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Polymers in regenerative medicine


Biomedical applications from nano- to macro-structures

Edited By

manuel monleoacuten PradasCentro de Biomateriales e Ingenieriacutea TisularUniversitat Politegravecnica de ValegravenciaCIBER de Bioingenieriacutea Biomateriales y Nanomedicina (CIBER-BBN)Valencia Spain

marIacutea J vicentPolymer Therapeutics Laboratory Centro de Investigacioacuten Priacutencipe FelipeValencia Spain

Copyright copy 2015 by John Wiley amp Sons Inc All rights reserved

Published by John Wiley amp Sons Inc Hoboken New JerseyPublished simultaneously in Canada

No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopying recording scanning or otherwise except as permitted under Section 107 or 108 of the 1976 United States Copyright Act without either the prior written permission of the Publisher or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center Inc 222 Rosewood Drive Danvers MA 01923 (978) 750-8400 fax (978) 750-4470 or on the web at wwwcopyrightcom Requests to the Publisher for permission should be addressed to the Permissions Department John Wiley amp Sons Inc 111 River Street Hoboken NJ 07030 (201) 748-6011 fax (201) 748-6008 or online at httpwwwwileycomgopermission

Limit of LiabilityDisclaimer of Warranty While the publisher and author have used their best efforts in preparing this book they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose No warranty may be created or extended by sales representatives or written sales materials The advice and strategies contained herein may not be suitable for your situation You should consult with a professional where appropriate Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages including but not limited to special incidental consequential or other damages

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Wiley also publishes its books in a variety of electronic formats Some content that appears in print may not be available in electronic formats For more information about Wiley products visit our web site at wwwwileycom

Library of Congress Cataloging-in-Publication Data

Polymers in regenerative medicine biomedical applications from nano- to macro-structures edited by Manuel Monleoacuten Pradas Maria J Vicent p cm Includes bibliographical references and index ISBN 978-0-470-59638-8 (hardback)I Monleoacuten Pradas Manuel editor II Vicent Maria J editor [DNLM 1 Polymers 2 Nanomedicinendashtrends 3 Regenerative Medicinendashtrends 4 Tissue Engineeringndashtrends QT 375P7] R857M3 61028prime4ndashdc23 2014017656

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

v

Contents

Preface xi

Contributors xvii

Part A Methods for synthetic extracellular Matrices and scaffolds 1

1 Polymers as Materials for tissue engineering scaffolds 3Ana Valleacutes Lluch Dunia Mercedes Garciacutea Cruz Jorge Luis Escobar Ivirico Cristina Martiacutenez Ramos and Manuel Monleoacuten Pradas

11 The Requirements Imposed by Application on Material Structures Intended as Tissue Engineering Scaffolds 3

12 Composition and Function 5121 General Considerations 5122 Some Families of Polymers for Tissue Engineering Scaffolds 8123 Composite Scaffold Matrices 12

13 Structure and Function 14131 General Considerations 14132 Structuring Polymer Matrices 15

14 Properties of Scaffolds Relevant for Tissue Engineering Applications 24141 Porous Architecture 24142 Solid State Properties Glass Transition Crystallinity 25143 Mechanical and Structural Properties 26144 Swelling Properties 28145 Degradation Properties 29146 Diffusion and Permeation 30

vi ConTEnTS

147 Surface Tension and Contact Angle 31148 Biological Properties 31

15 Compound Multicomponent Constructs 32151 Scaffold-Cum-Gel Constructs 32152 Scaffolds and Membranes Containing Microparticles 34153 other Multicomponent Scaffold Constructs 34

16 Questions Arising from Manipulation and Final Use 35161 Sterilization 35162 Cell Seeding Cell Culture Analysis 36163 In the Surgeonrsquos Hands 37

References 37

2 natural-Based and stimuli-Responsive Polymers for tissue engineering and Regenerative Medicine 49Mariana B Oliveira and Joatildeo F Mano

21 Introduction 4922 natural Polymers and Their Application in TE amp RM 52

221 Polysaccharides 52222 Protein-Based Polymers 60223 Polyesters 65

23 natural Polymers in Stimuli-Responsive Systems 65231 pH-Sensitive natural Polymers 67232 Temperature Sensitive natural Polymers 67233 natural Polymers Modified to Show Thermoresponsive

BehaviormdashModifying Responsive Polymers and Agents 71

234 Light-Sensitive PolymersmdashPotential Use of Azobenzeneα-Cyclodextrin Inclusion Complexes 72

24 Conclusions 73References 74

3 Matrix Proteins Interactions with synthetic surfaces 91Patricia Rico Marco Cantini George Altankov and Manuel Salmeroacuten-Saacutenchez

31 Introduction 9132 Protein Adsorption 92

321 Cell Adhesion Proteins 93322 Experimental Techniques to Follow Protein

Adsorption 94323 Effect of Surface Properties on Protein Adsorption 97

33 Cell Adhesion 109331 Experimental Techniques to Characterize Cell

Adhesion 112332 Cell Adhesion at CellndashMaterial Interface 115

34 Remodeling of the Adsorbed Proteins 122

ConTEnTS vii

341 Protein Reorganization and Secretion at the CellndashMaterial Interface 122

342 Proteolytic Remodeling at CellndashMaterials Interface 126References 128

4 Focal Adhesion Kinase in CellndashMaterial Interactions 147Cristina Gonzaacutelez-Garciacutea Manuel Salmeroacuten-Saacutenchez and Andreacutes J Garciacutea

41 Introduction 14742 Role of FAK in Cell Proliferation 14943 Role of FAK in Migratory and Mechanosensing Responses 15044 Role of FAK in the Generation of Adhesives Forces 15245 Influence of Material Surface Properties on FAK Signaling 156

451 Effect of Mechanical Properties on FAK Signaling 156452 Effect of Surface Topography on FAK Signaling 160453 Effect of Surface Chemistry on FAK Signaling 163454 Effect of Surface Functionalization in FAK Expression 165

References 168

5 Complex CellndashMaterials Microenvironments in Bioreactors 177Stergios C Dermenoudis and Yannis F Missirlis

51 Introduction 17752 CellndashECM Interactions 178

521 ECM Chemistry 179522 ECM Topography 181523 ECM Mechanical Properties 183524 ECM 3D Structure 184525 ECM-Induced Mechanical Stimuli 186

53 Cellndashnutrient Medium 187531 Composition and Volume-Related Phenomena 188532 Mechanical Stresses Induced by nutrient Medium 191

54 other Aspects of Interaction 194541 Co-Culture Systems 195542 Material Interactions 196


Part B nanostructures for tissue engineering 207

6 self-Curing systems for Regenerative Medicine 209Julio San Romaacuten Blanca Vaacutezquez and Mariacutea Rosa Aguilar

61 Introduction 20962 Self-Curing Systems for Hard Tissue Regeneration 210

621 Antimicrobial Self-Curing Formulations 211622 Self-Curing Formulations for osteoporotic Bone 214

viii ConTEnTS

623 Antineoplastic Drug-Loaded Self-Curing Formulations 216624 nonsteroidal Anti-Inflammatory Drug-Loaded Formulations 217625 Self-Curing Formulations with Biodegradable Components 218

63 Self-Curing Hydrogels for Soft Tissue Regeneration 219631 Chemically Cross-Linked Hydrogels 220632 Chemically and Physically Cross-Linked Hydrogels 225

64 Expectative and Future Directions 226References 226

7 self-Assembling Peptides as synthetic extracellular Matrices 235MT Fernandez Muintildeos and CE Semino

71 Introduction 23572 In Vitro Applications 23873 In Vivo Applications 242References 245

8 Polymer therapeutics as nano-sized Medicines for tissue Regeneration and Repair 249Ana Armintildeaacuten Pilar Sepuacutelveda and Mariacutea J Vicent

81 Polymer Therapeutics as nano-Sized Medicines 249811 The Concept and Biological Rationale behind Polymer

Therapeutics 249812 Current Status and Future Trends 252

82 Polymer Therapeutics for Tissue Regeneration and Repair 254821 IschemiaReperfusion Injuries 255822 Wound HealingRepair 260823 Musculoskeletal Disorders 263824 Diseases of the Central nervous System 267

83 Conclusions and Future Perspectives 272References 273

9 How Regenerative Medicine Can Benefit from nucleic Acids Delivery nanocarriers 285Erea Borrajo Anxo Vidal Maria J Alonso and Marcos Garcia-Fuentes

91 Introduction 285911 Learning from Viruses How to overcome Cellular Barriers 286

92 nanotechnology in Gene Delivery 292921 Lipid nanocarriers 292922 Polymeric nanocarriers 294923 Inorganic nanoparticles 300

93 nanotechnology in Regenerative Medicine 302931 Bone Regeneration 303932 Cartilage Regeneration 305933 Tendon Regeneration 308

ConTEnTS ix

934 Myocardium Regeneration 309935 neurological Tissue 311


10 Functionalized Mesoporous Materials with Gate-Like scaffoldings for Controlled Delivery 337Elena Aznar Estela Climent Laura Mondragon Feacutelix Sancenoacuten and Ramoacuten Martiacutenez-Maacutentildeez

101 Introduction 337102 Mesoporous Silica Materials with Gate-Like Scaffoldings 339

1021 Controlled Delivery by pH Changes 3391022 Controlled Delivery Using Redox Reactions 3451023 Controlled Delivery Using Photochemical Reactions 3491024 Controlled Delivery via Temperature Changes 3521025 Controlled Delivery Using Small Molecules 3551026 Controlled Delivery Using Biomolecules 356

103 Concluding Remarks 360References 361

11 Where Are We Going Future trends and Challenges 367Sang Jin Lee and Anthony Atala

111 Introduction 367112 Classification of Biomaterials in Tissue Engineering

and Regenerative Medicine 3681121 naturally Derived Materials 3681122 Biodegradable Synthetic Polymers 3701123 Tissue Matrices 372

113 Basic Principles of Biomaterials in Tissue Engineering 373114 Development of Smart Biomaterials 374115 Scaffold Fabrication Technologies 376

1151 Injectable Hydrogels 3761152 Electrospinning 3771153 Computer-Aided Scaffold Fabrication 3781154 Functionalization of Tissue-Engineered Biomaterial

Scaffolds 379116 Summary and Future Directions 381References 384

Index 391

xi

Preface

Life expectancy has been continuously increasing and consequently human pathol-ogies related to aging such as musculoskeletal disorders arthritis nonhealing wounds or neurodegenerative diseases are becoming major health problems Therefore there is a need to identify novel strategies to improve the current therapeutic armory This book presents a number of topics from polymer applications in the field of regenerative medicine with a span from polymeric nanostructures to scaffolds The full therapeutic potential of novel polymeric systems can only be developed through multidisciplinary collaborative research involving biologists chemists clinicians and industries This book tries to provide concepts and founda-tions to a general readership as well as current applications and an overview of this exponentially growing field for experts

Synthetic and natural polymers are compounds of great interest in many fields especially in biomedical applications In the past they have been extensively used as excipients in traditional dosage forms as materials for prostheses valves or contact lenses More recently their applications have been extended to sophisticated drug delivery systems and rationally designed scaffolds for cell therapy so that interesting polymer structures for a variety of applications now cover the nanoscale in polymer therapeutics the microscale in delivery systems and the macroscale in hybrid cell-material constructs for tissue regeneration

Polymeric materials are especially suited to interface with cells Polymers are long-chain molecules that share basic features with biological macromolecules both kinds of molecules deform with the inertial mechanism of conformational change and both are able to exhibit structure at a molecular level (the local sequence of different chemical monomers) and at a supramolecular and nano- to micrometer level (phase-separated domains crystalline domains) More complex multimolecule

xii Preface

arrangements leading to the macroscopic network structure of the extracellular matrix (ecM) represent a third level of structure with typical dimensions ranging from tens to hundreds of microns

The contributions in the first part of the book ldquoMethods for synthetic extracellular matrices and scaffoldsrdquo comprise those topics that are more directly related to the tissue engineering and regenerative applications of polymer structures where their micro- and macrostructures have more importance Key questions permitting a rational design (chapter 1) and selection of materials (chapters 2 and 6) for scaffolds with adequate interactions with the biological interphase (chapters 3ndash5) are addressed as well as specific techniques and applications where scaffolds drive the therapeutic output and organ replacement is discussed in chapter 11 a closer look is then given in Part B ldquoNanostructures for tissue engineeringrdquo to the effect of mod-ifications at the nanoscale a hot topic in the design of nanomedicines for tissue repair a field of exponential growth Here the selection of polymers as active com-ponents of nanostructures together with the understanding of the solution conforma-tion of natural and synthetic materials (chapter 8) with self-assembled properties at the nanoscale (chapter 7) is of crucial importance to better design therapies in regen-erative medicine These materials should be able to efficiently deliver to the targeted site the bioactive agents of different nature including small drugs peptides proteins (chapters 8 and 10) or even oligonucleotide sequences (chapter 9)

chapter 1 addresses the performance of polymers as materials for tissue engineering scaffolds These synthetic tridimensional structures provide grafted cells with a niche and with adequate mechanical and chemical stimuli and thus can promote the process of tissue regeneration Various mechanical physicochemical biological and struc-tural requirements posed on these structures are discussed and how to match them through bulk and surface chemistry and by means of different porogenic techniques are elaborated Questions arising from the interplay between composition function and structure are discussed and the most important parameters for a physical and biological characterization of scaffold performance are presented The possibilities afforded by polymerization chemistry andor subsequent processing or treatment make polymers such unique materials for tissue engineering scaffolds

Many polymers from natural sources have found application in tissue engineering and regenerative medicine chapter 2 presents a comprehensive overview of them as well as examples of their application and clinical use Their origin varies from marine crustacean and algae as well as mammalian plants and microorganism-processed products These polymers have good biodegradability usually low-inflammatory response and reduced cytotoxicity which make them so interesting The properties and main uses of naturally derived polyesters polysaccharides (chitosan agarose alginates starch hyaluronate and others) protein-based polymers (silk collagen fibrin and others) are discussed and emphasis is given to the responsive nature of these polymers and to their modification in order to obtain sensitive biomaterial systems for tissue engineering Stimulindashresponsive or ldquosmartrdquo polymeric systems are polymers that undergo strong physical or chemical property changes responding to small changes in environmental conditions of a physical (eg temperature light mechanical stress or electric field) or chemical (eg pH or ionic strength) nature

Preface xiii

Various aspects of the interaction between polymer surfaces and cells are covered in chapters 3 and 4 This is a central problem in the understanding of the regenera-tion process assisted by synthetic materials The recognition of an alien surface by a cell is mediated through its membrane receptors that interact with the adsorbed protein layer on the surface a thorough discussion of the processes of protein adsorption cell adhesion and matrix remodeling phenomena at the cellndashmaterial interface is presented in chapter 3 cell adhesion is the first step of the regeneration process and plays a fundamental role in subsequent cell differentiation growth via-bility and phenotype expression The nature of the adsorbed layer of proteins on a polymer surface dictates the initial cellular response and eventually the fate of a synthetic material when it is placed in a biological environment The chapters review the role of surface chemistry and patterning on the phenomenon of fibrillogenesis of adsorbed ecM proteins such as laminin and fibronectin and the different experi-mental techniques to follow protein adsorption The fundamentals of cell adhesion on synthetic polymers are also presented in these chapters The role of the different adhesion structures is examined especially of focal adhesions fibrillar adhesions and focal complexes These are multidomain molecules that can interact with several distinct partner molecules and they are decisive for the proliferative or migratory response of cells and for the generation of the forces governing the mechanosensory processes in cells The influence of mechanical topographical and chemical prop-erties of the synthetic surface on focal adhesion kinase a signaling protein contrib-uting to integrin control of cell motility survival and proliferation is specifically addressed in chapter 4

The processes of cellndashmaterial interaction in vivo though are much more com-plex than any of the experimental situations that can be reproduced in vitro Many cell types coexist in any tissue and the cross-talk processes between them through different kinds of signals are to a large extent unknown an attempt to come closer to more realistic scenarios involves the use of bioreactors where cells and materials can be combined with different signaling molecules under culture conditions that can be controlled in ways that try to resemble aspects of the natural cell microenvironment nutrient flow mechanical stresses concentration gradients different gas diffusion etc including coculture systems This problem is addressed in chapter 5 the last of the first ldquomacrordquo part of our book with emphasis given to the dynamic character of the processes that lead to the consideration of the bioreactor the cells and the soluble and synthetic materials as a hybrid system

The second part of the book ldquoNanostructures for tissue engineeringrdquo includes contributions addressing topics where the molecular and nanoscale dimensions of the materials play a dominant role as is the case of therapeutics Bioactive nanostruc-tures molecularly crafted to signal cells or carry therapeutic agents to specific cells have great potential to regenerate tissues and cure disease The chemistry of such nanostructures should allow them to interact specifically with cell receptors or intracellular structures

The first example of the importance of nanostructures shows the application of self-curing formulations for hard as well as soft tissue regeneration (chapter 6) which react chemically in the human body and allow targeting and controlled release

xiv Preface

of bioactive components Self-curing systems based on macromolecular architec-tures can be applied locally and can act as antibacterial antimicrobicide or anti-inflammatory agents although very promising steps have been already achieved to obtain real biomimetic systems that could be integrated in the natural ecM with adequate biofunctionality and biodegradability is still a challenge The ecM is there-fore a main target for consideration in future development of bioactive and biode-gradable formulations that should be able to act as controlled reservoirs of bioactive agents controlled release matrices and in addition as the adequate scaffolds for the development of natural regenerated tissues and organs The application of physical interactions between macromolecular systems and the selective chemical reactions will be key factors for the evolution of these active materials at the nanoscale

The organization of these nanostructures at larger length scales comparable to cells and large colonies of cells will also be critical to their function chapter 7 describes an extensive family of amphiphilic molecules that self-assemble into supra-molecular nanofibers with capacity to display a large diversity of signals to cells ldquoSelf-assemblyrdquo is the spontaneous arrangement of molecules into stable patterns by the driving force of noncovalent interactions such as hydrogen bonds ionic bonds electrostatic bonds and van der Waals interactions regular alternating hydrophobic and hydrophilic residues in short peptide molecules create two distinct surfaces one hydrophobic and the other hydrophilic resulting in β-sheet structures in water They are water soluble and form soft hydrogels when a change in ionic strength andor the pH of the solution occurs due to salts or buffers as a result a network of interweaving nanofibers of around 10 nm diameter is formed with many features in common with the ecM furthermore the versatility of the modification of these materials permits their functionalization with signaling sequences to instruct cells in different ways This chapter illustrates the use of these systems to regenerate axons in the central nervous system for spinal cord injuries bone and blood vessels in cardiovascular therapies With the appropriate supramolecular design these nanostructures could also be used in stem cell cancer and gene therapies

Nanomedicine has been defined as ldquothe use of nanosized tools for the diagnosis prevention and treatment of disease and to gain increased understanding of the com-plex underlying pathophysiology of disease The ultimate goal is improved quality-of-liferdquo currently about 40 nanoproducts for health care are in routine use among the nanotechnologies explained here ldquopolymer therapeuticsrdquo is blooming as the most successful first-generation nanomedicine (chapter 8) Polymer conjugates dif-fer from other nanopharmaceuticals that simply entrap solubilize or control drug release without resorting to chemical conjugation it sums the advantage of small size typically lt25 nm which enables better access to the biological targets that so many other nanocarriers cannot attain clinical proof-of-concept for polymerndash protein conjugates is already a fact but recent advances in polymer chemistry and the techniques available for physicochemical and biological characterization are enabling for the first time in-depth analysis of the critical polymer therapeutic characteristics governing their structurendashactivity relationships Initial studies to date cover a broad spectrum of pathologies trying to seek treatments for chronic and debilitating

Preface xv

diseases of increasing population of older age (ie diabetes hypertension infections digestive track diseases or rheumatoid arthritis) at present cimziareg (rheumatoid arthritis) Macugenreg (age-related macular degeneration) and Krystexxareg (chronic gout) are in routine clinical use Within this context a very promising research approach looks at polymer therapeutics as a tool to promote tissue repair applications in wound healing bone resorption or ischemiareperfusion injuries are described in chapter 8 More ambitious targets such as cardiac-tissue regener-ation or neurodegenerative disorders are the focus of ongoing research projects and first studies already in process should come to fruition in the near future although still in its infancy gene therapy can have a major role in this area Indeed stem cells can be genetically engineered by means of adequate nanovectors (viral or nonviral) to direct their ex vivo and in vivo behavior Gene therapy as regenerative medicine is still facing considerable delivery challenges (eg safety and ineffi-ciency) however the latest advances in gene nanocarrier design have led to a few nanoconstructs with acceptable toxicities and efficacies and is described chapter 9 This considerable progress has been fostered by new emerging materials designed in response to our deeper understanding of the biological barriers in gene delivery Moreover nanomaterials can now be used in combination with physical methods to increase further their efficacy in vitro and in vivo With current techniques and expecting further advances in the following years successful application of synthetic gene nanocarriers to regenerative medicine seems to be both a desirable and a reasonable goal

Organic polymeric materials can be combined with inorganic giving rise to new ldquointelligentrdquo hybrid materials possessing unique advantages chapter 10 discusses the use of mesoporous silica nanoparticles functionalized to become gated recepta-cles for controlled drug delivery These mesoporous supports can be capped and synthesized as nanometric particles resulting in suitable materials for the design of ldquonanodevicesrdquo for on-command delivery applications The molecular ldquogatesrdquo are sensitive to a variety of stimuli and the system is thus able to deliver active mole-cules or pharmaceuticals with high control

as a concluding overview chapter 11 ponders the challenges and opportunities in the field The chapter summarizes the requirements at the macro- and nanoscale for the polymers to be used in clinical applications and the technologies facilitating the pro-cess future advances in tissue engineering and regenerative medicine will depend on the development of ldquosmart biomaterialsrdquo that actively participate in functional tissue regeneration engineering the mechanical physical and biological properties of these materials requires unique experimental theoretical and computational approaches necessarily based on a profound understanding of their structure at the nanoscale

The volume of knowledge in these fields grows steadily and proposals alterna-tives and solutions for many problems accumulate in the pages of journals although some tissue engineering approaches have already demonstrated practical application clinical translation in regenerative medicine progresses only slowly for various reasons a much more dynamic translatory effort will surely result in real breakthroughs and one must hope actual advances in new health treatments

xvi Preface

The editors wish finally to express their deep gratitude to all authors and collaborators who have made this book possible

Mariacutea J Vicent Polymer Therapeutics Lab Centro de Investigacioacuten Priacutencipe Felipe Valencia Spain

Manuel Monleoacuten PradasUniversitat Politegravecnica de Valegravencia Spain

andCIBER de Bioingenieriacutea Biomateriales y Nanomedicina

(CIBER-BBN) Spain

xvii

Contributors

Mariacutea rosa Aguilar Biomaterials Group Polymeric Nanomaterials Biomaterials Department Institute of Polymer Science and Technology (CSIC) Madrid and Biomedical Research Networking Centre in Bioengineering Biomaterials and Nanomedicine (CIBER-BBN) Madrid Spain

Maria J Alonso Center for Research in Molecular Medicine and Chronic Diseases (CIMUS) Department of Pharmacy and Pharmaceutical Technology and Institute for Health Research (IDIS) University of Santiago de Compostela Campus Vida Santiago de Compostela Spain

George Altankov Institute for Bioengineering of Catalonia Barcelona and Institucioacute Catalana de Recerca i Estudis Avanccedilats (ICREA) Barcelona Spain

Ana Armintildeaacuten Polymer Therapeutics Lab Centro de Investigacioacuten Priacutencipe Felipe Valencia Spain

Anthony Atala Wake Forest Institute for Regenerative Medicine Wake Forest School of Medicine Winston-Salem NC USA

Elena Aznar Centro de Reconocimiento Molecular y Desarrollo Tecnoloacutegico (IDM) Unidad Mixta Universidad Politeacutecnica de Valencia-Universidad de Valencia Valencia and CIBER de Bioingenieriacutea Biomateriales y Nanomedicina (CIBER-BBN) Valencia Spain

Erea borrajo Center for Research in Molecular Medicine and Chronic Diseases (CIMUS) Department of Pharmacy and Pharmaceutical Technology Department of Physiology School of Medicine and Institute for Heath Research (IDIS) University of Santiago de Compostela Campus Vida Santiago de Compostela Spain

xviii CoNTRIBUToRS

Marco Cantini Biomedical Engineering Research Division School of Engineering University of Glasgow Glasgow UK

Estela Climent Centro de Reconocimiento Molecular y Desarrollo Tecnoloacutegico (IDM) Unidad Mixta Universidad Politeacutecnica de Valencia-Universidad de Valencia Valencia and CIBER de Bioingenieriacutea Biomateriales y Nanomedicina (CIBER-BBN) Valencia Spain

Dunia Mercedes Garciacutea Cruz Center for Biomaterials and Tissue Engineering Universitat Politegravecnica de Valencia Valencia Spain

stergios C Dermenoudis Laboratory of Biomechanics amp Biomedical Engineering Mechanical Engineering amp Aeronautics Department University of Patras Rion Greece

Mt Fernandez Muintildeos Department of Bioengineering IQS-School of Engineering Ramon Llull University Barcelona Spain

Andreacutes J Garciacutea Woodruff School of Mechanical Engineering Petit Institute for Bioengineering and Bioscience Georgia Institute of Technology Atlanta GA USA

Marcos Garcia-Fuentes Center for Research in Molecular Medicine and Chronic Diseases (CIMUS) Department of Pharmacy and Pharmaceutical Technology and Institute for Health Research (IDIS) University of Santiago de Compostela Campus Vida Santiago de Compostela Spain

Cristina Gonzaacutelez-Garciacutea Woodruff School of Mechanical Engineering Petit Institute for Bioengineering and Bioscience Georgia Institute of Technology Atlanta GA USA

Jorge Luis Escobar ivirico Center for Biomaterials and Tissue Engineering Universitat Politegravecnica de Valencia Valencia Spain

sang Jin Lee Wake Forest Institute for Regenerative Medicine Wake Forest School of Medicine Winston-Salem NC USA

Ana Valleacutes Lluch Center for Biomaterials and Tissue Engineering Universitat Politegravecnica de Valencia Valencia Spain

Joatildeo F Mano 3Bs Research Group-Biomaterials Biodegradables and Biomimetics University of Minho Guimaratildees and ICVS3Bs PT Government Associated Laboratory BragaGuimaratildees Portugal

ramoacuten Martiacutenez-Maacutentildeez Centro de Reconocimiento Molecular y Desarrollo Tecnoloacutegico (IDM) Unidad Mixta Universidad Politeacutecnica de Valencia-Universidad de Valencia Valencia Departamento de Quiacutemica Universidad Politeacutecnica de Valencia Valencia and CIBER de Bioingenieriacutea Biomateriales y Nanomedicina (CIBER-BBN) Valencia Spain

CoNTRIBUToRS xix

Yannis F Missirlis Laboratory of Biomechanics amp Biomedical Engineering Mechanical Engineering amp Aeronautics Department University of Patras Rion Greece

Laura Mondragon Centro de Reconocimiento Molecular y Desarrollo Tecnoloacutegico (IDM) Unidad Mixta Universidad Politeacutecnica de Valencia-Universidad de Valencia Valencia and CIBER de Bioingenieriacutea Biomateriales y Nanomedicina (CIBER-BBN) Valencia Spain

Mariana b oliveira 3Bs Research Group-Biomaterials Biodegradables and Biomimetics University of Minho Guimaratildees and ICVS3Bs PT Government Associated Laboratory BragaGuimaratildees Portugal

Manuel Monleoacuten Pradas Center for Biomaterials and Tissue Engineering Universitat Politegravecnica de Valencia Valencia and Biomedical Research Networking Center in Bioengineering Biomaterials and Nanomedicine (CIBER-BBN) Valencia Spain

Cristina Martiacutenez ramos Center for Biomaterials and Tissue Engineering Universitat Politegravecnica de Valencia Valencia Spain

Patricia rico Center for Biomaterials and Tissue Engineering Universitat Politegravecnica de Valegravencia Valencia and CIBER de Bioingenieriacutea Biomateriales y Nanomedicina Valencia Spain

Julio san romaacuten Biomaterials Group Polymeric Nanomaterials Biomaterials Department Institute of Polymer Science and Technology (CSIC) Madrid and Biomedical Research Networking Centre in Bioengineering Biomaterials and Nanomedicine (CIBER-BBN) Madrid Spain

Manuel salmeroacuten-saacutenchez Biomedical Engineering Research Division School of Engineering University of Glasgow Glasgow UK

Feacutelix sancenoacuten Centro de Reconocimiento Molecular y Desarrollo Tecnoloacutegico (IDM) Unidad Mixta Universidad Politeacutecnica de Valencia-Universidad de Valencia Valencia Departamento de Quiacutemica Universidad Politeacutecnica de Valencia Valencia and CIBER de Bioingenieriacutea Biomateriales y Nanomedicina (CIBER-BBN) Valencia Spain

CE semino Department of Bioengineering IQS-School of Engineering Ramon Llull University Barcelona Spain

Pilar sepuacutelveda Fundacioacuten Hospital La Fe Valencia Spain

blanca Vaacutezquez Biomaterials Group Polymeric Nanomaterials Biomaterials Department Institute of Polymer Science and Technology (CSIC) Madrid and Biomedical Research Networking Centre in Bioengineering Biomaterials and Nanomedicine (CIBER-BBN) Madrid Spain

xx CoNTRIBUToRS


Anxo Vidal Center for Research in Molecular Medicine and Chronic Diseases (CIMUS) Department of Physiology School of Medicine and Institute for Heath Research (IDIS) University of Santiago de Compostela Campus Vida Santiago de Compostela Spain

Methods for synthetic extracellular Matrices and scaffolds

part a

Polymers in Regenerative Medicine Biomedical Applications from Nano- to Macro-Structures First Edition Edited by Manuel Monleoacuten Pradas and Mariacutea J Vicent copy 2015 John Wiley amp Sons Inc Published 2015 by John Wiley amp Sons Inc

3

Polymers as materials for tissue engineering scaffolds

Ana Valleacutes Lluch1 Dunia Mercedes Garciacutea Cruz1 Jorge Luis Escobar Ivirico1 Cristina Martiacutenez Ramos1 and Manuel Monleoacuten Pradas12

1 Center for Biomaterials and Tissue Engineering Universitat Politegravecnica de Valencia Valencia Spain2 Biomedical Research Networking Center in Bioengineering Biomaterials and Nanomedicine (CIBER-BBN) Valencia Spain

1

11 the requirements imPosed by aPPlication on material structures intended as tissue engineering scaffolds

The discovery of the multipotent nature of different kinds of stem cells opened new horizons for therapeutics for surgery and for medicine in general Current treatments for diseased or injured tissues or organs range from transplantation from allogenic or xenogenic donors or reconstruction by transfer of autologous tissues to the use of nonbiological either implanted or extracorporeal devices None of these strategies is free of inconveniencies (shortage and effectiveness of donors clinical complications need of immunosuppressive drugs tumors formation etc) The hope to understand stem cells in their behavior up to the point of directing their differentiation toward desired lineages is at the base of the different new therapeutical strategies encom-passed by regenerative medicine

This process of cell differentiation is triggered and governed by a multiplicity of factors and stimuli which cells receive from an immediate environment made from

4 PoLyMERs As MATERIALs foR TIssuE ENGINEERING sCAffoLDs

other interacting cells and from the extracellular matrix (ECM) In cases of severe loss or degeneration of tissue the sites of intended regeneration have lost their basic structures and thus new grafted cells even if having the right properties in vitro fail to regenerate functional tissue in vivo At this point synthetic tridimensional struc-tures so-called scaffolds may be of help by providing grafted cells with a niche and adequate mechanical and chemical stimuli As an example cardiac tissue regenera-tion in cases of myocardial infarction and subsequent ventricular remodeling has been recently addressed by cell transplantation and cell sheet engineering with a variety of cell populations and supply methods [1 2] Common difficulties found include lack of functional integration and a low survival of the grafted cells These shortcomings could be overcome by means of biomaterials into which the cells would be encapsulated [3 4]

Generally speaking scaffolds must assist the regeneration process performing as an artificial cellular environment during some stages of the tissue regeneration [5 6] Either in vitro or in vivo they must replace as best as possible some of the functions of the ECM they must (i) contribute to the structural and mechanical integrity of the diseased tissue (ii) serve as a means of transport of nutrients and wastes and facili-tate vascularization (iii) act as a spatial guide for cell spreading and organization and (iv) transduce mechanical or biochemical stimuli and eventually transport store and deliver active molecules that effect the expression of the phenotype Besides these functions in defining the requirements on materials intended as scaffolds two other sets of factors must be taken into account those deriving from the specificity of the application (in vitro or in vivo temporal or permanent etc) and those related to processability and manufacture (sizes and shapes of the implants sterilization procedures)

function specific application and processability considerations thus define a number of requisite properties of mechanical physicochemical biological and struc-tural nature from the mechanical side strength (resistance to failure) and stiffness (characterized by shear tensile or compressive moduli) are the most important prop-erties to be addressed Modulus values as different as those of brain and bone deter-mine a wide interval of magnitude and mechanotransduction of signals to the cells depends significantly on this property especially on the surface moduli The most important physicochemical properties of scaffold materials are their degradable or stable nature their permeability and diffusivity to fluids and gases and their hydro-philic or hydrophobic nature Material surfaces possess also specific biologically rel-evant properties their chemical functionalities may be directly involved in the surface nucleation of compounds such as bone hydroxyapatite or they may adsorb ECM proteins in different conformations thus affecting cell adhesion spreading and pro-liferation Lastly microstructural properties of the materials such as their pore volume fraction pore connectivity and geometry (shape dimensions of the pores regularity) are critical for the scaffoldrsquos final performance The scaffoldrsquos ability to host cells in required numbers to allow vascularization throughout it or to guide and organize spatially cell growth in specific ways depends crucially on these properties

our ways to meet these mechanical physicochemical biological and structural requirements is through bulk and surface chemistry for the first three and through

CoMPosITIoN AND fuNCTIoN 5

different porogenic techniques for the fourth A material with a given overall chemical composition may be furthermore in very different physical states it may be a random or a block copolymer it may be an interpenetrated network it may be semi-crystalline or amorphous vitreous or rubbery under physiological conditions These possibilities are afforded by polymerization chemistry andor subsequent processing or treatment and make polymers such unique materials for tissue engineering applications

The intended end uses of the scaffolds are widely different scaffolds may be implanted empty (acellular) when they are expected to be invaded and colonized by the cells in a short period of time otherwise it is necessary to seed the scaffolds with the appropriate cells before implantation or even to preculture them in vitro within the scaffold [7] A bioreactor can be helpful in this stage to recreate in vitro some dynamic conditions and the mechanical andor chemical stimuli that the cells would receive in vivo scaffolds may be chemically modified in order to direct cell anchorage or differentiation through addition of proteins peptides growth factors (Gfs) hormones enzymes or other regulators of the cell behavior [8ndash11] several methods for the controlled release of factors from scaffolds have been developed [12ndash14]

Polymer materials are especially suited to interface with cells Being formed by long chain molecules they share some basic properties with biological macromole-cules At the most fundamental level both kinds of molecules deform with the inertial mechanism of conformational change which gives rise to molecular dynamics with characteristic relaxation phenomena at long time scales Moreover both biological and synthetic macromolecules are able to exhibit structure at a subnanometer molec-ular level (the local arrangement of different chemical monomers) and at a supramo-lecular nano- to micrometer level phase-separated domains crystalline domains And the more complex multimolecular associations leading to the macroscopic net-work structure of the ECM can be mimicked to some extent by the porous architecture of the polymer scaffolds This represents a third level of structure with typical dimensions ranging from tens to hundreds of microns

12 comPosition and function

121 general considerations

1211 The Influence of Surface ChemistryThe fate of an implant is determined by the host tissue reaction to it and this is mainly a matter of surface interactions chemical and topological [15 16] Cells react to events in their environment as a consequence of signaling processes transduced by cell membrane receptors These are large molecules that bind to or react with chemical functionalities of the environmental molecules in specific ways triggering a number of subsequent cellular processes [17] The usual foreign body reaction to implanted synthetic material consists in a number of processes ending in the iso-lation of the implant through its encapsulation in high-density fibrotic tissue This circumstance may in some applications imply the failure of the implant as it makes


impossible a functional continuous integration of the grafted cells in the site of regen-eration The first stages in the encapsulation process involve the adsorption of ECM proteins onto the foreign surface and the interaction of cellndashmembrane receptors with them [18] The conformation of the adsorbed proteins thus may play an impor-tant role in the fate of an implanted scaffold since the cellndashmaterial interaction is always mediated by the ECM proteins adsorbed on the materialrsquos surface the chemical and topological properties of the surface responsible for the adsorbed con-formation of the proteins will always be determinant for the biological performance of a scaffold [19] Cell adhesion and cell spreading especially at early stages of the process will depend on those properties

The features of surface chemistry having the greatest influence in this respect are the hydrophilicndashhydrophobic balance of functionalities the surface charges their spatial distribution on the surface and the surface stiffness

The more or less hydrophilic character of a surface is determined by the presence in its composition of hydrophilic functionalities (such as ndashoH ndashCooH ndashNH

2 ndashsH

polar groups or bound ions) and by their mobility (large in the rubber state very impeded in the glassy state) ECM proteins adsorb poorly onto highly hydrophilic surfaces and consequently the cells adhere with difficulty especially at the earliest times of contact Adsorption sites of this kind of surfaces are preferentially occupied by water molecules in a labile dynamic equilibrium that is difficult competing protein adsorption In this situation proteins adsorb if at all in small amounts and with a typically globular conformation which minimizes the area of their interface with the material and thus the free energy Correspondingly cells attach if at all in small amounts and with a rounded shape with a poorly developed cytoskeleton frequently preferring cell-to-cell associations over cellndashsurface contacts By contrast on more hydrophobic surfaces ECM proteins tend to adsorb in larger amounts and with more extended conformations now energetically preferred since the proteinndashmaterial inter-action destroys the waterndashsurface bond decreasing the free energy This causes cell-binding sequences in the adsorbed proteins to be more accessible and as a consequence cells attach to hydrophobic surfaces more and with more extended shapes numerous processes and larger focal adhesions and a developed cytoskeleton

The state of affairs just described for hydrophilic and for hydrophobic surfaces applies as a general rule to the early stages of the cellndashndashmaterial interaction process With time different situations may arise as a consequence of new processes com-peting with those just described such as the progressive build-up of proteinndashprotein interactions (fibrillogenesis) [20] and the cell remodeling of the ECM The early interactions however may be critical for the success of the implant since it is they who determine cell invasion neovascularization and the foreign-body response

The spatial distribution of the surface functionalities is also important for the processes just mentioned since proteins have both hydrophilic and hydrophobic domains the shapes they acquire upon adsorption may depend on the presence on the material surface of alternatingly distributed hydrophilic and hydrophobic domains at a scale that matches the separation of those domains in the proteins Block copoly-mers interpenetrating polymer networks blends or nanocomposite organicndashorganic or organicndashinorganic materials are systems whose phase distribution can be tailored


at the nanometer scale relevant for the proteinndashsurface and thus for cellndashsurface interaction The issue of surface topography is also controllable to some degree by factors such as the sizes of the phase-separated nanodomains in heterogeneous material surfaces These may include crystallites of different sizes alternating with amorphous domains in semicrystalline polymers or nanophases of different chemical and mechanical properties in block copolymers polymer blends or interpenetrated polymer networks Protein adsorption and hence cell early adhesion on the material is very sensitive to these purely physical features of a surface nano- and microrough-ness of the surface topography favors protein adsorption acting as nucleation points for the adsorption process by diminishing the interfacial surface tension

A different example of the import of surface chemistry is represented by the so-called ldquobioactivityrdquo of surfaces This term which arguably should be referring to a wider class of phenomena has come to be identified with the ability of certain synthetic materials to nucleate the growth of hydroxyapatite crystals at a rate relevant for physiological interaction [21] This is of great importance in bone tissue engineering where a hydroxyapatite layer grown on the surfaces of a scaffold may integrate it is hoped continuously with the surrounding bone tissue and avoid the formation of the aforementioned fibrous capsule which especially in the case of bone would have disastrous effects on the vascularization of the implant and on its load-bearing capacity Bioactivity thus understood is a process triggered by surface groups that may enter into exchange reactions in aqueous medium (blood or physiological fluids in vivo simulated body fluid (sBf) in vitro) to bind calcium ions which in their turn start the process of apatite crystallization in the presence of phosphate ions usually hybrid organicndashinorganic polymer composites exhibit this kind of bioactivity In them typi-cally a silica-based phase included in the polymer matrix exhibits silanol groups ndashsioH at the surface which upon dissociation ndashsiominus act as nucleation sites for the calcium ions acting as precursors for hydroxyapatite nucleation and growth

surface chemistry is important also from the mechanical point of view Mechanotransduction cell motility (migration) or the extension of cell processes (eg neurites) on the surface are all very sensitive to its stiffness

The issue of surface chemistry is to some extent independent from bulk composition of the material materials can be subjected to surface treatments and functionalized in the desired ways a surface layer with properties significantly different from the bulk can thus be achieved furthermore surface properties are interfacial properties this means that even in the case of nontreated surfaces the out-ermost layer of a material may possess effective properties different from the bulk due to the fact that interfacial interactions suffered during manufacture (eg contact with mould surfaces) or in situ (eg the hydrophobic interaction in the presence of water) have altered the conformation of the functionalities at the surface and thus affected their availability

1212 The Influence of Bulk Chemistryscaffolds besides hosting cells must in most applications withstand certain mechanical stresses The overall mechanical stiffness of an implant determines its manipulability and its success in stressed environments stiffness depends on the


cohesive energy density of the material and is thus a function of the bulk chemical composition on it depend also other important properties most singularly diffu-sivity and permeability to water and to small molecular weight species the hydrogel character the biostability or biodegradability of the material and in the latter case the degradation mechanism If degradation takes place following a hydrolytic route the material will degrade when in contact with water If its bulk chemistry is hydro-phobic the process will start at the materialrsquos surface and proceed gradually towards its interior In this case degradation erodes progressively thicker outer shells of the piece but an inner nucleus remains unaffected for a time which can preserve some of the mechanical properties of the piece By contrast in the case of hydrophilic chemistries bulk swelling occurs which allows the onset of hydrolysis at all points of the piece from the start Hydrogels and hydrophilic polymers will thus degrade more rapidly than hydrophobic polymers and their bulk properties will start reflecting degradation at a faster rate

122 some families of Polymers for tissue engineering scaffolds

According to their biological stability polymers may be classified as biodegradable or biostable and these characteristics condition their choice for applications from the point of view of final use biodegradable polymers offer the significant advantage of disappearing from the body in due time The problems associated with long-term extraneous implants thus disappear However certain applications require permanent implants corneal prostheses cerebral stimulation devices dental implants cardiac restraint devices and many others In all these cases one faces the impossibility of complete regeneration and the need to preserve function or the necessity to preserve the long-term stability of an implant Contrary to what may be a first thought on the matter research on biostable synthetic polymers possesses a substantive interest of its ownBiodegradable polymers If under specific in vivo conditions a polymer undergoes chemical reactions that decompose it into nontoxic products that can be completely removed or metabolized by the human body the material is regarded as biodegrad-able specifically when a biomaterial is implanted in the human body an inflammatory response to the foreign body occurs This process is the result of the action of differ-ent cell types such as leukocytes and macrophages Through oxidative reactions caused by reactive species secreted by the cells (H

2o

2 No o

2minus) the polymer chains

may suffer scission and hydrolytic degradation (chain scission through water-labile groups of the polymer structure see fig 11) Depending on the hydrophilicity of the material these degradation processes advance in a front-like manner from the outside to the interior of the material (in hydrophobic polymers) or take place more rapidly in a more homogeneous way in the bulk of the material (in more hydrophilic polymers) Hydrolytic degradation can be catalyzed by enzymes or by the fluids with high content of acidic or basic compounds in the body The degradation process results in the loss of physical chemical and mechanical properties of the material The kinetics of biodegradation is a matter of chemistry but also of shape size and topology of an implant In general large specific surface areas (ie porosity




Edited By












10 9 8 7 6 5 4 3 2 1

v

Contents

Preface xi

Contributors xvii







vi ConTEnTS




References 37















ConTEnTS vii






References 168











viii ConTEnTS















ConTEnTS ix













Index 391

xi

Preface




xii Preface





Preface xiii





xiv Preface




Preface xv





xvi Preface





(CIBER-BBN) Spain

xvii

Contributors








xviii CoNTRIBUToRS














CoNTRIBUToRS xix













xx CoNTRIBUToRS




part a


3




1




















2 ndashsH














2o

2 No o












10 9 8 7 6 5 4 3 2 1

v

Contents

Preface xi

Contributors xvii







vi ConTEnTS




References 37















ConTEnTS vii






References 168











viii ConTEnTS















ConTEnTS ix













Index 391

xi

Preface




xii Preface





Preface xiii





xiv Preface




Preface xv





xvi Preface





(CIBER-BBN) Spain

xvii

Contributors








xviii CoNTRIBUToRS














CoNTRIBUToRS xix













xx CoNTRIBUToRS




part a


3




1




















2 ndashsH














2o

2 No o



v

Contents

Preface xi

Contributors xvii







vi ConTEnTS




References 37















ConTEnTS vii






References 168











viii ConTEnTS















ConTEnTS ix













Index 391

xi

Preface




xii Preface





Preface xiii





xiv Preface




Preface xv





xvi Preface





(CIBER-BBN) Spain

xvii

Contributors








xviii CoNTRIBUToRS














CoNTRIBUToRS xix













xx CoNTRIBUToRS




part a


3




1




















2 ndashsH














2o

2 No o



vi ConTEnTS




References 37















ConTEnTS vii






References 168











viii ConTEnTS















ConTEnTS ix













Index 391

xi

Preface




xii Preface





Preface xiii





xiv Preface




Preface xv





xvi Preface





(CIBER-BBN) Spain

xvii

Contributors








xviii CoNTRIBUToRS














CoNTRIBUToRS xix













xx CoNTRIBUToRS




part a


3




1




















2 ndashsH














2o

2 No o



ConTEnTS vii






References 168











viii ConTEnTS















ConTEnTS ix













Index 391

xi

Preface




xii Preface





Preface xiii





xiv Preface




Preface xv





xvi Preface





(CIBER-BBN) Spain

xvii

Contributors








xviii CoNTRIBUToRS














CoNTRIBUToRS xix













xx CoNTRIBUToRS




part a


3




1




















2 ndashsH














2o

2 No o



viii ConTEnTS















ConTEnTS ix













Index 391

xi

Preface




xii Preface





Preface xiii





xiv Preface




Preface xv





xvi Preface





(CIBER-BBN) Spain

xvii

Contributors








xviii CoNTRIBUToRS














CoNTRIBUToRS xix













xx CoNTRIBUToRS




part a


3




1




















2 ndashsH














2o

2 No o



ConTEnTS ix













Index 391

xi

Preface




xii Preface





Preface xiii





xiv Preface




Preface xv





xvi Preface





(CIBER-BBN) Spain

xvii

Contributors








xviii CoNTRIBUToRS














CoNTRIBUToRS xix













xx CoNTRIBUToRS




part a


3




1




















2 ndashsH














2o

2 No o



xi

Preface




xii Preface





Preface xiii





xiv Preface




Preface xv





xvi Preface





(CIBER-BBN) Spain

xvii

Contributors








xviii CoNTRIBUToRS














CoNTRIBUToRS xix













xx CoNTRIBUToRS




part a


3




1




















2 ndashsH














2o

2 No o



xii Preface





Preface xiii





xiv Preface




Preface xv





xvi Preface





(CIBER-BBN) Spain

xvii

Contributors








xviii CoNTRIBUToRS














CoNTRIBUToRS xix













xx CoNTRIBUToRS




part a


3




1




















2 ndashsH














2o

2 No o



Preface xiii





xiv Preface




Preface xv





xvi Preface





(CIBER-BBN) Spain

xvii

Contributors








xviii CoNTRIBUToRS














CoNTRIBUToRS xix













xx CoNTRIBUToRS




part a


3




1




















2 ndashsH














2o

2 No o



xiv Preface




Preface xv





xvi Preface





(CIBER-BBN) Spain

xvii

Contributors








xviii CoNTRIBUToRS














CoNTRIBUToRS xix













xx CoNTRIBUToRS




part a


3




1




















2 ndashsH














2o

2 No o



Preface xv





xvi Preface





(CIBER-BBN) Spain

xvii

Contributors








xviii CoNTRIBUToRS














CoNTRIBUToRS xix













xx CoNTRIBUToRS




part a


3




1




















2 ndashsH














2o

2 No o



xvi Preface





(CIBER-BBN) Spain

xvii

Contributors








xviii CoNTRIBUToRS














CoNTRIBUToRS xix













xx CoNTRIBUToRS




part a


3




1




















2 ndashsH














2o

2 No o



xvii

Contributors








xviii CoNTRIBUToRS














CoNTRIBUToRS xix













xx CoNTRIBUToRS




part a


3




1




















2 ndashsH














2o

2 No o



xviii CoNTRIBUToRS














CoNTRIBUToRS xix













xx CoNTRIBUToRS




part a


3




1




















2 ndashsH














2o

2 No o



CoNTRIBUToRS xix













xx CoNTRIBUToRS




part a


3




1




















2 ndashsH














2o

2 No o



xx CoNTRIBUToRS




part a


3




1




















2 ndashsH














2o

2 No o




part a


3




1




















2 ndashsH














2o

2 No o




3




1




















2 ndashsH














2o

2 No o



















2 ndashsH














2o

2 No o













2o

2 No o



thumbnail - download.e-bookshelf.de€¦ · 7.3 in vivo applications, 242 references, 245 8 polymer...

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