Nuno M. Neves is Professor at the Department of Polymer Engineering of the University of Minho, Portugal, where he is Vice-Director of the 3B’s Research Group – Biomaterials, Biodegradables and Biomimetics. Nuno M. Neves received his PhD degree in Polymer Science and Engineering from the University of Minho in collaboration with the University of Twente, The Netherlands. His main area of research is the development of biomaterials from natural origin polymers. His research group focuses mainly on tissue engineering and regenerative medicine strategies using stem cells and advanced drug delivery scaffolds and medical devices.Rui L. Reis is Professor of Tissue Engineering, Regenerative Medicine, Biomaterials and Stem Cells at the Department of Polymer Engineering of the University of Minho, Portugal. He is the Vice-Rector for Research of the University of Minho, Director of the 3B’s Research Group and the Director of the Portuguese Government Associate Laboratory ICVS/3B’s. Rui L. Reis received his PhD degree in Polymer Engineering from the University of Minho in collaboration with Brunel University in London, UK. His main area of research is the development of biomaterials from natural origin polymers that his group proposes for a range of biomedical applications.

• CONTRIBUTORS xixPREFACE xxixPART I 1 Collagen-Based Porous Scaffolds for Tissue Engineering 3Guoping Chen and Naoki Kawazoe1.1 Introduction, 31.2 Collagen Sponges, 41.3 Collagen Sponges with Micropatterned Pore Structures, 71.4 Collagen Sponges with Controlled Bulk Structures, 101.5 Hybrid Scaffolds, 121.6 Conclusions, 13References, 142 Marine Collagen Isolation and Processing Envisaging Biomedical Applications 16Joana Moreira-Silva, Gabriela S. Diogo, Ana L. P. Marques, Tiago H. Silva, and Rui L. Reis2.1 Introduction, 162.2 Extraction of Collagen from Marine Sources, 182.3 Collagen Characterization, 222.4 Marine Collagen Wide Applications, 252.5 Final Remarks, 32Acknowledgements, 34References, 343 Gelatin-Based Biomaterials for Tissue Engineering and Stem Cell Bioengineering 37Mehdi Nikkhah, Mohsen Akbari, Arghya Paul, Adnan Memic, Alireza Dolatshahi-Pirouz, and Ali Khademhosseini3.1 Introduction, 373.2 Crosslinking of Gelatin, 383.3 Physical Properties of Gelatin, 393.4 Application of Gelatin-Based Biomaterials in Tissue Engineering, 403.5 Gelatin for Stem Cell Therapy, 453.6 Application of Gelatin in Delivery Systems, 493.7 Conclusion and Perspectives, 50Acknowledgements, 50Abbreviations, 50References, 514 Hyaluronic Acid-Based Hydrogels on a Micro and Macro Scale 63A. Borzacchiello, L. Russo, and L. Ambrosio4.1 Classification and Structure of Hydrogels, 634.2 Hyaluronic Acid, 654.3 Hydrogel Mechanical Properties, 664.4 HA-Based Hydrogel for Biomedical Applications, 70References, 755 Chondroitin Sulfate as a Bioactive Macromolecule for Advanced Biological Applications and Therapies 79Nicola Volpi5.1 CS Structure, 815.2 Biological Roles of CS, 815.3 Osteoarthritis Treatment, 845.4 Cardio-Cerebrovascular Disease, 845.5 Tissue Regeneration and Engineering, 855.6 Chondroitin Sulfate-Polymer Conjugates, 865.7 Conclusions and Future Perspectives, 87References, 886 Keratin 93Mark Van Dyke6.1 Introduction, 936.2 Preparation of Keratoses, 986.3 Preparation of Kerateines, 1006.4 Oxidative Sulfitolysis, 1016.5 Summary, 102References, 1027 Elastin-Like Polypeptides: Bio-Inspired Smart Polymers for Protein Purification, Drug Delivery and Tissue Engineering 106Jayanta Bhattacharyya, Joseph J. Bellucci, and Ashutosh Chilkoti7.1 Introduction, 1067.2 Recombinant Protein Production Using ELPs as Purification Tags, 1077.3 Delivery of Therapeutics with ELPs, 1137.4 Tissue Engineering with ELPs, 1197.5 Conclusions, 122Acknowledgements, 122Abbreviations, 122References, 1238 Silk: A Unique Family of Biopolymers 127A. Motta, M. Floren, and C. Migliaresi8.1 Introduction, 1278.2 Main Silk Polymers, 1298.3 Fibroin Basic Processing: Regenerated Silk Fibroin, 1318.4 Materials Fabrication of Silk Proteins, 1318.5 Advanced Material Applications of Silks, 1358.6 Conclusion, 136References, 1379 Silk Protein Sericin: Promising Biopolymer for Biological and Biomedical Applications 142Sunita Nayak and Subhas C. Kundu9.1 Introduction, 1429.2 Sericin Extraction and Processing, 1469.3 Potential Applications of Sericin, 1479.4 Immunogenicity and Toxicity of Sericin, 1529.5 Conclusion, 153Acknowledgements, 154References, 15410 Fibrin 159Markus Kerbl, Philipp Heher, James Ferguson, and Heinz Redl10.1 Introduction, 15910.2 Fibrin Clotting, 16010.3 Fibrin Degradation, 16010.4 Fibrin Glue, 16310.5 Conclusion, 170Acknowledgement, 171References, 17111 Casein Proteins 176Pranav K. Singh and Harjinder Singh11.1 Introduction, 17611.2 Structures and Properties of Casein, 17811.3 Interaction of Caseins with Metal Ions, 18411.4 Conclusions, 185References, 18612 Biomaterials from Decellularized Tissues 190Ricardo Londono and Stephen F. Badylak12.1 Introduction, 19012.2 Host Response to Implanted ECM-Derived Biomaterials, 196References, 19913 Demineralized Bone Matrix: A Morphogenetic Extracellular Matrix 211A. Hari Reddi and Ryosuke Sakata13.1 Introduction, 21113.2 Demineralized Bone Matrix (DBM), 21113.3 From DBM to Bone Morphogenetic Proteins (BMPs), 21313.4 BMPs Bind to Extracellular Matrix, 21613.5 BMP Receptors, 21613.6 Future Perspectives, 218Acknowledgements, 218References, 218PART II14 Recent Developments on Chitosan Applications in Regenerative Medicine 223Ana Rita C. Duarte, Vitor M. Correlo, Joaquim M. Oliveira, and Rui L. Reis14.1 Introduction, 22314.2 Chitosan and Derivatives, 22414.3 Regenerative Medicine Applications of Chitosan, 22714.4 Processing Methodologies, 23114.5 Final Remarks, 236Acknowledgments, 237References, 23715 Starch-Based Blends in Tissue Engineering 244P.P. Carvalho, M.T. Rodrigues, R.L. Reis, and M.E. Gomes15.1 Introduction, 24415.2 Starch, 24515.3 Modification of Starch for Biomedical Applications, 24715.4 Starch-Based Blends, 24815.5 Conclusions and Future Perspectives, 254References, 25516 Agarose Hydrogel Characterization for Regenerative Medicine Applications: Focus on Engineering Cartilage 258Brendan L. Roach, Adam B. Nover, Gerard A. Ateshian, and Clark T. Hung16.1 The Foundations of Agarose, 25816.2 Structure-Function Relationships of Agarose Hydrogels, 25916.3 Agarose as a Tissue Engineering Scaffold, 26116.4 Agarose in the Clinic, 26616.5 A Scaffold to Build On, 267Acknowledgements, 268References, 26817 Bioengineering Alginate for Regenerative Medicine Applications 274Emil Ruvinov and Smadar Cohen17.1 Introduction, 27417.2 Regenerative Medicine: Definition and Strategies, 27517.3 Alginate Biomaterial, 27717.4 Alginate Implant: First in Man Trial for Prevention of Heart Failure, 28117.5 Alginate Hydrogel as a Vehicle for Stem Cell Delivery and Retention, 28417.6 Engineering Alginate-Based Cell Microenvironments, 28717.7 Alginate Hydrogel Carrier for Growth Factor Delivery, 28917.8 Engineering Alginate for Affinity Binding and Presentation of Heparin-Binding Growth Factors, 292References, 30018 Dextran 307Rong Wang, Pieter J. Dijkstra, and Marcel Karperien18.1 Introduction, 30718.2 Structure and Properties, 30818.3 Dextran Derivatives, 31018.4 Dextran Copolymers, 31418.5 Degradation, 31618.6 Outlook, 316References, 31619 Gellan Gum-based Hydrogels for Tissue Engineering Applications 320Joana Silva-Correia, Joaquim Miguel Oliveira, and Rui Lu´ýs Reis19.1 Introduction, 32019.2 Gellan Gum and its Derivatives, 32219.3 Tissue Engineering Applications, 32519.4 Final Remarks, 331Acknowledgments, 332References, 332PART III20 Biomedical Applications of Polyhydroxyalkanoates 339L.R. Lizarraga-Valderrama, B. Panchal, C. Thomas, A.R. Boccaccini, and I. Roy20.1 Introduction, 33920.2 Skin Tissue Engineering, 34120.3 Nerve Tissue Engineering, 34420.4 Cardiac Tissue Engineering, 34820.5 Dental Tissue Engineering, 35620.6 Bone Tissue Engineering, 35820.7 Cartilage Tissue Engineering, 36620.8 Osteochondral Tissue Engineering, 36820.9 Drug Delivery, 37020.10 Conclusions and the Future Potential of PHAs in Biomedical Applications, 373References, 37321 Bacterial Cellulose 384Hernane S. Barud, Junkal Gutierrez, Wilton R. Lustri, Maristela F.S. Peres, Sidney J.L. Ribeiro, Sybele Saska, and Agniezska Tercjak21.1 Introduction, 38421.2 BC Dressings, 38521.3 Bacterial Cellulose for Tissue Engineering and Regenerative Medicine, 38821.4 Concluding Remarks, 393Acknowledgments, 394References, 394PART IV22 Molecularly Imprinted Cryogels for Protein Purification 403Müge Andac¸, Igor Yu Galaev, and Adil Denizli22.1 Introduction, 40322.2 Molecularly Imprinted Cryogels for Protein Purification, 40522.3 Some Selected Applications of Molecularly Imprinted Cryogels (MIC) for Macromolecules, 41422.4 Concluding Remarks and Future Perspectives, 421References, 42323 Immunogenic Reaction of Implanted Biomaterials from Nature 429Martijn Van Griensven and Elizabeth Rosado Balmayor23.1 Introduction, 42923.2 Implantation Leads to Tissue Injury, 43023.3 Inflammatory Responses, 43123.4 Foreign Body Reaction, 43323.5 Immunogenic Reactions Towards Natural Biomaterials, 43523.6 Final Remarks, 438References, 43824 Chemical Modification of Biomaterials from Nature 444J.C. Rodr´ýguez Cabello, I. Gonz´alez De Torre, M. Santos, A.M. Testera, and M. Alonso24.1 Protein Modification, 44424.2 Lipid Modifications, 45124.3 Polysaccharide Chemical Modifications, 457References, 466PART V25 Processing of Biomedical Devices for Tissue Engineering and Regenerative Medicine Applications 477Vitor M. Correlo, Albino Martins, Nuno M. Neves, and Rui L. Reis25.1 Introduction, 47725.2 Processing Techniques of Naturally Derived Biomaterial, 47825.3 Processing Techniques of Natural-Based Polymeric Blends, 483References, 48726 General Characterization of Physical Properties of Natural-Based Biomaterials 494Manuel Alatorre-Meda and Joäo F. Mano26.1 Introduction, 49426.2 Bulk Properties, 49526.3 Surface Properties, 50726.4 Concluding Remarks, 512Acknowledgments, 512References, 51227 General Characterization of Chemical Properties of Natural-Based Biomaterials 517Manuel Alatorre-Meda and Joäo F. Mano27.1 Introduction, 51727.2 Molecular Weight and Elemental Composition, 51827.3 Physiological Degradation, 52427.4 Concluding Remarks, 527Acknowledgments, 529References, 52928 In Vitro Biological Testing in the Development of New Devices 532Marta L. Alves Da Silva, Albino Martins, Ana Costa-Pinto, Rui L. Reis, and Nuno M. Neves28.1 Introduction, 53228.2 Cytotoxicity Assays, 53328.3 Evaluation of Cell Morphology and Distribution, 53328.4 Cell Viability Assays, 53528.5 Cell Proliferation Assays, 53628.6 Biochemical Analysis, 53728.7 Genotypic Expression Analysis, 54128.8 Histological Assessment, 54228.9 In Vitro Engineered Tissues, 54328.10 Concluding Remarks, 548References, 54829 Advanced In-Vitro Cell Culture Methods Using Natural Biomaterials 551Marta L. Alves Da Silva, Rui L. Reis, and Nuno M. Neves29.1 Introduction, 55129.2 Bioreactors, 55229.3 Hypoxia, 55329.4 Co-Cultures, 55529.5 Transfection, 55529.6 Nanoparticles and Related Systems, 55829.7 Concluding Remarks, 559References, 55930 Testing Natural Biomaterials in Animal Models 562Ana Costa-Pinto, Tírcia C. Santos, Nuno M. Neves, and Rui L. Reis30.1 Laboratory Animals as Tools in Biomaterials Testing, 56230.2 Inflammation and Host Reaction, 56430.3 Animal Models for Tissue Engineering, 56830.4 Final Remarks, 574References, 575PART VI31 Delivery Systems Made of Natural-Origin Polymers for Tissue Engineering and Regenerative Medicine Applications 583Albino Martins, Helena Ferreira, Rui L. Reis, and Nuno M. Neves31.1 Introduction, 58331.2 Advantages and Disadvantages of Natural Polymers-Based Delivery Systems, 58531.3 Fundamentals of Drug Delivery, 58631.4 In Vitro and In Vivo Applications of Natural-Based Delivery Systems, 59131.5 Concluding Remarks, 601References, 60232 Translational Research into New Clinical Applications 612M. David Harmon and Sangamesh G. Kumbar32.1 Introduction, 61232.2 Cardiovascular System Applications, 61332.3 Integumentary System Applications, 61632.4 Musculoskeletal System Applications, 61832.5 Nervous System Applications, 61932.6 Respiratory System Applications, 62132.7 Gastrointestinal System Applications, 62232.8 From Idea to Product, 624Acknowledgements, 626References, 62633 Challenges and Opportunities of Natural Biomaterials for Advanced Devices and Therapies 629R.L. Reis and N.M. Neves33.1 Introduction, 62933.2 Challenges of Natural Biomaterials, 63033.3 Opportunities of Natural Biomaterials, 63133.4 Final Remarks, 631References, 63234 Adhesives Inspired by Marine Mussels 634Courtney L. Jenkins, Heather J. Meredith, and Jonathan J. Wilker34.1 Introduction, 63434.2 Requirements for a Bioadhesive, 63534.3 Marine Mussels, 63634.4 Bulk Adhesion Testing, 63834.5 Extracted Mussel Adhesive Proteins, 64034.6 Mimics of Mussel Adhesive, 64134.7 Conclusions, 645Acknowledgement, 645References, 64535 Final Comments and Remarks 649R.L. Reis and N.M. NevesINDEX 651

Nuno M. Neves is Professor at the Department of Polymer Engineering of the University of Minho, Portugal, where he is Vice-Director of the 3B’s Research Group – Biomaterials, Biodegradables and Biomimetics. Nuno M. Neves received his PhD degree in Polymer Science and Engineering from the University of Minho in collaboration with the University of Twente, The Netherlands. His main area of research is the development of biomaterials from natural origin polymers. His research group focuses mainly on tissue engineering and regenerative medicine strategies using stem cells and advanced drug delivery scaffolds and medical devices.Rui L. Reis is Professor of Tissue Engineering, Regenerative Medicine, Biomaterials and Stem Cells at the Department of Polymer Engineering of the University of Minho, Portugal. He is the Vice-Rector for Research of the University of Minho, Director of the 3B’s Research Group and the Director of the Portuguese Government Associate Laboratory ICVS/3B’s. Rui L. Reis received his PhD degree in Polymer Engineering from the University of Minho in collaboration with Brunel University in London, UK. His main area of research is the development of biomaterials from natural origin polymers that his group proposes for a range of biomedical applications.

• CONTRIBUTORS xixPREFACE xxixPART I 1 Collagen-Based Porous Scaffolds for Tissue Engineering 3Guoping Chen and Naoki Kawazoe1.1 Introduction, 31.2 Collagen Sponges, 41.3 Collagen Sponges with Micropatterned Pore Structures, 71.4 Collagen Sponges with Controlled Bulk Structures, 101.5 Hybrid Scaffolds, 121.6 Conclusions, 13References, 142 Marine Collagen Isolation and Processing Envisaging Biomedical Applications 16Joana Moreira-Silva, Gabriela S. Diogo, Ana L. P. Marques, Tiago H. Silva, and Rui L. Reis2.1 Introduction, 162.2 Extraction of Collagen from Marine Sources, 182.3 Collagen Characterization, 222.4 Marine Collagen Wide Applications, 252.5 Final Remarks, 32Acknowledgements, 34References, 343 Gelatin-Based Biomaterials for Tissue Engineering and Stem Cell Bioengineering 37Mehdi Nikkhah, Mohsen Akbari, Arghya Paul, Adnan Memic, Alireza Dolatshahi-Pirouz, and Ali Khademhosseini3.1 Introduction, 373.2 Crosslinking of Gelatin, 383.3 Physical Properties of Gelatin, 393.4 Application of Gelatin-Based Biomaterials in Tissue Engineering, 403.5 Gelatin for Stem Cell Therapy, 453.6 Application of Gelatin in Delivery Systems, 493.7 Conclusion and Perspectives, 50Acknowledgements, 50Abbreviations, 50References, 514 Hyaluronic Acid-Based Hydrogels on a Micro and Macro Scale 63A. Borzacchiello, L. Russo, and L. Ambrosio4.1 Classification and Structure of Hydrogels, 634.2 Hyaluronic Acid, 654.3 Hydrogel Mechanical Properties, 664.4 HA-Based Hydrogel for Biomedical Applications, 70References, 755 Chondroitin Sulfate as a Bioactive Macromolecule for Advanced Biological Applications and Therapies 79Nicola Volpi5.1 CS Structure, 815.2 Biological Roles of CS, 815.3 Osteoarthritis Treatment, 845.4 Cardio-Cerebrovascular Disease, 845.5 Tissue Regeneration and Engineering, 855.6 Chondroitin Sulfate-Polymer Conjugates, 865.7 Conclusions and Future Perspectives, 87References, 886 Keratin 93Mark Van Dyke6.1 Introduction, 936.2 Preparation of Keratoses, 986.3 Preparation of Kerateines, 1006.4 Oxidative Sulfitolysis, 1016.5 Summary, 102References, 1027 Elastin-Like Polypeptides: Bio-Inspired Smart Polymers for Protein Purification, Drug Delivery and Tissue Engineering 106Jayanta Bhattacharyya, Joseph J. Bellucci, and Ashutosh Chilkoti7.1 Introduction, 1067.2 Recombinant Protein Production Using ELPs as Purification Tags, 1077.3 Delivery of Therapeutics with ELPs, 1137.4 Tissue Engineering with ELPs, 1197.5 Conclusions, 122Acknowledgements, 122Abbreviations, 122References, 1238 Silk: A Unique Family of Biopolymers 127A. Motta, M. Floren, and C. Migliaresi8.1 Introduction, 1278.2 Main Silk Polymers, 1298.3 Fibroin Basic Processing: Regenerated Silk Fibroin, 1318.4 Materials Fabrication of Silk Proteins, 1318.5 Advanced Material Applications of Silks, 1358.6 Conclusion, 136References, 1379 Silk Protein Sericin: Promising Biopolymer for Biological and Biomedical Applications 142Sunita Nayak and Subhas C. Kundu9.1 Introduction, 1429.2 Sericin Extraction and Processing, 1469.3 Potential Applications of Sericin, 1479.4 Immunogenicity and Toxicity of Sericin, 1529.5 Conclusion, 153Acknowledgements, 154References, 15410 Fibrin 159Markus Kerbl, Philipp Heher, James Ferguson, and Heinz Redl10.1 Introduction, 15910.2 Fibrin Clotting, 16010.3 Fibrin Degradation, 16010.4 Fibrin Glue, 16310.5 Conclusion, 170Acknowledgement, 171References, 17111 Casein Proteins 176Pranav K. Singh and Harjinder Singh11.1 Introduction, 17611.2 Structures and Properties of Casein, 17811.3 Interaction of Caseins with Metal Ions, 18411.4 Conclusions, 185References, 18612 Biomaterials from Decellularized Tissues 190Ricardo Londono and Stephen F. Badylak12.1 Introduction, 19012.2 Host Response to Implanted ECM-Derived Biomaterials, 196References, 19913 Demineralized Bone Matrix: A Morphogenetic Extracellular Matrix 211A. Hari Reddi and Ryosuke Sakata13.1 Introduction, 21113.2 Demineralized Bone Matrix (DBM), 21113.3 From DBM to Bone Morphogenetic Proteins (BMPs), 21313.4 BMPs Bind to Extracellular Matrix, 21613.5 BMP Receptors, 21613.6 Future Perspectives, 218Acknowledgements, 218References, 218PART II14 Recent Developments on Chitosan Applications in Regenerative Medicine 223Ana Rita C. Duarte, Vitor M. Correlo, Joaquim M. Oliveira, and Rui L. Reis14.1 Introduction, 22314.2 Chitosan and Derivatives, 22414.3 Regenerative Medicine Applications of Chitosan, 22714.4 Processing Methodologies, 23114.5 Final Remarks, 236Acknowledgments, 237References, 23715 Starch-Based Blends in Tissue Engineering 244P.P. Carvalho, M.T. Rodrigues, R.L. Reis, and M.E. Gomes15.1 Introduction, 24415.2 Starch, 24515.3 Modification of Starch for Biomedical Applications, 24715.4 Starch-Based Blends, 24815.5 Conclusions and Future Perspectives, 254References, 25516 Agarose Hydrogel Characterization for Regenerative Medicine Applications: Focus on Engineering Cartilage 258Brendan L. Roach, Adam B. Nover, Gerard A. Ateshian, and Clark T. Hung16.1 The Foundations of Agarose, 25816.2 Structure-Function Relationships of Agarose Hydrogels, 25916.3 Agarose as a Tissue Engineering Scaffold, 26116.4 Agarose in the Clinic, 26616.5 A Scaffold to Build On, 267Acknowledgements, 268References, 26817 Bioengineering Alginate for Regenerative Medicine Applications 274Emil Ruvinov and Smadar Cohen17.1 Introduction, 27417.2 Regenerative Medicine: Definition and Strategies, 27517.3 Alginate Biomaterial, 27717.4 Alginate Implant: First in Man Trial for Prevention of Heart Failure, 28117.5 Alginate Hydrogel as a Vehicle for Stem Cell Delivery and Retention, 28417.6 Engineering Alginate-Based Cell Microenvironments, 28717.7 Alginate Hydrogel Carrier for Growth Factor Delivery, 28917.8 Engineering Alginate for Affinity Binding and Presentation of Heparin-Binding Growth Factors, 292References, 30018 Dextran 307Rong Wang, Pieter J. Dijkstra, and Marcel Karperien18.1 Introduction, 30718.2 Structure and Properties, 30818.3 Dextran Derivatives, 31018.4 Dextran Copolymers, 31418.5 Degradation, 31618.6 Outlook, 316References, 31619 Gellan Gum-based Hydrogels for Tissue Engineering Applications 320Joana Silva-Correia, Joaquim Miguel Oliveira, and Rui Lu´ýs Reis19.1 Introduction, 32019.2 Gellan Gum and its Derivatives, 32219.3 Tissue Engineering Applications, 32519.4 Final Remarks, 331Acknowledgments, 332References, 332PART III20 Biomedical Applications of Polyhydroxyalkanoates 339L.R. Lizarraga-Valderrama, B. Panchal, C. Thomas, A.R. Boccaccini, and I. Roy20.1 Introduction, 33920.2 Skin Tissue Engineering, 34120.3 Nerve Tissue Engineering, 34420.4 Cardiac Tissue Engineering, 34820.5 Dental Tissue Engineering, 35620.6 Bone Tissue Engineering, 35820.7 Cartilage Tissue Engineering, 36620.8 Osteochondral Tissue Engineering, 36820.9 Drug Delivery, 37020.10 Conclusions and the Future Potential of PHAs in Biomedical Applications, 373References, 37321 Bacterial Cellulose 384Hernane S. Barud, Junkal Gutierrez, Wilton R. Lustri, Maristela F.S. Peres, Sidney J.L. Ribeiro, Sybele Saska, and Agniezska Tercjak21.1 Introduction, 38421.2 BC Dressings, 38521.3 Bacterial Cellulose for Tissue Engineering and Regenerative Medicine, 38821.4 Concluding Remarks, 393Acknowledgments, 394References, 394PART IV22 Molecularly Imprinted Cryogels for Protein Purification 403Müge Andac¸, Igor Yu Galaev, and Adil Denizli22.1 Introduction, 40322.2 Molecularly Imprinted Cryogels for Protein Purification, 40522.3 Some Selected Applications of Molecularly Imprinted Cryogels (MIC) for Macromolecules, 41422.4 Concluding Remarks and Future Perspectives, 421References, 42323 Immunogenic Reaction of Implanted Biomaterials from Nature 429Martijn Van Griensven and Elizabeth Rosado Balmayor23.1 Introduction, 42923.2 Implantation Leads to Tissue Injury, 43023.3 Inflammatory Responses, 43123.4 Foreign Body Reaction, 43323.5 Immunogenic Reactions Towards Natural Biomaterials, 43523.6 Final Remarks, 438References, 43824 Chemical Modification of Biomaterials from Nature 444J.C. Rodr´ýguez Cabello, I. Gonz´alez De Torre, M. Santos, A.M. Testera, and M. Alonso24.1 Protein Modification, 44424.2 Lipid Modifications, 45124.3 Polysaccharide Chemical Modifications, 457References, 466PART V25 Processing of Biomedical Devices for Tissue Engineering and Regenerative Medicine Applications 477Vitor M. Correlo, Albino Martins, Nuno M. Neves, and Rui L. Reis25.1 Introduction, 47725.2 Processing Techniques of Naturally Derived Biomaterial, 47825.3 Processing Techniques of Natural-Based Polymeric Blends, 483References, 48726 General Characterization of Physical Properties of Natural-Based Biomaterials 494Manuel Alatorre-Meda and Joäo F. Mano26.1 Introduction, 49426.2 Bulk Properties, 49526.3 Surface Properties, 50726.4 Concluding Remarks, 512Acknowledgments, 512References, 51227 General Characterization of Chemical Properties of Natural-Based Biomaterials 517Manuel Alatorre-Meda and Joäo F. Mano27.1 Introduction, 51727.2 Molecular Weight and Elemental Composition, 51827.3 Physiological Degradation, 52427.4 Concluding Remarks, 527Acknowledgments, 529References, 52928 In Vitro Biological Testing in the Development of New Devices 532Marta L. Alves Da Silva, Albino Martins, Ana Costa-Pinto, Rui L. Reis, and Nuno M. Neves28.1 Introduction, 53228.2 Cytotoxicity Assays, 53328.3 Evaluation of Cell Morphology and Distribution, 53328.4 Cell Viability Assays, 53528.5 Cell Proliferation Assays, 53628.6 Biochemical Analysis, 53728.7 Genotypic Expression Analysis, 54128.8 Histological Assessment, 54228.9 In Vitro Engineered Tissues, 54328.10 Concluding Remarks, 548References, 54829 Advanced In-Vitro Cell Culture Methods Using Natural Biomaterials 551Marta L. Alves Da Silva, Rui L. Reis, and Nuno M. Neves29.1 Introduction, 55129.2 Bioreactors, 55229.3 Hypoxia, 55329.4 Co-Cultures, 55529.5 Transfection, 55529.6 Nanoparticles and Related Systems, 55829.7 Concluding Remarks, 559References, 55930 Testing Natural Biomaterials in Animal Models 562Ana Costa-Pinto, Tírcia C. Santos, Nuno M. Neves, and Rui L. Reis30.1 Laboratory Animals as Tools in Biomaterials Testing, 56230.2 Inflammation and Host Reaction, 56430.3 Animal Models for Tissue Engineering, 56830.4 Final Remarks, 574References, 575PART VI31 Delivery Systems Made of Natural-Origin Polymers for Tissue Engineering and Regenerative Medicine Applications 583Albino Martins, Helena Ferreira, Rui L. Reis, and Nuno M. Neves31.1 Introduction, 58331.2 Advantages and Disadvantages of Natural Polymers-Based Delivery Systems, 58531.3 Fundamentals of Drug Delivery, 58631.4 In Vitro and In Vivo Applications of Natural-Based Delivery Systems, 59131.5 Concluding Remarks, 601References, 60232 Translational Research into New Clinical Applications 612M. David Harmon and Sangamesh G. Kumbar32.1 Introduction, 61232.2 Cardiovascular System Applications, 61332.3 Integumentary System Applications, 61632.4 Musculoskeletal System Applications, 61832.5 Nervous System Applications, 61932.6 Respiratory System Applications, 62132.7 Gastrointestinal System Applications, 62232.8 From Idea to Product, 624Acknowledgements, 626References, 62633 Challenges and Opportunities of Natural Biomaterials for Advanced Devices and Therapies 629R.L. Reis and N.M. Neves33.1 Introduction, 62933.2 Challenges of Natural Biomaterials, 63033.3 Opportunities of Natural Biomaterials, 63133.4 Final Remarks, 631References, 63234 Adhesives Inspired by Marine Mussels 634Courtney L. Jenkins, Heather J. Meredith, and Jonathan J. Wilker34.1 Introduction, 63434.2 Requirements for a Bioadhesive, 63534.3 Marine Mussels, 63634.4 Bulk Adhesion Testing, 63834.5 Extracted Mussel Adhesive Proteins, 64034.6 Mimics of Mussel Adhesive, 64134.7 Conclusions, 645Acknowledgement, 645References, 64535 Final Comments and Remarks 649R.L. Reis and N.M. NevesINDEX 651

Nuno M. Neves is Professor at the Department of Polymer Engineering of the University of Minho, Portugal, where he is Vice-Director of the 3B’s Research Group – Biomaterials, Biodegradables and Biomimetics. Nuno M. Neves received his PhD degree in Polymer Science and Engineering from the University of Minho in collaboration with the University of Twente, The Netherlands. His main area of research is the development of biomaterials from natural origin polymers. His research group focuses mainly on tissue engineering and regenerative medicine strategies using stem cells and advanced drug delivery scaffolds and medical devices.Rui L. Reis is Professor of Tissue Engineering, Regenerative Medicine, Biomaterials and Stem Cells at the Department of Polymer Engineering of the University of Minho, Portugal. He is the Vice-Rector for Research of the University of Minho, Director of the 3B’s Research Group and the Director of the Portuguese Government Associate Laboratory ICVS/3B’s. Rui L. Reis received his PhD degree in Polymer Engineering from the University of Minho in collaboration with Brunel University in London, UK. His main area of research is the development of biomaterials from natural origin polymers that his group proposes for a range of biomedical applications.

• CONTRIBUTORS xixPREFACE xxixPART I 1 Collagen-Based Porous Scaffolds for Tissue Engineering 3Guoping Chen and Naoki Kawazoe1.1 Introduction, 31.2 Collagen Sponges, 41.3 Collagen Sponges with Micropatterned Pore Structures, 71.4 Collagen Sponges with Controlled Bulk Structures, 101.5 Hybrid Scaffolds, 121.6 Conclusions, 13References, 142 Marine Collagen Isolation and Processing Envisaging Biomedical Applications 16Joana Moreira-Silva, Gabriela S. Diogo, Ana L. P. Marques, Tiago H. Silva, and Rui L. Reis2.1 Introduction, 162.2 Extraction of Collagen from Marine Sources, 182.3 Collagen Characterization, 222.4 Marine Collagen Wide Applications, 252.5 Final Remarks, 32Acknowledgements, 34References, 343 Gelatin-Based Biomaterials for Tissue Engineering and Stem Cell Bioengineering 37Mehdi Nikkhah, Mohsen Akbari, Arghya Paul, Adnan Memic, Alireza Dolatshahi-Pirouz, and Ali Khademhosseini3.1 Introduction, 373.2 Crosslinking of Gelatin, 383.3 Physical Properties of Gelatin, 393.4 Application of Gelatin-Based Biomaterials in Tissue Engineering, 403.5 Gelatin for Stem Cell Therapy, 453.6 Application of Gelatin in Delivery Systems, 493.7 Conclusion and Perspectives, 50Acknowledgements, 50Abbreviations, 50References, 514 Hyaluronic Acid-Based Hydrogels on a Micro and Macro Scale 63A. Borzacchiello, L. Russo, and L. Ambrosio4.1 Classification and Structure of Hydrogels, 634.2 Hyaluronic Acid, 654.3 Hydrogel Mechanical Properties, 664.4 HA-Based Hydrogel for Biomedical Applications, 70References, 755 Chondroitin Sulfate as a Bioactive Macromolecule for Advanced Biological Applications and Therapies 79Nicola Volpi5.1 CS Structure, 815.2 Biological Roles of CS, 815.3 Osteoarthritis Treatment, 845.4 Cardio-Cerebrovascular Disease, 845.5 Tissue Regeneration and Engineering, 855.6 Chondroitin Sulfate-Polymer Conjugates, 865.7 Conclusions and Future Perspectives, 87References, 886 Keratin 93Mark Van Dyke6.1 Introduction, 936.2 Preparation of Keratoses, 986.3 Preparation of Kerateines, 1006.4 Oxidative Sulfitolysis, 1016.5 Summary, 102References, 1027 Elastin-Like Polypeptides: Bio-Inspired Smart Polymers for Protein Purification, Drug Delivery and Tissue Engineering 106Jayanta Bhattacharyya, Joseph J. Bellucci, and Ashutosh Chilkoti7.1 Introduction, 1067.2 Recombinant Protein Production Using ELPs as Purification Tags, 1077.3 Delivery of Therapeutics with ELPs, 1137.4 Tissue Engineering with ELPs, 1197.5 Conclusions, 122Acknowledgements, 122Abbreviations, 122References, 1238 Silk: A Unique Family of Biopolymers 127A. Motta, M. Floren, and C. Migliaresi8.1 Introduction, 1278.2 Main Silk Polymers, 1298.3 Fibroin Basic Processing: Regenerated Silk Fibroin, 1318.4 Materials Fabrication of Silk Proteins, 1318.5 Advanced Material Applications of Silks, 1358.6 Conclusion, 136References, 1379 Silk Protein Sericin: Promising Biopolymer for Biological and Biomedical Applications 142Sunita Nayak and Subhas C. Kundu9.1 Introduction, 1429.2 Sericin Extraction and Processing, 1469.3 Potential Applications of Sericin, 1479.4 Immunogenicity and Toxicity of Sericin, 1529.5 Conclusion, 153Acknowledgements, 154References, 15410 Fibrin 159Markus Kerbl, Philipp Heher, James Ferguson, and Heinz Redl10.1 Introduction, 15910.2 Fibrin Clotting, 16010.3 Fibrin Degradation, 16010.4 Fibrin Glue, 16310.5 Conclusion, 170Acknowledgement, 171References, 17111 Casein Proteins 176Pranav K. Singh and Harjinder Singh11.1 Introduction, 17611.2 Structures and Properties of Casein, 17811.3 Interaction of Caseins with Metal Ions, 18411.4 Conclusions, 185References, 18612 Biomaterials from Decellularized Tissues 190Ricardo Londono and Stephen F. Badylak12.1 Introduction, 19012.2 Host Response to Implanted ECM-Derived Biomaterials, 196References, 19913 Demineralized Bone Matrix: A Morphogenetic Extracellular Matrix 211A. Hari Reddi and Ryosuke Sakata13.1 Introduction, 21113.2 Demineralized Bone Matrix (DBM), 21113.3 From DBM to Bone Morphogenetic Proteins (BMPs), 21313.4 BMPs Bind to Extracellular Matrix, 21613.5 BMP Receptors, 21613.6 Future Perspectives, 218Acknowledgements, 218References, 218PART II14 Recent Developments on Chitosan Applications in Regenerative Medicine 223Ana Rita C. Duarte, Vitor M. Correlo, Joaquim M. Oliveira, and Rui L. Reis14.1 Introduction, 22314.2 Chitosan and Derivatives, 22414.3 Regenerative Medicine Applications of Chitosan, 22714.4 Processing Methodologies, 23114.5 Final Remarks, 236Acknowledgments, 237References, 23715 Starch-Based Blends in Tissue Engineering 244P.P. Carvalho, M.T. Rodrigues, R.L. Reis, and M.E. Gomes15.1 Introduction, 24415.2 Starch, 24515.3 Modification of Starch for Biomedical Applications, 24715.4 Starch-Based Blends, 24815.5 Conclusions and Future Perspectives, 254References, 25516 Agarose Hydrogel Characterization for Regenerative Medicine Applications: Focus on Engineering Cartilage 258Brendan L. Roach, Adam B. Nover, Gerard A. Ateshian, and Clark T. Hung16.1 The Foundations of Agarose, 25816.2 Structure-Function Relationships of Agarose Hydrogels, 25916.3 Agarose as a Tissue Engineering Scaffold, 26116.4 Agarose in the Clinic, 26616.5 A Scaffold to Build On, 267Acknowledgements, 268References, 26817 Bioengineering Alginate for Regenerative Medicine Applications 274Emil Ruvinov and Smadar Cohen17.1 Introduction, 27417.2 Regenerative Medicine: Definition and Strategies, 27517.3 Alginate Biomaterial, 27717.4 Alginate Implant: First in Man Trial for Prevention of Heart Failure, 28117.5 Alginate Hydrogel as a Vehicle for Stem Cell Delivery and Retention, 28417.6 Engineering Alginate-Based Cell Microenvironments, 28717.7 Alginate Hydrogel Carrier for Growth Factor Delivery, 28917.8 Engineering Alginate for Affinity Binding and Presentation of Heparin-Binding Growth Factors, 292References, 30018 Dextran 307Rong Wang, Pieter J. Dijkstra, and Marcel Karperien18.1 Introduction, 30718.2 Structure and Properties, 30818.3 Dextran Derivatives, 31018.4 Dextran Copolymers, 31418.5 Degradation, 31618.6 Outlook, 316References, 31619 Gellan Gum-based Hydrogels for Tissue Engineering Applications 320Joana Silva-Correia, Joaquim Miguel Oliveira, and Rui Lu´ýs Reis19.1 Introduction, 32019.2 Gellan Gum and its Derivatives, 32219.3 Tissue Engineering Applications, 32519.4 Final Remarks, 331Acknowledgments, 332References, 332PART III20 Biomedical Applications of Polyhydroxyalkanoates 339L.R. Lizarraga-Valderrama, B. Panchal, C. Thomas, A.R. Boccaccini, and I. Roy20.1 Introduction, 33920.2 Skin Tissue Engineering, 34120.3 Nerve Tissue Engineering, 34420.4 Cardiac Tissue Engineering, 34820.5 Dental Tissue Engineering, 35620.6 Bone Tissue Engineering, 35820.7 Cartilage Tissue Engineering, 36620.8 Osteochondral Tissue Engineering, 36820.9 Drug Delivery, 37020.10 Conclusions and the Future Potential of PHAs in Biomedical Applications, 373References, 37321 Bacterial Cellulose 384Hernane S. Barud, Junkal Gutierrez, Wilton R. Lustri, Maristela F.S. Peres, Sidney J.L. Ribeiro, Sybele Saska, and Agniezska Tercjak21.1 Introduction, 38421.2 BC Dressings, 38521.3 Bacterial Cellulose for Tissue Engineering and Regenerative Medicine, 38821.4 Concluding Remarks, 393Acknowledgments, 394References, 394PART IV22 Molecularly Imprinted Cryogels for Protein Purification 403Müge Andac¸, Igor Yu Galaev, and Adil Denizli22.1 Introduction, 40322.2 Molecularly Imprinted Cryogels for Protein Purification, 40522.3 Some Selected Applications of Molecularly Imprinted Cryogels (MIC) for Macromolecules, 41422.4 Concluding Remarks and Future Perspectives, 421References, 42323 Immunogenic Reaction of Implanted Biomaterials from Nature 429Martijn Van Griensven and Elizabeth Rosado Balmayor23.1 Introduction, 42923.2 Implantation Leads to Tissue Injury, 43023.3 Inflammatory Responses, 43123.4 Foreign Body Reaction, 43323.5 Immunogenic Reactions Towards Natural Biomaterials, 43523.6 Final Remarks, 438References, 43824 Chemical Modification of Biomaterials from Nature 444J.C. Rodr´ýguez Cabello, I. Gonz´alez De Torre, M. Santos, A.M. Testera, and M. Alonso24.1 Protein Modification, 44424.2 Lipid Modifications, 45124.3 Polysaccharide Chemical Modifications, 457References, 466PART V25 Processing of Biomedical Devices for Tissue Engineering and Regenerative Medicine Applications 477Vitor M. Correlo, Albino Martins, Nuno M. Neves, and Rui L. Reis25.1 Introduction, 47725.2 Processing Techniques of Naturally Derived Biomaterial, 47825.3 Processing Techniques of Natural-Based Polymeric Blends, 483References, 48726 General Characterization of Physical Properties of Natural-Based Biomaterials 494Manuel Alatorre-Meda and Joäo F. Mano26.1 Introduction, 49426.2 Bulk Properties, 49526.3 Surface Properties, 50726.4 Concluding Remarks, 512Acknowledgments, 512References, 51227 General Characterization of Chemical Properties of Natural-Based Biomaterials 517Manuel Alatorre-Meda and Joäo F. Mano27.1 Introduction, 51727.2 Molecular Weight and Elemental Composition, 51827.3 Physiological Degradation, 52427.4 Concluding Remarks, 527Acknowledgments, 529References, 52928 In Vitro Biological Testing in the Development of New Devices 532Marta L. Alves Da Silva, Albino Martins, Ana Costa-Pinto, Rui L. Reis, and Nuno M. Neves28.1 Introduction, 53228.2 Cytotoxicity Assays, 53328.3 Evaluation of Cell Morphology and Distribution, 53328.4 Cell Viability Assays, 53528.5 Cell Proliferation Assays, 53628.6 Biochemical Analysis, 53728.7 Genotypic Expression Analysis, 54128.8 Histological Assessment, 54228.9 In Vitro Engineered Tissues, 54328.10 Concluding Remarks, 548References, 54829 Advanced In-Vitro Cell Culture Methods Using Natural Biomaterials 551Marta L. Alves Da Silva, Rui L. Reis, and Nuno M. Neves29.1 Introduction, 55129.2 Bioreactors, 55229.3 Hypoxia, 55329.4 Co-Cultures, 55529.5 Transfection, 55529.6 Nanoparticles and Related Systems, 55829.7 Concluding Remarks, 559References, 55930 Testing Natural Biomaterials in Animal Models 562Ana Costa-Pinto, Tírcia C. Santos, Nuno M. Neves, and Rui L. Reis30.1 Laboratory Animals as Tools in Biomaterials Testing, 56230.2 Inflammation and Host Reaction, 56430.3 Animal Models for Tissue Engineering, 56830.4 Final Remarks, 574References, 575PART VI31 Delivery Systems Made of Natural-Origin Polymers for Tissue Engineering and Regenerative Medicine Applications 583Albino Martins, Helena Ferreira, Rui L. Reis, and Nuno M. Neves31.1 Introduction, 58331.2 Advantages and Disadvantages of Natural Polymers-Based Delivery Systems, 58531.3 Fundamentals of Drug Delivery, 58631.4 In Vitro and In Vivo Applications of Natural-Based Delivery Systems, 59131.5 Concluding Remarks, 601References, 60232 Translational Research into New Clinical Applications 612M. David Harmon and Sangamesh G. Kumbar32.1 Introduction, 61232.2 Cardiovascular System Applications, 61332.3 Integumentary System Applications, 61632.4 Musculoskeletal System Applications, 61832.5 Nervous System Applications, 61932.6 Respiratory System Applications, 62132.7 Gastrointestinal System Applications, 62232.8 From Idea to Product, 624Acknowledgements, 626References, 62633 Challenges and Opportunities of Natural Biomaterials for Advanced Devices and Therapies 629R.L. Reis and N.M. Neves33.1 Introduction, 62933.2 Challenges of Natural Biomaterials, 63033.3 Opportunities of Natural Biomaterials, 63133.4 Final Remarks, 631References, 63234 Adhesives Inspired by Marine Mussels 634Courtney L. Jenkins, Heather J. Meredith, and Jonathan J. Wilker34.1 Introduction, 63434.2 Requirements for a Bioadhesive, 63534.3 Marine Mussels, 63634.4 Bulk Adhesion Testing, 63834.5 Extracted Mussel Adhesive Proteins, 64034.6 Mimics of Mussel Adhesive, 64134.7 Conclusions, 645Acknowledgement, 645References, 64535 Final Comments and Remarks 649R.L. Reis and N.M. NevesINDEX 651