix for effectively inducing osteogenic pathways in stem cells, Stem Cells Dev. 26 (1) (2016) 25e34. [134] G. Mbalaviele, S. Sheikh, J.P. Stains, V.S. Salazar, S.L. Cheng, D. Chen, R. Civitelli, Betacatenin and BMP-2 synergize to promote osteoblast differentiation and new bone formation, J. Cell. Biochem. 94 (2) (2005) 403e418. [135] M. Wan, C. Li, G. Zhen, K. Jiao, W. He, X. Jia, W. Wang, C. Shi, Q. Xing, Y.F. Chen, S. Jan De Beur, B. Yu, X. Cao, Injury-activated transforming growth factor beta controls mobilization of mesenchymal stem cells for tissue remodeling, Stem Cells 30 (11) (2012) 2498e2511. [136] J.R. Lieberman, A. Daluiski, T.A. Einhorn, The role of growth factors in the repair of bone. Biology and clinical applications, J. Bone Joint Surg. Am. 84-A (6) (2002) 1032. [137] V. Devescovi, E. Leonardi, G. Ciapetti, E. Cenni, Growth factors in bone repair, La Chirurgia degli organi di movimento 92 (3) (2008) 161e168.

Material characteristics, surface/interface Chapter | 1

77

[138] G.S. Zode, A. Sethi, A.-M. Brun-Zinkernagel, I.-F. Chang, A.F. Clark, R.J. Wordinger, Transforming growth factor-b2 increases extracellular matrix proteins in optic nerve head cells via activation of the Smad signaling pathway, Mol. Vis. 17 (2011) 1745e1758. [139] L.P. Singh, K. Green, M. Alexander, S. Bassly, E.D. Crook, Hexosamines and TGF-b1 use similar signaling pathways to mediate matrix protein synthesis in mesangial cells, Am. J. Physiol. Renal Physiol. 286 (2) (2004) F409eF416. [140] R. Derynck, R.J. Akhurst, Differentiation plasticity regulated by TGF-b family proteins in development and disease, Nat. Cell Biol. 9 (9) (2007) 1000. [141] A.G. Geiser, C.W. Hummel, M.W. Draper, J.W. Henck, I.R. Cohen, D.G. Rudmann, K.B. Donnelly, M.D. Adrian, T.A. Shepherd, O.B. Wallace, A new selective estrogen receptor modulator with potent uterine antagonist activity, agonist activity in bone, and minimal ovarian stimulation, Endocrinology 146 (10) (2005) 4524. [142] T. Yasui, Y. Kadono, M. Nakamura, Y. Oshima, T. Matsumoto, H. Masuda, J. Hirose, Y. Omata, H. Yasuda, T. Imamura, Regulation of RANKL-induced osteoclastogenesis by TGF-b through molecular interaction between Smad3 and Traf6, J. Bone Miner. Res. 26 (7) (2011) 1447e1456. [143] G. Sapkota, M. Knockaert, C. Alarco´n, E. Montalvo, A.H. Brivanlou, J. Massague´, Dephosphorylation of the linker regions of Smad1 and Smad2/3 by small C-terminal domain phosphatases has distinct outcomes for bone morphogenetic protein and transforming growth factor-beta pathways, J. Biol. Chem. 281 (52) (2006) 40412e40419. [144] C.C. Wurgler-Hauri, L.M. Dourte, T.C. Baradet, G.R. Williams, L.J. Soslowsky, Temporal expression of 8 growth factors in tendon-to-bone healing in a rat supraspinatus model, J. Shoulder Elb. Surg. 16 (5 Suppl. l) (2007) S198eS203. [145] R. Kathariya, N. Shah, Role of PDGF in Wound Healing: An Animal Study, Iadr Asia/ pacific Region, 2015. [146] X. Yang, L. Liaw, I. Prudovsky, P.C. Brooks, C. Vary, L. Oxburgh, R. Friesel, Fibroblast growth factor signaling in the vasculature, Curr. Atheroscler. Rep. 17 (6) (2015) 509. [147] N. Ferrara, H.P. Gerber, J. Lecouter, The biology of VEGF and its receptors, Nat. Med. 9 (6) (2003) 669e676. [148] K. Ksiazek, A comprehensive review on mesenchymal stem cell growth and senescence, Rejuvenation Res. 12 (2) (2009) 105. [149] G. Yushin, E.N. Hoffman, M.W. Barsoum, Y. Gogotsi, C.A. Howell, S.R. Sandeman, G.J. Phillips, A.W. Lloyd, S.V. Mikhalovsky, Mesoporous carbide-derived carbon with porosity tuned for efficient adsorption of cytokines, Biomaterials 27 (34) (2006) 5755e5762. [150] C.A. Howell, S.R. Sandeman, G.J. Phillips, A.W. Lloyd, J.G. Davies, S.V. Mikhalovsky, S.R. Tennison, A.P. Rawlinson, O.P. Kozynchenko, H.L. Owen, The in vitro adsorption of cytokines by polymer-pyrolysed carbon, Biomaterials 27 (30) (2006) 5286e5291. [151] J.D. Kimmel, G.A. Gibson, S.C. Watkins, J.A. Kellum, W.J. Federspiel, IL-6 adsorption dynamics in hemoadsorption beads studied using confocal laser scanning microscopy, J. Biomed. Mater. Res. B Appl. Biomater. 92 (2) (2010) 390e396. [152] T. Velnar, T. Bailey, V. Smrkolj, The wound healing process: an overview of the cellular and molecular mechanisms, J. Int. Med. Res. 37 (5) (2009) 1528e1542. [153] L.C. Gerstenfeld, T.J. Cho, T. Kon, T. Aizawa, A. Tsay, J. Fitch, G.L. Barnes, D.T. Graves, T.A. Einhorn, Impaired fracture healing in the absence of TNF-alpha signaling: the role of TNF-alpha in endochondral cartilage resorption, J. Bone Miner. Res. 18 (9) (2003) 1584.

78 Bioactive Materials for Bone Regeneration [154] W. Zou, I. Hakim, K. Tschoep, S. Endres, Z. Bar-Shavit, Tumor necrosis factor-a mediates RANK ligand stimulation of osteoclast differentiation by an autocrine mechanism & dagger, J. Cell. Biochem. 83 (1) (2001) 70e83. [155] N. Franchimont, S. Wertz, M. Malaise, Interleukin-6: an osteotropic factor influencing bone formation? Bone 37 (5) (2005) 601e606. [156] B.H. Fellah, B. Delorme, J. Sohier, D. Magne, P. Hardouin, P. Layrolle, Macrophage and osteoblast responses to biphasic calcium phosphate microparticles, J. Biomed. Mater. Res. A 93A (4) (2010) 1588e1595. [157] K.T. Steeve, P. Marc, T. Sandrine, H. Dominique, F. Yannick, IL-6, RANKL, TNF-alpha/ IL-1: interrelations in bone resorption pathophysiology, Cytokine Growth Factor Rev. 15 (1) (2004) 49e60. [158] J. Wang, D. Liu, B. Guo, X. Yang, X. Chen, X. Zhu, Y. Fan, X. Zhang, Role of biphasic calcium phosphate ceramic-mediated secretion of signaling molecules by macrophages in migration and osteoblastic differentiation of MSCs, Acta Biomaterialia 51 (2017) 447e460. [159] R.J. Miron, D.D. Bosshardt, OsteoMacs: key players around bone biomaterials, Biomaterials 82 (2015) 1. [160] P. Guihard, Y. Danger, B. Brounais, E. David, R. Brion, J. Delecrin, C.D. Richards, S. Chevalier, F. Re´dini, D. Heymann, Induction of osteogenesis in mesenchymal stem cells by activated monocytes/macrophages depends on oncostatin M signaling, Stem Cells 50 (4) (2012) 762e772. [161] T. Kizuki, T. Saito, M. Ohgaki, Y. Yokogawa, Effects of serum proteins in apatite layer formed in culture medium on initial adhesion and cell proliferation, Key Eng. Mater. 309e311 (2006) 283e288.

Material characteristics, surface/interface Chapter | 1

79

Chapter 1.4

Osteogenesis induced by bioactive porous materials and the related molecular mechanism Since the discovery of osteoinduction in the early 20th century, innovative bioactive materials with osteoinductive potential have emerged. It is generally accepted and well established that an osteoinductive material should induce bone formation upon implantation in nonosseous sites, also known as heterotopic or ectopic sites. This phenomenon is called “osteoinduction,” and the capacity for osteoinduction is called “osteoinductivity” or “osteoinductive potential.” Generally, osteoinduction has been observed with diverse calcium phosphate biomaterials in various forms such as (i) sintered bioceramics including HA [1], beta-tricalcium phosphate (b-TCP) [2], and biphasic calcium phosphate (BCP), (ii) cements [3], (iii) coatings as well as (iv) coralderived ceramics [4], in various animal models. Osteoinductivity of bioactive glass, alumina ceramics and titanium were also reported, which are able to form an apatite layer in vivo and in vitro when immersed in a supersaturated calcium-phosphate solution [5,6]. The only synthetic polymer to date to have shown osteoinductivity is poly-hydroxyethylmethacrylate (poly-HEMA) [7]. Based on the extensive research work surrounding these biomaterials, the intriguing phenomena have been categorized as those influenced by materials factors, and those by biological factors. It is well known that osteoinduction is highly dependent on several material parameters including porosity, granule size, phase composition, and even sintering temperature. In this chapter, we divide the osteoinduction phenomenon into three principles (Fig. 1.16): (1) angiogenesis induced by porous biomaterials and its role in osteoinduction; (2) osteogenesis related microenvironment and cell participant; (3) immunoresponse arose by implant-host interaction. The aim of this chapter is to gather the current knowledge on osteoinductivity of bone grafting materials for the effective development of new graft substitute that enhance bone regeneration.

1.4.1 Angiogenesis of bioactive materials and the involved molecular mechanism Vascularization played a critical role in bone repair and regeneration. In fetal bone development, matured vasculature provides nutrients, cytokines, and oxygen to various cells and takes away metabolic waste and carbon dioxide

80 Bioactive Materials for Bone Regeneration

FIGURE 1.16 schematic diagram for the osteoinductive mechanism of bioactive porous materials. Multiple factors including ionic environment, neovascularization, immunoresponse, cell recruitment and cytokine autocrine/paracrine effects are all involved and interact with each other.

[8]. Hausman et al. found that the inhibition of angiogenesis can completely abort fracture healing of rat femoral bone [9]. The repair of bone defects with bioactive implants is expected to experience similar cellular and molecular events as natural fracture healing, including hematoma, inflammation, bone formation accompanied with vascularization. The efficacy of neovascularization is thus essential for the repair of large bone defects. Several studies conducted by Jiang Chang’s research group have reported a strong ability to stimulate angiogenesis by silicate bioceramics, whose ion releasing ability and ion type were suggested to be essential [10e12]. Gorustovich et al. had also made a detailed review on the effect of bioactive glasses on angiogenesis [13]. As for the porous Ca-P-based bioceramics, new blood vessels, and matured Haversian canals in the inner pores were often observed after in vivo ectopic implantation even without addition of exogenous cells or growth factors [14e17]. In addition to the functions mentioned above, neovascularization inside bioactive material is also serves as a conduit for all kinds of progenitor cells. For example, the recruitment of MSCs to the site of implantation requires vasculature to initiate osteoinduction especially in large bone defect. Other than a direct recruitment, vascularization inside of the material also supplied all kinds of cytokines to transform other cell types into MSCs. Endothelial cells and pericytes are the cells mainly responsible for the vascularization. It is discovered by many studies that the endothelial cells are able to transit into osteoblasts under the regulation of inflammation process [18,19]. Moreover, these cells can secret cytokines like BMP-7 and BMP-2, which are positively

Material characteristics, surface/interface Chapter | 1

81

correlated with the intensity of inflammatory stress [20]. It is known that BMPs not only regulates osteogenesis but angiogenesis as well [21]. Under a certain concentration of BMPs, endothelial cells can transform into MSCs via a process defined as endothelialemesenchymal transition [22]. Recently, our research group has discovered that the neovascularization occurring in ectopic bone formation of Ca-P material is dependent on its phase composition (Fig. 1.17) [23]. The results indicated a more soluble b-TCP phase could induce more functional matured blood vessel than either HA or BCP bioceramics, with similar pore size and porosity. A higher Ca2þ concentration in the porous microenvironment may favor neovascularization, as confirmed in the in vitro test of capillary tube branch point counting using the conditioned media of three types of materials cocultured with HUVECs. Several studies have demonstrated that the Ca2þ concentration mediated vascularization and the involved angiogenic response of bone marrow progenitor cells is via fibroblast growth factor 2 (FGF2)-induced phosphorylation pathways [24e26]. Moreover, the porous structure of biomaterial is favorable for neovascularization to occur. On the one hand, the concavity provides a reservoir to maintain the required Ca2þ ions. On the other hand, the mechanical distortion created with porous structure works together with vascularization to bring and harbor MSCs, myoblasts, myoendothelial cells and pericytes to reside into the concavity [27].

FIGURE 1.17 H&E staining for the decalcified sections of porous Ca-P ceramics implanted into the thigh muscles of mice for 1, 2, and 4 weeks. Black arrows represent new blood vessels formed [42].

82 Bioactive Materials for Bone Regeneration

1.4.2 Osteogenesis of bioactive materials and materialmediated mesenchymal stem cell function 1.4.2.1 Osteogenic ionic environment created in the porous structure The specific physiochemical nature of the Ca-P ceramicsdi.e., the degraded Ca2þ, PO3 4 ions, and concavitydare one of the most critical factors associated with osteogenesis. The Ca2þ ions, on the one hand, are the most ubiquitous inorganic biological signal carriers which take part in the entire cell cycle, from fertilization to apoptosis [28]. On the other hand, inorganic PO3 4 ions (or Pi) either in the form of energy currency (i.e., ATP, cAMP, ADP, etc.) or participated in the universal phosphorylation process modulate cell fate in every possible way. The release of Ca2þand PO3 4 ions of Ca-P scaffolds or layers are related to both physical and biological degradations. Among various monophasic calcium phosphate materials, HA is the most stable and the least soluble phase [29,30]. It has a Ksp value of 2.9  1058 over a pH range of w3.5ew9.7 [31]. The primary advantages of HA implants are its mechanical stability, good osteoconductive and moderate osteoinductive abilities. However, for a faster osteogenic process, HA in combination with a more soluble b-TCP phase, known as BCP bioceramics, is commonly used in clinical bone repair. It was discovered that the osteoinduction of porous BCP bioceramics is strongly associated with its ability to develop bonelike apatite on the surface [32,33]. Further, the abundance of the bonelike apatite appears to be inversely correlated with the HA/b-TCP ratio of ceramics. Greater amounts of apatite are measured with lower ratios. It was proposed that the formation of bonelike apatite results from the partial dissolution of bioceramics to reach a supersaturation of the calcium and phosphate ions in the porous microenvironment. The reprecipitation of these ions incorporates the carbonate, then magnesium ions were presented in the biological fluid, and thus bonelike apatite is formed. In the aspect of biological degradation, specific acid-secreting cell types like osteoclast or macrophage would resorb the implant surface and free the ions on site.

1.4.2.1.1 Ca2þ gradient Calcium ion gradient is observed in extracellular microenvironments and recognized as one of the most prevalent chemical cues for cell differentiation and migration [34,35]. It is known to be a strong homing signal which recruits various types of cells to initiate bone formation, for instance, bone marrow progenitor cells or preosteoblast [34,36e39]. A sustained high concentration of Ca2þ is also able to stimulate the maturation of these cells into boneforming cells [38,39]. It is suspected that the calcium ions, being dynamically released or resorbed at the surface of a biomaterial, can recruit

Material characteristics, surface/interface Chapter | 1

83

mesenchymal stem cells (MSCs) chemotactically and promote their osteogenic differentiation in a concentration dependent way [40]. In addition, protein adsorption can be selectively facilitated by the ionic environment to promote osteogenesis. For example, our group previously discovered that bone morphogenetic protein 2 (BMP-2) can be adsorbed onto the (110) surface of HA in priority [41]. Furthermore, we found that the dynamically balanced ion concentration created in the porous bioceramics, which is greatly dependent on its solubility and phase composition, can affect protein adsorption behavior [42]. For example, the fibrinogen, type I collagen (Col-1), insulin proteins are prone to be adsorbed on the surface of BCP rather than HA bioceramics [42,43]. It is speculated that a more soluble b-TCP phase accounts for an increase of local ion concentrations and thus fastens the formation of bonelike apatite incorporated with various proteins in the body fluid [40]. Klar et al. in 2013 had correlated calcium ion concentration with BMP-2 expression and bone formation [44]. They suggested that the ionic environment is dependent on the chemical and physical feature of the porous structure. Another study, by Syed-Picard et al., showed that connexin 43 protein, which mediates gap junctions between cells, can be modulated by calcium ion concentration [45]. The consequent bone-forming cell functions and 3D special tissue differentiation pattern would be affected accordingly. To integrate extracellular calcium into cytoplasm, responses from variety of calcium channels/pump were observed. Jung et al. conducted a series of studies to study the bone forming ability of the osteoblastic MC3T3-E1 cells upon HA scaffold released Ca2þ ions [46]. An activated CaMK2a/CAM pathway was found when Ca2þ ions being transported inside of the cells via calcium channels and calcium sensing receptors (CaSR). Finally, the cAMPresponse element binding protein (CREB) promotor and/or extracellular signal-regulated kinase 1/2 (ERK1/2) pathway will be activated to upregulate osteoblastic differentiation [47]. Barradas et al. (2012) further revealed that the proliferation and morphological spreading pattern of MSCs would be positively regulated by high extracellular calcium level offered by calcium phosphate-based biomaterials [48]. Moreover, the process is accompanied with upregulated BMP-2 gene and protein expression. In their study, however, it is type L voltage-gated calcium channels rather than CaSR that played a role in mediating the extracellular calcium sensing and transportation. Moreover, the microarray analysis showed that MEK1/2 activity was essential. It is proposed that, the internalization of calcium ion, mainly by calcium channel, would activate PKC, MEK1/2 and ERK1/2 pathways in sequence. Finally, it entered the nucleus to positively regulate BMP-2 expression by activator protein 1 and Fos expression. This study was in accordance with another in vivo study that demonstrated that blockage of the calcium channel downregulates BMP-2 expression and results in less bone formation [45].

84 Bioactive Materials for Bone Regeneration

1.4.2.1.2 PO3 4 internalization In the recent decade, the role of PO3 4 ion concentration in osteogenesis gains growing attention. Firstly, PO3 4 is a participant of mineralization and maturation of bone matrix [34,49]. Beck et al. had reported upregulated mineralization-associated genes induced by higher PO3 4 concentration [50]. Later, they have confirmed that it is the activation of ERK1/2 and PKC pathways, not p38 pathway, that is crucial to the promoted matrix gla protein (MGP) and osteopontin (OPN) expressions stimulated by the material-released PO3 4 [51]. A study done by Khoshiat et al. had taken one step further to prove that the involvement of Ca2þ ions is indispensable in the activation of PO3 4 dependent ERK1/2 pathway to manipulate the MGP/OPN expression during osteogenesis [52]. They have also suggested that this bone-forming effect induced by Ca-P based material originated extracellularly and ended in cell nucleus as gene regulatory signal. During the transmembrane signal transduction, the integrity of the lipid rafts is required. Shih et al. (2014) have conducted a series of in vitro study using Ca-P bearing matrices to reveal that the released PO3 4 is translocated into the cell plasma by solute carrier family 20 member 1 (SLC20a1), a phosphate transporter on the cytoplasmic membrane [53]. Later, the internalized PO3 4 ions turn into activated Pi in the mitochondria compartment and turned adenosine diphosphate (ADP) into adenosine triphosphate (ATP), the basic energy source for cellular response. This autocrine and/or paracrine signal will promote the osteogenic differentiation of MSCs to osteoblasts. A perturbation of osteoblastic differentiation is observed when the aforementioned SLC20a1 transporter is blocked, resulting in undermined intramitochondrial Pi and ATP synthesis [53]. Together, it seems that the released Ca2þ or PO3 4 may activate different pathways and arose diverse cellular responses in the way of material induced osteogenesis. Whether Ca2þ or PO3 4 work alone or in combination would be required during osteogenic differentiation of bone-forming progenitor cells is controversial. However, it is certain that two are better than one in reprecipitation of bonelike apatite on the material surface [52]. In the combination, the osteogenesis might be enhanced via different signal pathways that one individual ion cannot achieve. 1.4.2.2 Cells of origin and cellular events in material-induced osteogenesis 1.4.2.2.1 Cells of origin At the beginning of de novo bone formation, fibrous connective tissue and microsized capillaries were observed in the inner pores of Ca-P ceramics [54]. Later, ALP positive-stained polymorphic cells were found to be aggregated adjacent to the ceramic surface associated with the microcapillaries [55]. Ripamonti et al. (1993) did laminin staining for vascular endothelial cells

Material characteristics, surface/interface Chapter | 1

85

using the HA samples being implanted extraskeletally to the baboons [56]. They also observed that the positive staining was localized in the vicinity of ceramic pores and around capillaries as well as the surrounding of the single cells that were migrating out of the vascular compartment. It is for long being presumed by our research group (2000) that the connective tissue and capillaries that invaded into the pores of Ca-P material did supply bone-forming progenitor cells for further differentiation with the appropriate ionic environment [57]. With the development of new technology, in a recent publication (2013), Song and his colleague used a sex-mismatched beagle dog model to study the origin of MSCs in the ectopic bone formation induced by BCP granules [58]. Bone marrow MSCs of male dogs were injected to female bone marrow cavity. At week 4 after the material implantation, cells with Y chromosomes were distributed in new bone matrix throughout the granules. At week 6, cells with Y chromosomes were still presented in mineralized bone adjacent to the pore surface of the granules. It is confirmed that MSCs can be recruited from bone marrow and homed to the ectopic site via blood circulation to serve as a cell origin for Ca-P-induced bone formation. Besides homing of MSCs, another prevalent hypothesis suggests that the endothelial cells or pericytes originate from the invaded capillary is able to differentiate into bone-forming progenitor cells. It is supported by the known fact that Ca-P induced osteogenesis undergoes intramembranous ossification [32]. Endothelial cells compose in inner lining of blood vessels and lymphatic vessels, which are similar to the epithelial cells. Like epithelial cell is capable of epithelial-mesenchymal transition (EMT) during would healing, endothelial cells can also undergo endothelial-mesenchymal transition (EndMT) in host emergency [18]. It is worth noticing that Medici et al. discovered that the vascular endothelial cells dedifferentiated into MSCs-liked progenitor cells, and then differentiated into chondrocytes and osteoblasts, under pathological heterotopic ossification condition [22]. This EndMT process was activated by intense inflammation or suspected to be regulated by other undefined changes in the microenvironment. In prevalent opinion, this process might be documented as a physiological reprogramming of the differentiated cells. Furthermore, an emerging role of myoendothelial stem cells was identified [59,60]. It is a special population of stem cells with both myoblastic and endothelial features, found in straited muscular tissue to offer continuous cell differentiation, proliferation, and replacement. Ripamonti et al. proposed that these specific population of stem cells can be a possible cell source to differentiate into bone-forming cells when encounter with bone graft materials [61]. Another potential cell of origin, pericytes, are subendothelial cells that ubiquitously exist in all-sized vessels ranging from microvasculature to aorta [59]. They are defined as regulator of the endothelial cells during its budding, proliferation, and differentiation [62]. Pericytes are also capable to transit into other cell types due to its stemlike nature. Most importantly, pericytes can secrete ALP and being suggested as a source of osteoblasts in periosteal

86 Bioactive Materials for Bone Regeneration

osteogenesis [63e65]. It is proposed by Barradas et al. that pericytes might have contributed to osteogenesis induced by porous bioactive materials [32].

1.4.2.2.2 Events at cellular level During the material-induced osteogenesis, bone-forming-associated genes and proteins are activated in sequence. One of our group’s recent publications (2017) compared the ectopic bone formation process at gene, protein, and tissue levels induced by varied phase compositions of Ca-P ceramics [66]. Instead of a steady increase, multiple peaks were observed in most of the temporal gene expression pattern (Fig. 1.18). The gene expression results were further confirmed by immunohistochemical staining of the corresponding proteins. Osterix (OSX) and Col-1 were upregulated successively. Higher peak values of BMP-2, bone morphogenetic protein receptor type IA (BMPR1A), and osteoprotegrin (OPG) expressions were found in the osteoinductive BCP group than in the nonosteoinductive groups. We concluded that the occurrence time and magnitude of the bone-forming-related gene expression peaks are critical during osteogenesis. Certain genes and proteins are considered markers of osteogenesis. These markers are BMP-2, BMP-4, BMP-7, Runx2/Cbfa1,

FIGURE 1.18 Expressions of osterix, osteoprotegerin, type I collagen, and BMP2 genes in cells grown into the inner pores of porous Ca-P bioceramics after implantation into the thigh muscles of mice for 1, 2, 3, 4, 8, 12, and 16 weeks; BCP1 denotes bioceramics with an HA to b-TCP ratio of 70/30; BCP2 denotes bioceramics with an HA to b-TCP ratio of 30/70.

Material characteristics, surface/interface Chapter | 1

87

osterix, Col-1, OPN, OCN, BSP, and ALP, listed in the figure below. Sometimes these markers were employed to predict the osteogenic ability of materials. For instance, human-bone-marrow-derived MSCs had significantly higher Col-1 and OCN gene expressions when cocultured with Ca-P bioceramics compared with tissue culture plastic control [67]. In a gene microarray study conducted by Xu et al. (2015), they have thoroughly investigated the gene expression profiling of human mesenchymal stem cells (hMSCs) cultured on mineralized collagen [68]. Their results indicated that mineralized collagen would promote gene expressions of BMP-2, Col-1, and cathepsin K precursor compared with nonosteoinductive high-temperature sintered HA control. The BMP family comprises potent osteogenic cytokines that have been researched extensively and applied clinically. The most studied, BMP-2, is discovered in bone and cartilage development and the healing process. With regard to bioactive material-induced osteogenesis, upregulated BMP-2 was first seen with decalcified bone matrix (DBM) and later observed by Ca-P ceramics intramuscular implantation [69e71]. Gene expression of BMP-4 was also found to be activated in vivo by BCP ceramic 3 days after implantation [72]. Moreover, Eyckmans et al. had confirmed the activation of BMP-2, BMP-4 and BMP-6 genes in BCP granules loaded with human periosteumderived cells (hPDCs) [70]. Previous work of our group (2014) and other groups had revealed that a higher BMP-2, BMP-4, and OCN expressions induced by the material were correlative with more bone formation in vivo [73e75]. Besides BMPs, other markers like Col-1 and ALP gene expressions were found to be elevate from day 3 to week 24 after intramuscular BCP implantation in beagle dogs [72]. OPN, OCN and osteonectin (ON) were detected in either HA, b-TCP or BCP group at 16 weeks after implantation in rat muscle, as observed by immunobiological staining [76]. Together, the aforementioned gene/protein markers offered fundamental evidence and cellular-level mechanism for the material-induced osteogenesis. There is similarity and difference of the markers activated during natural and materialinduced osteogenesis. A more comprehensive understanding of the molecularlevel mechanism, i.e., the pathways involved, will be presented in the next section. Cell adhesion on to the substrate material is essential in determining cell fates and activities. It is well known that cells adhere to substrate surface via integrins, a family of cell-surface transmembrane receptors, which consist of a and b subunits as a heterodimeric glycoprotein. The extracellular parts of integrin can selectively bind to ligands that are mainly adsorbed proteins on material surface, i.e., fibronectin and vitronectin [75,77]. These proteins are abundant in blood and situated on material surface immediately thus they provide a provisional matrix to support cell adhesion event. Together with integrin extracellular domain, these proteins cluster into focal adhesion plaque [78,79]. With focal contact, the intracellular domain of integrins can sense the

88 Bioactive Materials for Bone Regeneration

adhesion and arose a signal cascade inside of the cells. These “outside-in signal” are related to a series of biomolecular events begins with reorganization of actin-based cytoskeleton, and then the focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), BMP/Smad protein kinase C (PKC) pathways [80], etc. Matsuura et al. (2000) reported that the RGD domains of vitronectin and fibronectin on Ca-P and titanium surface is important for binding and spreading of the osteoblasts and suggested that it is positively correlated with osteoconductivity of the material [81]. Later, Kilpadi and his coworkers further confirmed that as compared with titanium and stainless steel, the osteogenic HA ceramic could adsorb more vitronectin and fibronectin [82]. The specific binding of integrin a5b3 and a5b1 was increased significantly with HA ceramics. There were more osteoblast precursor cells attaching to and proliferating on its surface as a result. FAK is the most direct intracellular pathway related to integrin-mediated binding. Early in 2007, a study done by Salsznyk et al. confirmed the FAK signaling pathway can regulate the osteogenic differentiation of hMSCs [83]. The study was conducted with hMSCs cocultured with purified Col-1 and vitronection coated surface, in presence or absence of FAK targeted siRNA. They found that the siRNA treatment could decrease several osteogenic markers, i.e., Runx2/Cbfa-1, osterix, ALP and a reduced calcium phosphate deposition and lower mineral: matrix ratio. Later, Marino et al. (2000) who cocultured human adipose-derived stem cells with b-TCP ceramic demonstrated a phosphorylated FAK and the resultant osteogenic differentiation [84]. Besides FAK signaling pathway, Lu et al. discovered that the osteoconductivity can be regulated by integrin a2b1 integrin mediated MAPK signaling pathway [85]. Blocking of integrin a2b1 or inhibition of MAPK pathway attenuated gene expression of BMP2 and its receptors and aborted the activation of Smad1/5-signaling pathway in human osteoblasts cultured on b-TCP ceramics. Liu et al. demonstrated the activating of the integrin-BMP/Smad signaling pathway with MSCs plated on nanohydroxyapatite-bearing chitosan scaffold [86]. Phosphorylated Smad1/5/8 in BMP pathway showed evident nuclear localization. Osteogenic markers like BMP-2/4, Runx2, ALP, OCN, collagen I and integrin subunits were significantly elevated in nanohydroxyapatite-cultured group. Recently, our research group (2016) further confirmed that porous BCP ceramics could activate MAPK signaling pathways (Figs. 1.19 and 1.20). Moreover, blockage of down-stream signals of MAPK, either ERK1/2 or P38, could dramatically attenuate BCP-induced osteogenesis [87].

1.4.2.3 Osteogenic mechanism of bioactive porous titanium Not until Fujibayashi et al. in 2004 discovered that porous titanium metal with a titanium oxide surface layer was osteoinductive, it was still uncertain

Material characteristics, surface/interface Chapter | 1

89

FIGURE 1.19 schematic illustration of the transduction of “outside-in signaling” mediated by integrins [87].

FIGURE 1.20 Osteoblastic differentiation of MSCs and markers for different stages.

whether porous metal biomaterials could possess osteoinductivity [88,89]. Since then, various kinds of surface modified porous titanium and its alloy were reported to be osteoinductive [90e94]. However, the mechanism of osteogenesis induced by porous titanium-based material is still controversial. One point of view suggested the bonelike apatite formation on their porous surface were essential for metallic biomaterials to form binding with surrounding living bone [89]. As mentioned in the previous section, bonelike apatite formation on surface of bioceramics is essential for them to achieve osteoinduction. Similarly, the ability to retain a bio-apatite layer on surface of

90 Bioactive Materials for Bone Regeneration

titanium-based porous biomaterials may also be a critical step for them to foster osteoinductivity. Previous studies have showed that calcium and phosphate ions have affinity to the titanium surface [95,96], which will be beneficial for the nucleation and precipitation of bonelike apatite layer on the surface [89]. This assumption was widely accepted and certain kinds of surface treatments are being developed to grant the surface of titanium ability to maintain more bonelike apatite layer, tested in simulated body fluid (SBF). By this approach, osteogenesis was greatly improved in vivo for these materials [90e94,97]. Furthermore, protein adsorption may also be one of the possible mechanisms for the osteoinduction of porous titanium-based materials. It was demonstrated the surface microstructured porous titanium could bind more protein than the unmodified one [98]. Besides apatite formation and protein adsorption, the direct influence of the titanium surface on stem cells or boneforming related cells might also be one reason to promote its osteoinduction. Yang et al. reported that surface microstructured porous titanium, modified with H2O2/TaCl5, could enhance the adhesion and osteoblastic differentiation of MSCs [98]. Kim et al. demonstrated that the SLA acid-etched titanium surfaces increased the mRNA expression of the Wnt3a, b-catenin and integrin a2b1 in periodontal ligament stem cells, which were associated with the higher mRNA expression of Runx2, osterix, FosB and Fra1 as well as the ALP activity [99]. Together, several signal pathways, including BMPs, TGF-b, Wnt, ERK1/2, Notch signaling, were reported to be involved in the osteogenesis of various surface-modified titanium or its alloys, using osteoblasts, boneforming precursor cells or stem cells [100e107]. So far, it seems that the molecular mechanisms for the osteogenesis of porous titanium-based materials were similar to that of the porous calcium phosphate ceramic.

1.4.3 Role of immunoresponse in the osteogenesis of bioactive materials The contribution of protein adsorption and ion accumulation inside of the porous structure to material osteogenic ability was extensively studied and discussed above. With the development of biotechnology, new mechanisms of material induced osteogenesis have been proposed. Cell-material interaction and cellecell cross-talk signal pathways have been prolific in the past decade. It is now generally agreed that not only gathering of factors from body fluid, but also a direct stimulation from bioactive material to trigger cell osteogenic differentiation leads to efficient bone formation. In this section, several prevalent pathways were acknowledged and documented below (Fig. 1.21).

1.4.3.1 Autocrine effect of mesenchymal stem cells Classic BMP-initiated osteogenic differentiation was mentioned previously. Although upregulated BMP-2/4 expressions induced by bioactive materials

Material characteristics, surface/interface Chapter | 1

91

FIGURE 1.21 Cells and signals related to bone building and repair: ①osteoblastic differentiation in endochondral ossification; ②osteoblastic differentiation in intramembranous ossification, and ③ osteoclast differentiation of monocytes in hematopoietic stem cells.

were widely confirmed, it is still unclear where the BMPs mainly came from. Eyckmans et al. (2010) utilized hPDCs seeded in a Collagraft scaffold and explored the mechanisms by which the osteogenesis is driven [70]. They found inhibition of endogenous BMP and Wnt signaling by antagonists Noggin and Frzb would abrogate osteoinduction. Intracellular BMP/Wnt signaling, including Smad 1/5/8 expression, is required for Ca-P material-induced osteogenesis. To further exclude exogenous cells and cytokines in the physiological environment, we did an in vitro experiment (2015) using nonosteogenic culture media to host MSCs on HA or BCP bioceramics [73]. These two bioceramics are proven to be osteogenic in previous work, and the BCP (HA: b-TCP ¼ 6:4%, 60% porosity) used had a higher osteogenic ability than the HA bioceramic [14]. Our results indicated that both Ca-P materials were able to induce osteogenic differentiation of MSCs via BMP-2 autocrine secretion, especially intense in the BCP group. Furthermore, Smad 1/4/5 and Dlx5, the main molecule signals on the BMP/Smad pathway were upregulated higher in the BCP than in the HA group. Since the stimulation of BMP-2 protein can upregulate other BMPs’ expression via autocrine pathway [69,108], we suspect that there might be BMPs amplification cascade induce by the bioceramics. Our conclusion was in line with Barradas’s in vitro study using HA and b-TCP bioceramics [74]. However, in the complicated in vivo environment, there are numerical other factors involved which may shield the autocrine effect. For example, one in vivo study using multiple inhibitors showed that the blockage of osteoclastogenesis or calcium channel would decrease BMP-2 secretion and bone formation [44]. Other than BMPs, immune cytokine might have also worked in an autocrine way in osteogenesis triggered by bioactive materials. Gene microarray

92 Bioactive Materials for Bone Regeneration

analysis done by Barradas et al. showed that G-protein coupled receptor (GPCR) 5A and regulator of G-protein signaling two in MSCs were strongly elevated by b-TCP bioceramic, more than 100 folds compared with that of the HA bioceramic cocultured group [74]. The earlies time point in their study was 12 h postcoculture. Therefore, the GPCR signaling pathway associated genes may herald the earliest response of MSCs to bioactive ceramics. Liu et al. in 2015 demonstrated that the composite scaffolds of HA and silk fibroin enhance MSCs mediated bone regeneration via the interleukin 1 a (IL-1a) autocrine/ paracrine signaling loop [109]. Their cDNA microarray results identified 247 differentially expressed genes in MSCs cultured on nanohydroxyapatite bearing scaffolds compared with the control. The greatest disparity in gene expression levels were observed with Il1a and Ilr2 (Interleukin 1 receptor type II). The results were validated by PCR. Furthermore, the addition of IL-1a into cultures of MSCs significantly increased both BMP-2 and Ilr2 expression. However, with MSCs alone, the Il1r2 expression increased substantially, whereas BMP-2 expression exhibited a decrease rather than increase. These data suggested that nanohydroxyapatite may exert osteoinductive effects on MSCs via the secretion of IL-1a in an autocrine/paracrine loop, and IL-1a activity could be regulated through the synthesis of IL1R2 by MSCs upon interaction with nanohydroxyapatite. Other autocrine effect includes adenosine-signaling pathway. As mentioned previously, it was shown by Shih et al. (2014), the phosphate released from Ca-P bioceramic participated in ADP/ATP synthetic pathway taken place in mitochondria [53]. Other suspected genes which might participated in MSCs autocrine loop in osteogenesis include but not limited to the transforming growth factor b (TGF-b), b-catenin, epidermal growth factor (EGF), tumor necrosis factor alpha (TNF-a), nuclear factor-kappa B (NF-kB) and Interleukin 6 (IL-6).

1.4.3.2 Paracrine effect from immune cells Immune reactions play important roles in determining the in vivo fate of bone substitute materials. As exogenous substance, bone substitute tends to be isolated or rejected by the human body. To avoid foreign body reactions, inert materials have been adopted for implantation for centuries. However, this approach not only limited the material’s bone regeneration but also sometimes resulted in fibrous tissue encapsulation [110]. The positive role of immune cells in bone healing was recognized with the recent development of osteoimmunology. Miramond et al. in 2014 designed experiments with nude mouse model to study the osteoinductivity of BCP ceramic under immunedeficient condition [111]. They found in nude mouse BCP alone has low, but nonzero, osteoinductive properties, while addition of human total bone marrow can largely improve osteoinduction. The immune and skeletal systems are closely related, sharing numerous cytokines, signaling molecules,

Material characteristics, surface/interface Chapter | 1

93

receptors, and transcription factors [112]. This knowledge led to a shift of research interest from inert to bioactive bone substitute, which is capable of immunomodulation in a positive way [113]. Thus, in vitro methods previously employed to assess bone implant materials based on the interactions between bone-forming cells and materials were insufficient. Chen et al. suggested that ignoring the importance of immune response of the host might lead to the discrepancies between in vitro and in vivo experiments that are often observed [114e116]. As the in vivo single-cell typed coculturing with material does not fully reflect the in vivo condition, which involves early immunomodulation upon transplantation. It is therefore of great importance to investigate the effects of immune cells on biomaterial-mediated osteogenesis. Firstly, it was observed that consistently sufficient Ca2þ concentration would cause circulating monocytes to fuse into macrophages, which is one of the most important effector cells that determines the outcome of long-term inflammatory reaction to materials [117]. Furthermore, under certain physiochemical stimulations from implant material, macrophages can promote the osteogenic differentiation of the MSCs in the environment [118,119]. De Bruijn et al. demonstrated that macrophages in response to the rough surfaced HA would secreted more prostaglandin E2 (PGE2), which is involved in adhesion behavior of osteoblasts, than to the smooth surfaced HA. The presence of macrophages is not merely indispensable for osteogenesis, furthermore, their heterogeneity and plasticity are of great significance for immune system modulation [120]. In a study conducted by Chen et al. (2014), macrophage secreted BMP-2 was significantly upregulated upon b-TCP stimulation [121]. Furthermore, b-TCP extracts were observed to promote macrophage phenotype transition to M2 extreme, with the activation of CaSR pathway. It is worth noticing that when macrophage conditioned b-TCP extracts were applied to MSCs, the osteogenic differentiation of MSCs was significantly enhanced, unveiling the important role of macrophages in biomaterial-induced osteogenesis. Therefore, a strategy to harness the power of macrophages/monocytes for enhanced bone tissue engineering has been proposed [122]. Osteotropic factors like IL-1b AND IL-4 secreted by macrophage/monocytes are believed to be beneficial in bone regeneration and the healing process. In addition to being bulk material, particles degraded from ceramics can also influence the cellular behavior of the macrophage, making it produce cytokines such as IL-6, IL-10, and TNF-a [123e125]. One of our recent works (2017) examined bulk BCP ceramic and its degradation products’ influences on the osteogenic differentiation and migration of MSCs (Fig. 1.22) [119]. The results of in vitro experiments confirmed that the expression of inflammatory factors IL-1, IL-6, and macrophage chemotactic protein 1 (MCP-1) as well as growth factors vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and EGF in macrophages were upregulated to varying degrees by BCP ceramic and its degradation products. Cell migration assay showed that the conditioned media secreted by macrophages induced by either

94 Bioactive Materials for Bone Regeneration

(A)

(B1)

(B2)

(C)

(D1)

(D2)

FIGURE 1.22 Upper: ELISA analysis for inflammatory cytokines secreted by mouse RAW 264.7 macrophages into culture media after 3 days of coculture with tissue culture plate (control), BCP, BCP-conditioned media (BCM), and BCP-degraded particles (BPs), respectively; Lower: SEM observation for the morphology and spreading of mouse RAW 264.7 macrophages cultured with BCP ceramic and its degradation products. (A) control group; (B1 and B2) BCP group with 2000 and 10,000 magnification; (C) BCM group; (D1 and D2) BP group with 2000 and 10,000 magnification [92].

BCP ceramic or its degradation products would promote the migration of MSCs. In sum, the ceramic and its degradation products can stimulate macrophages to express and secrete various signaling molecules that in turn recruit MSCs and promote osteogenesis (Fig. 1.23). Compared with BCP-conditioned

Material characteristics, surface/interface Chapter | 1

95

FIGURE 1.23 Schematic illustration of the involvement of paracrine-signaling factors secreted by macrophages in BCP-stimulated osteogenic differentiation of preosteoblasts [91].

media, degradation particles (5e30 mm) played a more critical role in this process (Fig. 1.22). Other than macrophages and monocytes, other immune cells, such as T cells and B cells, may also participate in the osteogenesis process [32,114]. Neuron-specific enolase (Nse) promoter (Nse-BMP4) mice, which lack mature B and T cells, showed smaller spreading and overall heterotopic ossification [126].

1.4.4 Summary In the past decade, our ability to describe biological processes accurately has been greatly improved with advances in cellular and molecular techniques. It is essential for researchers to investigate the biological response induced by scaffolds, since both the general and detailed conditions of bone repair are reflected as osteogenic activity. Osteoinduction implies the initial recruitment of immature cells and the stimulation of these cells to differentiate into preosteoblasts. The different material factors influencing osteoinductivity and the mechanisms behind this phenomenon are presented in this chapter. Interestingly, we have shown that some osteogenic agents including TGF-b, BMPs, Wnt, and other growth factors via related signaling pathways are fundamentally important throughout the material-induced osteoinduction process. With osteoinductivity, bone grafts are able to achieve better bone regeneration in vivo, even in a critical-sized defect, without the addition of cells or growth factors. As an ideal bone substitute, Ca-P with osteoinductive ability ensures sufficient bony tissue forms and grows on the surface of the scaffold. Another significant property, the biodegradation of Ca-P, refers to various fields that are so complex that researchers have not yet come to an agreement on the mechanism. With the development of in vivo labeling and tracking biotechnology and enhanced resolution of CT-based assessment, biodegradationassociated cellular events might be further elucidated.

96 Bioactive Materials for Bone Regeneration

References [1] H. Yuan, K. Kurashina, J.D. de Bruijn, Y. Li, K. de Groot, X. Zhang, A preliminary study on osteoinduction of two kinds of calcium phosphate ceramics, Biomaterials 20 (19) (1999) 1799e1806. [2] H. Yuan, J. De Bruijn, Y. Li, J. Feng, Z. Yang, K. De Groot, X. Zhang, Bone formation induced by calcium phosphate ceramics in soft tissue of dogs: a comparative study between porous a-TCP and b-TCP, J. Mater. Sci. Mater. Med. 12 (1) (2001) 7e13. [3] H. Yuan, Y. Li, J. De Bruijn, K. De Groot, X. Zhang, Tissue responses of calcium phosphate cement: a study in dogs, Biomaterials 21 (12) (2000) 1283e1290. [4] U. Ripamonti, J. Crooks, L. Khoali, L. Roden, The induction of bone formation by coralderived calcium carbonate/hydroxyapatite constructs, Biomaterials 30 (7) (2009) 1428e1439. [5] S.B. Klopcic, J. Kovac, T. Kosmac, Apatite-forming ability of alumina and zirconia ceramics in a supersaturated Ca/P solution, Biomol. Eng. 24 (5) (2007) 467e471. [6] H. Yuan, J.D. Bruijn, De, X. Zhang, C.A. Blitterswijk, K. Van, De Groot, Bone induction by porous glass ceramic made from Bioglass (45S5), J. Biomed. Mater. Res. B Appl. Biomater. 58 (3) (2001) 270e276. [7] G.D. Winter, B.J. Simpson, Heterotopic bone formed in a synthetic sponge in the skin of young pigs, Nature 223 (5201) (1969) 88e90. [8] J.M. Kanczler, R.O. Oreffo, Osteogenesis and angiogenesis: the potential for engineering bone, Eur. Cells Mater. 15 (1) (2008) 100e114. [9] M.R. Hausman, M.B. Schaffler, R.J. Majeska, Prevention of fracture healing in rats by an inhibitor of angiogenesis, Bone 29 (6) (2001) 560e564. [10] H. Li, J. Chang, Stimulation of proangiogenesis by calcium silicate bioactive ceramic, Acta Biomater. 9 (2) (2013) 5379e5389. [11] H. Li, J. Chang, Bioactive silicate materials stimulate angiogenesis in fibroblast and endothelial cell co-culture system through paracrine effect, Acta Biomater. 9 (6) (2013) 6981e6991. [12] W. Zhai, H. Lu, L. Chen, X. Lin, Y. Huang, K. Dai, K. Naoki, G. Chen, J. Chang, Silicate bioceramics induce angiogenesis during bone regeneration, Acta Biomater. 8 (1) (2012) 341e349. [13] A.A. Gorustovich, J.A. Roether, A.R. Boccaccini, Effect of bioactive glasses on angiogenesis: a review of in vitro and in vivo evidences, Tissue Eng. B Rev. 16 (2) (2010) 199e207. [14] H. Yuan, C. Van Blitterswijk, K. De Groot, J. De Bruijn, A comparison of bone formation in biphasic calcium phosphate (BCP) and hydroxyapatite (HA) implanted in muscle and bone of dogs at different time periods, J. Biomed. Mater. Res. A 78 (1) (2006) 139e147. [15] C.S. Chang, C.Y. Su, T.C. Lin, Scanning electron microscopy observation of vascularization around hydroxyapatite using vascular corrosion casts, J. Biomed. Mater. Res. 48 (4) (2015) 411e416. [16] M. Ru¨cker, M.W. Laschke, D. Junker, C. Carvalho, F. Tavassol, R. Mu¨lhaupt, N.C. Gellrich, M.D. Menger, Vascularization and biocompatibility of scaffolds consisting of different calcium phosphate compounds, J. Biomed. Mater. Res. A 86A (4) (2010) 1002e1011. [17] C. Zhang, P. Huang, J. Weng, W. Zhi, Y. Hu, H. Feng, Y. Yao, S. Li, T. Xia, Histomorphological researches on large porous hydroxyapatite cylinder tubes with polylactic

Material characteristics, surface/interface Chapter | 1

[18] [19] [20]

[21]

[22] [23]

[24]

[25] [26] [27] [28] [29] [30]

[31] [32]

[33] [34]

[35]

[36]

97

acid surface coating in different nonskeletal sites in vivo, J. Biomed. Mater. Res. A 100A (5) (2012) 1203e1208. D. Medici, R. Kalluri, Endothelial-mesenchymal transition and its contribution to the emergence of stem cell phenotype, Semin. Cancer Biol. 22 (5e6) (2012) 379e384. S. Potenta, E. Zeisberg, R. Kalluri, The role of endothelial-to-mesenchymal transition in cancer progression, Br. J. Canc. 99 (9) (2008) 1375e1379. M. Crisan, S. Yap, L. Casteilla, C.-W. Chen, M. Corselli, T.S. Park, G. Andriolo, B. Sun, B. Zheng, L. Zhang, A perivascular origin for mesenchymal stem cells in multiple human organs, Cell stem cell 3 (3) (2008) 301e313. V.A. de Jesus Perez, T.-P. Alastalo, J.C. Wu, J.D. Axelrod, J.P. Cooke, M. Amieva, M. Rabinovitch, Bone morphogenetic protein 2 induces pulmonary angiogenesis via Wnteb-catenin and WnteRhoAeRac1 pathways, J. Cell Biol. 184 (1) (2009) 83e99. D. Medici, B.R. Olsen, The role of endothelial-mesenchymal transition in heterotopic ossification, J. Bone Miner. Res. 27 (8) (2012) 1619e1622. Y. Chen, J. Wang, X.D. Zhu, Z.R. Tang, X. Yang, Y.F. Tan, Y.J. Fan, X.D. Zhang, Enhanced effect of b-tricalcium phosphate phase on neovascularization of porous calcium phosphate ceramics: in vitro and in vivo evidence, Acta Biomater. (2015). ´ . Castan˜o Linares, J.A. Planell Estany, E. Engel A. Aguirre, A. Gonzalez, M. Navarro, O Lo´pez, Control of Microenvironmental Cues with a Smart Biomaterial Composite Promotes Endothelial Progenitor Cell Angiogenesis, 2012. E.C. Kohn, R. Alessandro, J. Spoonster, R.P. Wersto, L.A. Liotta, Angiogenesis: role of calcium-mediated signal transduction, Proc. Natl. Acad. Sci. 92 (5) (1995) 1307e1311. L. Munaron, Intracellular calcium, endothelial cells and angiogenesis, Recent Pat. Anticancer Drug Discov. 1 (1) (2006) 105e119. U. Ripamonti, L.C. Roden, C. Ferretti, R.M. Klar, Biomimetic matrices self-initiating the induction of bone formation, J. Craniofac. Surg. 22 (5) (2011) 1859e1870. J. Krebs, M. Michalak, Calcium : a matter of life or death, Ann. Dermatol. Venereol. 136 (2) (2007) 125e132. P. Ducheyne, S. Radin, L. King, The effect of calcium phosphate ceramic composition and structure on in vitro behavior. I. Dissolution, J. Biomed. Mater. Res. 27 (1) (1993) 25. C.P. Klein, J.M. de Blieck-Hogervorst, J.G. Wolke, G.K. De, Studies of the solubility of different calcium phosphate ceramic particles in vitro, Biomaterials 11 (7) (1990) 509e512. L.C. Bell, H. Mika, B.J. Kruger, Synthetic hydroxyapatite-solubility product and stoichiometry of dissolution, Arch. Oral Biol. 23 (5) (1978) 329e336. A.M.C. Barradas, H.P. Yuan, C.A. van Blitterswijk, P. Habibovic, Osteoinductive biomaterials: current knowledge of properties, experimental models and biological mechanisms, Eur. Cells Mater. 21 (2011) 407e429. J.M. Bouler, P. Pilet, O. Gauthier, E. Verron, Biphasic calcium phosphate ceramics for bone reconstruction: a review of biological response, Acta Biomater. 53 (15) (2017) 1e12. Y.C. Chai, A. Carlier, J. Bolander, S.J. Roberts, L. Geris, J. Schrooten, H. Van Oosterwyck, F.P. Luyten, Current views on calcium phosphate osteogenicity and the translation into effective bone regeneration strategies, Acta Biomater. 8 (11) (2012) 3876e3887. T. Lobovkina, I. Go¨zen, Y. Erkan, J. Olofsson, S.G. Weber, O. Orwar, Protrusive growth and periodic contractile motion in surface-adhered vesicles induced by Ca2þ-gradients, Soft Matter 6 (2) (2010) 268e272. A. Aguirre, A. Gonza´lez, J. Planell, E. Engel, Extracellular calcium modulates in vitro bone marrow-derived Flk-1þ CD34þ progenitor cell chemotaxis and differentiation

98 Bioactive Materials for Bone Regeneration

[37] [38] [39] [40]

[41]

[42]

[43]

[44]

[45] [46] [47]

[48]

[49]

[50] [51] [52]

[53]

through a calcium-sensing receptor, Biochem. Biophys. Res. Commun. 393 (1) (2010) 156e161. G.E. Breitwieser, Extracellular calcium as an integrator of tissue function, Int. J. Biochem. Cell Biol. 40 (8) (2008) 1467e1480. M.M. Dvorak, D. Riccardi, Ca2þ as an extracellular signal in bone, Cell Calcium 35 (3) (2004) 249e255. A. Gonzalez-Vazquez, J.A. Planell, E. Engel, Extracellular calcium and CaSR drive osteoinduction in mesenchymal stromal cells, Acta Biomater. 10 (6) (2014) 2824e2833. Z. Tang, X. Li, Y. Tan, H. Fan, X. Zhang, The material and biological characteristics of osteoinductive calcium phosphate ceramics, Regenerative Biomaterials 5 (1) (2018) 43e59. K. Wang, M.H. Wang, Q.G. Wang, X. Lu, X.D. Zhang, Computer simulation of proteins adsorption on hydroxyapatite surfaces with calcium phosphate ions, J. Eur. Ceram. Soc. 37 (6) (2017). X. Zhu, H. Zhang, H. Fan, W. Li, X. Zhang, Effect of phase composition and microstructure of calcium phosphate ceramic particles on protein adsorption, Acta Biomaterialia 6 (4) (2010) 1536e1541. X. Zhu, H. Fan, D. Li, Y. Xiao, X. Zhang, Protein adsorption and zeta potentials of a biphasic calcium phosphate ceramic under various conditions, J. Biomed. Mater. Res. B Appl. Biomater. 82 (1) (2007) 65e73. R.M. Klar, R. Duarte, T. Dix-Peek, C. Dickens, C. Ferretti, U. Ripamonti, Calcium ions and osteoclastogenesis initiate the induction of bone formation by coral-derived macroporous constructs, J. Cell Mol. Med. 17 (11) (2013) 1444e1457. F.N. Syed-Picard, T. Jayaraman, R.S. Lam, E. Beniash, C. Sfeir, Osteoinductivity of calcium phosphate mediated by connexin 43, Biomaterials (2013). G.-Y. Jung, Y.-J. Park, J.-S. Han, Effects of HA released calcium ion on osteoblast differentiation, J. Mater. Sci. Mater. Med. 21 (5) (2010) 1649e1654. M. Zayzafoon, K. Fulzele, J.M. McDonald, Calmodulin and calmodulin-dependent kinase IIa regulate osteoblast differentiation by controlling c-fos expression, J. Biol. Chem. 280 (8) (2005) 7049e7059. A.M. Barradas, H.A. Fernandes, N. Groen, Y.C. Chai, J. Schrooten, J. van de Peppel, J.P. van Leeuwen, C.A. van Blitterswijk, J. de Boer, A calcium-induced signaling cascade leading to osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells, Biomaterials 33 (11) (2012) 3205e3215. M. Murshed, D. Harmey, J.L. Millan, M.D. McKee, G. Karsenty, Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone, Genes Dev. 19 (9) (2005) 1093e1104. G.R. Beck, B. Zerler, E. Moran, Phosphate is a specific signal for induction of osteopontin gene expression, Proc. Natl. Acad. Sci. 97 (15) (2000) 8352e8357. G.R. Beck, N. Knecht, Osteopontin regulation by inorganic phosphate is ERK1/2-, protein kinase C-, and proteasome-dependent, J. Biol. Chem. 278 (43) (2003) 41921e41929. S. Khoshniat, A. Bourgine, M. Julien, M. Petit, P. Pilet, T. Rouillon, M. Masson, M. Gatius, P. Weiss, J. Guicheux, L. Beck, Phosphate-dependent stimulation of MGP and OPN expression in osteoblasts via the ERK1/2 pathway is modulated by calcium, Bone 48 (4) (2011) 894e902. Y.-R.V. Shih, Y. Hwang, A. Phadke, H. Kang, N.S. Hwang, E.J. Caro, S. Nguyen, M. Siu, E.A. Theodorakis, N.C. Gianneschi, Calcium phosphate-bearing matrices induce

Material characteristics, surface/interface Chapter | 1

[54]

[55]

[56]

[57] [58]

[59]

[60]

[61]

[62] [63]

[64]

[65]

[66]

[67]

[68]

99

osteogenic differentiation of stem cells through adenosine signaling, Proc. Natl. Acad. Sci. 111 (3) (2014) 990e995. Z. Yang, H. Yuan, W. Tong, P. Zou, W. Chen, X. Zhang, Osteogenesis in extraskeletally implanted porous calcium phosphate ceramics: variability among different kinds of animals, Biomaterials 17 (22) (1996) 2131e2137. Z.J. Yang, H. Yuan, P. Zou, W. Tong, S. Qu, X.D. Zhang, Osteogenic responses to extraskeletally implanted synthetic porous calcium phosphate ceramics: an early stage histomorphological study in dogs, Journal of materials science, Materials in medicine 8 (11) (1997) 697e701. U. Ripamonti, B. Van den Heever, J. Van Wyk, Expression of the osteogenic phenotype in porous hydroxyapatite implanted extraskeletally in baboons, Matrix 13 (6) (1993) 491e502. Y.H. Zhang X, K. De Groot, Calcium Phosphate Biomaterials with Intrinsic Osteoinductivity, the 6th World Biomaterials Congress, Kamuela, Hawaii, USA, 2000, pp. 1e13. G. Song, P. Habibovic, C. Bao, J. Hu, C.A. van Blitterswijk, H. Yuan, W. Chen, H.H. Xu, The homing of bone marrow MSCs to non-osseous sites for ectopic bone formation induced by osteoinductive calcium phosphate, Biomaterials 34 (9) (2013) 2167e2176. J.C. Kovacic, M. Boehm, Resident vascular progenitor cells: an emerging role for nonterminally differentiated vessel-resident cells in vascular biology, Stem Cell Res. 2 (1) (2009) 2e15. B. Zheng, B. Cao, M. Crisan, B. Sun, G. Li, A. Logar, S. Yap, J.B. Pollett, L. Drowley, T. Cassino, B. Gharaibeh, B.M. Deasy, J. Huard, B. Peault, Prospective identification of myogenic endothelial cells in human skeletal muscle, Nat. Biotechnol. 25 (9) (2007) 1025e1034. U. Ripamonti, R.M. Klar, L.F. Renton, C. Ferretti, Synergistic induction of bone formation by hOP-1, hTGF-beta3 and inhibition by zoledronate in macroporous coral-derived hydroxyapatites, Biomaterials 31 (25) (2010) 6400e6410. C. Betsholtz, P. Lindblom, H. Gerhardt, Role of Pericytes in Vascular Morphogenesis, Mechanisms of Angiogenesis, Springer, 2005, pp. 115e125. A. Dellavalle, M. Sampaolesi, R. Tonlorenzi, E. Tagliafico, B. Sacchetti, L. Perani, A. Innocenzi, B.G. Galvez, G. Messina, R. Morosetti, Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells, Nat. Cell Biol. 9 (3) (2007) 255e267. L. Diaz-Flores, R. Gutierrez, A. Lopez-Alonso, R. Gonzalez, H. Varela, Pericytes as a supplementary source of osteoblasts in periosteal osteogenesis, Clin. Orthop. Relat. Res. 275 (1992) 280e286. B. Piault, M. Rudnicki, Y. Torrente, G. Cossu, J.P. Tremblay, T. Partridge, E. Gussoni, L.M. Kunkel, J. Huard, Stem and progenitor cells in skeletal muscle development, maintenance, and therapy, Mol. Ther. 15 (5) (2007) 867e877. Z. Tang, Y. Tan, Y. Ni, W. Jing, X. Zhu, Y. Fan, X. Chen, Y. Xiao, X. Zhang, Comparison of ectopic bone formation process induced by four calcium phosphate ceramics in mice, Mater Sci Eng C Mater Biol Appl 70 (2016) 1000e1010. P. Muller, U. Bulnheim, A. Diener, F. Luthen, M. Teller, E.D. Klinkenberg, H.G. Neumann, B. Nebe, A. Liebold, G. Steinhoff, J. Rychly, Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells, J. Cell Mol. Med. 12 (1) (2008) 281e291. J.H. Song, J.H. Kim, S. Park, W. Kang, H.W. Kim, H.E. Kim, J.H. Jang, Signaling responses of osteoblast cells to hydroxyapatite: the activation of ERK and SOX9, J. Bone Miner. Metab. 26 (2) (2008) 138e142.

100 Bioactive Materials for Bone Regeneration [69] D. Chen, M. Zhao, G.R. Mundy, Bone morphogenetic proteins, Growth Factors 22 (4) (2004) 233e241. [70] J. Eyckmans, S.J. Roberts, J. Schrooten, F.P. Luyten, A clinically relevant model of osteoinduction: a process requiring calcium phosphate and BMP/Wnt signalling, J. Cell Mol. Med. 14 (6B) (2010) 1845e1856. [71] P. Sibilla, A. Sereni, G. Aguiari, M. Banzi, E. Manzati, C. Mischiati, L. Trombelli, L. del Senno, Effects of a hydroxyapatite-based biomaterial on gene expression in osteoblast-like cells, J. Dent. Res. 85 (4) (2006) 354e358. [72] L. Sun, L. Wu, C. Bao, C. Fu, X. Wang, J. Yao, X. Zhang, C.A. van Blitterswijk, Gene expressions of Collagen type I, ALP and BMP-4 in osteo-inductive BCP implants show similar pattern to that of natural healing bones, Mater. Sci. Eng. C 29 (6) (2009) 1829e1834. [73] Z.R. Tang, Z. Wang, F.Z. Qing, Y.L. Ni, Y.J. Fan, Y.F. Tan, X.D. Zhang, Bone morphogenetic protein Smads signaling in mesenchymal stem cells affected by osteoinductive calcium phosphate ceramics, J. Biomed. Mater. Res. A 103 (3) (2015) 1001e1010. [74] A.M. Barradas, V. Monticone, M. Hulsman, C. Danoux, H. Fernandes, Z. Tahmasebi Birgani, F. Barrere-de Groot, H. Yuan, M. Reinders, P. Habibovic, C. van Blitterswijk, J. de Boer, Molecular mechanisms of biomaterial-driven osteogenic differentiation in human mesenchymal stromal cells, Integrative biology : quantitative biosciences from, Nano to Macro 5 (7) (2013) 920e931. [75] J. Wang, Y. Chen, X. Zhu, T. Yuan, Y. Tan, Y. Fan, X. Zhang, Effect of phase composition on protein adsorption and osteoinduction of porous calcium phosphate ceramics in mice, J. Biomed. Mater. Res. A (2014) (n/a-n/a). [76] L. Zheng, J. Sun, X. Chen, G. Wang, B. Jiang, H. Fan, X. Zhang, In vivo cartilage engineering with collagen hydrogel and allogenous chondrocytes after diffusion chamber implantation in immunocompetent host, Tissue Eng. A 15 (8) (2009) 2145e2153. [77] A.A. Sawyer, K.M. Hennessy, S.L. Bellis, Regulation of mesenchymal stem cell attachment and spreading on hydroxyapatite by RGD peptides and adsorbed serum proteins, Biomaterials 26 (13) (2005) 1467e1475. [78] R.O. Hynes, Integrins: bidirectional, allosteric signaling machines, Cell 110 (6) (2002) 673e687. [79] M.C. Siebers, P.J.T. Brugge, X.F. Walboomers, J.A. Jansen, Integrins as linker proteins between osteoblasts and bone replacing materials. A critical review, Biomaterials 26 (2) (2005) 137e146. [80] A. Shekaran, A.J. Garcia, Extracellular matrix-mimetic adhesive biomaterials for bone repair, J. Biomed. Mater. Res. A 96 (1) (2011) 261e272. [81] T. Matsuura, R. Hosokawa, K. Okamoto, T. Kimoto, Y. Akagawa, Diverse mechanisms of osteoblast spreading on hydroxyapatite and titanium, Biomaterials 21 (11) (2000) 1121e1127. [82] K.L. Kilpadi, P.L. Chang, S.L. Bellis, Hydroxylapatite binds more serum proteins, purified integrins, and osteoblast precursor cells than titanium or steel, J. Biomed. Mater. Res. 57 (2) (2001) 258e267. [83] R.M. Salasznyk, R.F. Klees, W.A. Williams, A. Boskey, G.E. Plopper, Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells, Exp. Cell Res. 313 (1) (2007) 22e37. [84] G. Marino, F. Rosso, G. Cafiero, C. Tortora, M. Moraci, M. Barbarisi, A. Barbarisi, b-Tricalcium phosphate 3D scaffold promote alone osteogenic differentiation of human adipose stem cells: in vitro study, J. Mater. Sci. Mater. Med. 21 (1) (2010) 353e363.

Material characteristics, surface/interface Chapter | 1

101

[85] Z. Lu, H. Zreiqat, The osteoconductivity of biomaterials is regulated by bone morphogenetic protein 2 autocrine loop involving a2b1 integrin and mitogen-activated protein kinase/extracellular related kinase signaling pathways, Tissue Eng. A 16 (10) (2010) 3075e3084. [86] H. Liu, H. Peng, Y. Wu, C. Zhang, Y. Cai, G. Xu, Q. Li, X. Chen, J. Ji, Y. Zhang, H.W. OuYang, The promotion of bone regeneration by nanofibrous hydroxyapatite/chitosan scaffolds by effects on integrin-BMP/Smad signaling pathway in BMSCs, Biomaterials 34 (18) (2013) 4404e4417. [87] X. Chen, J. Wang, Y. Chen, H. Cai, X. Yang, X. Zhu, Y. Fan, X. Zhang, Roles of calcium phosphate-mediated integrin expression and MAPK signaling pathways in the osteoblastic differentiation of mesenchymal stem cells, J. Mater. Chem. B 4 (13) (2016) 2280e2289. [88] S. Fujibayashi, M. Neo, H.M. Kim, T. Kokubo, T. Nakamura, Osteoinduction of porous bioactive titanium metal, Biomaterials 25 (3) (2004) 443e450. [89] T. Kokubo, S. Yamaguchi, Novel bioactive materials developed by SBF evaluation: surface-modified Ti metal and its alloys, Acta Biomater. 44 (2016) 16e30. [90] A. Fukuda, M. Takemoto, T. Saito, S. Fujibayashi, M. Neo, D.K. Pattanayak, T. Matsushita, K. Sasaki, N. Nishida, T. Kokubo, Osteoinduction of porous Ti implants with a channel structure fabricated by selective laser melting, Acta Biomater. 7 (5) (2011) 2327e2336. [91] T. Sjo¨stro¨m, L.E. Mcnamara, R.M. Meek, M.J. Dalby, B. Su, 2D and 3D nanopatterning of titanium for enhancing osteoinduction of stem cells at implant surfaces, Adv. Healthc. Mater. 2 (9) (2013) 1285. [92] Takemoto, M. *Fujibayashi, S. **Matsushita, T. **Suzuki, J. ***Kokubo, T. *Nakamura, Osteoinductive Ability of Porous Titanium Implants Following Three Types of Surface Treatment. [93] M. Takemoto, S. Fujibayashi, M. Neo, J. Suzuki, T. Matsushita, T. Kokubo, T. Nakamura, Osteoinductive porous titanium implants: effect of sodium removal by dilute HCl treatment, Biomaterials 27 (13) (2006) 2682e2691. [94] C. Zhao, X. Zhu, K. Liang, J. Ding, X. Zhou, H. Fan, X. Zhang, Osteoinduction of porous titanium: a comparative study between acid-alkali and chemical-thermal treatments, J. Biomed. Mater. Res. B Appl. Biomater. 95B (2) (2010) 387e396. [95] F. Barrere, C.A. van Blitterswijk, G.K. De, P. Layrolle, Influence of ionic strength and carbonate on the Ca-P coating formation from SBFx5 solution, Biomaterials 23 (9) (2002) 1921e1930. [96] K.E. Healy, P. Ducheyne, Hydration and preferential molecular adsorption on titanium in vitro, Biomaterials 13 (8) (1992) 553e561. [97] T. Kawai, M. Takemoto, S. Fujibayashi, H. Akiyama, M. Tanaka, S. Yamaguchi, D.K. Pattanayak, K. Doi, T. Matsushita, T. Nakamura, Osteoinduction on acid and heat treated porous Ti metal samples in canine muscle, PLoS One 9 (2) (2014) e88366. [98] J. Yang, J. Wang, T. Yuan, X.D. Zhu, Z. Xiang, Y.J. Fan, X.D. Zhang, The enhanced effect of surface microstructured porous titanium on adhesion and osteoblastic differentiation of mesenchymal stem cells, J. Mater. Sci. Mater. Med. 24 (9) (2013) 2235e2246. [99] S.Y. Kim, J.Y. Yoo, J.Y. Ohe, J.W. Lee, J.H. Moon, Y.D. Kwon, J.S. Heo, Differential expression of osteo-modulatory molecules in periodontal ligament stem cells in response to modified titanium surfaces, Biomed Res. Intl. 2014 (1) (2014) 452175. [100] N. Chakravorty, S. Hamlet, A. Jaiprakash, R. Crawford, A. Oloyede, M. Alfarsi, Y. Xiao, S. Ivanovski, Pro-osteogenic topographical cues promote early activation of

102 Bioactive Materials for Bone Regeneration

[101]

[102]

[103]

[104] [105]

[106]

[107]

[108] [109]

[110] [111]

[112]

[113]

[114] [115]

[116]

osteoprogenitor differentiation via enhanced TGFb, Wnt, and Notch signaling, Clin. Oral Implant. Res. 25 (4) (2014) 475e486. N. Chakravorty, S. Hamlet, Y. Xiao, S. Ivanovski, Surface Topography Upregulates TGF-b, Wnt and Notch Signalling in Osteoblasts, Iadr/aadr/cadr General Session and Exhibition, 2013. C.R. Larissa, F. Marcelo, T. Lucas, F. Emanuela, L. Helena, O. Paulo, H. Mohammad, R. Adalberto, B. Marcio, C.R. Larissa, Titanium with nanotopography induces osteoblast differentiation mediated by endogenous BMP-2/4, Front. Bioeng. Biotechnol. 4 (2016). J. Vlacic-Zischke, S.M. Hamlet, T. Friis, M.S. Tonetti, S. Ivanovski, The influence of surface microroughness and hydrophilicity of titanium on the up-regulation of TGFb/BMP signalling in osteoblasts, Biomaterials 32 (3) (2011) 665e671. L.I. Zhen-Chun, Expression of TGF-b1 on the interface of surface-prepared titanium implant and bone, Chin. J. Prac. Stomatol. (2010). L.F. Zhuang, H.H. Jiang, S.C. Qiao, C. Appert, M.S. Si, Y.X. Gu, H.C. Lai, The roles of extracellular signal-regulated kinase 1/2 pathway in regulating osteogenic differentiation of murine preosteoblasts MC3T3-E1 cells on roughened titanium surfaces, J. Biomed. Mater. Res. A 100A (1) (2011) 125e133. G. Yang, F. Wen, T. Liu, F. He, X. Chen, Z. Yi, X. Guan, Gene expression profiling of bone marrow-derived stromal cells seeded onto a sandblasted, large-grit, acid-etched-treated titanium implant surface: the role of the Wnt pathway, Arch. Oral Biol. 61 (2015) 71. M.S.C.R. Larissa, M.S. Francischini, L.N. Teixeira, E.P. Ferraz, H.B. Lopes, T.D.O. Paulo, M.Q. Hassan, A.L. Losa, M.M. Beloti, Titanium with nanotopography induces osteoblast differentiation by regulating endogenous bone morphogenetic protein expression and signaling pathway, J. Cell. Biochem. 117 (7) (2016) 1718e1726. F. Deschaseaux, L. Sense´be´, D. Heymann, Mechanisms of bone repair and regeneration, Trends Mol. Med. 15 (9) (2009) 417e429. H. Liu, G.W. Xu, Y.F. Wang, H.S. Zhao, S. Xiong, Y. Wu, B.C. Heng, C.R. An, G.H. Zhu, D.H. Xie, Composite scaffolds of nano-hydroxyapatite and silk fibroin enhance mesenchymal stem cell-based bone regeneration via the interleukin 1 alpha autocrine/paracrine signaling loop, Biomaterials 49 (2015) 103e112. J.D. Bryers, C.M. Giachelli, B.D. Ratner, Engineering biomaterials to integrate and heal: the biocompatibility paradigm shifts, Biotechnol. Bioeng. 109 (8) (2012) 1898e1911. T. Miramond, P. Corre, P. Borget, F. Moreau, J. Guicheux, G. Daculsi, P. Weiss, Osteoinduction of biphasic calcium phosphate scaffolds in a nude mouse model, J. Biomater. Appl. 29 (4) (2014) 595e604. M.C. Walsh, N. Kim, Y. Kadono, J. Rho, S.Y. Lee, J. Lorenzo, Y. Choi, Osteoimmunology: interplay between the immune system and bone metabolism, Annu. Rev. Immunol. 24 (24) (2006) 33e63. S. Franz, S. Rammelt, D. Scharnweber, J.C. Simon, Immune responses to implants e a review of the implications for the design of immunomodulatory biomaterials, Biomaterials 32 (28) (2011) 6692e6709. Z. Chen, T. Klein, R.Z. Murray, R. Crawford, J. Chang, C. Wu, Y. Xiao, Osteoimmunomodulation for the development of advanced bone biomaterials, Mater. Today (2015). Z. Chen, J. Yuen, R. Crawford, J. Chang, C. Wu, Y. Xiao, The effect of osteoimmunomodulation on the osteogenic effects of cobalt incorporated b-tricalcium phosphate, Biomaterials 61 (2015) 126e138. C. Wu, Z. Chen, Q. Wu, D. Yi, T. Friis, X. Zheng, J. Chang, X. Jiang, Y. Xiao, Clinoenstatite coatings have high bonding strength, bioactive ion release, and

Material characteristics, surface/interface Chapter | 1

[117]

[118]

[119]

[120]

[121]

[122] [123]

[124]

[125]

[126]

103

osteoimmunomodulatory effects that enhance in vivo osseointegration, Biomaterials 71 (2015) 35e47. M. Bartneck, K.H. Heffels, P. Yu, M. Bovi, G. Zwadlo-Klarwasser, J. Groll, Inducing healing-like human primary macrophage phenotypes by 3D hydrogel coated nanofibres, Biomaterials 33 (16) (2012) 4136e4146. X. Chen, J. Wang, X. Zhu, X. Yang, Y. Fan, X. Zhang, The positive role of macrophage secretion stimulated by BCP ceramic in the ceramic-induced osteogenic differentiation of pre-osteoblasts via Smad-related signaling pathways, RSC Adv. 6 (104) (2016) 102134e102141. J. Wang, D. Liu, B. Guo, X. Yang, X. Chen, X. Zhu, Y. Fan, X. Zhang, Role of BCP ceramic-mediated secretion of signalling molecules by macrophages in migration and osteoblastic differentiation of MSCs, Acta Biomater. 51 (2017). J. De Bruijn, K. Shankar, H. Yuan, P. Habibovic, Osteoinduction and its Evaluation, Bioceramics and Their Clinical Applications, Woodhead publishing Ltd, Cambridge, 2008, p. 199. Z. Chen, C. Wu, W. Gu, T. Klein, R. Crawford, Y. Xiao, Osteogenic differentiation of bone marrow MSCs by beta-tricalcium phosphate stimulating macrophages via BMP2 signalling pathway, Biomaterials 35 (5) (2014) 1507e1518. L. Dong, C. Wang, Harnessing the power of macrophages/monocytes for enhanced bone tissue engineering, Trends Biotechnol. 31 (6) (2013) 342e346. P. Laquerriere, A. Grandjean-Laquerriere, E. Jallot, G. Balossier, P. Frayssinet, M. Guenounou, Importance of hydroxyapatite particles characteristics on cytokines production by human monocytes in vitro, Biomaterials 24 (16) (2003) 2739e2747. D. Le Nihouannen, G. Daculsi, A. Saffarzadeh, O. Gauthier, S. Delplace, P. Pilet, P. Layrolle, Ectopic bone formation by microporous calcium phosphate ceramic particles in sheep muscles, Bone 36 (6) (2005) 1086e1093. D. Le Nihouannen, A. Saffarzadeh, O. Gauthier, F. Moreau, P. Pilet, R. Spaethe, P. Layrolle, G. Daculsi, Bone tissue formation in sheep muscles induced by a biphasic calcium phosphate ceramic and fibrin glue composite, J. Mater. Sci. Mater. Med. 19 (2) (2008) 667e675. L. Kan, Y. Liu, T.L. McGuire, D.M.P. Berger, R.B. Awatramani, S.M. Dymecki, J.A. Kessler, Dysregulation of local stem/progenitor cells as a common cellular mechanism for heterotopic ossification, Stem Cells 27 (1) (2009) 150e156.

Chapter 2

Biomaterial-induced microenvironment and host reaction in bone regeneration Chapter outline 2.1 Bioactive inorganic ions for the manipulation of osteoimmunomodulation to improve bone regeneration 2.1.1 Introduction 2.1.2 Application of bioactive ions in developing bone biomaterials and their possible application in manipulating osteoimmunomodulation 2.1.2.1 Strontium 2.1.2.2 Zinc 2.1.2.3 Magnesium 2.1.2.4 Calcium 2.1.2.5 Silicon 2.1.2.6 Cobalt 2.1.2.7 Copper 2.1.2.8 Europium 2.1.2.9 Fluorine 2.1.3 Combining bioactive elements to develop novel bone biomaterials with osteoimmunomodulatory properties as well as promote osteogenesis 2.1.4 Summaries and future prospects References 2.2 Silicate-based bone cements for hard tissue regeneration 2.2.1 Preparation of silicate-based bone cement

107 107

109 110 112 113 114 115 116 118 119 120

122 124 125 135

2.2.2 Self-setting properties and drug delivery performance of silicate-based bone cement 2.2.2.1 Setting time and mechanical strength 2.2.2.2 Injectability and washout resistance 2.2.2.3 Drug loading and release properties of silicate-based bone cements 2.2.3 In vitro and in vivo bioactivity and osteoinductivity of silicatebased bone cement References 2.3 Trace elementebased biomaterials for osteochondral regeneration 2.3.1 Introduction 2.3.2 Biomaterials for osteochondral regeneration 2.3.2.1 The clinical need for osteochondral regeneration 2.3.2.2 The anatomy and properties of osteochondral tissue 2.3.2.3 Current strategies for osteochondral regeneration

139 139 143

143

144 148

151 151 152

153

153

154

135

Bioactive Materials for Bone Regeneration. https://doi.org/10.1016/B978-0-12-813503-7.00002-9 Copyright © 2020 Higher Education Press. Published by Elsevier Ltd. All rights reserved.

105

106 Bioactive Materials for Bone Regeneration 2.3.2.4 Biomaterials for cartilage and bone regeneration 2.3.2.4.1 Biomaterials for cartilage regeneration 2.3.2.4.2 Biomaterials for bone regeneration 2.3.3 Trace elementebased biomaterials for osteochondral regeneration 2.3.3.1 Manganese-doped biomaterials for osteochondral regeneration 2.3.3.2 Molybdenum-doped biomaterials for osteochondral regeneration 2.3.3.3 Lithium-based biomaterials for osteochondral regeneration 2.3.3.4 Strontium-based biomaterials for

155

155

155

156

156

156

157

osteochondral regeneration 2.3.3.5 Other nutrient elements for osteochondral regeneration 2.3.4 Conclusions and perspectives References 2.4 Bioactive ions for bone tissue engineering design 2.4.1 Introduction 2.4.2 Effects of bioactive ions on cell proliferation and stemness maintenance 2.4.3 Effects of bioactive ions on osteogenesis and osteoclastogenesis 2.4.4 Effects of bioactive ions on angiogenesis 2.4.5 Design of bioactive ion composite biomaterials for bone tissue engineering 2.4.6 Conclusions and perspectives References

160

161 161 162 166 166

167

168 171

172 175 175

When biomaterials are implanted into human bone defects, they interact with surrounding tissues and cells and create a unique microenvironment in the tissue wound site, which plays a critical role in regulating bone tissue regeneration. In particular, bioactive ions released from bioceramics have shown specific bioactivities by creating a specific ion-based microenvironment during bone regeneration. This chapter focuses on the biomaterial-induced microenvironment and host reaction in bone regeneration including bioactive inorganic ions for the manipulation of osteoimmunomodulation to improve bone regeneration, silicate-based bone cements (CSCs) for bone tissue regeneration, and bone tissue engineering (BTE) strategy based on the synergistic effects of silicon and strontium ions.

Biomaterial-induced microenvironment and host reaction Chapter | 2

107

Chapter 2.1

Bioactive inorganic ions for the manipulation of osteoimmunomodulation to improve bone regeneration Osteoimmunomodulation is the interaction of the immune and skeletal systems during biomaterial-mediated osteogenesis. Based on this concept, biomaterials with osteoimmunomodulatory effects have been developed, and they have proven effective in improving the osteogenic capacity of bone biomaterials. Bioactive ions have shown significant effects on modulating osteoimmune response and have demonstrated highly tunable properties. The osteoimmunomodulatory effect differs from ionic concentration and different ion combinations. This suggests that bioactive ion-based strategy is effective for tuning the osteoimmune environment. This chapter summarizes related studies on bioactive ion-mediated osteoimmunomodulatory effects as well as the mechanisms for how these ions modulate local immune response and subsequently influence osteogenesis, which would provide valuable knowledge for the development of bone-substitute materials containing “osteoimmune-smart” bioactive ions.

2.1.1 Introduction Immune cells have been known to play vital roles in bone dynamics. It has been reported that macrophages and T lymphocytes are critical immune cells that play vital roles in osteogenesis. Macrophages have been demonstrated to present early in chondrogenic centers and persist until callus was formed during fracture repair. Elimination of macrophages will reduce or even block callus formation [1]. T lymphocytes regulate osteogenesis through the production of IFN-g, IL-17, TNF-a, and TGF-b, which affect the proliferation and differentiation of osteoblasts [2e5]. As for the process of osteoclastogenesis, immune cells function via the RANKL/RANK/osteoprotegerin (OPG) system. Overexpression of RANKL on T cells under inflammatory conditions leads to osteoclastogenesis and bone loss [6,7]. Meanwhile, B cells and dendritic cells can synthesize OPG to suppress RANKL/RANK binding, thus rescuing bone loss [8,9]. Immune cells can also release inflammatory cytokines (e.g., ILs 1, 6, and 17 as well as TNF-a) to regulate activation of the RANKL/RANK/OPG axis. Involvement of immune cells in the process of biomaterial-mediated bone formation has also been well recognized. An “osteoimmunomodulation”based strategy has been proposed to guide the design of bone biomaterials [10]. An ideal biomaterial should have a favorable osteoimmunomodulatory

108 Bioactive Materials for Bone Regeneration

effect that modulates the osteoimmune environment to direct osteogenic differentiation. To be specific, immune cells and regulatory molecules released from immune cells can significantly regulate the process of osteogenesis, osteoclastogenesis, and angiogenesis [11e14], which finally determine bone regeneration outcomes. Because of the complicated interaction network of the immune and bone systems involving various cells and regulators, it is difficult to precisely modulate the osteoimmune environment into a favorable one. Our previous studies have demonstrated that via tuning the physicochemical properties of biomaterials, immune cells can be well educated, thus modulating the osteoimmune environment in required directions [15e18]. Among all exploring strategies, we found that incorporating bone biomaterials with bioactive ions is one of the most viable osteoimmunomodulatory approaches [18]. A number of bioactive ions have been discovered with osteoimmunomodulatory effects including calcium (Ca) [19], magnesium (Mg) [20], strontium (Sr) [21], and silicon (Si) [22]. These bioactive ions have demonstrated a highly tunable feature in osteoimmunomodulation (Fig. 2.1). Via doping bioactive elements into bone biomaterials, the bioactivity of bone

FIGURE 2.1 Various bioactive ions have been found to possess osteoimmunomodulatory properties. Every single ion modulates the osteoimmune microenvironment in a concentrationdependent manner. In addition, several bioactive ions are able to combine together in variable proportions and components to elicit combined effects to modulate immune cell response and the expression of inflammatory cytokines, thereby affecting osteogenesis, osteoclastogenesis, angiogenesis, and fibrosis processes. Therefore, we can improve bone regeneration outcomes via regulation of ion concentrations and the combinination of various bioactive ions in different ways.

Biomaterial-induced microenvironment and host reaction Chapter | 2

109

biomaterials could be well upregulated, and significant effects would be elicited for regulating bone dynamics, thus affecting regeneration outcomes [23]. A bioactive ion-based osteoimmunomodulatory strategy is highly flexible and provides a broad-spectrum osteoimmune environment. To fully apply this strategy for osteoimmunomodulation, we should first understand each bioactive ion’s osteoimmunomodulatory effect and how ion combinations affect modulation outcomes. With this understanding, some “osteoimmune-smart” biomaterials have been successfully developed and are summarized in this chapter. It is the authors’ ambition that this chapter will set up a bioactive ion-based osteoimmunomodulation strategy for developing advanced bone biomaterials that favor bone regeneration.

2.1.2 Application of bioactive ions in developing bone biomaterials and their possible application in manipulating osteoimmunomodulation Bioactive ions perform great physiological functions in the sense that their incorporation into biomaterials, especially bone-substitute materials, strongly improves bioactivities and the osteogenic efficacy of implants. Over the past few decades, many inorganic ions have been found to have potential therapeutic effects that can be applied in bone biomaterials due to their stimulating effects on both osteogenesis and angiogenesis. In addition, some of them (e.g., Cu, Zn, and Ag) also exhibit the additional therapeutic effects of antiinflammatory and antibiotic, which is a crucial aspect for reducing or preventing a device-related infection and contributes to successful bone-repair process. Previous studies have revealed that a number of bioactive elements such as calcium (Ca), cobalt (Co), strontium (Sr), zinc (Zn), copper (Cu), silicon (Si), magnesium (Mg), europium (Eu), and fluorine (F) have potential in modulating bone dynamics. They could elicit significant effects on promoting the proliferation and differentiation of osteoblastic cells with the mechanism of activating multiple signal pathways, such as Wnt, OSM, and Akt kinaserelated signal pathway, on mesenchymal stem cells (MSCs) or osteoblasts. In addition, some ions also possess the ability to inhibit osteoclastogenesis, thus suppressing active bone resorption. They can reduce the formation and activity of osteoclasts by regulating the OPG/RANK/RANKL pathway or inhabiting osteoclastic activity related genesexpressingde.g., tartrate-resistant acid phosphatase (TRAP), cathepsin K, and CTR. When incorporated into bone biomaterials, such as TCP, mesoporous bioactive glass and mesoporous silica nanospheres, and sustained released into the surrounding environment in a certain concentration range, these bioactive ions exhibited significant osteogenic effects in vivo and in vitro. All of these suggest bioactive ions could be promising candidates for bone regenerative applications. Angiogenesis, an important process interlinked with osteogenesis, is of great importance for cell survival in bone regeneration process by supporting nutrient and oxygen exchange. A number of bioactive ions have been found to possess the ability for angiogenesis through enhancing angiogenic

110 Bioactive Materials for Bone Regeneration

factorsexpressing, such as vascular endothelial growth factor (VEGF) and angiotensin-1. Studies have demonstrated that some metallic ions such as Cu, Sr, Co, Mu and Ni, could facilitate angiogenesis by eliciting a hypoxic or hypoxic-like microenvironment to stabilize hypoxia-inducible factors (HIFs) and subsequently activate the HIF target genes such as VEGF, which is the most important factor for angiogenesis. Furthermore, bioactive ions can elicit proangiogenic effects by generating the activation of reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK). Because of the favorable proangiogenic effects, some ions have been applied in different kinds of bone biomaterials as effective angiogenic agents. In addition to the vital role on osteogenesis and angiogenesis, a number of studies have also revealed that the immune response of implanted materials can be modulated by the incorporation of bioactive ions (e.g., Ca, Mg, Sr, Cu, Co, and Zn). Many bioactive ions have shown the ability to influence the recruitment, maturation or activation of immune cells, such as macrophages, T cells, B cells and neutrophils, and subsequently modulate proinflammatory or antiinflammatory cytokine (e.g., TNF-a, IFN-g, IL-6, IL-1b, IL-10, and IL-1ra) production. The underlying mechanisms are closely related to toll-like receptor (TLR) pathway and the activation of NF-kB. These bioactive ions could elicit a series of effects on immune response, which vary with composition, concentration and release rate. However, the specific effects of each bioactive ion and their dose-dependent effect are still undefined. Related studies have been performed on several bioactive ions (listed below) to investigate their osteoimmunomodulatory effect as well as the mechanisms of how these ions modulate the local immune response and subsequently influence osteogenesis, which would provide valuable information for the development of “osteoimmune-smart” bioactive ions containing bone-substitute materials.

2.1.2.1 Strontium Strontium (Sr) is located in the second main group of the fourth cycle on the periodic table of chemical elements. In human body, 98% Sr can be found in bone tissue, which is the only metallic element that is related with compressive strength of bone [24]. It has similar chemical properties in comparison with Ca, because they locate in the same main group and have similar atomic radius. Somehow, Srmay take the place of Ca in the bone tissue [25]. Sr has been found to be an effective regulator in bone dynamics. It elicits significant effect in promoting osteogenesis, via activating multiple signal pathways including calcium sensing receptor (CaSR), Akt kinase-related pathway and Wnt signal pathway onMSCs or osteoblasts [26,27]. Sr can also regulate the ratio of OPG/RANK/RANKL to inhibit osteoclastogenesis [28,29]. Due to this antiosteoclastogenic effect, it has been applied widely in clinics as a promising agent in treating osteoporosis [30]. In addition, Sr promotes neovascularization through enhancing the expression of angiogenic factors, such as VEGFand Ang-1 [31,32].

Biomaterial-induced microenvironment and host reaction Chapter | 2

111

With these positive effects, Sr has been applied in improving the osteogenic capacity of biomaterials. Via incorporating Sr, bone-substitute materials including hydroxyapatites (HAs) and silicate glasses can enhance alkaline phosphate (ALP) activities and the expression of collagen type I (Col1), osteocalcin (OC), Runx2, and angiogenic factors, thus improving osteogenesis outcomes [33]. In addition, Sr can be applied for the optimization of guided bone regeneration barrier membrane to improve guided new bone formation. Through the addition of Sr HA, barrier membrane demonstrates higher elasticity and strength, and the slow release of Sr ion stimulates new bone formation [34]. Interestingly, Sr also has been found to have effect on regulating immune response. Sr changes the inflammatory cytokine expression profile. When Sr is applied to stimulate immune cells, the secretion of proinflammatory cytokines (TNFa, IFNg, etc.) is significantly inhibited while antiinflammatory cytokines is increased (IL-10 and others). The underlying mechanism is in the connection with the antagonizing of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) activation [35]. These indicate that Sr can be a promising candidate bioactive ion for the osteoimmunomodulation. Previous studies have proved that the incorporation of Sr can successfully endow bone biomaterials with favorable osteoimmunomodulatory property. Monodispersed Sr-contained bioactive glasses microspheres were found to promote the early angiogenesis through tuning the macrophage phenotype switch toward M2 extreme [36]. The incorporation of Sr can also endow CaP with an antiinflammatory effect, resulting in the inhibition of proinflammatory cytokine (TNF-a and others) release. The generated osteoimmune environment can slow down osteoclastogenesis [37,38]. These results collectively imply that the incorporation of Sr can significantly modulate the osteoimmune environment, thus regulating bone regeneration. However, this osteoimmunomodulatory effect appears to be different among different bone biomaterials. The underlying mechanism may lie in the change of Sr ionic concentration. When the Sr ionic concentration was set at high (500 mmol/L) and low (10 mmol/L) level, the release of TNF-a was both inhibited [37,38]. However, the correlation between the release of IL-6 and Sr ionic concentration was found to be different. The release of IL-6 was enhanced at high concentration (around 500 mmol/L), but suppressed at low concentration (10 mmol/L) [37,38]. When the concentration was increased to as high as 5100 mmol/L, proinflammatory cytokines (TNF-a, IFN-g, OSM) are all inhibited [39]. These prove the correlation between Sr ionic concentration and osteoimmunomodulation. However, no consensus has been reached in the most favorable ionic concentration range for osteoimmunomodulation. As a summary, Sr is an effective and safe agent for bone regenerative application. It can not only regulate the bone dynamic, but also modulate the osteoimmune microenvironment. These effects are closely related to Sr ionic concentration. Via tuning of Sr ionic concentration, different osteoimmune environments can be generated, thus affecting osteogenesis outcomes.

112 Bioactive Materials for Bone Regeneration

To obtain the most favorable osteoimmune environment, further research efforts should be put in finding out the optimum concentration range of Sr for osteoimmunomodulation.

2.1.2.2 Zinc Zinc is an essential trace metal element which is important in the growth and development of immune and skeletal systems [40,41]. Because of the similar mechanical properties with mammalian bone tissue, Zn and Zn alloy are potential biomaterials of bone substitution. In addition, Zn2þ ion possesses several physiological functions and can influence the growth of bone tissue [42]. In addition, due to its antibacterial properties, Zn ion can inhibit colonization of bacteria on the surface of biomaterials [43]. Zn is widely applied in skeletal and dental biomaterials because of these benefits [44]. Zn can promote osteoblastogenesis through improving osteoblastic activities, and the underlying mechanism may relate to the regulation of TGF-b/ Smad signal pathways [45]. Zn also improves the deposition of collagen and tissue mineralization to augment new bone formation via increasing the expression of ALP and osteopontin (OPN) [46]. Because of these positive regulatory effects on bone dynamic, Zn has been widely used in improving the osteogenic capacity of bone biomaterials. Zn has been applied for modifying guided bone regeneration barrier membrane to achieve a better regeneration outcome. The addition of Zn in HA coatings on gelatin membrane was found to improve osteogenesis effects and possess excellent antibacterial properties [47]. With the incorporation of Zn in TCP, the activity of TRAP and ALP are increased in human bone marrow stromal cells (hBMSCs) due to the releasing Zn2þ [48]. Moreover, via incorporating Zn, titanium implant coating such as titanium dioxide and calcium silicate coating demonstrate increasing expression of osteogenic genes in rabbit chondrocytes and mesenchymal stem cells (rBMSCs) and better osseointegration in vivo [49]. In addition to the regulatory effects on skeletal system, Zn is also an essential factor in maintaining normal immune function. Zn deficiency causes the impairment of the production and signaling of inflammatory cytokines, then affect the immunological functions [40]. The released Zn ion from Zn-contained TCP regulates the formation of multinuclear giant cell and the activities of macrophages [48]. Furthermore, Zn can influence the expression of inflammatory profiles. Through regulating TLR-4 pathway, Zn2þ enhances the release of antiinflammatory cytokine IL-10 while inhibits the expression of TNF-a and IL-1b [50]. Zn modulates osteoimmune microenvironment by releasing Zn ions to surrounding tissue, and the effects are corresponding with its ionic concentration. In homeostasis status, Zn concentration is maintained within a range of 10e18 mmol/L in plasma. We synthesized Zn-contained bioactive glass and coated it on barrier collagen membrane, the released Zn2þ (w100 mmol/L) in condition medium resulted in the upregulation of IL-1b on the macrophage

Biomaterial-induced microenvironment and host reaction Chapter | 2

113

stimulated by modified membrane [51]. In addition, previous study showed that Zn2þ over 100 mmol/L may elicit inhibition of the growth of PBMCs [52]. These indicates the dose-related effects of Zn2þ ionic on modulating osteoimmunomodulation. In conclusion, Zn has a multitude of effects when implanted in human body. It can promote bone regeneration via improving osteoblast activity and tissue mineralization and inhibit bone resorption by suppressing the formation and differentiation of osteoclasts. The application of Zn can also modulate the osteoimmune environment and elicits antiinflammation effects, thus promoting the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and subsequent new bone formation. Therefore, Zn can be considered as a promising agent for endowing favorable osteoimmunomodulatory properties of biomaterials.

2.1.2.3 Magnesium Magnesium (Mg) is an important nutrient elements for human body. As an essential intracellular cation, Mg participates in more than 300 enzymatic reactions [53]. In physical condition, 60%e65% Mg is stored in the bone and teeth. Because of its similar mechanical properties with bone tissue, Mg metal can eliminate the effects of stress shielding when implanted in bone [54,55]. In addition, Mg is a biodegradable metal and can be replaced by the new formed bone [54], which makes it a “star” bone-substitute metal material. Mg palys an essential role in skeleton metabolism. The deficiency of Mg has negative effects on all stages of skeletal metabolism, resulting in retardation of bone growth and abnormal osteoblastic and osteoclastic cells behaviors [56,57]. Previous study showed that Mg ion promote osteoblasts proliferation and the expression of osteogenesis-related marker in vitro [58]. In addition, Mg suppresses the formation and polarization of osteoclasts. It can also inhibit osteoclast activity to suppress bone resorption. The mechanism is in connection with the inhibition of RANKL-induced cytokines expressiondc-Src, modulating metalloproteinase (MMP)-9, etc.dand the expression of genes related to osteoclastic activity (TRAP, cathepsin K, and CTR) [58]. Due to the excellent properties just mentioned, Mg- and Mg-alloye incorporated bone substitutes are widely used in BTE. It was found that Mg alloy can promote the formation of hard callous via enhancing osteoblast adhesion and subsequently stimulating new bone formation [59]. It is proved that Mg2þ at a controlled concentration (w50ppm) can effectively enhance osteoblastic activity in vitro and significantly promote in situ bone regeneration [60]. In addition to a regulatory effect in bone dynamics, Mg can also elicit significant effects in modulating immune response [61]. Mg was found to reverse the effects of LPS/IFN-g stimulation on macrophage and increase the production of M2 phenotype macrophage, thereby promoting the bone-repair

114 Bioactive Materials for Bone Regeneration

process [62]. Mg can also suppress proinflammatory cytokine (IL-1b, IL-6, TNF-a, etc.) production via inhibition of the TLR pathway [62,63]. In addition, Mg inhibits the activation of NF-kB by slowing down the degradation of IkBa and subsequent NF-kB nuclear translocation [62,63]. Considering its effects on the immune system, Mg has proved to be a feasible supplement for optimizing the osteoimmunomodulatory effects of biomaterials. The Mg-containing calcium phosphate cement (MCPC) downregulated proinflammatory cytokines (TNF-a, IL-6, etc.) and upregulated bone-repair related cytokine TGF-b1. The osteogenic capacity of BMSCs and angiogenic potential of HUVECs were enhanced in the MCPC-induced immune microenvironment [64]. In addition, previous study has demonstrated that Mg leach liquor suppresses the expression and autoamplification of activated T nuclear factor 1 protein (NFATc1) by inhibiting activation of the NF-kB signaling pathway and inhibiting Ca2þ-dependent calcineurin signaling, thus downregulating osteoclastogenic gene (TRAP, CTR, MMP9, NFATc1, c-Src, etc.) expression, eventually blocking osteoclast differentiation and bone resorption [58]. Mg regulates immune response in a dose-dependent manner. Mg2þ at high concentration (20 mM) has a stronger inhibitory effect on polarization toward M1 of macrophage than that of low concentrations (5e10 mM) [62]. IL-1b and IL-6 decreased while IL-10 increased in macrophage in a 5 mM Mg2þ culture medium [65]. These results showed the correlation between ionic concentration and the osteoimmunomodulatory properties of Mg2þ. In summary, Mg is usually considered a promising biomaterial that possesses suitable mechanical properties, favorable biodegradation properties, and osteoinductive and osteoconductive effects; it is thus widely applied in bone regeneration. Current studies further prove its osteoimmunomodulatory properties. Mg acts on the immune system by regulating the polarization of macrophage and expression profile of inflammation cytokines in a dosedependent manner. However, rapid degradation of Mg scaffold in vivo triggers an acute and unfavorable inflammatory response. Therefore, further studies are needed to adjust the biodegradable properties of Mg-based materials and control the release of Mg2þ to overcome its disadvantages and achieve the optimal osteoimmunomodulatory property.

2.1.2.4 Calcium Ca is the most abundant metal in the human body and is primarily stored in the skeletal system [66]. In physical condition, Ca homeostasis is tightly regulated by the parathyroid hormone and calcitonin, which are the positive (parathyroid hormone) and negative (calcitonin) regulators of osteoclast-mediated bone resorption. In addition, as one of the major components of CaPebased bone biomaterials, Ca is widely applied in bone regeneration, orthopedics, and dentistry [67].

Biomaterial-induced microenvironment and host reaction Chapter | 2

115

Ca plays a significant role in bone dynamic as it is an essential component of bone tissue. It is proved that extracellular Ca2þ enhanced proliferation, osteogenic differentiation and chemotaxis ofBMSCs via activating calcium sensor receptor (CaSR) [68]. In addition to CaSR, store operated calcium entry and voltage gated Ca2þ channels can also mediate the extracellular Ca2þ entry into osteoblast and osteogenic differentiation of osteoprogenitors [69e71]. In addition, it is reported that the decreased extracellular Ca2þ concentration can lead to the upregulation of Wnt signaling pathway and enhanced new bone formation [72]. According to the similar chemical component with natural bone, various calcium-containing biomaterial including CaP and calcium silicate are widely applied for BTE. For instance, TCP (b-TCP) has been well recognized as an osteoconductive bone substitution and is used for bone augmentation. b-TCP can promote the production of RANKL and Wnt in osteocyte, which enhances the degradation of material by osteoclast and osteogenesis by osteoblastic cells respectively, and the release Ca2þ plays an important role in this process [73]. Nevertheless, the participation of Ca in certain inflammatory signaling pathways is well documented. It is reported that Ca2þ regulates inflammation through the noncanonical Wnt 5A/Ca2þ signaling pathway [74]. In general, the Wnt 5A/Ca2þ signaling pathway is known to enhance inflammation, and Wnt 5a is recognized as an inflammatory marker. However, high concentrations of extracellular Ca2þ have been found to promote the secretion of Wnt 5a by activating the CaSR signaling pathway, thereby reducing the expression of TNF-a by inhibiting NF-kB and decreasing TNFR1 via the Wnt 5a/Ror2 signaling pathway, finally relieving inflammation response [74,75]. This proves that Ca2þ can modulate the osteoimmune microenvironment surrounding implanted bone biomaterial. Some studies have proved the beneficial osteoimmunomodulatory properties of Ca2þ in bone regeneration. When exposed to b-TCP extracts, macrophage switched toward the M2 phenotype, and the induced osteoimmune microenvironment enhanced the osteogenic differentiation of BMSCs, which was related to the activation of CaSR by Ca2þ [76]. Moreover, the application of a Ca2þ-containing biomaterial coating (b-TCP, etc.) can endow Mg scaffold with favorable osteoimmunomodulatory properties, creating an immune microenvironment that facilitates osteogenesis [77]. In conclusion, Ca is an important mineral element in natural bone tissue, and Ca-containing bone substitutions perform well in clinical application. It elicits significant modulation of the osteimmune environment, thus regulating bone regeneration.

2.1.2.5 Silicon Silicon (Si) is a metalloid element (number 29 in the periodic table) and almost always exists as a complex silicate or silica rather than as an element

116 Bioactive Materials for Bone Regeneration

due to its great affinity for oxygen. It is the third most abundant trace element in the human body derived predominantly from diet and has a close relationship with connective tissues [78,79]. Maintaining sufficient dietary silicon intake is important for health maintenance. A number of studies have proved the important role of Si in bone formation. Si is involved in bone calcification during the early mineralization stage of bone regeneration and could promote osteoblast proliferation, extracellular matrix (ECM) synthesis, ALP activity, and OC synthesis [80,81]. Si-containing biomaterials such as bioceramics bioactive glass and coatings, which can slowly release Si ions, have showed significant regulatory effects on the proliferation and differentiation of osteoblastic cells [82e84]. Si may also elicit a significant influence on immune reaction. It is well known that the silica particle is closely related to pneumoconiosis involved with immune reaction such as the recruitment of polymorphonuclear leukocytes and macrophages and the production of proinflammatory cytokines [85]. Si could inhibit the immune reaction of monocytes by suppressing the activity of succinic dehydrogenase [86]. It was reported that Si based quantum dots triggered an inflammatory response in human lung fibroblasts and caused the production of the proinflammatory cytokines IL-6 and IL-8, which are responsible for stimulating the production of not only NO but also MMPs [87]. The immune-stimulatory effects of Si may have a close relationship with the particle size of materials. Study has shown that nanometer-sized silica caused less fibrogenic reaction than micrometer-sized silica, probably because nanoparticles tend to be diffused and translocated due to their ultrafine particle size compared with that of microsized particles [88]. All of these suggest the potential effect of Si on modulating the osteoimmune environment to regulate bone dynamics. Until now, however, no direct research been reported on Si ionemediated osteoimmunomodulation. To sum up, Si is an important trace element for humans and is especially associated with connective tissue formation. It possesses the ability to enhance bone calcification and facilitate osteogensis, which can be applied in bonesubstitution materials to promote bone formation. Potential immunomodulatory effects enable Si to be applied as a promising osteoimmunomodulatory agent. To achieve this aim, further research is required to evaluate its dosedependent osteoimmunomodulatory effect to obtain the optimum dose range.

2.1.2.6 Cobalt Co is a relatively stable metallic element located in the eighth group of the fourth cycle on the periodic table. Co-based alloys, such as Ca-chromiummolybdenum (Mo), have excellent corrosion and biodegradation resistance and mechanical properties and have been widely used in biomedical research and clinical applications [89,90]. Co is also an essential trace element in the human body and an important component of Vitamin B12. It participates in the

Biomaterial-induced microenvironment and host reaction Chapter | 2

117

process of human hematopoiesis and promotes erythropoiesis, which makes it a drug for treating anemia. Co can potently elicit a hypoxic or hypoxic-like microenvironment to stabilize HIFs [91,92]. HIFs are the primary transcription factors involved in hypoxia that allow cells to adapt to a hypoxic or an inflammatory state. HIF-1 is composed of a and b subunits as a heterodimeric transcription factor, and a major regulatory factor contributing to the adaption of individual cells to a hypoxic environment [93]. In a hypoxic state, HIF-1a subunit stabilizes, dimerizes with HIF-1b, and translocates to the nucleus, causing the activate transcriptional activation of target genes like VEGF, glucose transporter-1, and erythropoietin and finally promoting angiogenesis, erythropoiesis, and anaerobic metabolism [92,94]. In addition, HIFs are reported to contribute to signaling of the Wnt and OSM pathways, both of which benefit osteogenesis. Due to its positive effect in improving the expression of VEGF and angiogenesis, Co has been applied widely in bone-substitute materials to endow them with angiogenic effects. It is found that low amounts of Co (10 mm) [3], as a result, causes the fusion of macrophages to form the multinucleated macrophages, also named FBGCs amd induced mainly by IL-4 and IL-13, were found in clinical trials to have localization of the receptor at the fusion area [44]. Despite of the phagocytosis of pathogen and debris, macrophages and FBGCs can also release active oxygen intermediates (ROIs, oxygen free radicals), some enzymes, acids and other degradation mediators on the surface of cell membranes and biomaterials to reduce their own inhibition [45,46]. However, FBGCs exhibit poor phagocytic abilities though terminally differentiated. The major function of FBGCs is excretion of high levels of lysosomes [47] causing osteolysis and biomaterial degradation [48]. Nevertheless, there is an imaginable limit of material size that even the FBGCs fail to engulf, and thus macrophages undergo “frustrated phagocytosis” during which a series of cytokines, reactive oxygen species (ROS), and proteinases are released contributing to the degradation of the biomaterial [3] (Fig. 3.2). The last stage of host responses to most implanted biomaterials is generally a fibrous capsule or fibrosis. The VEGF produced by M2 macrophages, FBGCs, and other cells can activate fibroblasts to synthesize collagen. As a result, prolongation of fibroblast activation and excessive biomass-relevant matrix deposition can be performed [49,50]. Then a cascade follows such as regeneration of connective tissues and remodeling of organs on the biomaterial surface, leading to the wound-healing process [22]. However, activation of macrophages not only contributes to the phagocytosis of debris and pathogens, but also can lead to some adverse effects for

A bone regeneration concept Chapter | 3

191

FIGURE 3.2 Classification and the development of the macrophage.

192 Bioactive Materials for Bone Regeneration

bone regeneration. The most common type is aseptic loosening, driven by a certain kind of particle, which causes the release of TNF-a from the macrophages. As a result, osteolysis may be present due to activation of osteoclasts by TNF-a. In this aspect, the participation of macrophages must be reduced in order to a better prognosis [51].

3.1.3 T cells and B cells in bone-relevant physiological and pathological processes The lymphocyte population is composed of T lymphocytes (T cells), B lymphocytes (B cells), and NK cells. There are many response mechanisms to stimulation. After foreign implantation in the body, lymphocytes perform a vital role in immune response. From their initial response to biomaterials, lymphocytes can release inflammatory mediators according to different subtypes [52].

3.1.3.1 T lymphocyte activation and features of immunological function T lymphocytes, which account for the largest percentage, are divided into cytotoxic (CD8þ) and T-helper (CD4þ) subpopulations. CD8þ cells destroy cells in a similar manner as that of NK cells. CD4þ T cells are further separated into type 1 (Th1) that secretes IFN-g and TNF-b and the type 2 (Th2) T helper that produces the IL-4, IL-5, IL-10, and IL-13 subsets [53]. These T cells can directly or indirectly communicate and guide other cell types through soluble factors (i.e., cytokines), thus inducing a variety of responses, which are also known as juxtacrine (cellecell) and paracrine (cytokineecell) interactions, activating the adhesion of macrophages and fusion into FBGCs. As for the process of T-lymphocyte functioning, activation begins with APCs, such as macrophages, that provide antigen-specific signals (signal 1), costimulation (signal 2), and surrounding cytokine milieu (signal 3) in the condition of MHC class II. These signals include both biochemical and physical abilities, stimulating T lymphocytes. As a result, a synapse between T lymphocytes and APCs is formed and is available for a series of combinations of ligands and receptors: the TCR/CD3 or CD4 with peptide-bearing major histocompatibility complex and CD28 binding to CD80/CD86 [54]. In addition, macrophages can directly drive cellecell interactions with lymphocytes without antigen, inducing the formation of different subtypes: Th1 and Th2 lymphocytes [55]. Th1 polarization is associated with a proinflammatory response, while Th2 polarization is associated with regulatory, anti-inflammatory, wound-healing, and constructive remodeling

A bone regeneration concept Chapter | 3

193

responses [56]. In the acute inflammatory phase of host response, Th1 cells induce inflammation against foreign antigens and promote the release of IL2, IFN-g, and TNF-b, which are of significance to the activation of macrophage. Meanwhile, IFN-g, also released by NK cells and CD8þ Th1 cells, acts as an inducer of Th1 as well as IL-12. Furthermore, IL-4 is stimulated by the presence of IFN-g via the STAT1 pathway, promoting the formation and activation of Th2 cells. Th2 cells contribute to a strong immunoglobulin E response that induces the stimulation of eosinophils and mast cells against infection [57]. Thus a series of cell factors, such as IL-5, IL-6, IL-9, IL-10, and IL-13, are released by the Th2 cells, which build up the vital function of defense against foreign body infection. IL-4 and IL-13 can be inducers of fusion of biomaterial-adherent macrophages into FBGCs. Th2 cells also contribute to B-cell differentiation and are involved in the identification of foreign substances and producing antibodies against microbes and antigens [58]. Furthermore, other types of T lymphocytes gradually go into the sight of studies because effective inflammation and immunity are performed. TH17 lymphocyte is one subtype able to release IL-17 and IL-22, mediating the production of GM-CSF and leading to differentiation of monocytes and macrophages [59]. In addition, T lymphocytes can suppress immunity by the participation of regulatory T cells, another subtype of T cells that promotes inhibition via reducing IL-2 [60]. However, CD8þ T cells, the cytotoxic T cells producing interferon-g/tumor necrosis factorea, inhibit osteogenic differentiation according to strengthened regeneration of depletion of CD8þ cells in studies [61]. Specifically, T lymphocytes cannot be activated by synthetic nonbiodegradable biomaterials [54]. Despite the proinflammatory function of T lymphocytes and cytokines, the effect after implantation also involves the regulation of bone regeneration. In a study, IL-12 and IL-23 are controlled by IL-12P40 depletion in mice, which is an inducer of both cytokines in response to the implanted biomaterial. As a result, the level of IL-12 and IL-23are decreased in the IL-12p40/ mice. Furthermore, the level of IFN-g and IL-17, which are upregulated by the IL-12 family and contribute to the apoptosis of bone marrow mesenchymal stem cells, is reduced as well with a better bone regeneration in these mice. What is interesting is that, a large amount of immune cellsdresident macrophages, dendritic cells (DCs), T cells, and B cellsdaccumulate to the damaged tissue when severe inflammation occurs in the body, which are able to release a large number of proinflammatory cytokines, such as IL-12 and IL-23. Therefore, the inhibition of immune cells like lymphocytes that release excessive amount of these cytokines and the depletion of IL-12p40 may be a potent method of promoting the bone repair after implantation. [62].

194 Bioactive Materials for Bone Regeneration

In addition, T lymphocyte and macrophage interaction not only involves activation of each other but also induces suppression of the immune effect, a way to establish peripheral tolerance. IL-10 acts as an antiinflammatory cytokine capable of inducing antigen specific disability of CD4þ T cells. Similarly, TNF-a can reduce T-cell response as well as apoptosis [63].

3.1.3.2 B-lymphocyte response to antigens B cells are involved in the recognition of foreign substances and producing antibodies for the elimination of the antigens [64]. Resting B cells can be stimulated by the existence of macrophages and activated T cells, leading to the activated form of B lymphocytes [65]. B lymphocytes have a potent function in immune homeostasis. They can produce cytokines like IFN-g, IL-6, IL-10, which regulates other immune cells in the host response; they are in charge of the wound healing, lymphoid tissue organogenesis, tumor immunity, and so on; also, the costimulation and antigen presenting of T lymphocytes can be induced by the B lymphocytes, which also regulates the balance of Th1/Th2 cytokines [66]. In addition, B lymphocytes have autoregulation of immunity via regulatory B cells, which promotes the release of IL-10, inhibiting proinflammatory cytokines and supporting regulatory T-cell differentiation [67]. In conclusion, lymphocytes account for a great portion of the immunity of foreign biomaterial, indicating close correlation with macrophages of various subtypes in both direct and indirect interaction. A huge number of cytokines are promoted in this process to initiate the immunity function, which has been widely studied.

3.1.4 Dendritic and natural killer cells in bone-relevant physiological and pathological processes DCs are important antigen-presenting cells in the process of immunogical response after implantation of biomaterials, derived from peripheral blood mononuclear cells in the stimulating of granulocyte macrophage colonystimulating factor and IL-4 [68], which are classified by plasmacytoid DCs, traditional DCs, Langerhans cells, etc. In the host response, immature DCs transfer into mature DCs under the stimulation of pathogen-associated molecular patterns (PAMPs), by a series of substances of the biomaterialsd extracellular matrix (ECM) proteins and integrinsdand activated mainly by the innate immune receptor, TLR, that modulates specific lymphocytic responses [69e71]. As for lymphocytes affected by DCs, naive T cells can be polarized to Th1 by IL-12 expressed by DCs, and Th2 cells are induced by interferon

A bone regeneration concept Chapter | 3

195

regulatory factor 4 in DC initiation. Th17 can also be promoted by transforming growth factor-b (TGF-b), IL-23, and IL-1b. After the recognition of the immune receptor and microbial structure, the synapse between the antigen-presenting cells (DCs) and T lymphocytes begins to form, leading to a series of signals between them including (1) adhesive mediators (e.g., b1 and b2 integrins), (2) “signal one” of the peptideeMHC complex and T-cell receptor (TCR), (3) “signal two,” which constitutes costimulatory molecules on the DC (e.g., CD80, CD86, CD40) that connect their respective receptors to the surfaces of T cells (e.g., CD28 and CD40L), and (4) “signal three” or the “polarizing” signal, like IL-12 [71,72]. B cells as well are activated by DCs, to a certain extent, by the release of cytokines and transportation of antigens to give cell-bound signals with or without the participation of CD40 [73e75]. The activation of natural killer cells is also regulated by DCderived IL-27 [76]. In conclude, DCs are critical in the host response of biomaterial, also, can infiltrate into the biomaterial as the macrophages, and be activated and release different cytokines dependent to the material of implantation [77]. On the other hand, not all biomaterials lead to the strong activation of DCs [78]. DCs are closely relevant to macrophages as well. Since macrophages release plenty of cytokines, they influence the activation and suppression of DCs. As we know, macrophages can release IL-6 and IL-1b along with MMPmediated release of ECM fragments and TLR ligands, which are essential for the activation of DCs. However, the release of IL-10 and TGF-b resists the activation of DCs, functioning as controlling feedback [71] (Fig. 3.3). NK cells represent crucial effectors of congenital immunity in the protection of an individual from microbes and are innate immune cells whose function is critical in the first-line of defense, recognizing pathogens by many types of receptors, such as TLR. In turn, NK cells can express different functions of TLRs (TLR-2, TLR-3, TLR-7, TLR-8, TLR-9 .) and are responsible for different recognitions such as LPS, bacterial lipoproteins and lipoteichoic acids, double-stranded RNA, single-stranded RNA, and so on, also known as PAMPs [79]. Also, NK cells have a close relationship with DC function. Myeloid DCs, specialized APCs, have been proved to be involved in promoting cytokine production and cytotoxicity of NK cells. In addition, natural interferon-producing cells/plasmacytoid DCs play an additional role in NK cell activation. In turn, IFN-g is induced by NK cells to regulate the function of DCs, further promoting DC-mediated Th1 priming and the release of proinflammatory cytokines [80,81]. Moreover, Th1 and cytotoxic T lymphocyte can also be activated by the presence of NK cells, resulting in a stronger response to CD4þ/CD8þ cells [80]. Thus, the cross talk of NKs with other types of immune cells is of great significance in the process of host response.

196 Bioactive Materials for Bone Regeneration

FIGURE 3.3 Process of dendritic cells activition by the biomaterial implantation.

References [1] H.T. Aiyelabegan, E. Sadroddiny, Fundamentals of protein and cell interactions in biomaterials, Biomed. Pharmacother. 88 (Complete) (2017) 956e970. [2] S. Zeeshan, B. Patricia, B. Oriyah, et al., Macrophages, foreign body giant cells and their response to implantable biomaterials, Materials 8 (9) (2015) 5671e5701. [3] J.M. Anderson, A. Rodriguez, D.T. Chang, Foreign body reaction to biomaterials, Semin. Immunol. 20 (2) (2008) 86e100. [4] S. Franz, S. Rammelt, D. Scharnweber, et al., Immune responses to implants e a review of the implications for the design of immunomodulatory biomaterials, Biomaterials 32 (28) (2011) 6692e6709. [5] M. Kenny, J. Lin-Hua, F. Richard, et al., Immunological responses to total hip arthroplasty, J. Funct. Biomater. 8 (3) (2017) 33. [6] A. Uzman, B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, P. Walter, Molecular biology of the cell (fourth ed.), Biochem. Mol. Biol. Educ. 31 (4) (2010) 212e214. [7] A.H. Morris, D.K. Stamer, T.R. Kyriakides, The host response to naturally-derived extracellular matrix biomaterials, Semin. Immunol. (February 2017) 72e91. S1044532316301105. [8] J.M. Anderson, Biocompatibility and bioresponse to biomaterials, Principles of Regenerative Medicine (2008) 704e723. [9] M.B. Gorbet, M.V. Sefton, Biomaterial-associated thrombosis: roles of coagulation factors, complement, platelets and leukocytes, Biomaterials 25 (26) (2004) 5681e5703.

A bone regeneration concept Chapter | 3 [10] [11]

[12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]

[26] [27] [28] [29] [30] [31]

197

P.R.T. Kuzyk, E.H. Schemitsch, The basic science of peri-implant bone healing, Indian J. Orthop. 45 (2011) 108e115, https://doi.org/10.4103/0019-5413.77129. S. Al-Maawi, A. Orlowska, R. Sader, et al., In vivo, cellular reactions to different biomaterialsdphysiological and pathological aspects and their consequences, Semin. Immunol. (2017). S1044532317300118. J.R. Alhamdi, P. Tao, I.M. Al-Naggar, et al., Controlled M1-to-M2 transition of aged macrophages by calcium phosphate coatings, Biomaterials (2018). S0142961218304897. B. Nilsson, K.N. Ekdahl, T.E. Mollnes, et al., The role of complement in biomaterialinduced inflammation, Mol. Immunol. 44 (1) (2007) 82e94. R.O. Hynes, Integrins: bidirectional, allosteric signaling machines, Cell 110 (6) (2002) 673e687. Z. Julier, A.J. Park, P.S. Briquez, et al., Promoting tissue regeneration by modulating the immune system, Acta Biomater. (15 April 2017) 13e28. S1742706117300661. T.A. Wilgus, S. Roy, J.C. Mcdaniel, Neutrophils and wound repair: positive actions and negative reactions, Adv. Wound Care 2 (7) (2013) 379e388. S.D. Kobayashi, J.M. Voyich, C. Burlak, et al., Neutrophils in the innate immune response, Arch. Immunol. Ther. Exp. 53 (6) (2005) 505e517. G.C. Gurtner, S. Werner, Y. Barrandon, et al., Wound repair and regeneration, Wound Repair Regen. 11 (6) (2010) 5Ae8A. F. Geissmann, S. Jung, D.R. Littman, Blood monocytes consist of two principal subsets with distinct migratory properties, Immunity 19 (1) (2003) 0e82. G. Broughton, J.E. Janis, C.E. Attinger, The basic science of wound healing, Plast. Reconstr. Surg. 117 (Suppl. ment) (2006) 12Se34S. A. Celada, Evidence for a gamma-interferon receptor that regulates macrophage tumoricidal activity, J. Exp. Med. 160 (1) (1984) 55e74. C. Townsend, R.D. Beauchamp, B.M. Evers, et al., Sabiston Textbook of Surgery[M]// Sabiston Textbook of Surgery, 2004. M. Waters, P. Vandevord, M. Van Dyke, Keratin biomaterials augment anti-inflammatory macrophage phenotype in-vitro, Acta Biomater. (15 January 2018) 213e223. S1742706117306700. T.A. Wynn, K.M. Vannella, Macrophages in tissue repair, regeneration, and fibrosis, Immunity 44 (3) (2016) 450e462. K. Garg, N.A. Pullen, C.A. Oskeritzian, et al., Macrophage functional polarization (M1/M2) in response to varying fiber and pore dimensions of electrospun scaffolds, Biomaterials 34 (18) (2013) 4439e4451. Z. Guoying, G. Thomas, Host responses to biomaterials and anti-inflammatory design-a brief review, Macromol. Biosci. (2018) 1800112. S.A. Eming, T. Krieg, J.M. Davidson, Gene therapy and wound healing, Clin. Dermatol. 25 (1) (2007) 79e92. M.T. O’Hollaren, A review of middleton’s allergy: principles and practice, 6th edition, with online updates, Medscape Gen. Med. 7 (2005). D.M. Mosser, J.P. Edwards, Exploring the full spectrum of macrophage activation, Nat. Rev. Immunol. 8 (12) (2008) 958e969. F.O. Martinez, A. Sica, A. Mantovani, et al., Macrophage activation and polarization, Front. Biosci. 13 (2) (2008) 453e461. M.A, Al E. Lucas, The relationship of fibrinogen structure to plasminogen activation and plasmin activity during fibrinolysis, Ann. N. Y. Acad. Sci. 408 (1) (2010) 71e91.

198 Bioactive Materials for Bone Regeneration [32] Sequential delivery of immunomodulatory cytokines to facilitate the M1-to-M2 transition of macrophages and enhance vascularization of bone scaffolds, Biomaterials 37 (2015) 194e207. [33] R. Klopfleisch, Macrophage reaction against biomaterials in the mouse model e phenotypes, functions and markers, Acta Biomater. (1 October 2016) 3e13. S1742706116303191. [34] H.B. Cohen, D.M. Mosser, Extrinsic and intrinsic control of macrophage inflammatory responses, J. Leukoc. Biol. 94 (5) (2013) 913e919. [35] P.M. Kou, J.E. Babensee, Macrophage and dendritic cell phenotypic diversity in the context of biomaterials, J. Biomed. Mater. Res. A 96A (1) (2015) 239e260. [36] K.R. Nakazawa, B.A. Walter, D.M. Laudier, et al., Accumulation and localization of macrophage phenotypes with human intervertebral disc degeneration, Spine J. 18 (2) (February 2018) 343e356. [37] F.O. Martinez, S. Gordon, The M1 and M2 paradigm of macrophage activation: time for reassessment, F1000 Prime Rep. (2014) 6. [38] R.B. Nussenblatt, R.W.J. Lee, E. Chew, et al., Immune responses in age-related macular degeneration and a possible long-term therapeutic strategy for prevention, Am. J. Ophthalmol. 158 (1) (2014), 5-11.e2. [39] V. Lee, Q. Cao, Y. Wang, et al., Role of macrophages in renal injury, repair and regeneration, Reg. Nephrol. (2011) 125e139. [40] K. Liu, C. Mohan, What do mouse models teach us about human systemic lupus erythematosus? Syst. Lupus Erythematosus (2016) 265e271. [41] N. Kawakami, Live imaging of effector cell trafficking and autoantigen recognition within the unfolding autoimmune encephalomyelitis lesion, J. Exp. Med. 201 (11) (2005) 1805e1814. [42] D.C. Ifrim, J. Quintin, L.A.B. Joosten, et al., Trained immunity or tolerance: opposing functional programs induced in human monocytes after engagement of various pattern recognition receptors, Clin. Vaccine Immunol. Cvi 21 (4) (2014) 534. [43] Z. Xia, J.T. Triffitt, A review on macrophage responses to biomaterials, Biomed. Mater. 1 (1) (2006) R1. [44] K.M. Defife, C.R. Jenney, A.K. Mcnally, et al., Interleukin-13 induces human monocyte/ macrophage fusion and macrophage mannose receptor expression, J. Immunol. 158 (7) (1997) 3385. [45] P.M. Henson, The immunologic release of constituents from neutrophil leukocytes. II. Mechanisms of release during phagocytosis, and adherence to nonphagocytosable surfaces, J. Immunol. 107 (6) (1971) 1547e1557. [46] P.M. Henson, The immunologic release of constituents from neutrophil leukocytes. I. The role of antibody and complement on nonphagocytosable surfaces or phagocytosable particles, J. Immunol. 107 (6) (1971) 1535. [47] B. Burke, C.E. Lewis, The Macrophage, Oxford University Press, Oxford, UK, 2002. [48] E. Solheim, B. Sudmann, G. Bang, E. Sudmann, Biocompatibility and effect on osteogenesis of poly(orthoester) compared to poly(dl-lactic acid), J. Biomed. Mater. Res. 49 (2000) 257e263. [49] W.K. Ward, J. Diabetes Sci. Technol. 2 (2008) 768. [50] R.F. Diegelmann, M.C. Evans, Wound healing: an overview of acute, fibrotic and delayed healing, Front. Biosci. 9 (71) (2003) 283e289. [51] E. Ingham, J. Fisher, The role of macrophages in osteolysis of total joint replacement, Biomaterials 26 (11) (2005) 1271e1286.

A bone regeneration concept Chapter | 3 [52]

[53]

[54] [55]

[56] [57] [58] [59] [60] [61]

[62] [63] [64]

[65]

[66] [67] [68]

[69] [70] [71]

199

D.T. Chang, E. Colton, J.M. Anderson, Paracrine and juxtacrine lymphocyte enhancement of adherent macrophage and foreign body giant cell activation, J. Biomed. Mater. Res. A 89 (2) (2009) 490e498. D.T. Chang, E. Colton, T. Matsuda, et al., Lymphocyte adhesion and interactions with biomaterial adherent macrophages and foreign body giant cells, J. Biomed. Mater. Res. A 91A (4) (2009) 1210e1220. A. Rodriguez, J.M. Anderson, Evaluation of clinical biomaterial surface effects on T lymphocyte activation, J. Biomed. Mater. Res. A 92A (1) (2010) 214e220. W.G. Brodbeck, M. Macewan, E. Colton, H. Meyerson, J.M. Anderson, Lymphocytes and the foreign body response: lymphocyte enhancement of macrophage adhesion and fusion, J. Biomed. Mater. Res. A 74 (2005). T.J. Keane, S.F. Badylak, The host response to allogeneic and xenogeneic biological scaff;old materials, J. Tissue Eng. Regenerat. Med. 9 (5) (2015) 504e511. C.A. Thaiss, N. Zmora, M. Levy, et al., The microbiome and innate immunity, Nature 535 (7610) (2016) 65e74. J.R. McGhee, The world of Th1/Th2 subsets: first proof, J. Immunol. 175 (1) (2005) 3e4. L. Wu, S. Ong, M.V. Talor, et al., Cardiac fibroblasts mediate IL-17A-driven inflammatory dilated cardiomyopathy, J. Exp. Med. 211 (7) (2014) 1449e1464. P.C. Schr?Der, S. Illi, V.I. Casaca, et al., A switch in regulatory T cells through farmexposure during immune maturation in childhood, Allergy (2016) 604e615. S. Reinke, S. Geissler, W.R. Taylor, et al., Terminally differentiated CD8þ T cells negatively affect bone regeneration in humans, Sci. Transl. Med. 5 (177) (2013), 177ra36177ra36. J. Xu, Y. Wang, J. Li, et al., IL-12p40 impairs mesenchymal stem cell-mediated bone regeneration via CD4þ T cells, Cell Death Differ. 23 (12) (2016) 1941e1951. J.M. Anderson, A.K. Mcnally, Biocompatibility of implants: lymphocyte/macrophage interactions, Semin. Immunopathol. 33 (3) (2011) 221e233. D.T. Chang, J.A. Jones, H. Meyerson, et al., Lymphocyte/macrophage interactions: biomaterial surface-dependent cytokine, chemokine, and matrix protein production, J. Biomed. Mater. Res. A 87 (3) (2008) 676e687. N.J. Hallab, M. Caicedo, R. Epstein, et al., In vitro reactivity to implant metals demonstrates a person-dependent association with both T-cell and B-cell activation, J. Biomed. Mater. Res. A 92A (2) (2010), 0-0. T.W. Lebien, T.F. Tedder, B lymphocytes: how they develop and function, Blood 112 (5) (2008) 1570e1580. C. Mauri, A. Bosma, Immune regulatory function of B cells, Annu. Rev. Immunol. 30 (1) (2012) 221e241. S.P. Shankar, J.E. Babensee, Comparative characterization of cultures of primary human macrophages or dendritic cells relevant to biomaterial studies, J. Biomed. Mater. Res. A 92A (2) (2010) 791e800. P. Matzinger, An innate sense of danger, Semin. Immunol. 961 (1) (2010) 341e342. P.S. Hume, J. He, K. Haskins, et al., Strategies to reduce dendritic cell activation through functional biomaterial design, Biomaterials 33 (14) (2012) 3615e3625. C.A. Leifer, Dendritic cells in host response to biologic scaffolds, Semin. Immunol. (February 2017) 41e48. S1044532316300835.

200 Bioactive Materials for Bone Regeneration [72] J. Banchereau, R.M. Steinman, Dendritic cells and the control of immunity, Nature 392 (6673) (March 19, 1998) 245e252. [73] L. Song, G. Dong, L. Guo, et al., The function of dendritic cells in modulating the host response, Mol. Oral Microbiol. (2017) 13e21. [74] M. Wykes, A. Pombo, C. Jenkins, et al., Dendritic cells interact directly with naive B lymphocytes to transfer antigen and initiate Class switching in a primary T-dependent response, J. Immunol. 161 (3) (1998) 1313e1319. [75] M. Wykes, G. Macpherson, Dendritic celleB-cell interaction: dendritic cells provide B cells with CD40-independent proliferation signals and CD40-dependent survival signals, Immunology 100 (1) (2010) 1e3. [76] J. Wei, S. Xia, H. Sun, et al., Critical role of dendritic cell-derived IL-27 in antitumor immunity through regulating the recruitment and activation of NK and NKT cells, J. Immunol. 191 (1) (2013) 500e508. [77] B.G. Keselowsky, J.S. Lewis, Dendritic cells in the host response to implanted materials, Semin. Immunol. 29 (2017). [78] S.F. Badylak, Xenogeneic extracellular matrix as a scaffold for tissue reconstruction, Semin. Cell Dev. Biol. 13 (5) (2004) 377e383. [79] S. Akira, S. Uematsu, O. Takeuchi, Pathogen recognition and innate immunity, Cell 124 (2006) 783e801. [80] P. Kalinski, R.B. Mailliard, A. Giermasz, et al., Natural killeredendritic cell cross-talk in cancer immunotherapy, Expert Opin. Biol. Ther. 5 (10) (2005) 1303e1315. [81] M. Della Chiesa, E. Marcenaro, S. Sivori, et al., Human NK cell response to pathogens, Semin. Immunol. 26 (2) (2014) 152e160.

A bone regeneration concept Chapter | 3

201

Chapter 3.2

Biomaterials and their degradation products in the immune microenvironment and regeneration 3.2.1 Metallic implants, the immune microenvironment, and regeneration The use of metals in treatment can be traced back several centuries. In the 20th century, stainless steel and cobaltechromium alloys were the first metal materials successfully used in orthopedics [1]. Ti and Ti alloys were introduced in the 1940s. Metallic biomaterials are mainly used in weight-bearing systems, such as hip and knee prostheses and for the fixation of internal and external bone fractures. At present, Ti-based and CoeCr-based alloys, because they exhibit excellent mechanical and anticorrosion properties, are widely used in surgery. Although they have strong anticorrosion properties, implants and their degradations still can be part of the immune microenvironment and generate proinflammatory response and aseptic loosening.

3.2.1.1 Immune response to titanium implants Titanium is widely used as an orthopedic implant material. Its advantages include high biocompatibility, increased resistance to corrosion, lack of toxicity on macrophages and fibroblasts, and diminished inflammatory response in peri-implant tissues. Its surface is composed of an oxide layer that provides the ability to repair itself by reoxidation when damaged [2,3]. Although Ti has better corrosion resistance, implantation can still generate Ti particles and ions that have an impact on the immune microenvironment. Study indicates that the complement level around Ti changes from a response to traumatic injury (related to C3) to a more reparative phase (more related to C5 and ARG1) [4]. Some in vitro studies have addressed the effect of Ti microparticles on bone remodeling. Osteoblasts incubated with very high concentrations of Ti microparticles (30,000 mg/L) for 48 h led to increased production of osteoclastogenesis factors, such as receptor activator of nuclear factor kappa-B ligand (RANKL) and CSF-1 [5].

3.2.1.1.1 Macrophage role in immune response to titanium implants In macrophages, Ti ions appear to increase the production of proinflammatory cytokines (IL-1b, IL-6, and TNF-a) with concomitant downregulation of

202 Bioactive Materials for Bone Regeneration

TGF-b expression [6]. Similar to other types of particles, Ti microparticles may be phagocytosed by macrophages. This process is reported to involve scavenger receptors [7]. Exposure to titanium alloy (Ti6Al4V) led to higher concentrations of those cytokines: IL-1b, IL-a, MCP-1, GM-CSF, and IL-10 [8]. Concentration of Ti particles is critical, as IL-1b production was found to be concentration-dependent [9]. Moreover, the interaction between Ti microparticles and macrophages involves cellular receptors TLR-2, TLR-3, TLR-4, and TLR-9, and the intracellular adaptor myeloid differentiation factor 88 (MyD88), TIR-domain-containing adapter-inducing interferon-b and NF-k B, whose involvement in particle-induced inflammation has been reported. It is known that activation of NF-kB increases secretion of proinflammatory cytokines, such as TNF-a, IL1-b, and IL-6 [10].

3.2.1.1.2 T cell and dendritic cell roles in immune response to titanium implants Ti ions activate T cells, resulting in increased expression of CD69, CCR4, and RANKL in a concentration-dependent manner [11,12]. Recent in vitro experiments have shown that Ti4þions promote adaptive immune responses; the authors show that human DCs exposed to Ti4þ can select specific T cells targeting Ti4þ while these T cells do not respond to Ti3þ ions [13]. Ti 4þ alters DC properties, resulting in strengthened T lymphocyte reactivity and deviation toward a Th1-type immune response [14]. Ti-induced delayed hypersensitivity may occur due to long-term exposure to Ti ions or TiO2 particles. 3.2.1.1.3 Influence of implant surface characteristics on immune response to titanium implants The interaction of early cell and tissue with titanium implants is very important, especially the interface structure. Research shows that nanotopography downregulates the inflammatory process and strengthens the osteogenic response during the early stage of osseointegration. With nanotopography, the results showed downregulated expression of MCP-1 and a relatively lower number of recruited CD68-positive macrophages [15]. Also, titanium with a hydrophilic rough surface induced macrophage activation similar to that of the antiinflammatory M2-like state, increasing IL-4 and IL-10 levels. The synergistic effect of hydrophilicity strengthening and roughness strengthening can form a microenvironment suitable for shortening healing time and enhancing osseointegration, which may lead to a higher level of implant success [16]. In addition, compared with planar titanium, rough surface titanium strengthens endothelial cell response (monocyte adhesion, migration, and nitric acid/endothelin-1 secretion) of nano-rough titanium to submicron-rough titanium. Compared with the planar surface, macrophage adhesion and proinflammatory cytokine release on nano- and submicron titanium surfaces decreased [17].

A bone regeneration concept Chapter | 3

203

3.2.1.1.4 Aseptic loosening in titanium implants Aseptic implant loosening is the main complication. It is caused by wear debris and characterized by osteolysis at the boneeprosthesis interface [18]. Studies have shown that titanium debris can destroy the balance between bone formation and bone resorption in two ways: directly by differentially activating osteoclasts and osteoblasts [20] or indirectly by Ti ions forming particles that can be used as secondary stimuli to activate cytokines produced by inflammatory macrophages and lymphocytes, such as release of IL-1b from human macrophages [19]. IL-1b released from macrophages can induce inflammation and lead to imbalance in host response by coacting infections. IL-1b is one of the most effective cytokines and initiates inflammation by a long chain of effects: it promotes the migration of white blood cells from the blood to infected tissues; it induces fever and increased expression of adhesion molecules; and it induces the expression of genes encoding IL-6 and nitric oxide synthase [21e23]. In addition, bone marrow stromal/stem cells (BMSCs) exposed to Ti microparticles showed impaired osteogenic differentiation and increased production of IL-8 [24], a chemokine that is involved in the recruitment of neutrophils and overexpressed in periprosthetic tissues of patients with aseptic loosening [25]. Finally, it causes aseptic loosening of orthopedic implants [26]. But in this case, Patterson and other research companies neutralized the Ti-induced activation and release of IL-1b human macrophages in vitro. Cobaltechromium alloy implants may inhibit inflammation of periimplant macrophages induced by Ti [27]. 3.2.1.1.5 Immune response to cobaltechromium alloy implants Apart from Ti, there are more than one million metal-on-metal hip replacement prostheses made of cobaltechromium alloy worldwide. After surgery, a considerable number of Co and Cr ions are released into synovial fluid. [28]. Co ions stimulate the release of profibrotic signals from macrophages and then promote collagen synthesis and increase the number of alpha-SMA-positive fibroblasts. At first, macrophages induced by Co have a positive influence on bone regeneration [29]. Co and macrophages interact synergistically, affecting the functional properties of fibroblasts and stability of ECM. Study shows that Co and Cr are capable of stimulating migration of T lymphocytes in the absence of cytokines [30]. T lymphocytes are closely relevant to the recurrence of early symptoms including marked joint effusion and the development of osteolysis, which may indicate the possibility of this reaction after initial implantation [31]. 3.2.1.1.6 Aseptic loosening in cobalt-chromium alloy implants With the passage of time, Co and Cr ions are continuously released into periprosthetic tissues, inducing cytotoxicity, apoptosis and necrosis, and

204 Bioactive Materials for Bone Regeneration

FIGURE 3.4 Ti implants regulate immune microenvironment through multiple pathways and involve immune cells(macrophages, DC cell, T cell). During the process immune cells produce different kinds of cytokines.

upregulation of various cytokines and chemokines [32]. This leads to aseptic loosening, pain, implant wear and metal debris adverse reactions, tissue remodeling, and pseudotumor formation [33,34]. The mechanism of these side effects is relevant to accumulation of macrophages around the prosthesis by producing ROS to downregulate the RhoA signal. The presence of macrophages will release a variety of inflammatory mediators, such as IL-1b and TNF-a [35], that can affect ECM turnover and the function of homeostasis and strengthen fibroblasts in tissues [36,37]. In addition to macrophages participating in CoeCr granule (Ps)-induced aseptic loosening, fibroblast-like synovial cells (FLSs) play an important role in osteolysis. De Li et al. found that the MyD88-independent TLR signaling pathway in FLSs mediates CoCr-Ps-induced RANKL expression in fibroblasts [38]. In order to provide better implant biocompatibility, synthetic biodegradable polymer coatings and materials can be used [39].

3.2.2 Inorganic materials implants, immune microenvironment, and regeneration Alumina zirconia and some porous ceramics are commonly used in firstgeneration ceramic biomaterials. The structure of these biomaterials is closely related to their manufacturing processes, resulting in the formulation being limited to certain conditions and having a critical impact on the biological and mechanical properties of the materials, especially the lack of biological activity [40].

A bone regeneration concept Chapter | 3

205

Ceramics and cement, as the most common ceramic materials at present, play an important role in filling bone defects, such as BGs, glass ceramics, and calcium phosphate [41]. Because the structure and surface characteristics of bone mineral phase are similar, the biological activity of bone is better. Although the interface of fibrous connective tissue is not regulated, it can promote its binding to bone [42,43]. In addition, bioactive ceramics show excellent performance in biological compatibility and electrical conductivity.

3.2.2.1 General immune response to bioceramic implants Generally, bioceramics produce a series of subsequent inflammatory characteristics during implantation due to the interaction between immune cells and the material surface, which is always accompanied by surgical injury. These inflammatory features occur after blood protein adsorption and coagulation cascade as well as complement and platelet activation [44,45]. Host inflammatory characteristics, also known as foreign body characteristics, have a fundamental impact on the results of biomaterial implantation [46,47]. As far as we know, implanted monocytes will attach to the surface of the implant and then differentiate into adhesive macrophages, secreting cytokines to participate in other cells that produce inflammatory characteristics, causing mediated inflammation and wound healing [48,49]. Normally, macrophages fuse with each other to produce FBGCs, which only exist on the tissueeimplant surface and release oxygen free radicals, degrading enzymes and acids and resulting in the failure of implant degradation [50,51]. In the process of host response, macrophages are sent to the cortex of the biomaterial to recognize foreign substances. The macrophage is smart enough that once it passes a certain size, it spontaneously coalesces and fuses with the FBGCs [52]. However, fibrocystic cells and FBGCs may change their survival sites with the change of conditions during the formation process and may only exist at the tissue-implant interface, making the implantation plan and regeneration plan fail, while releasing oxygen free radicals, degradation enzymes, and certain acids. Macrophage chemotactic protein-1 is a chemokine that stimulates fusion between FBGCs and induces macrophages to promote cell migration [53]. Macrophage inflammatory protein-1 is also a major cytokine that produces macrophages and plays an important role in immune system response to infection and inflammatory episodes [54]. According to studies, Mg2þ, Ca2þ, and Si2þ inhibit MAPK and NFeB pathways, while caspase stimulates macrophage apoptosis through the pathway, therefore lessening the production of inflammatory cytokines, FBGCs, and fiber tissues and exerting a certain inhibitory effect on silicate biological ceramics (YanHuang et al. 2018) [55].

206 Bioactive Materials for Bone Regeneration

3.2.2.2 Calcium phosphate ceramics in bone regeneration Nowadays, the most commonly used inorganic implant materials are bioactive ceramics and bioactive grass. For bioactive ceramics, there are several types, like hydroxyapatite (HA) [Ca10(PO4)6(OH)2], b-tricalcium phosphate (b-TCP, Ca3(PO4)2), TCPs, a mixture of HA and b-TCP that is called biphasic calcium phosphate (BCP), and others. Although their microstructures are different, their core elements of Ca and P are not changed. As the most representative bioactive ceramics, CaP ceramics have compositions similar to that of natural bone [56]. A Wang J et al. study found that BCP ceramic and its degradation products promoted the secretion of inflammatory cytokines and growth factors from macrophages, and compared with the ionic microenvironment, the degradation particles had a stronger effect. The results of cellular experiments confirmed that the gene expression of most inflammatory factors (IL-1, IL-6, and MCP-1) and growth factors (VEGF, PDGF, and EGF) by macrophages were upregulated to varying degrees by BCP ceramic and its degradation products [57].

3.2.2.2.1 Calcium ion immune microenvironment regulation through inflammation adjustment Calcium ions play a key role in the growth of bones and blood vessels by participating in certain inflammatory signaling pathways, and atypical Wnt5A/ Ca2þ signaling pathways strengthen inflammatory responses (A. De 2011). When Wnt5A binds to Fz5, the Wnt/Ca2þ signaling pathway is activated by Ca2þ/calmodulin (CaM)-dependent protein kinase II (CaM-KII) and protein kinase C, and the downstream inflammatory factor gene is upregulated by the transcription factor NF-kB. CaM-KII interacts with the cyclic AMP-response element binding protein in macrophages, which activates cyclooxygenase-2 to produce the proinflammatory hormone prostaglandin E2 [70]. High concentrations of extracellular calcium have also been found to activate the calciumsensitive receptor (CaSR) signaling cascade, leading to production of Wnt5A, which can reduce TNF-a expression by downregulating TNF-a via the Wnt5A/Ror2 signaling pathway [59]. 3.2.2.2.2 Positive effects of phosphate ions on bone regeneration Phosphate ions are also involved in the regeneration of new bone. Studies have found that phosphate ion degradation products can induce differentiation of stem cells into osteoblasts through absorption of stem cells from these biomaterials by phosphate ions to form important metabolic molecules (e.g., ATP), followed by ATP metabolites (adenosine) that signal stem cells and induce them to differentiate into osteoblasts. Alkaline phosphatase is an exogenous enzyme involved in the degradation of inorganic pyrophosphates and provides sufficient local phosphate concentration for mineralization [60e62]. Therefore, CaP degradation products play an important role in influencing the the microenvironment and bone regeneration [71].

A bone regeneration concept Chapter | 3

207

3.2.2.3 Other ions that regulate the immune microenvironment In recent years, the development of calcium-silica ceramics mainly includes wollastonite (CaSiO3), aqmanite (Ca2MgSiO7), diopside (CaMgeSi2O6), hard titanium (Ca2ZnSi2O7), bredigite (Ca7MgeSi4O16), merwinite (Ca3MgSi2O8), and the like [72]. Therefore, in some studies, zinc has been incorporated into CaP biomaterials to strengthen their osteogenic capacity. Bioactive glass is mainly composed of silica (SiO2), phosphorus pentoxide (P2O5) and calcium oxide (CaO), potassium oxide (K2O) and/or magnesium oxide (MgO) [73]. In addition to the calcium and phosphorus ions released by bioceramics such as Si and Zn, Mg participates in the regulation of the immune microenvironment through different mechanisms.

3.2.2.3.1 Silicon regulation of the immune microenvironment through macrophage inhibition Compared with untreated TCP, MgO/sr-doped TCP promotes bone formation through good early bone remodeling [75]. In vivo, silicon is usually absorbed in the form of metasilicates and widely distributed in connective tissue [74]. Silicon also inhibits the activity of macrophages and osteoclasts [76]. In this in vitro comparative study, the nanocrystallinity of SiHA affects the cell/ biomaterial interface by loss of cell anchorage (anoikis), delayed osteoclast-like cell differentiation (differentiated from original 264.7 cells), and reduction of this cell type [77]. Absorption activity induces osteocyte apoptosis. Composites were incubated with J744A. Mouse macrophages demonstrated that porous silicon did not induce immune response and might even inhibit it [78]. 3.2.2.3.2 Positive effect of silicon on bone regeneration Silicon (Si) is a basic element in the formation of bone and cartilage and is present in the region with the strongest osteogenic activity during bone growth [79e81]. This element is essential for normal development of the glycosaminoglycan network in the ECM [79] and can increase collagen content [82]. Bioactive silicate materials upregulate the expression of VEGF (H participates in the formation of blood vessels and bone) [83]. Some studies have shown that when expression of collagen and proteoglycan, which can synthesize proteins, is activated, activation of bone and transforming growth factor stimulates osteoblast proliferation and differentiation of b1 (TGF-b1 should be undertaken) [84]. The results indicate that higher soluble silicates improve the biochemical and mechanical properties of bones through stimulation of osteoblastogenesis (runt -relevant transcription factors) and inhibition of relevant osteoclastogenesis (peroxidase-activated receptor-g) [85].

208 Bioactive Materials for Bone Regeneration

3.2.2.3.3 Zinc regulation of the immune microenvironment through changes in tumor necrosis factor alpha and interleukin-1 beta levels Zinc (Zn) has a role in promoting bone formation and mineralization [86]. Therefore, zinc has been incorporated into cap biomaterials to strengthen its osteogenic capacity. However, in addition to its positive effect on osteogenesis, it also modulates immune response [65]. Zn-substituted ceramics can increase the release of antiinflammatory cytokine IL-10 while reducing the expression of TNF-a and IL-1b, which may be due to regulatory pathways [66e68]. Zinc Concentration Modulates Immune Cell Response: In addition, zinc stimulated IL-1 beta secretion in a concentration-dependent manner and had no effect on leukocyte IL-6 release (Easier to Peak 120/L) [69]. 3.2.2.3.4 Magnesium regulation of the immune microenvironment by inhibition of the toll-like receptor pathway Bioactive glass is mainly composed of silicon dioxide (SiO2), phosphorus pentoxide (P2O5), and CaO, some of which also contain sodium oxide (Na2O), K2O, and/or MgO. Among the components of bioactive glass, SiO2 and P2O5 are also used as network formers, while alkaline earth metallics (such as MgO or CaO) or alkaline earth metallics (such as Na2O or K2O) act as network modifiers through ion exchange to internal interfaces. The environment is modified. Magnesium (Mg) is a biodegradable and biocompatible metal mechanically similar to bone, thus eliminating the effects of stress shielding and improving in vivo degradation properties [87]. Mg ions inhibit the production of inflammatory cytokines by inhibiting the TLR pathway [44]. Macrophages recognize foreign bodies through the TLR pathway and induce innate immune responses, so Mg ions reduce the degradation and rejection processes of the implant [88]. 3.2.2.3.5 Different immune responses to hydroxyapatite and strontium-doped calcium phosphate Both HA and strontium-doped calcium phosphate (SCP) can cause similar bone formation in trabecular bone defects, but they cause different inflammatory responses and bone remodeling reactions. Current data indicate that HA particles are completely transplanted with TNF-a expression in OVX and non-OVX mice, but only 6 days after early implantation. On the other hand, SCP particles upregulate IL-6 in both early and late stages [89].

A bone regeneration concept Chapter | 3

FIGURE 3.5 Property of biocreamic implants, such as different ions, pore size, particle size etc, can regulate immune cell (macrophages) and related cytokines.

209

210 Bioactive Materials for Bone Regeneration

3.2.2.4 Appropriate pore and particle sizes for influencing the immune microenvironment to strengthen bone regeneration Structurally, natural bones have a three-dimensional structure of multiscale porous structures ranging from nanometers to submicron and microscale, [90] providing a microenvironment for cell and tissue growth. Multiscale porous structures not only provide a large number of binding sites for cell membrane receptors but also determine and maintain cellular functions. Cells can exhibit distinctly different differentiation characteristics by sensing structural information [92]. The pore diameter of several hundred micrometers (150e800 - mm) can provide a transport channel for nutrients and metabolites, which is beneficial to the formation of new bone tissue and blood vessels [91]. The study of submicron structure of tricalcium phosphate (TCP-S) gradient grain size (0.77  0.21 mm TCP-S) triggered the most acceptance of bone formation and absorption compared with TCP ceramics with large crystal particles due to high calcium and phosphate ion release And protein adsorption and phagocytic phagocytic granules [93]. Controlling the particle size of the bioactive glass particles also has a positive effect on bone regeneration. The interfacial reaction of the bioactive glass 45S5 can form two surface layers: an inner silica layer and an outer calcium-rich phosphate layer [95]. The particle size is about twice the thickness of the reaction layer and is completely reactive. These reactions produce a phase change which, in turn, can cause dimensional changes and cause cracks. Macrophages enter the granules through these fissures and help remove internal silica by phagocytosis [42, 94]. What remains is a layer of calcium phosphate shell that was previously formed as a biological reaction product. The osteoprogenitor cells migrate to the protective space of these in situ formed cavities and differentiate into osteoblasts. When the gap between the larger particles is more easily filled, it hinders the repair of the tissue. The size range of the restriction is also important to allow for optimal vascular development [96].

3.2.3 Organic materials implants, immune microenvironment, and regeneration Hydrogels can be used in biodegradable polymer materials for bone repair and regeneration, such as polyethylene glycol, polyvinyl alcohol, polyacrylamide, polylactic acid, and copolymerization thereof [97]. Compared with other biomaterials, these polymers have many advantages, such as extensive mechanical stiffness, controlled degradation rate, hydrogel absorbability, and good fusion with surrounding tissue, thus avoiding the complexity of surgical resection and reducing the possibility of inflammatory reactions [98].

A bone regeneration concept Chapter | 3

211

3.2.3.1 Immune response to ultra-high-molecular-weight polyethylene Ultra-high-molecular-weight polyethylene (UHMWPE), a material used for joint replacement, consists essentially of long-chain hydrocarbons that further cross each other by irradiation sterilization and/or increased wear resistance [99]. PE is considered to be a relatively inert biomaterial, but over time, particle fragments are produced by motion of the implant surface [100]. We have previously reported that the breakdown of UHMWPE also produces short-chain alkane polymers [101]. Side-chain-modified alkane polymers consisting of aldehydes, ketone groups, and hydroxyl groups interact directly through different membrane receptors such as TRLs 1/2, CD11b, and CD14 activation [102] [103]. TLR1/2 contact-induced inflammatory transcriptional programs are induced by the NFkB signaling pathway, pro-IL-1b, and pro-IL-18 [104]. The inflammatory response results in the release of various proinflammatory cytokines (il-1, il-6 TNF-a), growth factors (macrophage colony-stimulating factor-1), and chemokines (MIP-1a/MCP-1) that will eventually lead to systematic recruitment of more macrophage regions [105e108].

3.2.3.2 Ultra-high-molecular-weight polyethylene particle activation of the inflammasome After initial activation, UHMWPE particles are engulfed by local cells, causing instability in the body and activation of inflammatory bodies. It has previously been reported that the immune system considers lysosomal instability/destruction and cathepsin release an endogenous risk signal that induces activation of NALP3 inflammatory bodies [109]. The NALP3 inflammasome is a multicomplex inflammatory cell that regulates caspase-1-dependent processing and secretion of proinflammatory cytokines such as IL-1b) and IL-18 (earthquake). The phagocytic granules released by endosome rupture and cell necrosis are rephagocytosed by other cells, producing a persistent inflammatory state that leads to continuation of the inflammatory process [104].

3.2.3.3 Polylactic acid regulation of the immune microenvironment by macrophage Polylactic acid (PLA) and its copolymers are commonly used for a wide variety of applications. Immune reactions that are targeted against implanted biomaterials can affect their biocompatibility. PLA-based implants can cause inflammatory complications. Macrophages are key innate immune cells that control inflammation. In acute phase [110], implantation induces secretion of additional cytokines [111] such as IL-1b. Together, IL-1b and TNF-a are strong stimulators of fibroblast growth [109]. Also, PLA was found to decrease

212 Bioactive Materials for Bone Regeneration

TNF-a secretion (a marker for M1 activation) and increase stabilin-1 expression (as a marker for M2 activation) [112].

3.2.3.4 Polylactic acid degradation product influences on the microenvironment PLA and its copolymers are toxic due to accumulation of their acidic degradation products such as lactic acid and glycolic acid. However, the acidity of lactic acid or glycolic acid is between pH 4 and 5 [113] and is usually rapidly neutralized by local body fluids along the surface of the device. This inflammatory complication occurs when surrounding tissue does not eliminate the accumulation of acidic by-products that are formed by rapid degradation of the implant [115]. A decrease in pH also significantly affects distribution of inflammatory cytokines and endothelial cells in the vicinity of the implant. For example, vascular endothelial growth factor secreted by osteoblasts decreases at lower pH [114].

3.2.3.5 Surface property regulation of the immune microenvironment The surface properties of the implant material are relevant to the host’s foreign body reaction. Different types of polymers have different surface properties, such as: poly(benzyl N, N-diethyldithiocarbamate-co-styrene), polyacrylamide, poly(acrylic acid) sodium salt, and poly(dimethylaminoa crylamide) methylthione, which provide hydrophobic, hydrophilic, anionic, and cationic surface chemistry, respectively. Studies have shown that polymer scanning of hydrophilic and anionic surfaces inhibits monocyte adhesion and IL-4-mediated macrophage fusion into FBGCs. Thus, hydrophilic and anionic surfaces promote antiinflammatory-type responses by indicating that biomaterials adhere to monocytes and macrophages to produce selective cytokines. In particular, hydrophobic polystyrene has been shown to inhibit the expression of M1-associated markers and cytokines to promote M2-associated labeling.

3.2.3.6 Topography property regulation of macrophage On the other hand, highly hydrophilic O2 plasma-etched polystyrene (O2-ps40) has the opposite effect [117]. In order to limit the adhesion of macrophages, topographically induced changes in macrophage behavior are effective. In this study, parallel gratings (250 nm-2 mm linewidth) were characterized on polycaprolactone (PCL), PLA, and polydimethylsiloxane. The results showed that the topography had a greater effect on the adherent cell density of macrophages in vivo than in the planar control group, especially on the 2 mm PCL grating. Therefore, larger topographical cues (2 mm gratings) may physically

A bone regeneration concept Chapter | 3

213

disrupt or constrain the formation of fibrous scar tissue, reduce cell adhesion density, and limit FBGC fusion on larger gratings compared with planar controls [63].

3.2.3.7 Geometry property regulation of inflammation response In fact, recent studies have shown that inflammatory molecularly modified materials can affect the behavior of human natural killer cells and lead to increased recruitment of mesenchymal stem/mesenchymal cells (MSCs) [64]. Chitosan causes an increase in the secretion of TNF-a, while PLA-based scaffolds induce higher production of IL-6, IL-12/23, and IL-10. The material itself induces the greatest difference, and the geometry of the scaffold also affects the production of TNF-a and IL-12/23. The three-dimensional platform of different pore geometries also responds differently to macrophages. Chitosan scaffolds have larger pores and wider angles, resulting in higher secretion of these proinflammatory cytokines [64].

3.2.3.8 Delivering bioactive factors through polymers In addition, scientists believe that the transfer of exogenous bioactive factors into bone defects through hydrogel nanoparticles to accelerate bone repair, the most widely studied in the field of bone regeneration is insulin-like growth factor, VEGF, stromal cell-derived factor-1a, and fibroblast growth factor [118]. IL-4 released from the layer of coated polypropylene mesh is biologically active and promotes the polarization of macrophages to the M2 phenotype. The polarization of early macrophages at the tissue-implant interface shifts from proinflammatory (M1) to antiinflammatory/regulatory (M2) phenotype, reducing the host’s inflammatory response to nondegradable polypropylene mesh material and improving planting integration downstream of the body [119].

References [1] Biomaterials in Orthopaedic Surgery Chapter One Introduction to Biomaterials in Orthopaedic Surgery (#05233G) [2] J. Breme, E. Steinhauser, G. Paulus, Commercially pure titanium Steinhauser plate-screw system for maxillofa- cial surgery, Biomaterials 9 (1988) 310e313. [3] M. Browne, P.J. Gregson, Effect of mechanical surface pretreatment on metal ion release, Biomaterials 21 (2000) 385e392. [4] Trindade R1, Albrektsson T1,2, Galli S1, Prgomet Z3, Tengvall P2, Wennerberg A4.Osseointegration and Foreign Body Reaction: Titanium Implants Activate the Immune System and Suppress Bone Resorption during the First 4 Weeks after Implantation DOI: 10.1111/cid.12578. [5] D.P. Pioletti, A. Kottelat, The influence of wear particles in the expression of osteoclastogenesis factors by osteoblasts, Biomaterials 25 (2004) 5803e5808.

214 Bioactive Materials for Bone Regeneration [6] J.Y. Wang, B.H. Wicklund, R.B. Gustilo, D.T. Tsukayama, Titanium, chromium and cobalt ions modulate the release of bone-associated cytokines by human monocytes/macrophages in vitro, Biomaterials 17 (1996) 2233e2240. [7] D.S. Rakshit, J.T. Lim, K. Ly, L.B. Ivashkiv, B.J. Nestor, T.P. Sculco, et al., Involvement of complement receptor 3 (CR3) and scavenger receptor in macrophage responses to wear debris, J. Orthop. Res. 24 (2006) 2036e2044. [8] A.M. Kaufman, C.I. Alabre, H.E. Rubash, A.S. Shanbhag, Human macrophage response to UHMWPE, TiAlV, CoCr, and alumina particles: analysis of multiple cytokines using protein arrays, J. Biomed. Mater. Res. Part A 84 (2008) 464e474. [9] C.A. St Pierre, M. Chan, Y. Iwakura, D.C. Ayers, E.A. Kurt-Jones, R.W. Finberg, Periprosthetic osteolysis: characterizing the innate immune response to titanium wearparticles, J. Orthop. Res. 28 (2010) 1418e1424. [10] G.A. Obando-Pereda, L. Fischer, D.R. Stach-Machado, Titanium and zirconia particleinduced pro-inflammatory gene expression in cultured macro- phages and osteolysis, inflammatory hyperalgesia and edema in vivo, Life Sci. 97 (2014) 96e106. [11] E.P. Chan, A. Mhawi, P. Clode, M. Saunders, L. Filgueira, Effects of titanium(iv) ions on human monocyte-derived dendritic cells, Metallomics 1 (2009) 166e174. [12] D. Cadosch, M. Sutanto, E. Chan, A. Mhawi, O.P. Gautschi, B. von Katterfeld, et al., Titanium uptake, induction of RANK-L expression, and enhanced proliferation of human T-lymphocytes, J. Orthop. Res. 28 (2010) 341e347. [13] E. Chan, D. Cadosch, O.P. Gautschi, K. Sprengel, L. Filgueira, Influence of metal ions on human lymphocytes and the generation of titanium-specific T- lymphocytes, J. Appl. Biomater. Biomech. 9 (2011) 137e143. [14] D.1 Karazisis, S.2 Petronis, H.3 Agheli, L.3 Emanuelsson, B.3 Norlindh, A.3 Johansson, L.4 Rasmusson, P.3 Thomsen, O.5 Omar, The influence of controlled surface nanotopography on the early biological events of osseointegration, Acta Biomater. 53 (April 15, 2017) 559e571, https://doi.org/10.1016/j.actbio.2017.02.026. Epub 2017 Feb 21. [15] K.M. Hotchkissa, G.B. Reddya, S.L. Hyzya, Z. Schwartza, D. Barbara, Boyanab, and rene olivares-navarretea titanium surface characteristics, including topography and wettability, alter macrophage activation, Acta Biomater. 31 (February 2016) 425e434, https://doi.org/ 10.1016/j.actbio.2015.12.003. [16] J. Lu, T.J. Webster, Reduced immune cell responses on nano and submicron rough titanium, Acta Biomater. 16 (2015) 223e231. [17] H.1 Koivu, Z. Mackiewicz, Y. Takakubo, N. Trokovic, J. Pajarinen, Y.T. Konttinen, RANKL in the osteolysis of AES total ankle replacement implants, Bone 51 (3) (September 2012) 546e552, doi: 10.1016/. [18] M. Pettersson, P. Kelk, G.N. Belibasakis, D. Bylund, M. Molin Thoren, A. Johansson, Titanium ions form particles that activate and exe- cute interleukin-1beta release from lipopolysaccharide-primed macrophages, J. Periodontal. Res. 52 (2017) 21e32. [19] W.1 Wang, D.J. Ferguson, J.M. Quinn, A.H. Simpson, N.A. Athanasou, J Biomaterial particle phagocytosis by bone-resorbing osteoclasts, Bone Joint Surg Br 79 (5) (September 1997) 849e856 (Biomaterial particle phagocytosis by bone-resorbing osteoclasts). [20] C.A. Dinarello, Biologic basis for interleukin-1 in disease, Blood 87 (1996) 2095e2147. [21] L.A.J. O’Neill, C.A. Dinarello, The IL-1 receptor/toll-like receptor superfamily: crucial receptors for inflammation and host defense, Immunol. Today 21 (2000) 206e209. [22] B.K. Davis, H.T. Wen, J.P.Y. Ting, The inflammasome NLRs in immu- nity, inflammation, and associated diseases, in: W.E. Paul, D.R. Littman, W.M. Yokoyama (Eds.), Annu Rev Immunol vol. 29, 2011, pp. 707e735.

A bone regeneration concept Chapter | 3

215

[23] H. Haleem-Smith, E. Argintar, C. Bush, D. Hampton, W.F. Postma, F.H. Chen, et al., Biological responses of human mesenchymal stem cells to titanium wear debris particles, J. Orthop. Res. 30 (2012) 853e863. [24] E. Jamsen, V.P. Kouri, J. Olkkonen, A. Cor, S.B. Goodman, Y.T. Konttinen, et al., Characterization of macrophage polarizing cytokines in the aseptic loosening of total hip replacements, J. Orthop. Res. 32 (2014) 1241e1246. [25] W.H. Harris, A.L. Schiller, J.M. Scholler, R.A. Freiberg, R. Scott, Exten- sive localized bone resorption in the femur following total hip replacement, J. Bone Joint Surg. Am. 58 (1976) 612e618. [26] M. Pettersson,1 J. Pettersson,2 M. Molin Thoren,1 Anders Johansson Effect of Cobalt Ions on the Interaction between Macrophages and Titanium DOI10.1002. [27] K. Davda, F.V. Lali, B. Sampson, J.A. Skinner, A.J. Hart, An analysis of metal ion levels in the joint fluid of symptomatic patients with metal-on-metal hip replacements, J. Bone Joint Surg. Br. 93 (2011) 738e745. [28] T.A. Wynn, Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases, J. Clin. Invest. 117 (2007) 524e529. [29] S.J. Baskey, E.A. Lehoux, I. Catelas, Effects of Cobalt and Chromium Ions on Lymphocyte Migration Published Online, Wiley Online Library, July 7, 2016, https:// doi.org/10.1002/jor.23336. [30] H.G. Willert, G.H. Buchhorn, A. Fayyazi, et al., Metal-on- metal bearings and hypersensitivity in patients with artifi- cial hip joints. A clinical and histomorphological study, J. Bone Joint Surg. Am. 87 (2005) 28e36. [31] I. Catelas, A. Petit, D.J. Zukor, O.L. Huk, Cytotoxic and apoptotic effects of cobalt andchromium ions on J774 macrophages - Implication of caspase-3 in the apoptotic pathway, J. Mater. Sci. Mater. Med. 12 (10e12) (2001 Oct-Dec) 949e953. [32] W.a.N.I, National Joint Registry for England, 13rd Annual Report, 2016. [33] P. Campbell, E. Ebramzadeh, S. Nelson, K. Takamura, K. De Smet, H.C. Amstutz, Histological features of pseudotumor-like tissues from metal-on- metal hips, Clin. Orthop. Relat. Res. 468 (2010) 2321e2327. [34] S.B. Goodman, Wear particles, periprosthetic osteolysis and the immune system, Biomaterials 28 (2007) 5044e5048. [35] J. Xu, A. Nyga, W. Li, X. Zhang1, N. Gavara, M.M. Knight1, J.C. Shelton, Cobalt ions stimulate a fibrotic response through matrix remodelling, fibroblast contraction and release of pro-fibrotic signals from macrophages, Eur. Cells Mater. 36 (2018) 142e155, https://doi.org/10.22203/eCM.v036a11. [36] X. Jing, Y. Junyao, N. Agata, M. Ehteramyan, A. Moraga, W. Yuanhao, L. Zeng, M.M. Knight, J.C. Shelton, Cobalt (II) ions and nanoparticles induce macrophage retention by ROS-mediated down-regulation of RhoA expression, Acta Biomater. 72 (2018) 434e446. [37] L. De, H. Wang, Z. Li, C. Wang, F. Xiao, Y. Gao, X. Zhang, P. Wang, J. Peng, G. Cai, B. Zuo, Y. Shen, J. Qi, N. Qian, L. Deng, W. Song, X. Zhang, L. Shen, X. Chen, The inhibition of RANKL expression in fibroblasts attenuate CoCr particles induced aseptic prosthesis loosening via the MyD88- independent TLR signaling pathway, Biochem. Biophys. Res. Commun. 503 (2018) 1115e1122. [38] H. Tian, Z. Tang, X. Zhuang, X. Chen, Xiabin Jing Biodegradable Synthetic Polymers: Preparation, Functionalization and Biomedical Application Progress in Polymer Science, vol. 37, February 2012, pp. 237e280. Issue 2.

216 Bioactive Materials for Bone Regeneration [39] [40]

[41] [42] [43] [44]

[45]

[46] [47] [48] [49]

[50]

[51] [52]

[53]

[54]

[55]

M. Navarro, A. Michiardi, O. Castano, J.A. Planell, J.R. Soc, Biomaterials orthopaed. Inter. 5 (2008) 1137e1158. M. Vogel, C. Voigt, U. Gross, C. Mu¨ller-Mai, In vivo comparison of bioactive glass particles in rabbits, BioMaterials 22 (2001) 357e362, https://doi.org/10.1016/S01429612(00)00191-5. See also pages 26, 4, 359e371. R. Meffert, J. Thomas, K. Hamilton, C. Brownstein, Hydroxylapatite as an alloplastic graft in the treatment of periodontal osseous defect, J. Periodontol. 56 (1985) 63e73. E. Schepers, M. de Clercq, P. Ducheyne, R. Kempeneers, Bioactive glass particulate material as a filler for bone lesions, J. Oral Rehabil. 18 (1991) 439e452. B.D. Ratner, S.J. Bryant, Biomaterials: where we have been and where we are going, Annu. Rev. Biomed. Eng. 6 (2004) 41e75. S. Franz, S. Rammelt, D. Scharnweber, J.C. Simon, Immune responses to implants a review of the implications for the design of immunomodulatory biomaterials, Biomaterials 32 (2011) 6692e6709. M.R. Major, V.W. Wong, E.R. Nelson, M.T. Longaker, G.C. Gurtner, The foreign body response: at the interface of surgery and bioengineering, Plastic Reconstr. Surg. 135 (2015) 1489e1498. J.M. Anderson, A. Rodriguez, D.T. Chang, Foreign body reaction to biomaterials, Semin. Immunol. 20 (2008) 86e100. J.M. Morais, F. Papadimitrakopoulos, D.J. Burgess, Biomaterials/tissue interactions: possible solutions to overcome foreign body response, AAPS J. 12 (2010) 188e196. R.B. Johnston Jr., Current concepts: immunology. Monocytes and macrophages, N. Engl. J. Med. 318 (1988) 747e752. R. Trindade, T. Albrektsson, P. Tengvall, A. Wennerberg, Foreign body reaction to biomaterials: on mechanisms for buildup and breakdown of osseointegration, Clin. Implant Dent. Relat. Res. 18 (2016) 192e203. W.J. Kao, Evaluation of protein-modulated macrophage behavior on biomaterials: designing biomimetic materials for cellular engineering, Biomaterials 20 (1999) 2213e2221. S. Franz, et al., Immune responses to implants e a review of the implications for the design of immunomodulatory biomaterials, Biomaterials 32 (28) (2011) 6692e6709. T.R. Kyriakides, M.J. Foster, G.E. Keeney, A. Tsai, C.M. Giachelli, I. Clark-Lewis, B.J. Rollins, P. Bornstein, The CC chemokine ligand, CCL2/MCP1, participates in macrophage fusion and foreign body giant cell formation, Am. J. Pathol. 165 (2004) 2157e2166. B. Sherry, P. Tekamp-Olson, C. Gallegos, D. Bauer, G. Davatelis, S.D. Wolpe, F. Masiarz, D. Coit, A. Cerami, Resolution of the two components of macrophage inflammatory protein 1, and cloning and characterization of one of those components, macrophage inflammatory protein 1 beta, J. Exp. Med. 168 (1988) 2251e2259. Y. Huang, C. Wu, X. Zhang, J. Chang, K. Dai, Regulation of immune response by bioactive ions released from silicate bioceramics for bone regeneration, Acta Biomater. 66 (January 15, 2018) 81e92, https://doi.org/10.1016/j.actbio.2017.08.044. Epub 2017 Aug 30. C. He, X. Jin, P.X. Ma, Calcium phosphate deposition rate, structure and osteoconductivity on electrospun poly (L-lactic acid) matrix using electrodeposition or simulated body fluid incubation, Acta Biomater. 10 (2014) 419e427.

A bone regeneration concept Chapter | 3

217

[56] J. Wang, D. Liu, B. Guo, et al., Role of biphasic calcium phosphate ceramic- mediated secretion of signaling molecules by macrophages in migration and osteoblastic differentiation of MSCs, Acta Biomater. 51 (2017) 447e460. [57] A. De, Wnt/Ca2þ signaling pathway: a brief overview, ABBS (10 October 2011), 43. [58] R.J. MacLeod, et al., Am. Wnt5a secretion stimulated by the extracellular calciumsensing receptor inhibits defective Wnt signaling in colon cancer cells, J. Physiol. Gastrointest. Liver Physiol. 293 (1) (2007) G403eG411. [59] S.S. Kim, C.A. Sundback, S. Kaihara, M.S. Benvenuto, B.S. Kim, D.J. Mooney, et al., Dynamic seeding and in vitro culture of hepatocytes in a flow perfusion system, Tissue Eng. 6 (1) (2000) 39e44. [60] U. Stucki, J. Schmid, C.F. Hammerle, N.P. Lang, Temporal and local appearance of alkaline phosphatase activity in early stages of guided bone regeneration. A descriptive histochemical study in humans, Clin Oral Implants Res 12 (2) (2001) 121e127. [61] R. Marom, I. Shur, R. Solomon, D. Benayahu, Characterization of adhesion and differentiation markers of osteogenic marrow stromal cells, J. Cell. Physiol. 202 (1) (2005) 41e48. [62] Sulin Chen, Jacqueline A. Jones, Yongan Xu, Hong-Yee Low, James M. Anderson, Kam W. Leong, Characterization of Topographical Effects on Macrophage Behavior in a Foreign Body Response Model Biomaterials, vol. 31, 2010, pp. 3479e3491. [63] C.R. Almeida, D.P. Vasconcelos, R.M. Gonc¸alves, M.A. Barbosa, Enhanced mesenchymal stromal cell recruitment via natural killer cells by incorporation of inflammatory signals in biomaterials, J. R. Soc. Interface 9 (2012) 261e271. [64] Z. Chen, Y. Deliang, Z. Xuebin, C. Jiang, W. Chengtie, X. Yin, Nutrient element-based bioceramic coatings on titanium alloy stimulating osteogenesis by inducing beneficial osteoimmmunomodulation, J. Mater. Chem. B 2 (36) (2014) 6030e6043. [65] R.M. Day, A.R. Boccaccini, Effect of particulate bioactive glasses on human macrophages and monocytes in vitro, J. Biomed. Mater. Res. A 73 (1) (2005) 73e79. [66] H. Haase, L. Rink, Signal transduction in monocytes: the role of zinc ions, Biometals 20 (3e4) (2007) 579e585. [67] F. Velard, et al., Inflammatory cell response to calcium phosphate biomaterial particles, An overviewActa Biomater 9 (2) (2013) 4956e4963. [68] P. Scuderi, Differential effects of copper and zinc on human peripheral blood monocyte cytokine secretionCell Immunol 126 (2) (1990) 391e405. [69] X. Zhou, et al., Immunology 143 (2) (2014) 287e299. [70] Z. Chen,T. Klein, R.Z. Murray1, R. Crawford, Jiang C., Chengtie W., and Yin X. Osteoimmunomodulation for the Development of Advanced Bone Biomaterials Materials Today vol. 19, Number 6 July/August 2016. [71] H. Elsayed, A. Rinco´n Romero, L. Ferroni, et al., Bioactive glass-ceramic scaffolds from novel ‘inorganic gel casting’and sinter-crystallization, Materials 10 (2017) 171. [72] Y. Rezaei, F. Moztarzadeh, S. Shahabi, et al., Synthesis, characterization, and in vitro bioactivity of sol-gel-derived SiO2-CaO-P2O5-MgO-SrO bioactive glass, Synth React Inorg Met-Org Nano-Met Chem 44 (2014) 692e701. [73] J.R. Henstock, L.T. Canham, S.I. Anderson, Silicon: the evolution of its use in biomaterials, Acta Biomater. 11 (2015) 17e26. [74] S. Bose, S. Tarafder, S.S. Banerjee, et al., Understanding in vivo response and mechanical property variation in MgO, SrO and SiO2 doped b- TCP, Bone 48 (2011) 1282e1290.

218 Bioactive Materials for Bone Regeneration [75]

[76]

[77]

[78] [79] [80] [81]

[82]

[83] [84] [85] [86] [87] [88]

[89]

[90] [91] [92]

[93]

Z. Mladenovic , A. Johansson, B. Willman, K. Shahabi, E. Bjo¨rn, M. Ransjo¨, Soluble silica inhibits osteoclast formation and bone resorption in vitro, Acta Biomater. 10 (2014) 406e418. L. Casarrubios, M. Concepcio´n Matesanz, S. Sa´nchez-Salcedo, Daniel Arcos, Marı´a Vallet-Regı´ Marı´a Teresa Portole´s Nanocrystallinity effects on osteoblast and osteoclast response to silicon substituted hydroxyapatite, J. Colloid Interface Sci. 482 (2016) 112e120. J.R. Henstock, R. RuktanonchaiL, T. Canham, S.I. Anderson, Porous silicon confers bioactivity to polycaprolactone composites in vitro, J. Mater. Sci. 25 (4) (April 2014) 1087e1097. E.M. Carlisle, Silicon as a trace nutrient, Sci. Total Environ. 73 (1998) 95e106. E.M. Carlisle, In vivo requirement for silicon in articular cartilage and connective tissue formation in the chick, J. Nutr. 106 (1976) 478e484. K. Schwarz, A bound form of silicon in glycosaminoglycans and polyuronides, Proc. Natl. Acad. Sci. 70 (1973) 1608e1612. D.M. Reffitt, D.N. Ogston, R. Jugdaohsingh, H.F. Cheung, B.A. Evans, R.F. Thompson, J.J. Powell, G.N. Hampson, Orthosilicic acid stimulates collagen type 1 synthesis and osteoblastic differentiation in human osteoblast-like cells in vitro, Bone 32 (2003) 127e135. H. Li, J. Chang, Bioactive silicate materials stimulate angiogenesis in fibroblast and endothelial cell co-culture system through paracrine effect, Acta Biomater. 9 (2013) 6981e6991. P.E. Keep, M.J. Oursler, K.E. Wiegand, S.K. Bonde, T.C. Spelsberg, B.L. Riggs, J. Bone Miner. Res. 7 (11) (November 1992) 1281e1289. F. Maehira, I. Miyagi, Y. Eguchi, Effects of calcium sources and soluble silicate on bone metabolism and the related gene expression in mice, Nutrition 25 (2009) 581e589. J.K. Park, et al., The topographic effect of zinc oxide nanoflowers on osteoblast growth and osseointegration, Adv. Mater 22 (43) (2010) 4857e4861. M.P. Staiger, et al., Immune responses to implants e a review of the implications for the design of immunomodulatory biomaterials, Biomaterials 27 (9) (2006) 1728e1734. J. Sugimoto, et al., J. Immunol. 188 (12) (2012) 6338e6346. Carina Cardemil, Ibrahim Elgali, Wei Xia, Lena Emanuelsson, Birgitta Norlindh, Omar Omar, Peter Thomsen, Strontium-doped calcium phosphate and hydroxyapatite granules promote different inflammatory and bone remodelling responses in normal and ovariectomised rats, PLoS One 8 (12) (December 1, 2013). www.plosone.org. Y. Wu, G. Gao, G. Wu, Self-assembled three-dimensional hierarchical porous V2O5/ graphene hybrid aerogels for supercapacitors with high energy density and long cycle life, J. Mater. Chem. 3 (2015) 1828e1832. C. Gao, Y. Deng, P. Feng, et al., Current progress in bioactive ceramic scaffolds for bone repair and regeneration, Int. J. Mol. Sci. 15 (2014) 4714e4732. H. Wolfenson, I. Lavelin, B. Geiger, Dynamic regulation of the structure and functions of integrin adhesions, Dev. Cell 24 (2013) 447e458. R. Duan, D. Barbieri, Florence de Groot, D. Joost, de Bruijn, H. Yuan, Modulating bone regeneration in rabbit condyle defects with three surface-structured tricalcium phosphate ceramics, ACS Biomater. Sci. Eng. 4 (2018) 3347e3355. E. Schepers, P. Ducheyne, Bioactive glass particles of narrow size range for the treatment of oral bone defects: a 1}24 month experiment with several materials and particle sizes and size ranges, J. Oral. Rehab. 24 (3) (1997) 171e181.

A bone regeneration concept Chapter | 3 [94a] [94b] [95] [96] [97] [98]

[99] [100]

[101]

[102]

[103]

[104] [105]

[106]

[107]

[108]

[109]

219

L.L. Hench, E.C. Ethridge, Biomaterials: An Interfacial Approach, Plenum Press, New York, 1982. E.J.G. Schepers, P. Ducheyne, L. Barbier, S. Schepers, Bioactive glass particles of narrow size range: a new material for the repair of bone defects, Implant Dent. 2 (1993) 151e156. P. Ducheyne, Q. Qiu, Bioactive ceramics: the e!ect of surface reactivity on bone formation and bone cell function, Biomaterials 20 (1999) 2287e2303. M.I. Sabir, X. Xu, L. Li, A review on biodegradable polymeric materials for bone tissue engineering applications, J. Mater. Sci. 44 (2009) 5713e5724. R. Silva, B. Fabry, A.R. Boccaccini, Fibrous protein-based hydrogels for cell encapsulation, Biomaterials 35 (2014) 6727e6738. C.H. Geerdink, B. Grimm, W. Vencken, I.C. Heyligers, A.J. Tonino, Cross-linked compared with historical polyethylene in THA: an 8-year clinical study, Clin. Orthop. Relat. Res. (2008). H. Baudriller, P. Chabrand, D. Moukoko, Modeling UHMWPE wear debris generation, J. Biomed. Mater. Res. B Appl. Biomater. 80 (2) (2007) 479e485 [PubMed: 16862559]. R. Maitra, C.C. Clement, G.M. Crisi, N. Cobelli, L. Santambrogio, Immunogenecity of modified alkane polymers is mediated through TLR1/2 activation, PLoS One 3 (6) (2008) e2438 [PubMed: 18560588]. S. Xing, J.E. Waddell, E.L. Boynton, Changes in macrophage morphology and prolonged cell viability following exposure to polyethylene particulate in vitro, Microsc. Res. Technol. 57 (2002) 523e529. R.S. Tuan, F.Y. Lee, YTK, J.M. Wilkinson, R.L. Smith, What are the local and systemic biologic reactions and mediators to wear debris, and what host factors determine or modulate the biologic response to wear particles? J. Am. Acad. Orthop. Surg. 16 (Suppl. 1) (2008) S42eS48 [PubMed: 18612013]. R. Maitra, C.C. Clement, Brian Scharf, Giovanna M. Crisi, Sriram Chitta, Daniel Paget, P. Edward Purdue, Neil Cobelli, Laura Santambrogio, Endosomal damage and TLR2 mediated inflammasome activation by alkane particles in the generation of aseptic osteolysis, Mol. Immunol. 47 (2e3) (December 2009) 175e184. S.B. Goodman, T. Ma, Cellular chemotaxis induced by wear particles from joint replacements, Biomaterials 31 (2010) 5045e5050 [PubMed: 20398931]. P.G. Ren, Z. Huang, T. Ma, S. Biswal, R.L. Smith, S.B. Goodman, Surveillance of systemic trafficking of macrophages induced by UHMWPE particles in nude mice by noninvasive imaging, J. Biomed. Mater. Res. A 94 (2010) 706e711 [PubMed: 20213815]. P.G. Ren, A. Irani, Z. Huang, T. Ma, S. Biswal, S.B. Goodman, Continuous infusion of UHMWPE particles induces increased bone macrophages and osteolysis, Clin. Orthop. Relat. Res. 469 (2011) 113e122 [PubMed: 21042895]. E. Gibon, T. Ma, P.-G. Ren, K. Fritton, S. Biswal, Z. Yao, L. Smith, S.B. Goodman, Selective inhibition of the MCP-1-CCR2 ligand-receptor axis decreases systemic trafficking of macrophages in the presence of UHMWPE particles, J. Orthop. Res. 30 (2012) 547e553 [PubMed: 21913218]. H. Witkiewicz, T. Vidovszky, R.T. Turner, M.G. Rock, B.F. Morrey, M.E. Bolander, Fate of ultrahigh molecular weight polyethylene (UHMW-PE) wear debris in patients with hip implants, Tech. Orthop. 8 (4) (1993) 254e261 [PubMed: 11539273]. T.A. Wynn, A. Chawla, J.W. Pollard, Nature 496 (2013) 445e455.

220 Bioactive Materials for Bone Regeneration [110] A. Serafim, R. Mallet, F. Pascaretti-Grizon, I.C. Stancu, D. Chappard, Osteoblast-like cell behavior on porous scaffolds based on poly(styrene) fibers, BioMed Res. Int. 2014 (2014) 609319. [111] S.J. Yoon, S.H. Kim, H.J. Ha, Y.K. Ko, J.W. So, M.S. Kim, Y.I. Yang, G. Khang, J.M. Rhee, H.B. Lee, Reduction of inflammatory reaction of poly(D,L-lactic-co-glycolic acid) using demineralized bone particles, Tissue Eng. A 14 (2008) 539e547. [112] K.S. Stankevich, A. Gudima, V.D. Filimonov, H. Kluter, E.M. Mamontova, S.I. Tverdokhlebov, J. Kzhyshkowska, Surface modification of biomaterials based on highmolecular polylactic acid and their effect on inflammatory reactions of pri- mary human monocyte-derived macrophages: perspective for personalized thera- py, Mater. Sci. Eng. C 51 (2015) 117e126 (A.R. Amini, J.S). [113] K.A. Athanasiou, G.G. Niederauer, C.M. Agrawal, Sterilization, toxicity, biocompatibility and clinical applications of polylactic acid/polyglycolic acid copolymers, BioMaterials 17 (1996) 93e102. [114] J.A. Spector, B.J. Mehrara, J.A. Greenwald, P.B. Saadeh, D.S. Steinbrech, P.J. Bouletreau, L.P. Smith, M.T. Longaker, Osteoblast expression of vascular endothelial growth factor is modulated by the extracellular microenvironment, Am. J. Phys. Cell Physiol. 280 (2001) C72eC80. [115] J. Suganuma, H. Alexander, Biological response of intramedullary bone to poly-L-lactic acid, J. Appl. Biomater. 4 (1993) 13e27. [116] W. G. Brodbeck,Y. Nakayama, T. Matsuda, E. Colton, N. P. Ziats, J. M. Anderson Biomaterial Surface Chemistry Dictates Adherent Monocyte/Macrophage Cytokine Expression in Vitro doi:10.1006/cyto.2002.1048. [117] H.M. Rostama, S. Singha, F. Salazara, P. Magennisb, A. Hookb, T. Singhb, E. Nihal, d Vranac, M.R. Alexanderb, A.M. Ghaemmaghamia, The impact of surface chemistry modification on macrophage polarisation, Immunobiology 221 (2016) 1237e1246. [118] X. Bai, M. Gao, S. Syed, J. Zhuang, X. Xu, Xue-Qing Zhang Bioactive hydrogels for bone regeneration, Bioactive Mat. 3 (2018) 401e417. [119] Melba Navarro impact of 3-D printed PLA- and chitosan-based scaffolds on human monocyte/macrophage responses: unraveling the effect of 3-D structures on inflammation, Acta Biomater. 10 (2014) 613e622.

A bone regeneration concept Chapter | 3

221

Chapter 3.3

Biomaterial research and development aim to produce a “good” bone immune microenvironment by regulating immune cell response and bone regeneration With the rapid development of bone biology, people gradually realize that bone formation is not only the behavior of the bone system but also involves the pathophysiological processes of multiple systems such as the coagulation and immune systems. Studies have shown that the immune system is closely linked to the skeletal system and shares many cytokines and regulatory factors [1,2]. Therefore, the local microenvironment, especially the immune microenvironment, plays an important role in regulating the osteogenesis process. Therefore, it is necessary to develop biological materials capable of regulating the immune microenvironment. At present, the commonly used metallics for orthopedic implants mainly include medical stainless steel, titanium alloy, tantalum alloy, and cobalte chromiumemolybdenum alloy. Nonmetallics mainly include bioactive ceramics, polymethyl methacrylate, and hydroxyapatite. Coating implants or integrated surface bioactive particles implanted on the surface and/or inside [3], the concept of implanted materials has changed from traditional inert materials to new composite biomaterials with immunomodulatory capabilities, which regulate direct osteogenesis differentiation into osteoblasts, and more importantly regulate the bone immune microenvironment. At the same time, however, the mechanical properties of bone biomaterials still need to be guaranteed, requiring modification of the surface properties of biomaterials (such as surface topography, adhesion, hydrophilicity, and hydrophobicity), particle size, microporosity and ion release biomaterials. It is often the rule of participation in immune response (Fig. 3.6).

3.3.1 Surface property regulation of immune-mediated osteogenesis When the implant enters the body, including trauma during surgery, it activates the coagulation and immune systems, and various protein molecules adhere to the surface of the biomaterial. This is the underlying mechanism by which the different surface properties of biomaterials regulate immune response. Complement, fibrinogen, fibronectin, and vitronectin were all found to be attached

222 Bioactive Materials for Bone Regeneration

FIGURE 3.6 Bone formation is regulated in different ways.

to the surface of the implant. Due to the influence of the implant, these proteins were denatured, recognized by the immune system [4] and activated signal pathways, which had a significant impact on immune response [5]. At present, the commonly used method is to coat the implant surface with thin layer by physical, chemical, biochemical, or electrochemical methods and conduct biochemical modification to regulate the biological behavior of immune cells by changing the surface topography, adhesion charge, hydrophilicity, and hydrophobicity. Bai’s research [6] shows that hybrid micro-nano morphology, superhydrophilicity, and highly crystalline hydroxyapatite nanoparticles formed by a coating produced with MAO-650 (microarc oxidization with an annealing temperature of 650 C) not only support proliferation and differentiation of osteoblasts but also inhibit inflammatory response and enable favorable osteoimmunomodulation to facilitate osteogenesis.

3.3.1.1 Topography The surface nanotopography differences of biomaterials affect the biological behavior of immune cells [7]. For example, titanium is widely used in orthopedic surgery, and its surface roughness has a significant effect on macrophages. Macrophages can be adsorbed on the surface of various roughness levels of titanium, and more and more macrophages are attached to the surface over time [8]. Fortunately, macrophages on a somewhat rough titanium surface can be easily converted into M2 phenotypes [9]. The activated M2 macrophage can reduce the inflammatory response and prevent TNFa-induced bone loss [10]. In addition, the rough surface of biomaterials can significantly stimulate the production of inflammatory cytokines and

A bone regeneration concept Chapter | 3

223

chemokines. Inflammatory cytokines IL-6, IL-23, and OSM are also important regulators of osteoclast formation. IL-6 and IL-23 can increase the expression of RANKL, OSM can stimulate the production of RANKL by synergistic effect with IL-6 [11], and all three inflammatory cytokines can promote the production of osteoclasts by affecting the RANKL/RANK/OPG system [12,13]. It is generally believed that the surface roughness of bone is about 32 nm, so the nanomaterials are closest to the normal physiological environment. Meanwhile, there is evidence that the nanoscale microstructure can stimulate human BMSCs to produce bone mineral in vitro [14]. However, differences also exist in the nanoscale. Wang et al. [15] found that when the diameter of TiO2 NTs is 80e100 nm, it is likely to promote macrophages to the M1 phenotype, while it is easier to promote the M2 phenotype when the diameter is less than 30 nm. This indicates that the nanotopography can produce a good bone immune microenvironment.

3.3.1.2 Charge The charge on the surface also has a significant effect on immune response, but the mechanism is complicated and the influencing factors are various. Both positive and negative surface charges can adhere to the protein and promote osteoblast maturation, but it is not clear which charges work better [16]. Some studies have shown that positive particles are more likely to cause inflammation than negative and neutral ones [17]. This is because most mammalian cells, including immune cells, have a negative surface charge. And the positive ions on the surface of the biomaterial are combined with the negative charges on the surface of the cell membrane, resulting in the loss of negative charges on the surface of the cell membrane, which, under normal circumstances, induces signal transduction into the cytoplasm, leading to biological reactions including inflammatory reactions. It is important to note that T cells activated in chronic inflammatory response strengthen the osteogenic differentiation of bone marrow mesenchymal stem cells, and thereby participate in the strengthening of bone regeneration under chronic inflammation [18]. Rifas et al. [19] also believe that T-cell cytokines released from local inflammatory sites may be the driving force for the differentiation of local BMSCs into the osteoblast phenotype.

3.3.1.3 Hydrophilicity and hydrophobicity Hydrophilicity and hydrophobicity of biomaterials are additional important characteristics that affect the biological behavior of immune cells. In general, hydrophobic surfaces usually have more proteins attached to them than hydrophilic surfaces [16], and hydrophobic materials are also more likely than hydrophilic materials to strengthen monocytes adhesion, leading to local

224 Bioactive Materials for Bone Regeneration

immune responses [20]. The surface of hydrophilic/neutral copolymer inhibits the adhesion and fusion of macrophages to form FBGCs. Increasing titanium surface hydrophilicity can weaken the proinflammatory response of macrophages and thus accelerate implant bone integration [21]. Compared with the hydrophobic and hydrophilic smooth titanium, and the hydrophobic and hydrophilic rough titanium, some researchers found that m1-type macrophages on the surface of smooth titanium were activated along with TNF mutation, il-6 and il-1b levels were induced. Compared with hydrophobicity, hydrophilic macrophages are more inclined to induce antiinflammatory M2-type macrophages, and the hydrophilic rough titanium inducement can produce synergistic effect, which significantly increases the levels of IL-4 and IL-10 [22].

3.3.2 Particle size regulation of immune-mediated osteogenesis Studies have found that the degree of foreign body immune response is relevant to the size and shape of the implant, indicating the significance of particle shape and size characteristics in regulating immune response [23]. For the same number of particles, the smaller the particle size, the larger the surface area and the greater the possibility of immune response [24]. The smallest hydroxyapatite granules (1e30m) stimulated the maximum number of proinflammatory cytokines produced by immune cells (TNF-a, IL-1b, IL-6) [25]. Large blocks of gold are inert, while gold nanoparticles are highly reactive to immune response and even produce ROS [26]. However, this does not mean that smaller particles necessarily mediate more severe immune responses. It has been found that Th1 reaction can be induced if the particle is larger than 1 micron, and Th2 reaction can be induced if the particle is less than 0.5 microns [17,27]. In vivo studies have shown that reducing the size of HA particles can reduce inflammation [28]. The nanoscale HA particle coating can significantly promote bone formation and osseointegration and strengthen the interaction between bone cells and the implant surface [29]. Therefore, it is still necessary to explore the effects of different types and particle sizes of biological materials on the immune system.

3.3.3 Microporosity regulation of immune-mediated osteogenesis The significance of pore size and porosity of biomaterials is confirmed in the interaction between implants and host immune systems. Some studies suggest that higher porosity can promote osteogenesis, but at the same time, it should be noted that high porosity often leads to low strength of bone materials and easily leads to implant collapse [30]. Therefore, some scholars suggest that the porosity can be controlled between 40% and 80% according to different implants [31]. It seems

A bone regeneration concept Chapter | 3

225

that the larger the pore size, the lower the reactivity of the foreign body [32]. The mechanism may be relevant to macrophage polarization. However, it should be noted that it is beneficial to reduce the pore size appropriately. Although small pore size can block blood flow in and out, especially in the center of the biomaterials, they can lead to local anoxic conditions and inflammatory reactions [33]. However, hypoxia-inducible factors (HIFs) are easy to produce in hypoxic environments, which is also conducive to angiogenesis. Therefore, when inducing angiogenesis, appropriate pore size should be adopted to induce angiogenesis and avoid obvious inflammatory reaction. Studies have found that 80%e88% of the porosity and pore size of >50 mm are more conducive to bone growth [34]. Another study came to a basically consistent conclusion: when the porosity is 40%e80%, the optimal pore size is usually within the range of 200e500 mm [31].

3.3.4 Ion regulation of immune-mediated osteogenesis After bone biomaterials are implanted in the body, they undergo biochemical degradation in different degrees in the body [35]. Ions released from biomaterials during degradation can change local ion concentration and have a significant impact on local microenvironment. For example, a coating of clinoenstatite (CLT, MgSiO3) strengthens osteogenesis and inhibits osteoclast formation by releasing Mg and Si ions; it is expressed by downregulating proinflammatory cytokines [36]. Calcium (Ca) is a main component of calcium phosphate bone biomaterials and has been proved to be involved in some inflammatory signaling pathways [37]. Activation of the atypical Wnt5A/Ca2þ signaling way is known to strengthen the inflammatory response. High concentration of extracellular calcium activates the cascade of CaSR signaling, which produces Wnt5A and reduces the expression of TNF ligation, thus alleviating inflammation [37,38]. Silicon (Si) is an important microelement relevant to bone development. Ions released from silica bioactive glass and bioceramics can promote the proliferation and differentiation of osteoblasts [39,40]. When the Si level is lower than 30 ppm, the development of osteoclasts will be stimulated, while higher Si levels will inhibit osteoclast development and bone resorption activity [41]. Magnesium (Mg), a biodegradable biocompatible metal, has been proposed as a biodegradable metal bone biomaterials for application in orthopedics. Mg inhibits the production of proinflammatory cytokines by inhibiting TLR pathways [42]. Cobalt (Co) can promote angiogenesis by stabilizing HIF and stimulating HIF target genes such as VEGF [43]. However, Co ions are toxic and related to the invalid joint prostheses, so their application in biological materials is controversial [44]. In addition, a study has reported that the combination of Co and b-tricalcium can promote macrophage polarization toward M1-type [45].

226 Bioactive Materials for Bone Regeneration

Zinc (Zn) promotes bone formation and mineralization [46] and also affects the immune system. Zinc-substituted ceramics can promote antiinflammatory cytokine IL-10 release and reduce TNF-a and proinflammatory cytokine IL-1b expression [47,48]. In addition, in vitro and in vivo experiments proved that Zn-coated sulfonated polyetheretherketone can significantly activate M2-type macrophages and secrete the highest levels of BMP-2 and VEGF to improve local bone integration and angiogenesis, and the two synergistically promote bone integration [49]. Strontium (Sr), a physiological microelement that promotes osteogenesis and inhibits the formation of osteoclasts, has been used in the treatment of osteoporosis [50,51]. Some studies have shown that Ca and Sr often mix to make immunomodulatory coatings, and different ratios can produce synergistic or competitive effects. By acting on macrophages, Ca/Sr mixed with 2:1 can achieve the optimal effect and induce macrophages to polarize into the m2-phenotype [52,53]. This leads to an increase in BMP2, VEGF, and IL-10 production, promotes osteogenic differentiation of bone marrow mesenchymal stem cells, and promotes bone formation, angiogenesis, and tissue repair [54].

3.3.5 Summary and expectations Bone regeneration is a complicated process requiring synergistic action of cells from different systems. We proposed the concept of “bone immune microenvironment regulation” which not only reflected the host immune response to implant, but also emphasized the significance of creating a “good” bone immune microenvironment for bone regeneration by regulating immune cells. At present, bone biomaterials can be used to regulate the biological behavior of immune cells and manage the local bone immune microenvironment. Specifically, bone biomaterials with “good” bone immunoregulation can have a significant impact on local immune cells, produce appropriate inflammatory responses and release biomolecules, strengthen the accumulation and osteogenic differentiation of MSCs, balance the process of osteogenesis and osteoclasts, and form new bone integrated with biomaterials. The diversity and high plasticity of immune cells make it possible to regulate bone immune response, thus regulating bone immune environment and bone regeneration. Different biological materials and surface properties have different regulatory effects on bone immune response. By developing bone materials with biological activity and modifying the surface characteristics of bone biological materials, bone immune microenvironment can be regulated. Currently, the best method is to cover the surface of biological materials with bioregulatory coating, modify the nano structure of coating, or integrate the appropriate concentration of active ions on the coating surface or the material inside to regulate the local immune cell reaction, thus generating the necessary immune environment to promote functional bone regeneration.

A bone regeneration concept Chapter | 3

227

However, due to the complexity of the immune environment, the bone formation mechanism of immune-mediated regulation by various factors has not been clearly defined. At the same time, it is necessary to further study the surface coating process and ionic sustained release mode and optimal ion concentration in materials. The research and development of biomaterials based on bone immune microenvironment regulation aims to improve the clinical conversion rate of biomaterials and solve the scientific problem of bone regeneration and repair.

References [1] [2] [3] [4] [5] [6]

[7]

[8]

[9] [10]

[11] [12]

[13]

[14]

R.J. Schutte, L. Xie, B. Klitzman, W.M. Reichert, In vivo cytokine-associated responses to biomaterials, Biomaterials 30 (2009) 160e168. J.R. Arron, Y. Choi, Bone versus immune system, Nature 408 (2000) 535e536. R. Agarwal, A.J. Garcı´a, Biomaterial strategies for engineering implants for enhanced osseointegration and bone repair, Adv. Drug Deliv. Rev. 94 (2015) 53e62. R. Paul, A. David Farrar, C.C. Perryy, Interpretation of protein Adsorption: surface-induced conformational changes, J. Am. Chem. Soc. 84 (1994) 8168e8173. B. Nilsson, K.N. Ekdahl, T.E. Mollnes, J.D. Lambris, The role of complement in biomaterial-induced inflammation, Mol. Immunol. 44 (2007) 82e94. L. Bai, Z. Du, J. Du, W. Yao, J. Zhang, Z. Weng, S. Liu, Y. Zhao, Y. Liu, X. Zhang, A multifaceted coating on titanium dictates osteoimmunomodulation and osteo/angiogenesis towards ameliorative osseointegration, Biomaterials 162 (2018) 154. A.K. Refai, M. Textor, D.M. Brunette, J.D. Waterfield, Effect of titanium surface topography on macrophage activation and secretion of proinflammatory cytokines and chemokines, J. Biomed. Mater. Res. A 70 (2004) 194e205. J. Takebe, C. Champagne, S. Offenbacher, K. Ishibashi, L. Cooper, Titanium surface topography alters cell shape and modulates bone morphogenetic protein 2 expression in the J774A. 1 macrophage cell line, J. Biomed. Mater. Res. Part A 64 (2003) 207e216. K.A. Barth, J.D. Waterfield, D.M. Brunette, The effect of surface roughness on RAW 264.7 macrophage phenotype, J. Biomed. Mater. Res. A 101A (2013) 2679e2688. A. Das, C.E. Segar, B.B. Hughley, D.T. Bowers, E.A. Botchwey, The promotion of mandibular defect healing by the targeting of S1P receptors and the recruitment of alternatively activated macrophages, Biomaterials 34 (2013) 9853e9862. N.A. Sims, J.M. Quinn, Osteoimmunology: oncostatin M as a pleiotropic regulator of bone formation and resorption in health and disease, BoneKEy Rep. 3 (2014) 527. M.H.A. Meguid, Y.H. Hamad, R.S. Swilam, M.S. Barakat, Relation of interleukin-6 in rheumatoid arthritis patients to systemic bone loss and structural bone damage, Rheumatol. Int. 33 (2013) 697e703. I.E. Adamopoulos, M. Tessmer, C.C. Chao, S. Adda, D. Gorman, M. Petro, C.C. Chou, R.H. Pierce, W. Yao, N.E. Lane, IL-23 is critical for induction of arthritis, osteoclast formation, and maintenance of bone mass, J. Immunol. 187 (2011) 951. M.J. Dalby, N. Gadegaard, R. Tare, A. Andar, M.O. Riehle, P. Herzyk, C.D. Wilkinson, R.O. Oreffo, The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder, Nat. Mater. 6 (2007) 997e1003.

228 Bioactive Materials for Bone Regeneration [15] J. Wang, F. Meng, W. Song, J. Jin, Q. Ma, D. Fei, L. Fang, L. Chen, Q. Wang, Y. Zhang, Nanostructured titanium regulates osseointegration via influencing macrophage polarization in the osteogenic environment, Int. J. Nanomed. 13 (2018) 4029. [16] C.J. Wilson, R.E. Clegg, D.I. Leavesley, M.J. Pearcy, Mediation of biomaterialecell interactions by adsorbed proteins: a review, Tissue Engineering 11 (2005) 1e18. [17] M.A. Dobrovolskaia, S.E. McNeil, Immunological properties of engineered nanomaterials, Nat. Nanotechnol. 2 (2007) 469e478. [18] L. Rifas, S. Arackal, M.N. Weitzmann, Inflammatory T cells rapidly induce differentiation of human bone marrow stromal cells into mature osteoblasts, J. Cell. Biochem. 88 (2003) 650e659. [19] L. Rifas, T-cell cytokine induction of BMP-2 regulates human mesenchymal stromal cell differentiation and mineralization, J. Cell. Biochem. 98 (2010) 706e714. [20] R.M. Boehler, J.G. Graham, L.D. Shea, Tissue engineering tools for modulation of the immune response, Biotechniques 51 (2011) 239. [21] M.A. Alfarsi, S.M. Hamlet, S. Ivanovski, Titanium surface hydrophilicity modulates the human macrophage inflammatory cytokine response, J. Biomed. Mater. Res. A 102 (2013) 60e67. [22] K.M. Hotchkiss, G.B. Reddy, S.L. Hyzy, Z. Schwartz, B.D. Boyan, R. Olivares-Navarrete, Titanium surface characteristics, including topography and wettability, alter macrophage activation, Acta Biomaterialia 31 (2016) 425e434. [23] O. Veiseh, J.C. Doloff, M. Ma, A.J. Vegas, H.H. Tam, A.R. Bader, J. Li, E. Langan, J. Wyckoff, W.S. Loo, Size- and shape-dependent foreign body immune response to materials implanted in rodents and non-human primates, Nat. Mater. 14 (2015) 643e651. [24] W.K. Oh, S. Kim, M. Choi, C. Kim, Y.S. Jeong, B.R. Cho, J.S. Hahn, J. Jang, Cellular uptake, cytotoxicity, and innate immune response of silica-titania hollow nanoparticles based on size and surface functionality, ACS Nano 4 (2010) 5301e5313. [25] P. Laquerriere, A. Grandjeanlaquerriere, E. Jallot, G. Balossier, P. Frayssinet, M. Guenounou, Importance of hydroxyapatite particles characteristics on cytokines production by human monocytes in vitro, Biomaterials 24 (2003) 2739e2747. [26] B.R. Cuenya, Synthesis and catalytic properties of metal nanoparticles: size, shape, support, composition, and oxidation state effects, Thin Solid Films 518 (2010) 3127e3150. [27] M.O. Oyewumi, A. Kumar, Z. Cui, Nano-microparticles as immune adjuvants: correlating particle sizes and the resultant immune responses, Expert Rev. Vaccines 9 (2010) 1095e1107. [28] O. Malard, J.M. Bouler, J. Guicheux, D. Heymann, P. Pilet, C. Coquard, G. Daculsi, Influence of biphasic calcium phosphate granulometry on bone ingrowth, ceramic resorption, and inflammatory reactions: Preliminary in vitro and in vivo study, J. Biomed. Mater. Res. A 46 (2015) 103e111. [29] L. Bai, Y. Liu, Z. Du, Z. Weng, W. Yao, X. Zhang, X. Huang, X. Yao, R. Crawford, R. Hang, Differential effect of hydroxyapatite nano-particle versus nano-rod decorated titanium micro-surface on osseointegration, Acta Biomater. (2018). [30] Z. Wang, Z. Tang, F. Qing, Y. Hong, X. Zhang, Applications of calcium phosphate nanoparticles in porous hard tissue engineering scaffolds, Nano 7 (2012) 1230004. [31] Z. Tang, X. Li, Y. Tan, H. Fan, X. Zhang, The material and biological characteristics of osteoinductive calcium phosphate ceramics, Regenerat. Biomat. 5 (2017) 43e59. [32] D.W. I S, O.B. R G, M. KM, V.Z. WU, Experimental comparison of monofile light and heavy polypropylene meshes: less weight does not mean less biological response, World J. Surg. 31 (2006) 1586e1591.

A bone regeneration concept Chapter | 3 [33]

[34] [35] [36]

[37] [38]

[39]

[40]

[41] [42]

[43]

[44]

[45]

[46]

[47] [48] [49]

229

M.W. Laschke, Y. Harder, M. Amon, I. Martin, J. Farhadi, A. Ring, N. Torio-Padron, R. Schramm, M. Ru¨cker, D. Junker, Angiogenesis in tissue engineering: breathing life into constructed tissue substitutes, Tissue Eng. A 12 (2006) 2093. V. Karageorgiou, D. Kaplan, Porosity of 3D biomaterial scaffolds and osteogenesis, Biomaterials 26 (2005) 5474e5491. M. Bohner, L. Galea, N. Doebelin, Calcium phosphate bone graft substitutes: failures and hopes, J. Eur. Ceram. Soc. 32 (2012) 2663e2671. C. Wu, Z. Chen, Q. Wu, D. Yi, T. Friis, X. Zheng, C. Jiang, X. Jiang, X. Yin, Clinoenstatite coatings have high bonding strength, bioactive ion release, and osteoimmunomodulatory effects that enhance in vivo osseointegration, Biomaterials 71 (2015) 35e47. A. De, Wnt/Ca2þ signaling pathway: a brief overview, Acta Biochim. Biophys. Sin. 43 (2011) 745. R.J. Macleod, M. Hayes, I. Pacheco, Wnt5a secretion stimulated by the extracellular calcium-sensing receptor inhibits defective Wnt signaling in colon cancer cells, Am. J. Physiol. Gastrointest. Liver Physiol. 293 (2007) G403. A.K. Gaharwar, S.M. Mihaila, A. Swami, A. Patel, S. Sant, R.L. Reis, A.P. Marques, M.E. Gomes, A. Khademhosseini, Bioactive silicate nanoplatelets for osteogenic differentiation of human mesenchymal stem cells, Adv. Mater. 25 (2013) 3329e3336. C. Wu, P. Han, M. Xu, X. Zhang, Y. Zhou, G. Xue, J. Chang, Y. Xiao, Nagelschmidtite bioceramics with osteostimulation properties: material chemistry activating osteogenic genes and WNT signalling pathway of human bone marrow stromal cells, J. Mater. Chem. B 1 (2013) 876e885. A.M. Pietak, J.W. Reid, M.J. Stott, M. Sayer, Silicon substitution in the calcium phosphate bioceramics, Biomaterials 28 (2007) 4023e4032. S. Jun, A.M. Romani, A.M. Valentin-Torres, A.A. Luciano, C.M. Kitchen, Ramirez, F. Nicholas, M. Sam, H.B. Bernstein, Magnesium decreases inflammatory cytokine production: a novel innate immunomodulatory mechanism, J. Immunol. 206 (2012). S361S361. T.S. Li, K. Hamano, K. Suzuki, H. Ito, N. Zempo, M. Matsuzaki, Improved angiogenic potency by implantation of ex vivo hypoxia prestimulated bone marrow cells in rats, Am. J. Physiol. Heart Circ. Physiol. 283 (2002) 468e473. Z. Chen, Z. Wang, Q. Wang, W. Cui, F. Liu, W. Fan, Changes in early serum metal ion levels and impact on liver, kidney, and immune markers following metal-on-metal total hip arthroplasty, J. Arthroplast. 29 (2014) 612e616. Z. Chen, J. Yuen, R. Crawford, J. Chang, C. Wu, Y. Xiao, The effect of osteoimmunomodulation on the osteogenic effects of cobalt incorporated b-tricalcium phosphate, Biomaterials 61 (2015) 126e138. J.K. Park, Y.J. Kim, J. Yeom, J.H. Jeon, G.C. Yi, J.H. Je, S.K. Hahn, The topographic effect of zinc oxide nanoflowers on osteoblast growth and osseointegration, Adv. Mater. 22 (2010) 4857e4861. H. Haase, L. Rink, Signal transduction in monocytes: the role of zinc ions, Biometals 20 (2007) 579. V. F, B. J, A. J, L. P, Inflammatory cell response to calcium phosphate biomaterial particles: an overview, Acta Biomater. 9 (2013) 4956e4963. W. Liu, J. Li, M. Cheng, Q. Wang, K.W. Yeung, P.K. Chu, X. Zhang, Zinc-modified sulfonated polyetheretherketone surface with immunomodulatory function for guiding cell fate and bone regeneration, Adv. Sci. (2018) 1800749.

230 Bioactive Materials for Bone Regeneration [50] W. C, Z. Y, L. C, C. J, X. Y, Strontium-containing mesoporous bioactive glass scaffolds with improved osteogenic/cementogenic differentiation of periodontal ligament cells for periodontal tissue engineering, Acta Biomater. 8 (2012) 3805e3815. [51] Y. Zhang, L. Wei, J. Chang, R.J. Miron, B. Shi, S. Yi, C. Wu, Strontium-incorporated mesoporous bioactive glass scaffolds stimulating in vitro proliferation and differentiation of bone marrow stromal cells and in vivo regeneration of osteoporotic bone defects, J. Mater. Chem. B 1 (2013) 5711e5722. [52] A. Lode, C. Heiss, G. Knapp, J. Thomas, B. Nies, M. Gelinsky, M. Schumacher, Strontiummodified premixed calcium phosphate cements for the therapy of osteoporotic bone defects, Acta Biomaterialia 65 (2018) 475e485. [53] Y. Li, E. Luo, S. Zhu, J. Li, L. Zhang, J. Hu, Cancellous bone response to strontium-doped hydroxyapatite in osteoporotic rats, J. Appl. Biomater. Funct. Mater. 13 (2015) 28e34. [54] X. Yuan, H. Cao, J. Wang, K. Tang, B. Li, Y. Zhao, M. Cheng, H. Qin, X. Liu, X. Zhang, Immunomodulatory effects of calcium and strontium co-doped titanium oxides on osteogenesis, Front. Immunol. 8 (2017) 1196.

Index Note: ‘Page numbers followed by “t” indicate tables, “f” indicate figures and “b” indicate boxes’.

A Acquired host immune response, 186 Akermanite (AKT), 122, 143 Alkaline phosphatase, 206 Amber force field-based simulation, 55 Angiogenesis, 109e110, 117, 171e172 Articular cartilage, 153 Aseptic loosening cobalt-chromium alloy implants, 203e204 titanium implants, 203 Autologous bone grafting, 153

B Bioactive glass, 56, 207 Bioactive ion-based osteoimmunomodulation strategy angiogenesis, 109e110 applications, 109e122 calcium, 114e115 cobalt, 116e118 copper, 118e119 europium (Eu), 119e120 fluorine, 120e122 magnesium, 113e114 osteoblastic cell proliferation and differentiation, 109 physiological functions, 109 silicon (Si), 115e116 strontium (Sr), 110e112 therapeutic effects, 109 zinc, 112e113 Bioactive materials bonelike apatite formation, 14 challenges and prospects, 12e14 chemical composition, 4 grain size. See Grain size micropores, 5 nanoscale topography, 14e15 osteogenesis. See Osteogenesis porous initial nanopowder and precursor, 6e7 molding, 7e9

sintering process, 9e11 structure, 4e5 surface modification methods, 11e12 protein adsorption. See Protein adsorption trace ion doping, 16 whisker reinforcement, 15e16, 15f Bioceramics, 152 Biodegradable ceramics, 13 Biomaterial-specific tissue response, 186 Biphasic calcium phosphate (BCP), 4 grain sizes, 36e39 surface modification, 11e12 B-lymphocyte response, 194 Bonelike apatite formation, 14 Bone morphogenetic proteins (BMPs), 56e57, 65e66, 87 Bone-relevant physiological and pathological processes B-lymphocyte response, 194 dendritic cells (DCs) host response, 194 lymphocytes, 194e195 macrophages, 195 macrophage aseptic loosening, 190e192 immune functions, 188e190 initiating factors, 187e188 organ injury, 187 phagocytic capacity, 190 platelets, 187e188 subtypes, 188e190 wound-healing process, 190 natural killer (NK) cells, 195 T lymphocytes cytotoxic (CD8+), 192 IL-12 and IL-23 control, 193 signals, 192e193 Th1 and Th2 lymphocyte polarization, 192e193 T-helper (CD4+), 192 Bone-replacement-material research, 185

231

232 Index Bone tissue engineering (BTE) bioactive ions angiogenesis, 171e172 cell proliferation, 167e168 composite biomaterials, 172e174, 174f osteoclastogenesis, 168e171 osteogenesis, 168e171 stemness maintenance, 167e168 challenges, 166 factors affecting, 166e167 neovascularization, 166e167

C Ca-deficient hydroxyapatite (CDHA), 4 Calcium bone dynamics, 115 CaP and calcium silicate, 115 homeostasis, 114 inflammatory signaling pathways, 115 osteoimmunomodulatory properties, 115 Calcium phosphate cements (CPCs), 36, 135 Calcium phosphate (Ca-P) ceramics, 4 bonelike apatite formation, 14 dense, 4e5 3D printing methods, 9, 9f gas-foaming method, 7e8, 8f grain size microscale and submicroscale, 36e40, 37te38t nanoscale grain size, 40e43 immune microenvironment inflammation adjustment, 206 phosphate ion effects, 206 incubation environment, 60 micropores, 5 microwave sintering, 10e11 nanoceramics, 42e43 nanopowder and precursor synthesis, 6e7 nanoscale topography, 14e15 orderly micropatterned surface morphology, 26e27, 28f osteogenesis. See Osteogenesis porous, 4e5 protein adsorption. See Protein adsorption randomly structured surface morphology, 29e33 fabrication methods, 33 hydrothermal treatment, 29e31, 30fe31f simulated body fluid immersion and inducing, 31e33 surface topography, 5e6, 34f

Calcium sensing receptors (CaSR), 83 cAMP-response element binding protein (CREB) promotor, 83 Cartilage, 151e152 cDNA microarray, 91e92 Cell migration assay, 93e95 Ceramic-based composite, 13 Charmm force field-based simulations, 55 Chemoattractive-signalling molecules, 188e190 Chitosan scaffolds, 61 Chronic inflammatory phase, 188e190 Clinoenstatite (CLT) coating, 122e124 Cobalt (Co) angiogenesis, 117 bone-substitute materials, 117 Ca-chromium-molybdenum (Mo), 116e117 hypoxic-like microenvironment, 117 immunomodulatory effect, 117e118 Cobalt-chromium alloy implants aseptic loosening, 203e204 immune response, 203 Collagen (Col), 63e64 Congenital immunity, 186 Conventional biphasic calcium phosphate (cBCP) ceramics, 43 Copper extraantimicrobial effect, 119 hypoxia-mimicking element, 118 osteogenic differentiation, 118 osteoimmunomodulation, 118e119 Copper-containing mesoporous silica nanospheres (Cu-MSNs), 118e119 Cytokine adsorption, 67e68

D Danger signals, 187e188 Dendritic cells (DCs) host response, 194 lymphocytes, 194e195 macrophages, 195 Dentin sialophosphoprotein (DSPP), 145 Dicalcium silicate (C2S), 135, 136f, 137, 138f preparation, 135e139 self-setting properties and drug delivery performance 3D printing-hydration method, 137e139 drug loading and release properties, 143e144 foaming-hydration method, 137e139 injectability and washout resistance, 143

Index optical micrographs, 141f particle size distribution, average size, and specific surface area, 139t setting time, compressive strength, and porosity, 139e143, 141t XRD patterns and SEM images, 137, 138f Direct laser interference patterning (DLIP), 11e12 3D printing, 8t

E Electrospinning, 8t Ellipsometry, 53e54 Endothelial cells, 85e86 Endothelial-mesenchymal transition (EndMT), 85e86 Europium (Eu), 119e120 Extracellular protein adsorption collagen (Col), 63e64 fibronectin (FN), 64e65 human umbilical vein endothelial cell (HUVEC)-derived, 63 integrin expression, 63 laminin (LN), 65 mesenchymal stem cells (MSCs), 63 vitronectin (VN), 65 Extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, 83

F Fibroblast-like synovial cells (FLSs), 204 Fibronectin (FN), 64e65 Fluorine bone formation, 122 bone physiological activity modulation, 120 concentrations, 120 osteoimmunomodulatory effect, 120e122, 121f Focal adhesion kinase (FAK), 88 Foreign-body giant cells (FBGCs), 186 macrophage fusion, 205 phagocytic abilities, 190 Fourier transform infrared attenuated total internal reflection technique, 53e54 Freeze-drying, 8t Frustrated phagocytosis, 190

233

G Gas-foaming, 8t Gene microarray analysis, 91e92 G-protein coupled receptor (GPCR) signaling pathway, 91e92 Grain size microscale and submicroscale grain sizes biphasic calcium phosphate (BCP), 36e39 Ca-P-based bioceramics, 36e39, 37te38t sintering temperature, 36e39 TCP bioceramics, 40 nanoscale grain size bionic manufacturing, 40e41 bone biomineralization, 42 calcium orthophosphates, 42 calcium phosphate (Ca-P) nanoceramics, 42e43 hydroxyapatite (HA) ceramics, 42 protein interactions, 41e42 soft hydrogel, 40e41

H Hot pressure sintering, 10t Human bone marrow stromal cells (hBMSCs), 40 Hyaline cartilage, 151e152, 154 Hydrophobicity, 60e61 Hydroxyapatite (HA) ceramics, 4 muffle sintering, 10e11 nanoscale grain size, 42 orderly micropatterned surface morphology, 26e27 SEM images, 10e11, 11f surface micro- and nanostructure, 5e6 surface modification, 11e12 Hydroxyapatite (HA) whisker-reinforced biphasic calcium phosphate (BCP) ceramic composite, 15e16 Hypoxia-inducible factors (HIFs), 67e68, 109e110

I Immune cells, 195 Immune-mediated osteogenesis ion regulation calcium (Ca), 68e69

234 Index Immune-mediated osteogenesis (Continued ) cobalt (Co), 80e81 magnesium (Mg), 79e80 silicon (Si), 68e69, 79 strontium (Sr), 82 zinc (Zn), 81 microporosity regulation, 82e90 particle size regulation, 68e69 surface charge, 67 surface nanotopography, 65e67 Immune microenvironment inorganic materials implants bioactive glass, 207 bioceramic implants, 205 calcium phosphate ceramics, 206 calcium-silica ceramics, 207 hydroxyapatite and strontium-doped calcium phosphate, 208 immune response, 186 magnesium regulation, 208 pore and particle sizes, 53e54 silicon regulation, 207 zinc regulation, 208 organic materials implants bioactive factor delivery, 63e65 geometry property regulation, 62e68 hydrogels, 56 polylactic acid regulation, 56e60 surface property regulation, 60e61 topography property regulation, 61e62 ultra-high-molecular-weight polyethylene (UHMWPE), 56e61 Immunoglobulin E, 192e193 Immunoresponse, osteogenesis autocrine effect, mesenchymal stem cells adenosine-signaling pathway, 92 BMP-initiated osteogenic differentiation, 90e91 gene microarray analysis, 91e92 G-protein coupled receptor (GPCR) signaling pathway, 91e92 cells and signals, 91f paracrine effect bone implant materials, 92e93 calcium concentration, 93 ELISA analysis, 93e95, 94f in vitro methods, 92e93 in vivo single-cell typed coculturingmacrophages, 92e93

T cells and B cells, 95 Inflammasome, 56e61 Innate immune response, 186 Integrins, 187e188 Interleukin (IL-6), 67e68

L Laminin (LN), 65 Lipoproteins, 195 Lipoteichoic acids, 195 Lithium-based biomaterials, 157e158 Lymphocytes, 186 Lysozyme (LSZ), 58e59

M Macromolecular foreign bodies, 188e190 Macrophage, 67e68, 186 aseptic loosening, 190e192 immune functions, 188e190 initiating factors, 187e188 organ injury, 187 phagocytic capacity, 190 platelets, 187e188 polylactic acid regulation, 56e57 subtypes, 188e190 surface property regulation, 60e61 titanium implants, 201e202 topography property regulation, 61e62 wound-healing process, 190 Macrophage inflammatory protein-1, 205 Magnesium (Mg) immune response modulation, 113e114 osteoimmunomodulatory effects, 114 properties, 113 skeleton metabolism, 113 Magnesium (Mg)-containing calcium phosphate cement (MCPC), 114 Magnesium phosphate (MPC), 139e143 Manganese-doped biomaterials, 156 Mast cells, 192e193 Mesenchymal stem cells autocrine effect adenosine-signaling pathway, 92 BMP-initiated osteogenic differentiation, 90e91 gene microarray analysis, 91e92 G-protein coupled receptor (GPCR) signaling pathway, 91e92 biphasic calcium phosphate (BCP) bioceramics, 82 calcium ion gradient, 82e83

Index ionic environment, 82e84 osteoblastic differentiation, 89f PO3-4 internalization, 84 Metallic implants cobalt-chromium alloy implants aseptic loosening, 203e204 immune response, 203 titanium implants aseptic loosening, 203 implantation, 201 implant surface characteristics, 202 macrophage role, 201e202 T cell and dendritic cell roles, 202 Microgrooves, 11e12 Micromachining method, 11e12 Microsphere-sintering, 8t Microwave sintering, 10e11, 10t Mitogen-activated protein kinase (MAPK), 88 Molecular dynamics (MD) HSA adsorption, 54e55 limitation, 55 protein interactions, 55 Molybdenum-doped biomaterials, 156e157 Monocytes, 187e188 Mononuclear phagocyte cells, 186 Muffle sintering, 10t

N Nagelschmidtite (NAGEL), 122 Natural killer (NK) cells, 195

O Organic foam impregnation, 8t Osteochondral defects, 151e152 Osteochondral regeneration auto/allograft, 152 cartilage and bone regeneration, 155 clinical need, 153 minimally invasive therapy, 154e155 osteochondral tissue, 152e154 restorative treatments, 154e155 trace element-based biomaterials lithium-based biomaterials, 157e158 manganese-doped biomaterials, 156 molybdenum-doped biomaterials, 156e157 nutrient elements, 161 strontium-based biomaterials, 160e161, 160f

235

Osteogenesis bioactive material angiogenesis BMP-7 and BMP-2, 80e81 endothelial cells and pericytes, 80e81 MSCs recruitment, 80e81 neo-vascularization, 79e80 porous Ca-P-based bioceramics, 79e80 porous Ca-P ceramics, 81, 81f cells of origin, 84e86 cellular level events bone morphogenetic proteins (BMPs), 87 cell adhesion, 87e88 ectopic bone formation process, 86e87 focal adhesion kinase (FAK), 88 gene expression, 86e87, 86f mitogen-activated protein kinase (MAPK), 88 mesenchymal stem cell function biphasic calcium phosphate (BCP) bioceramics, 82 calcium ion gradient, 82e83 ionic environment, 82e84 osteoblastic differentiation, 89f PO3-4 internalization, 84 osteoinduction, 79, 80f porous titanium, 88e90 Osteoimmunomodulation B cells and dendritic cells, 107 bioactive elements incorporation akermanite and nagelschmidtite, 122, 123f Ca- and Sr-containing coatings, 124 clinoenstatite (CLT) coating, 122e124 Sr2MgSi2O7 (SMS) ceramic coatings, 122e124 Sr2ZnSi2O7 (SMS) ceramic coatings, 124 bioactive ion-based strategy, 107 bioactive ions, 108e109, 108f. See also Bioactive ion-based osteoimmunomodulation strategy ideal biomaterial, 107e108 T lymphocytes, 107 Osteoinduction, 79 Osteoinductivity, 79 Osteoprotegrin (OPG) expressions, 86e87, 86f Osterix (OSX), 86e87, 86f

236 Index

P Pericytes, 85e86 Platelet-derived growth factor (PDGF), 67 Platelets, 187e188 Polydopamine-assisted HA crystallization, 33 Polyetherketoneketone (PEKK) materials, 14 Polylactic acid (PLA), 56e60 Polymeric additives, 143 Polymethyl methacrylate (PMMA), 143 Polymorphonuclear cells, 186 Polymorphonuclear leukocytes (PMNs), 187e188 Porous bioactive materials initial nanopowder and precursor, 6e7 molding fabrication methods, 8t gas-foaming method, 7e8, 8f macropores, 7 micropores, 7 microsphere-sintering, 7e8 three-dimensional printing method, 9f sintering process, 9e11 structure, 4e5 surface modification methods, 11e12 Proinflammatory acute phase cytokines, 188e190 Protein adsorption bioactive materials bone morphogenetic proteins (BMPs), 65e66 cytokine adsorption, 67e68 electrostatic attraction, 62 extracellular protein adsorption, 63e65 IGF, 67 interactions, 61e62 osteogenesis, 62e68 platelet-derived growth factor (PDGF), 67 transcription growth factor beta (TGF-b), 66e67 vascular endothelial growth factor (VEGF), 67 bone-relative proteins, 53 chemical properties BSA, 59e60 incubation environment, 60 lysozyme adsorption, 58e59 proline-rich acidic salivary protein (PRP1), 59e60 protein-surface interactions, 58e59 transforming growth factor-b1 (TGF-b1), 58e59 computing methods, 54e55

experimental methods, 53e54 hydrophobicity, 60e61 surface topography particle size, 56e57 porosity, 56e57 roughness, 56

Q Quercetin (QUE)-tuned hydrothermal system, 33

S Silicate-based bioceramic scaffolds (SiCPs), 161 Silicate-based bone cements osteoinductivity, 145e148, 146f polymeric additives, 143 polymerization and solidification, 139e143 preparation, 135e139 setting time and mechanical strength, 139e143, 142t in vitro and in vivo bioactivity, 144e148 Silicon (Si), 4 bone formation, 116 immune reaction, 116 immune-stimulatory effects, 116 immunomodulatory effects, 115e116 Simulated body fluid (SBF) immersing method, 14 Sintering process advantages and disadvantages, 10t brittleness, 9e10 higher temperature, 10e11 microwave sintering, 10e11 Solute carrier family 20 member 1 (SLC20a1), 84 Spark plasma sintering, 10t Strontium (Sr) antiosteoclastogenic effect, 110 biomaterial osteogenic capacity improvement, 111 immune response regulation, 111 ionic concentration, 111e112 monodispersed Sr-contained bioactive glasses microspheres, 111 neovascularization, 110 osteochondral regeneration, 160e161, 160f osteogenesis, 110 osteoimmunomodulation, 110e112

Index Strontium-doped calcium phosphate (SCP), 208 Subchondral bone, 154 Surface micro- and nanostructure, 5e6 calcium phosphate-based bioceramics, 26e27 cellular response, 26 grain size microscale and submicroscale, 36e40 nanoscale grain size, 40e43 orderly micropatterned surface morphology, 26e27 orthopedic implant surface, 26 porosity calcium phosphate cement (CPC) scaffolds, 36 macroporosity, 35e36 micropores, 35 pore size, 35 tangent elastic modulus, 36 Ti6Al4V scaffolds, 36 randomly structured surface morphology, 29e33 Surface modification methods, 11e12

T T cells, 186, 202 Three-dimensional printing method, 8t, 9f Titanium implants, 204f advantages, 201 immune response aseptic loosening, 203 implantation, 201 implant surface characteristics, 202 macrophage role, 201e202 T cell and dendritic cell roles, 202 Trace ion doping, 16 Transcription growth factor beta (TGF-b), 66e67 Tricalcium phosphate (TCP-S) ceramics, 55

237

Tricalcium silicate (C3S), 135, 137, 138f preparation, 135e139 self-setting properties and drug delivery performance 3D printing-hydration method, 137e139 drug loading and release properties, 143e144 foaming-hydration method, 137e139 injectability and washout resistance, 143 optical micrographs, 141f particle size distribution, average size, and specific surface area, 139t setting time, compressive strength, and porosity, 139e143, 141t XRD patterns and SEM images, 137, 138f Tumor necrosis factor-a (TNF-a), 67e68 Two-step sintering, 10t

U Ultra-high-molecular-weight polyethylene (UHMWPE), 56e61

V Vacuum sintering, 10t Vascular endothelial growth factor (VEGF), 67, 187e188 Vitronectin (VN), 65

Z Zinc bone regeneration, 113 deficiency, 112 osteoblastogenesis, 112 osteoimmune microenvironment modulation, 112e113 Zinc-substituted hydroxyapatite (ZnHA) nanocrystals, 56e57