Octacalcium Phosphate Biomaterials 9780081025116, 0081025114, 9780081025123

Table of contents :
Content: 1. Octacalcium Phosphate Biomaterials - Past present and Future 2. Octacalcium phosphate effects on the systematic and local factors that regulate bone cell activity 3. Octacalcium phosphate and the Biomineralisation of bone, cellular interactions and their role in bone repair 4. Octacalcium Phosphate bone replacement and drug delivery 5. Dissolution and growth of Octacalcium Phosphate in the presence of tooth proteins 6. The influence of sterilization on Octacalcium phosphate for clinical applications 7. An osteoinductive biomaterial Octacalcium Phosphate and its mechanism for bone formation 8. Novel scaffold composites containing Octacalcium Phosphate and their role in bone repair 9. Synthesis methodologies options for large scale manufacture of Octacalcium Phosphate 10. Biomimetic synthesis of calcium phosphate-based biomaterials for hard tissue regeneration 11. Future analysis techniques to assess mechanical and physiological behavior of novel calcium phosphate materials including Octacalcium Phosphate - In-vivo human clinical model 12. Octacalcium phosphate for implant coating 13. Calcium phosphate-based bone graft substitutes and the special roles of Octacalcium phosphate materials 14. Unique compositional and structural properties of OCP among calcium phosphate 15. Octacalcium phosphate collagen composites and their clinical application

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Octacalcium Phosphate Biomaterials

Woodhead Publishing Series in Biomaterials

Octacalcium Phosphate Biomaterials Understanding of Bioactive Properties and Application

Edited by

Osamu Suzuki Gerard Insley

Woodhead Publishing is an imprint of Elsevier The Officers’ Mess Business Centre, Royston Road, Duxford, CB22 4QH, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, OX5 1GB, United Kingdom Copyright © 2020 Elsevier Ltd. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-08-102511-6 (print) ISBN: 978-0-08-102512-3 (online) For information on all Woodhead Publishing publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Matthew Deans Acquisition Editor: Sabrina Webber Editorial Project Manager: Gabriela Capille Production Project Manager: Swapna Srinivasan Cover Designer: Alan Studholme Typeset by MPS Limited, Chennai, India

Contents

List of contributors About the editors Preface 1

2

Evolution of octacalcium phosphate biomaterials Osamu Suzuki 1.1 Introduction 1.2 Selection of suitable materials to promote bone tissue repair 1.2.1 Octacalcium phosphate: materials science and relevance for biomaterials 1.3 Major findings and advances 1.3.1 Hypothesis and experimental support for the biological significance of octacalcium phosphate 1.3.2 Initial evidence that octacalcium phosphate provides a nucleation site for bone deposition 1.3.3 Elucidation of the mechanism of OCP-mediated bone formation and its association with OCP hydrolysis to HA 1.4 Development of octacalcium phosphate based bone substitute materials 1.5 Octacalcium phosphate biomaterials 1.6 Concluding remarks Acknowledgments References Octacalcium phosphate effects on the systemic and local factors that regulate bone-cell activity Yukari Shiwaku and Osamu Suzuki 2.1 Introduction 2.1.1 The effect of octacalcium phosphate on osteoblastic differentiation 2.1.2 Application of mesenchymal stem cells for bone regeneration 2.1.3 Octacalcium phosphate-mediated promotion of the transition from osteoblasts to osteocytes 2.1.4 Induction of osteoclast formation by octacalcium phosphate

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17 17 18 22 23 24

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2.1.5

Osteoblast osteoclast cross talk mediated by octacalcium phosphate 2.1.6 Enhancement of macrophage migration and regulation of immune response by octacalcium phosphate 2.1.7 Suppression of chondrogenic differentiation by octacalcium phosphate 2.1.8 Reparative dentin formation via odontoblast differentiation promoted by octacalcium phosphate 2.2 Conclusion Acknowledgment References 3

4

Functionalization of octacalcium phosphate for bone replacement Adriana Bigi and Elisa Boanini 3.1 Introduction 3.2 Structure and chemistry of octacalcium phosphate 3.3 Interaction with carboxylic acids 3.4 Interaction with biological molecules 3.5 Ion-substituted/doped octacalcium phosphate 3.6 Octacalcium phosphate for drug delivery 3.7 Concluding remarks References The influence of sterilization on octacalcium phosphate for clinical applications Kieran A. Murray, Nicola Do¨belin, Ahmad B. Albadarin, Jarosław Sadło, Guang Ren, Maurice N. Collins and Cathriona O’Neill 4.1 Introduction 4.2 Materials and methods 4.2.1 Material preparation 4.2.2 Packaging 4.2.3 Gamma irradiation 4.2.4 Annealing, accelerated aging, and sample description 4.2.5 Characterization techniques 4.3 Results and discussion 4.3.1 Electron spin resonance 4.3.2 X-ray diffraction 4.3.3 Ca:P ratio 4.3.4 Fourier-transform infrared spectroscopy 4.3.5 Scanning electron microscopy 4.3.6 Thermogravimetric analysis 4.4 Conclusion Acknowledgments References

26 27 29 31 31 33 33 37 37 38 41 43 45 47 49 50

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55 57 57 58 58 59 59 62 62 66 73 76 76 77 78 79 79

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Bioactivity and effect of bone formation for octacalcium phosphate ceramics Vladimir S. Komlev, Ilya I. Bozo, Roman V. Deev and Alex N. Gurin 5.1 Introduction 5.2 Synthesis of octacalcium phosphate powders 5.3 Octacalcium phosphate bulk scaffold preparation 5.4 Octacalcium phosphate effect on bone tissue cells in vitro 5.4.1 Octacalcium phosphate cytotoxicity and biocompatibility 5.4.2 Specific activity of octacalcium phosphate 5.5 Octacalcium phosphate stimulatory effect on reparative osteogenesis in vivo 5.5.1 Octacalcium phosphate bioactivity in the heterotopic conditions 5.5.2 Octacalcium phosphate bioactivity in the orthotopic conditions 5.6 Clinical experience of bone grafting with octacalcium phosphate 5.7 Octacalcium phosphate potential for activated bone substitutes development 5.7.1 Octacalcium phosphate based cell delivery 5.7.2 Octacalcium phosphate based growth factor delivery 5.7.3 Octacalcium phosphate based gene delivery 5.8 Conclusion References Novel scaffold composites containing octacalcium phosphate and their role in bone repair Ryo Hamai, Takahisa Anada and Osamu Suzuki 6.1 Introduction 6.2 Octacalcium phosphate/gelatin composites 6.2.1 Gelatin for bone substitute applications 6.2.2 Methods to prepare octacalcium phosphate/gelatin composites 6.2.3 Characterization of octacalcium phosphate/gelatin composites and octacalcium phosphate crystals precipitated in the presence of gelatin 6.2.4 Capacity of Octacalcium phosphate/gelatin composites to regenerate bone tissue in vivo 6.3 Octacalcium phosphate/collagen composites 6.3.1 Collagen for bone substitute applications 6.3.2 Fabrication and characterization of octacalcium phosphate/collagen composites 6.3.3 Bone regeneration capacity and biodegradation of octacalcium phosphate/collagen

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85 85 86 87 90 91 91 94 94 96 106 112 113 113 115 116 116

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6.4

Octacalcium phosphate/alginate composites 6.4.1 Application of alginate for preparation of scaffolds 6.4.2 Preparation of octacalcium phosphate/alginate composites 6.4.3 Characterization of octacalcium phosphate/alginate composites prepared by different methods 6.4.4 Capacity of octacalcium phosphate/alginate composites to regenerate bone tissue in vivo and stimulate cells in vitro 6.4.5 Octacalcium phosphate/alginate microbeads to deliver osteoblastic cells for tissue regeneration 6.5 Octacalcium phosphate/hyaluronic acid composites 6.5.1 Characteristics of hyaluronic acid and applications for bone repair 6.5.2 Preparation and characterization of octacalcium phosphate/hyaluronic acid composites 6.5.3 Capacity of octacalcium phosphate/hyaluronic acid composites to regenerate bone tissue in vivo and stimulate cells in vitro 6.6 Octacalcium phosphate based composites for other applications 6.7 Conclusion Acknowledgment References 7

Synthesis methodologies options for large-scale manufacturer of octacalcium phosphate Regina O’Sullivan and David Kelly 7.1 Introduction 7.2 Precipitation 7.3 Hydrolysis 7.4 Ion substitution 7.5 Aging 7.6 Parameters influencing large-scale manufacture 7.6.1 Dose rate 7.6.2 Molarity 7.6.3 pH 7.6.4 Temperature 7.6.5 Order of addition 7.6.6 Stir rate 7.7 Factors influenced by manufacturing parameters 7.7.1 Purity 7.7.2 Yield 7.8 Concluding remarks References Further reading

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Synthesis and physical chemical characterizations of octacalcium phosphate based biomaterials for hard-tissue regeneration Christian C. Rey, Christe`le Combes and Christophe Drouet Abbreviations 8.1 Introduction 8.2 Different types of octacalcium phosphates and related compounds 8.2.1 Triclinic octacalcium phosphate pentahydrate 8.2.2 Amorphous octacalcium phosphate 8.2.3 Apatitic octacalcium phosphate 8.2.4 What about carbonated octacalcium phosphate? 8.2.5 Biological apatites and octacalcium phosphate 8.2.6 Biomimetic nanocrystalline apatites and octacalcium phosphate 8.3 Synthesis routes of octacalcium phosphates 8.3.1 Hydrolysis 8.3.2 Precipitations 8.3.3 Constant composition crystal growth 8.4 Evolution of the different octacalcium phosphate phases and related compounds in aqueous media 8.4.1 Triclinic octacalcium phosphate 8.4.2 Amorphous octacalcium phosphate 8.4.3 Apatitic octacalcium phosphate 8.4.4 Biomimetic nanocrystalline apatites 8.5 Physical chemical characterization 8.5.1 Chemical analyses 8.5.2 X-ray diffraction 8.5.3 Vibrational spectroscopies 8.5.4 Solid-state nuclear magnetic resonance 8.6 Perspectives and conclusion References Calcium orthophosphate (CaPO4) based bone-graft substitutes and the special roles of octacalcium phosphate materials Sergey V. Dorozhkin 9.1 Introduction 9.2 General knowledge and definitions 9.3 CaPO4-based bone-graft substitutes 9.3.1 History 9.3.2 Chemical composition and preparation 9.3.3 Forming and shaping 9.4 The major properties of the calcium orthophosphate based biomaterials 9.4.1 Mechanical properties 9.4.2 Porosity

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213 213 214 216 216 216 218 220 220 222

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9.5

Biomedical applications 9.5.1 CaPO4 deposits (coatings, films, and layers) 9.5.2 Octacalcium phosphate containing biocomposites 9.5.3 Dental applications of octacalcium phosphate 9.6 Biological properties and in vivo behavior 9.6.1 Interactions with surrounding tissues and the host responses 9.6.2 Osteoconduction and osteoinduction 9.6.3 Biodegradation 9.6.4 Cellular response 9.7 CaPO4-based formulations in tissue engineering 9.7.1 Scaffolds and their properties 9.7.2 Scaffolds from CaPO4 9.8 A clinical experience 9.9 Conclusion and outlook References

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Development and clinical application of octacalcium phosphate/collagen composites Shinji Kamakura 10.1 Introduction 10.1.1 Necessity of bone regeneration 10.1.2 Bone regeneration by octacalcium phosphate 10.2 Development of octacalcium phosphate/collagen 10.3 Features of octacalcium phosphate/collagen from preclinical studies 10.3.1 Preclinical studies of octacalcium phosphate/collagen in small animals 10.3.2 Preclinical studies of octacalcium phosphate/collagen in large animals 10.4 Clinical research on octacalcium phosphate/collagen 10.4.1 Doctor-initiated clinical studies of octacalcium phosphate/collagen 10.4.2 Sponsor-initiated multicenter clinical trial of octacalcium phosphate/collagen 10.5 Future outlook of octacalcium phosphate/collagen Acknowledgments References Modification of octacalcium phosphate growth by enamel proteins, fluoride, and substrate materials and influence of morphology on the performance of octacalcium phosphate biomaterials Mayumi Iijima 11.1 Modification of octacalcium phosphate growth by enamel proteins and fluoride 11.1.1 Octacalcium phosphate growth in the presence of enamel proteins 11.1.2 Octacalcium phosphate growth in the presence of fluoride

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Octacalcium phosphate growth on β-tricalcium phosphate substrates with different particle size 11.2.1 Experimental 11.2.2 Octacalcium phosphate growth on micro tricalcium phosphate substrate 11.2.3 Octacalcium phosphate growth on nano tricalcium phosphate substrate 11.2.4 Octacalcium phosphate growth in basic solution 11.3 Morphology of octacalcium phosphate and mechanical property of its compression molding 11.3.1 Synthesis of octacalcium phosphate crystal 11.3.2 Measurement of the mechanical properties 11.3.3 Mechanical properties of octacalcium phosphate compact References

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Index

325 326 326 327 328 330 331 333 334 342 349

List of contributors

Ahmad B. Albadarin Stokes Laboratories, Bernal Institute, University of Limerick, Limerick, Ireland Takahisa Anada Division of Craniofacial Function Engineering, Tohoku University Graduate School of Dentistry, Sendai, Japan; Soft Materials Chemistry, Institute for Materials Chemistry and Engineering, Kyushu University, Fukuoka, Japan Adriana Bigi Department of Chemistry “Giacomo Ciamician”, University of Bologna, Bologna, Italy Elisa Boanini Department of Chemistry “Giacomo Ciamician”, University of Bologna, Bologna, Italy Ilya I. Bozo Human Stem Cells Institute, Moscow, Russia; State Research Centre A.I. Burnazyan Federal Medical Biophysical Centre of the FMBA of Russia, Moscow, Russia Christian C. Rey National Polytechnique Institute of Toulouse, Materials Engineering Department, CIRIMAT, University of Toulouse, CNRS, Toulouse INP – ENSIACET, Toulouse, France Maurice N. Collins Stokes Laboratories, Bernal Institute, University of Limerick, Limerick, Ireland Christe`le Combes National Polytechnique Institute of Toulouse, Materials Engineering Department, CIRIMAT, University of Toulouse, CNRS, Toulouse INP – ENSIACET, Toulouse, France Roman V. Deev Human Stem Cells Institute, Moscow, Russia Nicola Do¨belin RMS Foundation, Bettlach, Switzerland Sergey V. Dorozhkin Kudrinskaja square, Moscow, Russia Christophe Drouet National Polytechnique Institute of Toulouse, Materials Engineering Department, CIRIMAT, University of Toulouse, CNRS, Toulouse INP – ENSIACET, Toulouse, France

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List of contributors

Alex N. Gurin Central Scientific Research Institute of Dentistry and Maxillofacial Surgery, Moscow, Russia; I.M. Sechenov First Moscow State Medical University, Moscow, Russia Ryo Hamai Division of Craniofacial Function Engineering, Tohoku University Graduate School of Dentistry, Sendai, Japan Mayumi Iijima Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan Shinji Kamakura Bone Regenerative Engineering Laboratory, Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan David Kelly PBC BioMed, Shannon, Ireland Vladimir S. Komlev A.A. Baikov Institute of Metallurgy and Materials Science, Russian Academy of Sciences, Moscow, Russia; Federal Research Center “Crystallography and Photonics” of the Russian Academy of Sciences, Moscow, Russia Kieran A. Murray PBC BioMed, Shannon, Ireland Cathriona O’Neill Bemis Healthcare Packaging, Clara, Ireland Regina O’Sullivan Chanelle Pharma, Galway, Ireland Guang Ren Stokes Laboratories, Bernal Institute, University of Limerick, Limerick, Ireland Jarosław Sadło Institute of Nuclear Chemistry and Technology, Warsaw, Poland Yukari Shiwaku Division of Craniofacial Function Engineering, Tohoku University Graduate School of Dentistry, Sendai, Japan Osamu Suzuki Division of Craniofacial Function Engineering, Tohoku University Graduate School of Dentistry, Sendai, Japan

About the editors

Gerard Insley is a Chief Scientific Officer at PBC Biomed, a development company of commercial biomaterials with facilities based in Ireland. He has a PhD in Bioceramics and is affiliated with the Angstrom Laboratory, University of Uppsala, Sweden. He has over 20 years of experience in developing biomaterials for bone repair in traumatology, arthroplasty, and cranial clinical areas. As a researcher, Gerard was involved in developing the first commercialized solution deposited hydroxyapatite coatings for ingrowth surfaces. Gerard also developed and commercialized the first bone replacement material that could be used in load-bearing applications and went onto develop calcium phosphate cements that are injectable and delivered premixed to the surgeon. Recently his research has been focused on the next generation of calcium-based bone materials that are bioactive and osteostimulative. Osamu Suzuki is a Professor and Chair of Division of Craniofacial Function Engineering, Tohoku University Graduate School of Dentistry, Sendai, Japan since 2004. He is in charge of education and research in the field of biomaterials science and skeletal tissue engineering. He was a Researcher in Japan Fine Ceramics Co. Ltd., Sendai and a Senior Researcher in JGC Corp., Yokohama/Oarai, Japan from 1986 to 2004. He was the first to found an osteoconductivity of synthetic octacalcium phosphate (OCP) in comparison with other calcium phosphate materials, such as nonsintered hydroxyapatite, in his PhD study, carried out until 1991, in Tohoku University School of Medicine, Sendai, Japan. He has also been developing OCP bone substitute materials, combined with natural polymers, such as collagen, gelatin, alginate, and hyaluronic acid, together with his collaborators since 2004. He received his MSc degree in Yamagata University Graduate School of Engineering, Yonezawa, Japan on the strength design of bioceramic materials in 1986. He joined a visiting scientist program in Forsyth Dental Center, Physical Chemistry Department, Boston, USA from 1992 to 1994 to study the synthesis and the physicochemical properties of OCP and its related calcium phosphate materials. He has also been focusing on the study to elucidate the mechanism on how the property of OCP materials relates to activating bone regeneration associated with the cellular function involved in the bone repair by OCP. He received the Award of Japanese Society for Biomaterials (JSB) in 2015 by a series of studies “Elucidation of osteoconductivity and establishment of biomaterial science of OCP bone substitute materials.” He is an Executive Committee Member of the JSB and the Japanese Society for Dental Materials and Devices (JSDMD) and Editor-in-Chief of Dental Materials Journal since 2018.

Preface

Almost exactly a century ago in 1920, the first study on using a synthetic calcium phosphate for bone defect repair was published by Albee and Morrison [1]. They reported that bone fractures showed a more rapid bone growth and union than did the controls when a triple calcium phosphate slurry was injected into the gap between the bone ends. Although not all modern concepts on biocompatibility, osteoconduction, bioresorption, and so on were clearly defined at the time, this study established the groundwork for using calcium phosphate compounds, especially those with compositions similar to that of bone mineral, for stimulating the bone growth in the repair process. Much progress has occurred in the last several decades, leading to the development of a wide range of calcium phosphate-based bone graft materials in clinical use today. Although calcium phosphate salts have been known since ancient times, it was the work conducted by researchers in agricultural sciences, biomineralization, and chemical industries during a good part of the 20th century, which led to significantly increased understanding of the crystal structures, thermodynamic solubilities, and other fundamental properties of all known calcium (ortho)phosphate compounds. This knowledge forms the foundation for the modern, current-day biomineralization research including the development of calcium phosphate biomaterials. Due to its elusive, highly transient nature, octacalcium phosphate (OCP) until recently was the least well-understood calcium phosphate compound. Walter Brown postulated in 1957 [2] that OCP is a critical precursor of biologically formed apatites. It was not until the 1990s that studies have confirmed OCP to be the first mineral phase to form in many mineralized tissues. OCP is unique in many ways including that one half of OCP’s crystal unit cell is nearly identical to that of hydroxyapatite (HA), allowing OCP to convert to HA in situ. Another interesting finding is that human serum is approximately saturated with respect to OCP while it is highly supersaturated with HA [3]. A 2001 Karger Monograph focused on the OCP structure, chemistry, and other unique properties. Since then, many studies have been conducted on OCP by researchers throughout the world, and one area that has seen significant advances is the development of OCP-based materials for bone defect repairs. The current book, which covers OCP as a next-generation scaffold for repair and regeneration applications in compromised bone, is co-edited by Dr. Gerard Insley and Professor Dr. Osamu Suzuki. Dr. Insley is a world-renowned scientist on calcium phosphate-based biomaterials. His expertise spans from basic chemistry to materials science to clinical development of calcium phosphate materials. Dr. Suzuki pioneered the work on OCP biomaterials that started in the 1980s and extends from basic properties to

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cell-material interactions to animal and clinical evaluations of OCP materials. They have compiled an extremely comprehensive list of chapters contributed by leading researchers in their respective fields relating to the OCP-based biomaterials. This book will, for the first time, allow readers with a wide range of scientific background to understand the most up-to-date work on OCP. The nature of human bone defects varies widely. It is unlikely that one or a handful of graft materials will be optimum for repairing all types of defects. An ideal graft material for a given kind of defect will need to have a specific set of chemical, biological, mechanical, clinical, and so on attributes best suited for the repair. With progressively better understanding of the properties of the graft materials and the material tissue interactions, it can be expected that synthetic calcium phosphate-based materials and biocomposites will practically replace autografts in all bone repair procedures in the foreseeable future. There is little doubt that OCP will play an important role in this process. Laurence C. Chow Volpe Research Center, American Dental Association Foundation, National Institute of Standards and Technology, Gaithersburg, MD, USA

References [1] Albee F, Morrison H. Studies in bone growth: triple calcium phosphate as a stimulus to osteogenesis. Ann Surg 1920;71:32 8. [2] Brown WE, Lehr JR, Smith JP, Frazier AW. Crystallography of octacalcium phosphate. J Am Chem Soc 1957;79(19):5318 19. [3] Eidelman N, Chow LC, Brown WE. Calcium phosphate saturation levels in ultrafiltered serum. Calcif Tissue Int 1987 Feb;40(2):71 8.

Evolution of octacalcium phosphate biomaterials

1

Osamu Suzuki Division of Craniofacial Function Engineering, Tohoku University Graduate School of Dentistry, Sendai, Japan

1.1

Introduction

Octacalcium phosphate (OCP, Ca8H2(PO4)6
5H2O) can be used as a bone substitute material due to its highly osteoconductive and bioactive properties [1 3]. This chapter provides an introduction to and overview of OCP biomaterials. The driving forces behind selection and use of OCP for bone tissue repair, significant research findings, and clinical applications of OCP, as well as factors that are crucial for novel bone healing treatments involving OCP are also discussed.

1.2

Selection of suitable materials to promote bone tissue repair

1.2.1 Octacalcium phosphate: materials science and relevance for biomaterials The crystal structure of OCP determined by Dr. Brown revealed an alternating stack of apatite and hydrated layers [4]. The refined OCP structure further studied by Mathew et al. showed the nonstoichiometric composition regarding hydrogen in the structure [5]. Tung et al. determined the solubility of OCP from 4 C to 37 C that included physiological temperatures and introduced a method to minimize OCP hydrolysis during the measurement [6]. OCP is a precursor of hydroxyapatite (HA) that can be formed from a supersaturated calcium and phosphate solution [7] and thus has been considered a precursor to bone apatite crystals [4,8]. The plate-like morphology also supports OCP as a bone apatite mineral precursor [8]. A recent study showed that bone crystals can induce OCP formation through citric acid incorporation [9]. Despite these findings, whether OCP is in fact a precursor to bone apatite crystals remains controversial [10]. The detection of OCP in human dentin [11], but not bone, may be due to one or more factors: (1) biological apatite crystals, especially newly formed crystals, will likely be very small [12]; (2) a crystal phase referred to as the OCP-like phase may be present that has similar solubility as OCP [13] but not the primary X-ray characteristics such that the X-ray diffraction (XRD) patterns are similar to that of the apatitic phase [7,13]; and Octacalcium Phosphate Biomaterials. DOI: https://doi.org/10.1016/B978-0-08-102511-6.00001-7 Copyright © 2020 Elsevier Ltd. All rights reserved.

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Octacalcium Phosphate Biomaterials

(3) newly formed bone crystals are composed of low-crystalline nano-HA [14]. In solution chemistry the HA forms via an OCP-like phase hydrolyzed from amorphous calcium phosphate (ACP) that precipitates from a supersaturated metastable calcium phosphate solution at physiological pH [7]. Indeed, chemical analyses indicated that human serum is almost saturated with respect to OCP [15,16], which does not exclude the possible hydrolysis of OCP under supersaturation with respect to HA in a physiological setting. Furthermore, the cluster structure of OCP is maintained throughout HA formation [17] and biomineralization [18]. In human enamel the presence of OCP was evidenced by a central dark line structure [19], and in developing mice, it was present at the calvaria suture [20], although whether this finding in mice actually reflects the presence of OCP is unclear [10,21]. Nonetheless, from the perspective of biomaterial applications, OCP likely has essential roles during biomineralization and bone formation. As such, there is a growing body of information regarding the usefulness of OCP as a biomaterial [1,2,22 25], particularly given that the superior osteoconductive property of OCP was first found in an onlay graft of OCP granules on mouse calvaria in a comparison with the performance of various nonsintered calcium phosphate materials [22].

1.3

Major findings and advances

1.3.1 Hypothesis and experimental support for the biological significance of octacalcium phosphate During bone mineralization, ACP, OCP, and dicalcium phosphate dihydrate (DCPD) are thought to be precursors for the formation of bone apatite crystals based on the crystal structure and chemical properties of these compounds in a physiological setting [16,26 28]. From the perspective of biomaterials, one hypothesis contends that OCP may be actively involved in bone formation processes but does not act simply as a crystalline substrate that is intermediate to bone apatite crystals [22]. However, a second hypothesis states that bone formation may be more efficient if synthetic OCP is placed directly at the site of bone formation [22,23]. To test this latter hypothesis an experiment was conducted to assess the appearance of bone tissue around implanted materials including OCP and the crystallized calcium phosphate form of HA [22]. For that study, DCP (the anhydrous form of DCPD), ACP (Ca/P molar ratio 1.5), OCP, and stoichiometric HA (Ca/P molar ratio 1.67) or nonstoichiometric Ca-deficient HA having a lower Ca/P molar ratio (1.5) were wet synthesized. The HA materials were obtained directly without any precursor phases. The granule form of the calcium phosphate materials was implanted onto the subperiosteal region of mouse calvaria, and the rate of bone tissue appearance and changes in the crystal phase of the implanted materials were examined in undecalcified specimens over the subsequent 15 weeks by in situ microbeam XRD analysis of the corresponding area with implanted OCP [22]. Although bone tissue appeared regardless of which material was implanted, the precursor materials DCP, ACP, and OCP promoted earlier appearance of bone tissue

Evolution of octacalcium phosphate biomaterials

3

compared to that of HA materials (from 5 weeks). In particular, among the precursors, OCP had the earliest time of bone tissue appearance (at 1 week compared to 3 weeks for DCP and ACP). Microbeam XRD indicated that while the HA phases were maintained, OCP and ACP tended to convert to HA from between 1 and 5 weeks, whereas DCP tended to convert to HA slowly until 15 weeks. These results suggested that the introduction of synthetic OCP positively promotes bone formation [22]. These findings that supported this hypothesis formed the foundation for our subsequent studies on the use of OCP in biomaterials and research into the mechanisms by which OCP enhances bone formation.

1.3.2 Initial evidence that octacalcium phosphate provides a nucleation site for bone deposition Implantation of OCP granules having a diameter of several hundred micrometers and consisting of an aggregate of OCP crystals several micrometers in length onto the subperiosteal region of mouse calvaria resulted in osteoblasts localizing to the OCP granule surface to initiate bone deposition and also new bone ingrowth from existing original bone as shown by histological and ultrastructural examinations [22,29,30]. Transmission electron microscopy of the ultrastructure of decalcified specimens indicated that osteoblasts attached directly to the OCP surface to form bone matrix [22,29] and that the tissue structure surrounding the OCP implant comprised fine filaments and granular materials (noncollagenous proteins) that were highly similar to the components of the starting locus of intramembranous osteogenesis or so-called bone nodules [22,31]. Such bone nodules were later assigned as the calcospherite, a mineralized structure that forms from osteoblast secretions after initial mineral deposition within the matrix vesicle [31 33]. Secondary (additional) collagenous mineralization begins after matrix vesicle mineralization [34]. The initial mineral phase deposited within the matrix vesicle is thought to include OCP [35]. The bone nodule like ultrastructure indicates that noncollagenous proteins indeed accumulate around one OCP crystal in the aggregate (granule) form of implanted OCP (Fig. 1.1) [22]. Furthermore, the results suggest that OCP can form a bone nodule like structure that includes extracellular serum proteins and therefore can serve as a nucleus that initiates new bone deposition [1,22 24]. Histochemical and biochemical analyses showed that the accumulated noncollagenous proteins are lectin maclura pomifera agglutinin (MPA) binding glycoconjugates having a molecular weight 56 kDa and probably correspond to α2HSglycoproteins [23]. Subsequent proteome analysis detected that serum included osteoblast- and osteoclast-related proteins as well as αHS-glycoproteins that are involved in the bone metabolism [36]. In fact, assessment of undecalcified specimens showed the presence of osteoblasts, which form osteoids and mineralized bone matrices, whereas OCP granules implanted in the rabbit femur resulted in the recruitment of multinucleated osteoclast-like cells that attach directly to the OCP surface where they actively resorb the material (Fig. 1.2) [37]. Together these results indicate that OCP is a biodegradable material that can be remodeled by new

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Octacalcium Phosphate Biomaterials

Figure 1.1 Ultrastructure of noncollagenous proteins accumulated around a remnant of an OCP particle from OCP granules (500 1000 μm diameter) 1 week after implantation onto mouse calvaria. Newly formed collagen fibers that were distinct from the OCP particle were also observed. Multiple fine filaments and granular materials are localized around the OCP particle (arrowheads). Bar 5 0.2 μm. OCP, octacalcium phosphate. Source: Reproduced from Suzuki O, Nakamura M, Miyasaka Y, Kagayama M, Sakurai M. Bone formation on synthetic precursors of hydroxyapatite. Tohoku J Exp Med 1991;164:37 50 [22] with permission from Tohoku University Medical Press (the original source of this figure), and also from Suzuki O, Kamakura S, Katagiri T. Surface chemistry and biological responses to synthetic octacalcium phosphate. J Biomed Mater Res B: Appl Biomater 2006;77:201 212 [40] with permission from John Wiley & Sons.

bone through coupling-like metabolic mechanisms in which osteoblasts collaborate with osteoclast-like cells [37 39].

1.3.3 Elucidation of the mechanism of OCP-mediated bone formation and its association with OCP hydrolysis to HA One prominent characteristic of OCP that we confirmed is its progressive conversion to an apatitic structure both in vitro in a physiological environment but also in vivo in implanted sites [4,22 24,41,42]. The conversion of OCP to HA in in vivo conditions was expected to be based on its in vitro chemical behavior and predictions related to its properties [4,15,16,40,43,44]. Although OCP has been postulated to be a precursor to bone apatite crystals as a metastable salt [8], the rate of OCP conversion can be controlled by the degree of supersaturation that can be determined by the chemical composition in the solution environment [40,44,45] and the inclusion of accelerators and inhibitors of OCP hydrolysis [42,46,47]. As mentioned above, human serum is almost saturated with respect to OCP and

Evolution of octacalcium phosphate biomaterials

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Figure 1.2 Undecalcified histological sections stained with HE 3 weeks after implantation of OCP granules in rabbit femurs. The newly formed mineralized bone and unmineralized osteoid tissues that were HE- and non-HE-stained, respectively, were formed by osteoblasts on the OCP implant surface. Meanwhile, a multinuclear osteoclast-like cell (arrows) provided a direct attachment between the OCP surface and the newly formed bone. Asterisk ( ) OCP. Bars 5 50 μm. The details of the experiments have been reported by Imaizumi et al. [37]. All procedures in the experiment were approved by the Animal Research Committee of Tohoku University. The photograph was provided by Dr. Hideki Imaizumi, Department of Orthopedic Surgery, Osaki Citizen Hospital, Osaki, Japan. HE, Hematoxylin and eosin; OCP, octacalcium phosphate.

supersaturated with respect to HA [15,16]. Meanwhile, fluoride ions can promote OCP hydrolysis even at low concentrations [46], whereas ubiquitous magnesium ions and carbonate ions in the body inhibit OCP hydrolysis [47,48]. Microbeam XRD analysis of undecalcified tissue specimens from mouse calvaria and anhydrous tissue preparations of undecalcified specimens confirmed that OCP implanted onto mouse calvaria and subcutaneous tissue, respectively, did indeed convert to an apatitic phase over time [22,23]. Notably, the anhydrous tissue preparation was developed as a method that does not alter the mineral structure of the water [49]. These results showing that OCP can be converted to an apatite structure in vivo suggested that OCP should participate in some mechanisms that enhance bone formation during hydrolysis of OCP to a HA structure [22,23,42,50].

1.3.3.1 Hydrolysis of octacalcium phosphate and protein adsorption In vitro experiments were designed to investigate whether OCP works as an adsorbent for serum protein, which could be a model structure for previously observed bone nodule like formation following OCP implantation [22], and to assess how OCP hydrolysis affects protein accumulation when implanted in bone [42]. For these experiments, materials that promote various degrees of hydrolysis from OCP

6

Octacalcium Phosphate Biomaterials

to HA were prepared as model adsorbents to simulate in vivo hydrolysis of OCP [42]. There are no crystal phases other than OCP and HA between the starting material OCP and the final hydrolysis product HA, and therefore, these two phases could be used to determine the adsorption affinity of adsorbate proteins. The most hydrolyzed product (OCP hydrolyzate) was Ca-deficient HA, which has a lower Ca/P molar ratio and resembles bone apatite crystals in terms of its nonstoichiometric composition (Ca/P molar ratio 1.47). The maximum number of adsorption sites and adsorption affinity progressively changes as OCP hydrolysis advances. A recent study involving proteome analysis substantiated that serum proteins adsorbed onto OCP include αHS-glycoproteins as well as apolipoprotein E and complement component 3 [36], which have both been reported to be involved in controlling osteoblast and osteoclast activities in bone remodeling [51,52]. These findings suggest that the bone nodule like structure induced by OCP implantation could be formed through serum-derived extracellular matrix proteins [22,23] and the crystals during OCP hydrolysis. Moreover, OCP hydrolysis itself may be involved in enhancing bone formation processes [24,38,44].

1.3.3.2 Bone formation on octacalcium phosphate and its hydrolyzed product Ca-deficient hydroxyapatite The apparent osteoconductivity of calcium phosphate material can be compared among existing calcium phosphate materials using the same animal model. However, since the determination of the actual osteoconductivity of a specific calcium phosphate material can be challenging, osteoconductive properties should be compared under conditions, wherein the materials have similar physicochemical properties. These conditions can be achieved by subjecting the materials to similar processing methods. To determine the osteoconductivity of OCP relative to HA, wherein both are in a granule form consisting of aggregates of similar crystals, HA was obtained via hydrolysis of the original OCP, to produce a hydrolyzed apatitic product that is nonstoichiometric Ca-deficient HA (Ca/P molar ratio 1.46), and has similar characteristics to OCP in terms of crystal morphology, specific surface area, granule size, and porosity [24,38,42]. The rate of new bone formation (n-Bone%) was compared after implantation of either OCP granules or Ca-deficient HA (obtained from OCP) in critical-sized defects in rat calvaria over a 12-week period [24]. Histomorphometric analysis showed that the n-Bone% value was significantly higher for OCP than Ca-deficient HA. XRD analysis of OCP taken from the implanted site 10 and 21 days after implantation showed that OCP had progressively converted to an apatite structure [24]. Overall, these results strongly support that OCP has superior osteoconductivity to Ca-deficient HA in the presence of compatible material characteristics [1,24].

1.3.3.3 Hydrolysis of octacalcium phosphate and its relation to bone formation The mechanism characterized by us by which OCP enhances bone formation has two factors: (1) OCP provides a structure that can serve as a nucleus for initial bone

Evolution of octacalcium phosphate biomaterials

7

deposition [22,23]; and (2) conversion of OCP to HA is crucial for enhancing bone formation [22 24,42]. Two different experiments were performed to examine the mechanism of OCP conversion to HA and analyze the role of hydrolysis in this process [38,44]. Partially hydrolyzed OCP was prepared by incubating nonstoichiometeric OCP (Ca/P molar ratio 1.26) in hot water to yield a Ca/P molar ratio of 1.37, which is slightly higher than the theoretical Ca/P molar ratio 1.33, but the OCP structure has a lower degree of crystallinity than the original OCP as shown by its broad XRD pattern. The newly formed bone% (n-Bone%) of the partially hydrolyzed OCP was histomorphometrically compared to OCP and Ca-deficient HA (obtained from OCP hydrolysis) implanted in granule form in a standardized defect in rat tibia [38]. A Ca/P molar ratio of 1.37 for OCP enhanced more bone formation than did the original OCP and fully hydrolyzed apatitic product Ca-deficient HA in intramedullary canal spaces. This change was accompanied by increased expression levels of various genes, including osteogenic markers, osteoclast markers, and immune-related markers. The results suggest that OCP hydrolysis, and hydrolysis that occurs soon after implantation, positively affects bone formation and bone resorption [38]. Another experiment investigated how the rate of OCP hydrolysis affects bone formation [44]. Because OCP hydrolysis can be accelerated by increasing the degree of solution supersaturation with respect to HA around OCP crystals, adding ACP that has higher solubility than OCP may increase the saturation level around OCP crystals. An in vitro immersion experiment involving OCP and OCP/ACP in simulated body fluid, a mixture of synthetic ACP, and OCP (OCP/ACP) prepared in granule form, confirmed that the presence of ACP increased the solution supersaturation and resulted in faster conversion of OCP to an apatite structure. This acceleration in the rate of conversion was also observed for in vivo implantation in critical-sized calvaria defects in rats as detected by XRD analysis of OCP/ACP retrieved from the implantation site. Moreover, OCP/ACP enhanced the rate of bone formation more than did OCP or ACP alone. Together these results indicate that the promotion of OCP hydrolysis is essential for OCP-mediated augmentation in bone formation [38,44].

1.3.3.4 Ionic environment induced by octacalcium phosphate during hydrolysis and cellular activation One physicochemical change detected during hydrolysis of OCP to HA is ionic diffusion between the crystal surface/lattice and the surrounding solution [4,40,50]. OCP hydrolysis is irreversible [40,42,50] and induces a unique environment around the crystals in terms of calcium and phosphate ion concentrations that slightly acidify the local pH [53]. In particular, specific ionic dissolution conditions associated with OCP hydrolysis induced enhancements in (1) differentiation of mouse bone marrow derived stromal ST-2 cells to osteoblastic cells [24,54]; (2) osteoclast formation from macrophages in the presence of osteoblasts that express the osteoclast differentiation factor receptor activator of NF-κB ligand (RANKL) [55]; (3) macrophage migration [56]; and (4) differentiation of the clonal cell line IDG-SW3 to osteocytes [25]. This enhancement in osteoblastic cell differentiation and osteoclast formation showed OCP dose dependence [54,55]. These in vitro results support in vivo findings

8

Octacalcium Phosphate Biomaterials

Figure 1.3 Schematic view of the mechanism by which could OCP stimulate osteoblastic cells and osteoclast-like cells in vivo based on in vitro results [24,25,54 56]. Osteoblastic differentiation is enhanced during the conversion of OCP to HA. Upregulated expression of osteoblast differentiation markers such as ALP and osterix, as well as osteocyte differentiation markers, such as FGF23 and SOST/sclerostin, is induced by OCP crystals. Osteoclast formation is induced by coculturing the cells with osteoblastic cells and bone marrow cells even in the absence of 1,25(OH)2D3, an agent that promotes RANKL expression in osteoblasts, suggesting that OCP crystals themselves may induce upregulation of RANKL expression. OCP also enhances the migration of macrophages, which may fuse to form osteoclasts around OCP. Calcium ions are known to regulate osteoblastic differentiation, macrophage migration, and osteoclast formation [55 57]. Phosphate ions are involved in enhancing osteoblastic cell differentiation toward late osteocytes [25]. Similar ionic environments are induced during OCP hydrolysis that is accompanied by adsorption of specific serum proteins [36,42]. ALP, Alkaline phosphatase; HA, hydroxyapatite; OCP, octacalcium phosphate.

that OCP can augment bone formation by activating osteoblastic cells and induce the appearance of osteoclast-like cells, which are related to the higher osteoconductivity and higher biodegradability of OCP compared to other calcium phosphate materials [37,38]. The mechanism by which OCP enhances bone-related cellular activity and in turn augments osteoconductivity is summarized in Fig. 1.3.

1.4

Development of octacalcium phosphate based bone substitute materials

Due to the large number of water molecules in the OCP structure, OCP cannot be sintered into a form suitable for implantation without decomposing the single OCP

Evolution of octacalcium phosphate biomaterials

9

phase [58]. Capitalizing on the intrinsic bioactive property of OCP while avoiding thermal damage in the structure, we developed various composites with natural polymers that combine OCP with various natural polymers such as collagen, gelatin, alginate (Alg), and hyaluronic acid (HyA) [2,59 62], as have been examined with other calcium phosphate materials. Collagen is a cell-attaching extracellular matrix material that is widely used in implantable biomaterials [63]. OCP/collagen (OCP/Col) composites consist of OCP granules and reconstituted cross-linked sponge and has higher osteoconductivity compared to OCP alone [59]. The osteoconductivity increases with OCP dose [64], which also corresponds to observed in vitro cellular responses [54]. OCP/Col has been tested in animal models, then undergone clinical trial and a company-initiative clinical trial, and recently approved in Japan for the use in oral surgery [65]. Gelatin is a denatured collagen that, like collagen, includes cell-attachment molecular motifs and is therefore also widely used as an implantable biomaterial [66,67]. OCP/gelatin (OCP/Gel) composites can be produced by coprecipitation of OCP with gelatin molecules in hot water [60,68] or by mixing OCP granules with gelatin [69]. OCP/Gel undergoes rapid biodegradation, in part because gelatin is highly biodegradable, which is immediately followed by new bone formation [60,68]. Implantation of OCP/Gel composites in critical-sized defects in rats promoted repair of up to 70% and in rabbit tibia defects OCP/Gel composites accelerated formation of new bone tissue at a faster rate than did the highly porous β-TCP [70]. Unlike gelatin and collagen, Alg molecules have no sites for cell attachment [71,72]. OCP/Alg composites can also be prepared by coprecipitation of OCP with Alg molecules in aqueous solution [61]. OCP/Alg composites induced proliferation of osteoblasts in vitro and bone regeneration in critical-sized defects in mouse calvaria [61]. HyA is an extracellular matrix component present in most connective tissues in the human body [73,74]. As a linear natural polymer, HyA functions in several biological processes, such as osteoconduction and wound healing [75 79]. OCP/HyA composites prepared by mixing OCP granules with sodium HyAs of distinct molecular weights had superior injectability and osteoconductivity relative to OCP alone and also enhanced osteoclastic cell activity that could be due a synergistic effect of HyA and OCP [62].

1.5

Octacalcium phosphate biomaterials

OCP has been investigated as a bone substitute material in various forms, such as coatings on metallic implants [80 86], and clinically examined in humans in the granule form [87]. Moreover, osteoinductive properties of calcium phosphate materials, including OCP, have been suggested [80]. Calcium phosphate cement that can promote formation of OCP has also been studied [81,82]. OCP nucleation on β-TCP granules, which have crystallographic matching between these materials,

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Octacalcium Phosphate Biomaterials

have been developed to confer OCP properties to β-TCP [83]. Chemical factors that include OCP have been developed to control cellular responses [84]. A 3D form of OCP [85], or block form [86], has also been described, whereas the composites OCP/polycaprolactone and OCP/silk fibroin have been fabricated for the use as bone substitute materials [88,89]. However, clinical application has just started through some clinical trials in the forms of granules and disks made as a composite with collagen, which were applied to jaw bones in the field of oral surgery [65,87]. The highly osteoconductive properties of OCP [22,24,25,54 56] highlight the wide-ranging potential uses of OCP for bone tissue engineering.

1.6

Concluding remarks

The osteoconductive and bioactive performance of OCP and subsequent application studies of OCP biomaterials have significantly impacted biomaterials science and the development of bone substitutes based on calcium phosphate materials. Crucial issues to address to encourage the use of OCP will be to determine how OCP promotes osteogenic activity, that is, whether OCP can acquire the capacity of autologous bone, which induces bone induction if used as filling in bone defects. The combination of OCP with exogenous cells and/or growth factors may also enhance bone growth. From the perspective of materials science, knowledge concerning intrinsic bioactive properties of OCP, which has been shown to be controlled by its stoichiometry and microstructure [1,38,90], as well as an understanding of mechanisms associated with its osteoconductivity, is critical. Future multidisciplinary studies ranging from basic sciences to clinical evaluations will facilitate the application of OCP biomaterials to treat a variety of conditions.

Acknowledgments This study was supported in part by grants-in-aid (23106010, 17K19740, and 18H02981) from the Ministry of Education, Science, Sports and Culture of Japan (MEXT).

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[80] Habibovic P, van der Valk CM, van Blitterswijk CA, De Groot K, Meijer G. Influence of octacalcium phosphate coating on osteoinductive properties of biomaterials. J Mater Sci Mater Med 2004;15:373 80. [81] Imamura Y, Tanaka Y, Nagai A, Yamashita K, Takagi Y. Self-sealing ability of OCPmediated cement as a deciduous root canal filling materia. Dent Mater J 2010;29:582 8. [82] Markovic M, Chow LC. An octacalcium phosphate forming cement. J Res Natl Inst Stand Technol 2010;115:257 65. [83] Onuma K, Iijima M. Nanoparticles in β-tricalcium phosphate substrate enhance modulation of structure and composition of an octacalcium phosphate grown layer. CrystEngComm 2017;19:6660 72. [84] Forte L, Torricelli P, Boanini E, Gazzano M, Fini M, Bigi A. Antiresorptive and antiangiogenetic octacalcium phosphate functionalized with bisphosphonates: an in vitro tri-culture study. Acta Biomater 2017;54:419 28. [85] Komlev VS, Popov VK, Mironov AV, Fedotov AY, Teterina AY, Smirnov IV, et al. 3D printing of octacalcium phosphate bone substitutes. Front Bioeng Biotechnol 2015;3:81. [86] Sugiura Y, Munar ML, Ishikawa K. Fabrication of octacalcium phosphate block through a dissolution-precipitation reaction using a calcium sulphate hemihydrate block as a precursor. J Mater Sci Mater Med 2018;29:151. [87] Komlev VS, Barinov SM, Bozo II, Deev RV, Eremin II, Fedotov AY, et al. Bioceramics composed of octacalcium phosphate demonstrate enhanced biological behavior. ACS Appl Mater Interfaces 2014;6:16610 20. [88] Heydari Z, Mohebbi-Kalhori D, Afarani MS. Engineered electrospun polycaprolactone (PCL)/octacalcium phosphate (OCP) scaffold for bone tissue engineering. Mater Sci Eng C Mater Biol Appl 2017;81:127 32. [89] Yang Y, Wang H, Yan FY, Qi Y, Lai YK, Zeng DM, et al. Bioinspired porous octacalcium phosphate/silk fibroin composite coating materials prepared by electrochemical deposition. ACS Appl Mater Interfaces 2015;7:5634 42. [90] Honda Y, Anada T, Kamakura S, Morimoto S, Kuriyagawa T, Suzuki O. The effect of microstructure of octacalcium phosphate on the bone regenerative property. Tissue Eng A 2009;15:1965 73.

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Yukari Shiwaku and Osamu Suzuki Division of Craniofacial Function Engineering, Tohoku University Graduate School of Dentistry, Sendai, Japan

2.1

Introduction

Octacalcium phosphate (OCP) is a biodegradable and osteoconductive calcium phosphate (CaP) that stimulates bone cells and promotes bone-remodeling processes [1 8]. OCP can enhance new bone formation by osteoblasts and biodegradation by osteoclast-like cells. Histological analysis of tissues from animals implanted with OCP granules showed the presence of both osteoblasts and osteoclast-like cells adjacent to OCP granules [8]. Hematoxylin and eosin (HE) staining of histological sections of rabbit femur implanted with OCP granules indicated active cuboidal osteoblasts that were directly aligned on the osteoid surface (Fig. 2.1). In addition, osteoclast-like multinuclear cells were directly attached to the concave surface of OCP granules, indicative of direct cellular biodegradation of the OCP. Thus OCPinduced bone formation is typically accompanied by biological resorption of OCP by osteoclast-like cells. Bone and bone marrow have many different cell types. Osteoblasts, osteocytes, and osteoclasts are the main cells that regulate bone metabolism. OCP can facilitate osteoblastic differentiation [1,2] and promote the transition from osteoblasts to osteocytes [3]. In addition, OCP promotes osteoclast formation in the presence of bone marrow stromal cells (BMSCs) [4], whereas osteoclasts cultured on OCP can regulate osteoblastic differentiation driven by osteoclast-derived coupling factors [9]. OCP has unique physicochemical properties, including conversion to hydroxyapatite (HA) to stimulate cellular responses. On the other hand, immune responses are important factors in determining the success of bone regeneration. There are several immune cell types in bone marrow, which include granulocytes, macrophages, dendritic cells, T cells, and B cells. Among these, macrophages are key regulators of initial inflammation. OCP enhances macrophage migration to the implantation site and production of proinflammatory cytokines and chemokines during the initial stages of bone formation [5]. Chondrocytes secrete several matrix proteins, including type II collagen (Col) and proteoglycans to produce cartilage. During endochondral bone formation, Octacalcium Phosphate Biomaterials. DOI: https://doi.org/10.1016/B978-0-08-102511-6.00002-9 Copyright © 2020 Elsevier Ltd. All rights reserved.

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Figure 2.1 Undecalcified, HE staining of a rabbit femur implanted with OCP granules at 4 weeks. Aligned cuboidal osteoblasts formed noncalcified osteoid bone matrix around the OCP granule. Osteoclasts (arrow and double arrows) were resorbed directly on the surface of the OCP granule.  : OCP; B: newly formed bone. Bar 5 100 μm. HE, Hematoxylin and eosin; OCP, octacalcium phosphate. Source: Reproduced from Suzuki O. Octacalcium phosphate (OCP)-based bone substitute materials. Jpn Dent Sci Rev 2013;49:58 71 with permission from Elsevier, Ltd.

cartilage is replaced by bone tissue. Indeed, differentiation of chondrogenic ATDC5 cells cultured with OCP is inhibited [6]. Dental cells also react with OCP. Odontoblasts differentiate from dental pulp stem cells and produce dentin. In an earlier study, OCP was shown to have the potential to promote odontoblastic differentiation [7]. This chapter summarizes various cellular responses induced by OCP that has excellent osteoconductivity and biodegradability through its ability to stimulate activation of various bone cells (e.g., osteoblasts, osteocytes, osteoclasts, macrophages, and chondrocytes) [1 6]. These cellular responses are also affected by the unique physicochemical properties of OCP. Investigation of the mechanisms associated with OCP activation of surrounding cells is important to understand bone regeneration induced by implanted CaP materials.

2.1.1 The effect of octacalcium phosphate on osteoblastic differentiation To investigate the proliferation and osteoblastic differentiation induced by OCP, mouse bone marrow stromal ST-2 cells and primary calvarial osteoblastic cells were cultured on dishes precoated with OCP or HA [1,2]. Attachment and proliferation of cells cultured on OCP-coated plates were initially inhibited in an OCP dosedependent fashion but at later stages were higher compared to HA-coated plates or the control (plastic) surface.

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Figure 2.2 ALP activity curves of ST-2 cells cultured on OCP- or HA-coated plates or tissue culture plates (control) (A). ALP activity after 21 days of culture, (B) (n 5 3). aP , .01 compared with control. bP , .01 compared with 0.3 mg/well coating of OCP. cP , .01 compared with 1.5 mg/well coating of OCP. dP , .01 compared with 3.0 mg/well coating of OCP. ALP, Alkaline phosphatase; HA, hydroxyapatite; OCP, octacalcium phosphate. Source: Reproduced from Anada T, Kumagai T, Honda Y, Masuda T, Kamijo R, Kamakura S, et al. Dose-dependent osteogenic effect of octacalcium phosphate on mouse bone marrow stromal cells. Tissue Eng, A 2008;14:965 78 with permission from Mary Ann Liebert, Inc.

Osteoblastic differentiation of ST-2 cells was measured by alkaline phosphatase (ALP) activity and mRNA expression levels of Runx2, osterix (Osx), Col type I (Col-1), ALP, and osteopontin (OPN). ALP staining was more intense in OCPcoated wells than that in HA-coated [1]. Enzymatic ALP activity of cells cultured on OCP-coated wells increased progressively with increasing OCP concentration, but for the HA-coated group the activity was constant regardless of HA content (Fig. 2.2) [2]. Furthermore, OCP enhanced the expression of osteogenic markers, including Osx, Col-1, and ALP, by day 21 of culture [2]. Runx2 expression also tended to increase with increasing OCP dose, although this increase was not statistically significant. To clarify the mechanism the effect of mitogen-activated protein kinase (MAPK) signaling on osteoblastic differentiation by OCP was analyzed [10]. When ST-2 cells were cultured on OCP-coated plates in the presence of the p38 MAPK inhibitor SB203580, cell proliferation was suppressed after 14 days of culture. SB203580 also inhibited OCP-induced ALP activity and mRNA expression of osteoblastic differentiation markers, such as OPN and osteocalcin [10]. These results suggest that OCP promotes osteoblastic differentiation via the p38 MAPK signaling pathway. During cell culture, OCP converted to HA gradually, and this physicochemical property influenced the function of osteoblastic cells. Transformation of OCP to HA is accompanied by the incorporation of calcium ions (Ca21) and the release of inorganic phosphate (Pi) ions [11]. Chemical analysis of α-MEM after 3 days of

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Figure 2.3 Changes in Ca21 (A) and Pi (B) concentrations in α-MEM at 37 C after 3 days of incubation of ST-2 cells in OCP- or HA-coated wells (n 5 3). Ca21, Calcium ions; HA, hydroxyapatite; OCP, octacalcium phosphate; Pi, inorganic phosphate. Source: Reproduced from Anada T, Kumagai T, Honda Y, Masuda T, Kamijo R, Kamakura S, et al. Dose-dependent osteogenic effect of octacalcium phosphate on mouse bone marrow stromal cells. Tissue Eng, A 2008;14:965 78 with permission from Mary Ann Liebert, Inc.

incubation of ST-2 cells in OCP-coated wells revealed that the Ca21 concentration decreased with increasing of OCP content (about 0.13 mM; Fig. 2.3). On the contrary, the Pi concentration of supernatants from OCP-coated wells increased up to 1.90 mM depending on the OCP dose. The unique change in Ca21 and Pi concentrations, particularly that of Ca21, would affect the culture of osteoblastic cells as Ca21 play an important role in cell adhesion [12,13] and proliferation [14]. Meanwhile, Ca21 depletion by ethylenediaminetetraacetic acid inhibited ST-2 cell proliferation and ALP activity (Fig. 2.4) [10], indicating that Ca21 consumption mediated by OCP HA conversion influences ST-2 cell characteristics. Pi ions initiate mineralization in osteoblast-like cell culture [15] and elevations in Pi levels induce apoptosis of osteoblast-like cells [16]. However, changes in Pi concentration were unlikely to affect ST-2 cell proliferation following culture on OCP-coated plates, because the concentration threshold needed for induction of osteoblast cell apoptosis exceeds 5 mM. OCP also promotes osteoblastic differentiation not only in two-dimensional (2D) but also in three-dimensional (3D) culture [17]. Recently, 3D cell culture models have attracted significant attention in the tissue-engineering field, because 3D culture can better mimic cellular microenvironments compared to 2D culture. To investigate the effect of CaPs on osteoblastic differentiation in 3D culture, hybrids of mouse bone marrow mesenchymal stromal D1 cell spheroids and CaPs were cultured in osteogenic media. OCP was compared to both HA and beta-tricalcium

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Figure 2.4 Cell proliferation (A) and ALP activity (B) of ST-2 cells cultured with disodium hydrogen phosphate (1.2, 2.5, 4.7, or 7.5 mM) and 2 mM EGTA to mimic OCP-induced Ca21 and Pi concentration changes after 7 days of culture. ALP, Alkaline phosphatase; Ca21, calcium ions; EGTA, ethylene glycol tetraacetic acid; OCP, octacalcium phosphate; Pi, inorganic phosphate. Source: Reproduced from Nishikawa R, Anada T, Ishiko-Uzuka R, Suzuki O. Osteoblastic differentiation of stromal ST-2 cells from octacalcium phosphate exposure via p38 signaling pathway. Dent Mater J 2014;33:242 51 with permission from The Japanese Society of Dental Materials and Devices.

phosphate (β-TCP), which have broad clinical applications as bone-substitute materials. The results showed that osteoblastic differentiation of D1 cell spheroids was promoted in the presence of CaPs (Fig. 2.5). OCP showed the greatest capacity to increase ALP activity of D1 cell spheroids among all CaPs. These results indicate that 3D cell spheroids incorporate OCP, which may be an effective implantable cell/material hybrid for bone regeneration.

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Figure 2.5 The ALP activity of D1 cell spheroids and CaP/cell spheroids after 7 days of culture. ALP, Alkaline phosphatase; CaP, calcium phosphate ( P , .01). Source: Reproduced from Anada T, Sato T, Kamoya T, Shiwaku Y, Tsuchiya K, TakanoYamamoto T, et al. Evaluation of bioactivity of octacalcium phosphate using osteoblastic cell aggregates on a spheroid culture device. Regener Ther 2016;3:58 62 with permission from Elsevier B.V.

2.1.2 Application of mesenchymal stem cells for bone regeneration For tissue engineering the number of basic and clinical studies on the use of BMSCs for the repair of bone defects without autologous bone transplantation has increased remarkably [18,19]. BMSCs are a source of mesenchymal stem cells (MSCs) that can differentiate into osteoblastic cells [20]. In addition, the osteogenic ability of MSCs can be combined with suitable scaffold materials for in vivo bone regeneration [21,22]. To clarify whether OCP can induce bone regeneration in vivo when implanted with MSCs, the effect of MSCs seeded on OCP/collagen (OCP/Col) composites on bone formation was investigated in critical-sized calvarial defects in rats [23]. MSCs isolated from 4-week-old Wistar rat’s long bones were preincubated in osteogenic or maintenance medium in the presence or absence of basic fibroblast growth factor (bFGF). OCP/Col disks were seeded with MSCs that had been preincubated in osteogenic medium containing bFGF for 1 day before implantation. After the disks were implanted in critical-sized calvarial defects, the specimens were analyzed radiographically, histologically, and histomorphometrically. Soft X-ray analysis revealed that OCP/Col disks seeded on MSCs (OCP/Col
MSCs) and OCP/Col disks implanted in rat calvaria defects showed radiopacity at 4 and 8 weeks post-implantation. Bone mineral density of the OCP/Col
MSC group was significantly higher than that of the OCP/Col group. In histological observations,

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Figure 2.6 HE staining of calvarial defects implanted with OCP/Col
MSCs, OCP/Col, Col
MSCs, or Col. Bars 5 500 μm.  , remaining OCP; B, newly formed bone; Col, collagen; HE, hematoxylin and eosin; MSCs, mesenchymal stem cells; OCP, octacalcium phosphate. Source: Reproduced from Kawai T, Anada T, Masuda T, Honda Y, Sakai Y, Kato Y, et al. The effect of synthetic octacalcium phosphate in a collagen scaffold on the osteogenicity of mesenchymal stem cells. Eur Cell Mater 2011;22:124 36 with permission from AO Research Institute Davos.

newly formed bone could be clearly observed in the defect implanted with an OCP/ Col
MSC disk even at 4 weeks, whereas very little new bone formation occurred in the OCP/Col group (Fig. 2.6). The percentage of newly formed bone in animals implanted with OCP/Col
MSC disks was significantly higher in all groups at both 4 and 8 weeks. These results suggest that OCP crystals in a Col matrix can support exogenous MSC-mediated bone regeneration in an OCP/Col scaffold implanted in rats with critical-sized calvarial defects.

2.1.3 Octacalcium phosphate-mediated promotion of the transition from osteoblasts to osteocytes Osteocytes can be terminally differentiated from osteoblasts and embedded in the bone matrix. To investigate the effect of OCP on differentiation of osteoblasts to late osteocytes, mouse IDG-SW3 cells were cultured in transwells with OCP, commercially available β-TCP, or HA [3]. IDG-SW3 cells are useful for the analysis of osteoblast-to-osteocyte transition, because over time in culture these cells express the entire profile from late osteoblastic to late osteocytic marker genes, including SOST/sclerostin and FGF23 [24,25]. In a transwell culture system, OCP increased the activity of ALP, an osteoblastic marker, of IDG-SW3 cells compared to β-TCP and HA. In addition, the expression of dentin matrix protein-1 (DMP-1), a marker

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Figure 2.7 Real-time quantitative RT-PCR analyses of SOST/sclerostin at day 28 (A) and day 35 (B) in IDG-SW3 cells ( P , .05). RT-PCR, Real-time reverse transcription with the polymerase chain reaction. Source: Reproduced from Sai Y, Shiwaku Y, Anada T, Tsuchiya K, Takahashi T, Suzuki O. Capacity of octacalcium phosphate to promote osteoblastic differentiation toward osteocytes in vitro. Acta Biomater 2018;69:362 71 with permission from Elsevier, Ltd.

of mineralizing osteocytes, was promoted in the presence of OCP relative to other treatment groups. OCP also enhanced the expression of mature osteocyte markers SOST/sclerostin and FGF23 after 35 days of culture (Figs. 2.7 and 2.8). The hydrolysis of OCP to HA advanced in culture and was accompanied by release of Pi and incorporation of Ca21. In IDG-SW3 cells, increasing the Pi concentration in the culture media to 1.5 mM enhanced ALP activity, which could be eliminated by the addition of the Pi-transport inhibitor phosphonoformic acid to the media [3]. On the other hand, the ALP activity was lower in cells incubated with 4.0 mM Ca21 compared to the other groups (0 or 2.0 mM Ca21), indicating that the Ca21 concentration of culture media did not promote ALP activity of IDG-SW3 cells [3]. However, a recent paper reported that high concentration (5 mM) of Ca21 in the conditioned media promotes osteoblastic differentiation of MSCs [26]. Thus further studies are necessary to clarify the effect of Ca21 on differentiation from osteoblasts to osteocytes.

2.1.4 Induction of osteoclast formation by octacalcium phosphate The effect of OCP on osteoclast differentiation was examined using cocultures of mouse bone marrow macrophages and UAMS-32 mouse stromal cells grown on cell culture plates with and without OCP coating [4]. The coculture system induces receptor activator of nuclear factor-kappa B ligand (RANKL) expression in UAMS32 cells and promotes osteoclast differentiation of bone marrow macrophages even

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Figure 2.8 Real-time quantitative RT-PCR analyses of FGF23 levels at day 28 (A) and day 35 (B) of culture for IDG-SW3 cells ( P , .05). RT-PCR, Real-time reverse transcription with the polymerase chain reaction. Source: Reproduced from Sai Y, Shiwaku Y, Anada T, Tsuchiya K, Takahashi T, Suzuki O. Capacity of octacalcium phosphate to promote osteoblastic differentiation toward osteocytes in vitro. Acta Biomater 2018;69:362 71 with permission from Elsevier, Ltd.

in the absence of 1,25(OH)2D3, an essential factor that upregulates RANKL expression in osteoblasts. Compared with uncoated culture plates, OCP-coated plates dose-dependently enhanced the frequency of TRAP-positive cells after 4 6 days of culture (Fig. 2.9). Furthermore, real-time reverse transcription with the polymerase chain reaction analysis showed that the expression of TRAP and cathepsin K, typical osteoclast markers, was also increased in cells cultured on OCP-coated culture plates, indicating that the TRAP-positive cells were osteoclasts. BMP-2, a strong inducer of osteoblast differentiation, can also stimulate RANKL-dependent osteoclast differentiation and survival [27]. When recombinant BMP-2 protein was added to the coculture media, osteoclast formation by UAMS32 cells was significantly enhanced in OCP-coated, but not uncoated, culture plates. TRAP and cathepsin K expression in bone marrow cells was also upregulated in cultures treated with BMP-2 in the presence of OCP. Addition of BMP-2 enhanced RANKL expression by UAMS-32 cells only in the presence of OCP, suggesting that OCP stimulates RANKL expression of osteoblasts and promotes osteoclast differentiation. Previous studies revealed that changes in extracellular Ca21 concentrations induced RANKL expression in osteoblasts and promoted osteoclast formation [28,29]. The presence of OCP in UAMS-32 cell culture was associated with dose-dependent decreases in the Ca21 concentration of the medium [4]. This result indicates that changes in extracellular Ca21 levels followed by OCP HA conversion increase RANKL expression in osteoblasts and stimulate osteoclast formation.

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Figure 2.9 Osteoclast formation induced by OCP. (A) TRAP staining of bone marrow cells of mouse cocultured with UAMS-32 cells for 6 days on plates coated with or without OCP (1.5 mg/well). Arrows indicate TRAP-positive cells. (B) TRAP and cathepsin K mRNA expression levels in cells cocultured for 6 days on OCP-coated and uncoated plates (1.5 mg/ well) (n 5 4). (C) The number of TRAP-positive cells after 6 days of culture. Cells were cocultured on plates coated with various amounts of OCP (n 5 4). (D) Time-course study of osteoclast formation by cells cultured on uncoated (open circle) or OCP-coated (closed circle) plates (1.5 mg/well; n 5 4). OCP, Octacalcium phosphate. Source: Reproduced from Takami M, Mochizuki A, Yamada A, Tachi K, Zhao B, Miyamoto Y, et al. Osteoclast differentiation induced by synthetic octacalcium phosphate through receptor activator of NF-kappaB ligand expression in osteoblasts. Tissue Eng, A 2009;15:3991 4000 with permission from Mary Ann Liebert, Inc.

2.1.5 Osteoblast osteoclast cross talk mediated by octacalcium phosphate OCP can stimulate bone regeneration by promoting bone remodeling, which involves the coupling of bone resorption by osteoclasts with bone formation [8]. Recent studies showed that osteoclasts can coordinate osteoblast differentiation locally through several coupling factors [30,31]. Activation of EphrinB2 (EfnB2) ligands on osteoclasts coupled with EphB4 receptors on osteoblasts promotes osteoblast differentiation [32]. Sphingosine-1-phosphate produced by sphingosine kinase

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in osteoclasts enhances osteoblast differentiation and mineralization [33 35], whereas collagen triple helix repeat containing 1 (Cthrc1) and complement component 3a (C3a) are secreted by mature osteoclasts to enhance osteoblast differentiation [36,37]. In contrast, Semaphorin4D inhibits osteoblast differentiation [38]. To clarify whether different kinds of CaPs having diverse chemical compositions affect osteoclast formation and osteoclast osteoblast crosstalk, mouse bone marrow macrophages were cultured on disks coated with OCP, HA, β-TCP, and mixtures of these compounds [9]. The frequency of large TRAP-positive cells, indicating multinucleated osteoclasts, was significantly increased by culture on OCP or β-TCP disks compared to HA disks. Osteoclast formation in the presence of OCP was almost the same as that for β-TCP. Meanwhile, the expression patterns of coupling factors varied depending on the type of CaP. β-TCP and the HA/β-TCP mixture induced EfnB2 and Cthrc1 expression, whereas OCP and HA/OCP mixtures promoted C3a expression. These results suggest that different CaPs can influence the regulation of coupling factor expression in osteoclasts.

2.1.6 Enhancement of macrophage migration and regulation of immune response by octacalcium phosphate Immune responses are key regulators that determine the osteogenic capacity of biomaterials [39]. In particular, macrophages play an important role in regulating initial immune responses. Therefore immune responses induced by culture with OCP and other CaPs in relation to biodegradation and subsequent bone formation were investigated [5]. OCP was compared to Ca-deficient HA, which is an OCP hydrolysate (HL) that has plate-like morphology similar to that of OCP. In a macrophage migration assay using the mouse macrophage cell line J774.1, cells were cultured in transwells with OCP or HL or were left untreated (control). A higher number of mouse macrophages migrated in the presence of OCP and HL compared to the control group. Furthermore, the number of migrating cells tended to increase in the OCP group compared to the HL group when the amount of CaP was increased. Release of IL-6, an inflammatory cytokine, was significantly increased for up to 48 h when OCP or HL was added to the cell culture, whereas the control group cells produced almost no IL-6. Relative to HL treatment, IL-6 production dosedependently increased with OCP treatment for 12 h. It is known that OCP converts to HA under physiological conditions by incorporating Ca21 into the crystals. In order to investigate whether changes in Ca21 concentration affect macrophage migration, a cell migration assay using culture media containing various Ca21 concentrations was performed [5]. The number of migrating cells tended to increase as the Ca21 concentration increased up to 0.6 mM but decreased at concentrations above 0.6 mM. This result suggests that appropriate Ca21 concentration could induce migration of macrophages. In vivo analysis in rat tibias showed that the initial immune response differed between animals implanted with OCP or HL [5]. Histomorphometric analysis showed that CD68-positive cells, indicative of macrophages, accumulated around

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OCP implants to a greater extent than for HL within 5 days of implantation (Fig. 2.10). Osteoclasts are derived from a monocyte/macrophage lineage following M-CSF and RANKL stimulation. The number of cells positive for the osteoclast differentiation marker TRAP also increased around OCP implants compared

Figure 2.10 Immunohistochemical staining for CD68 at day 5 and 14 after implantation. (A and B) No materials, (C and D) OCP, (E and F) HL, bars 5 200 μm. White arrow: OCP granules; black arrow: HL granules. (G) Quantitative analysis of CD68-positive cells.  P , .01 and  P , .05 significant difference (n 5 3). HL, Hydrolysate; OCP, octacalcium phosphate. Source: Reproduced from Hirayama B, Anada T, Shiwaku Y, Miyatake N, Tsuchiya K, Nakamura M, et al. Immune cell response and subsequent bone formation induced by implantation of octacalcium phosphate in a rat tibia defect. RSC Adv 2016;6:S7475 84 with permission from The Royal Society of Chemistry.

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to HL or the sham-treated control group. Differences in the accumulation of macrophages around CaP materials were also affected by inflammatory cytokines. The chemokines CXCL2 and CCL3 are secreted mainly by macrophages and regulate bone remodeling. The number of CXCL2-positive cells was significantly higher in animals treated with OCP compared to those by HL or left untreated. CCL3-positive cells were also more frequent in the OCP treatment group. Taken together with the results in vitro and in vivo, it is reasonable to assume that migrated macrophages and inflammatory chemokines may be involved in promoting bone formation induced by OCP. It is considered that chemical ionic changes around OCP, such as local increase of Ca21 concentration, may influence early immune response, although the stoichiometry [40], the microstructure [41], and macrostructure [42] of OCP should also be factors that regulate the osteoconductivity of the OCP-based materials [43].

2.1.7 Suppression of chondrogenic differentiation by octacalcium phosphate Proliferation and differentiation of chondrocytes are required for endochondral ossification in long bones. Furthermore, BMP-2 is known to promote chondrocyte differentiation. As such, the effect of OCP on chondrogenic differentiation as well as the proliferation and apoptosis of the mouse chondrogenic cell line ATDC5 was examined in the presence or absence of BMP-2 [6]. ATDC5 cells cultured on untreated plastic plates in the presence of BMP-2 (100 ng/mL) showed significantly suppressed proliferation compared to that of cells cultured on OCP-coated plates, suggesting that OCP could abrogate the antiproliferative effect of BMP-2. Furthermore, the activity of the apoptosis marker caspase-3 was not enhanced by the presence of OCP. The effect of OCP on ATDC5 cell differentiation was examined by analyzing the expression of several marker genes for chondrogenic differentiation, including Col2a1, Acan, Col10a1, Sox9, Sox5, and Sox6 (Fig. 2.11). BMP-2 enhanced the expression of all of the genes previously mentioned on untreated plastic plates after 2 days of culture. In contrast, treatment with OCP markedly suppressed the expression of Sox6 in the presence of BMP-2. The concentration of both Ca21 and Pi increased upon culture of ATDC5 cells on OCP, but increases in Ca21 and Pi had little effect on differentiation of these cells. To investigate how direct contact with OCP affected chondrogenic differentiation, ATDC5 cells were cultured on plates in which one side was coated with OCP. Col2a1 expression was markedly suppressed in cells cultured on OCP and slightly inhibited in cells in the uncoated areas of the plates. Moreover, Sox6 expression in cells cultured on OCP was potently inhibited, whereas that of cells cultured on the uncoated area was the same as that for control cells cultured on untreated plates. These results indicate that direct contact of ATDC5 cells with OCP is required to suppress chondrocyte differentiation.

Figure 2.11 Effects of OCP on ATDC5 cell differentiation. ATDC5 cells were cultured in 6-well plastic plates with (OCP) or without (none) OCP coating for 2 days in the presence or absence of 100 ng/mL BMP-2. mRNA expression of Col2a1 (A), Acan (B), Col10a1 (C), Sox9 (D), Sox5 (E), Sox6 (F), and Gapdh was analyzed by real-time reverse transcription with the polymerase chain reaction (RT-PCR). The results are relative to values obtained for cells cultured in the absence of BMP-2 on normal plates (n 5 4).  Significant difference (P , .05). OCP, Octacalcium phosphate; RT-PCR, real-time reverse transcription with the polymerase chain reaction. Source: Reproduced from Shibuya I, Yoshimura K, Miyamoto Y, Yamada A, Takami M, Suzawa T, et al. Octacalcium phosphate suppresses chondrogenic differentiation of ATDC5 cells. Cell Tissue Res 2013;352:401 12 with permission from Springer-Verlag, Berlin Heidelberg.

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2.1.8 Reparative dentin formation via odontoblast differentiation promoted by octacalcium phosphate Dentin is formed by odontoblasts in dental pulp tissue and is the primary mineralized tissue in teeth. In terms of mechanism of formation and composition, dentin closely resembles bone [44]. Dental pulp tissue contains some stem cells, which retain the potential to differentiate into odontoblasts that possess the capacity to form new dentin throughout the lifetime of an organism. Dentin consists of poorly crystallized calcium-deficient HA that has various inorganic impurities, such as carbonate and fluoride ions [45 47]. Although it has been reported that OCP is contained in human calcifying dentin [48], whether it can induce dentin formation by dental pulp is unknown. To investigate the biomineralization of dental pulp exposed to synthetic OCP in vitro, primary dental pulp cells of rat were cultured in the presence or absence of varying amounts of OCP [7]. OCP- and HA-coated plates inhibited cell proliferation of rat dental pulp cells compared to noncoated plates after 7 days of culture as measured by a BrdUincorporation assay. Moreover, ALP activity was significantly higher on OCPcoated plates than on control and HA-coated plates throughout the culture period. OCP also significantly augmented the extent of mineralization and promoted the expression of odontoblast markers, such as dentin sialophosphoprotein (DSPP) and DMP-1 on days 3 and 7 (Fig. 2.12). Downregulation of Runx2 is closely related to odontoblast differentiation. In OCP-coated plates, Runx2 expression in pulp cells was dramatically decreased compared with that of noncoated and HA-coated plates. To clarify the mechanism of pulp cell OCP interactions, rat pulp cells were cultured on plates where one-half of the plate was coated with OCP. The expression of odontoblast marker genes, such as DSPP, DMP-1, ALP, and Col1a in pulp cells grown on the OCP-coated area, was similar to that of plates coated entirely with OCP. On the other hand, there was no significant difference in the levels of markergene expression between pulp cells grown on the noncoated areas of the plates. These results indicate that direct contact between cells and OCP is crucial for the effect of OCP on odontoblast differentiation from pulp stem cells.

2.2

Conclusion

The studies described in this chapter clearly show that OCP can enhance the activity of various bone cells, including osteoblasts, osteocytes, osteoclasts, and macrophages, during the conversion to HA [1 3,5]. Alterations to the physiochemical properties of OCP crystals, such as changes in ion concentration, can influence the proliferation of osteoblastic cells and the differentiation of osteocytes [3], as well as formation of osteoclasts [4]. On the other hand, in cultures of chondrocytes and odontoblasts, direct contact of OCP with cells is needed to stimulate cell differentiation. This result suggests that morphological features of OCP crystals could affect cellular responses. This accumulated knowledge about the mechanism by which

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* 10 5

0.2 0

0 7

3

**

25

*

Alizarin red (μg/104 cells)

BrdU-positive ratio (%)

Control HA OCP

ALP activity (arbitrary unit)

40

14 (days)

Control HA OCP

* p