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Bio-inorganic Hybrid Nanomaterials

Strategies, Syntheses, Characterization and Applications

Edited byEduardo Ruiz-Hitzky, Katsuhiko Ariga and Yuri Lvov

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Edited byEduardo Ruiz-Hitzky,Katsuhiko Ariga and Yuri Lvov

Further Reading

Vollath, D.

NanomaterialsAn Introdution to Synthesis, Properties and Applications

2008

ISBN: 978-3-527-31531-4

Willner, I., Katz, E. (Eds.)

BionanomaterialsSynthesis and Applications for Sensors, Electronics and Medicine

2008

ISBN: 978-3-527-31454-6

Ajayan, P. M., Schadler, L. S., Braun, P. V., Keblinski, P.

Nanocomposite Science and TechnologySecond Completely Revised Edition

2008

ISBN: 978-3-527-31248-1

Rao, C. N. R., Müller, A., Cheetham, A. K. (Eds.)

Nanomaterials ChemistryRecent Developments and New Directions

2007

ISBN: 978-3-527-31664-9

Kickelbick, G. (Ed.)

Hybrid MaterialsSynthesis, Characterization, and Applications

2007

ISBN: 978-3-527-31299-3

Kumar, Challa S. S. R. (Ed.)

Nanotechnologies for the Life Sciences10 Volume Set

2007

ISBN: 978-3-527-31301-3


Strategies, Syntheses, Characterization and Applications

Edited byEduardo Ruiz-Hitzky, Katsuhiko Ariga and Yuri Lvov

The Editors

Prof. Dr. Eduardo Ruiz-HitzkyInstituto de Ciencia de Materialesde MadridConsejo Superior de Investigaciones CientificasCantoblanco28049 MadridSpain

Dr. Katsuhiko ArigaNational Institute for Material Science1-1 Namiki305-0044 Tsukuba, IbarakiJapan

Prof. Yuri LvovInstitute for MicromanufacturingLouisiana Technical University911 Hergot AvenueRuston, LA 71272USA

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Library of Congress Card No.: applied for

British Library Cataloguing-in-Publication DataA catalogue record for this book is available from theBritish Library.

Bibliographic information published bythe Deutsche NationalbibliothekDie Deutsche Nationalbibliothek lists thispublication in the Deutsche Nationalbibliografie;detailed bibliographic data are available on theInternet at .

# 2008 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

All rights reserved (including those of translation intoother languages). No part of this book may bereproduced in any form – by photoprinting, micro-film, or any other means – nor transmitted or trans-lated into a machine language without writtenpermission from the publishers. Registered names,trademarks, etc. used in this book, even when notspecifically marked as such, are not to be consideredunprotected by law.

Printed in the Federal Republic of GermanyPrinted on acid-free paper

Cover Design WMX-Design, HeidelbergTypesetting Thomson Digital, IndiaPrinting Strauss GmbH, MörlenbachBinding Litges & Dopf GmbH, Heppenheim

ISBN: 978-3-527-31718-9

Contents

Preface XIII

Contributors XV

1 An Introduction to Bio-nanohybrid Materials 1Eduardo Ruiz-Hitzky, Margarita Darder, Pilar Aranda

1.1 Introduction: The Assembly of Biological Speciesto Inorganic Solids 1

1.2 Bio-nanohybrids Based on Silica Particles and SiloxaneNetworks 4

1.3 Calcium Phosphates and Carbonates in Bioinspiredand Biomimetic Materials 9

1.4 Clay Minerals and Organoclay Bio-nanocomposites 131.5 Bio-Nanohybrids Based on Metal and Metal Oxide

Nanoparticles 201.6 Carbon-based Bio-nanohybrids 221.7 Bio-nanohybrids Based on Layered Transition Metal Solids 281.8 Trends and Perspectives 31

References 32

2 Biomimetic Nanohybrids Based on Organosiloxane Units 41Kazuko Fujii, Jonathan P. Hill, Katsuhiko Ariga

2.1 Introduction 412.2 Monolayer on Solid Support 452.3 Layered Alkylsiloxane 532.4 Organic–Inorganic Hybrid Vesicle Cerasome 592.5 Mesoporous Silica Prepared by the Lizard Template Method 652.6 Future Perspectives 69

References 71

V

3 Entrapment of Biopolymers into Sol–Gel-derived SilicaNanonocomposites 75Yury A. Shchipunov

3.1 Introduction 753.2 Sol–Gel Processes 773.2.1 Chemistry 773.2.1.1 Hydrolysis 773.2.1.2 Condensation 783.2.1.3 Sol–Gel Transition 783.2.2 Silica Precursors 793.2.2.1 Orthosilicic Acid 803.2.2.2 Sodium Metasilicate 803.2.2.3 Alkoxides 803.2.3 Two-Stage Approach to Biopolymer Entrapment 823.3 Biocompatible Approaches 843.3.1 Modified Sol–Gel Processing 843.3.1.1 Method of Gill and Ballesteros 843.3.1.2 Low-Molecular and Polymeric Organic Additives 853.3.2 Organically-modified Precursors 863.3.3 Biocompatible Precursors by Brennan et al. 873.4 One-Stage Approach Based on a Silica Precursor

with Ethylene Glycol Residues 883.4.1 Precursor 883.4.2 Role of Biopolymers in Sol–Gel Processing 893.4.3 Advantages of One-Stage Processes 963.4.4 Hybrid Biopolymer–Silica Nanocomposite Materials 983.4.5 Enzyme Immobilization 993.5 Perspectives 102

References 103

4 Immobilization of Biomolecules on Mesoporous StructuredMaterials 113Ajayan Vinu, Narasimhan Gokulakrishnan, Toshiyuki Mori,Katsuhiko Ariga

4.1 Introduction 1134.2 Immobilization of Protein on Mesoporous Silica 1164.3 Immobilization of Protein on Mesoporous Carbon

and Related Materials 1244.4 Immobilization of Other Biopolymers on

Mesoporous Materials 1334.5 Immobilization of Small Biomolecules on Mesoporous Materials 1374.6 Advanced Functions of Nanohybrids of Biomolecules

and Mesoporous Materials 1414.7 Future Perspectives 149

References 150

VI Contents

5 Bio-controlled Growth of Oxides and Metallic Nanoparticles 159Thibaud Coradin, Roberta Brayner, Fernand Fiévet, Jacques Livage

5.1 Introduction 1595.2 Biomimetic Approaches 1605.3 In vitro Synthesis of Hybrid Nanomaterials 1655.3.1 Polysaccharides 1655.3.1.1 Alginates 1655.3.1.2 Carrageenans 1695.3.1.3 Chitosan 1715.3.2 Proteins 1745.3.2.1 Gelatin 1745.3.2.2 Collagen 1755.3.2.3 Protein Cages and Viral Capsids 1775.3.3 Lipids 1805.3.4 DNA Scaffolds 1815.4 Perspectives: Towards a Green Nanochemistry 183

References 184

6 Biomineralization of Hydrogels Based on Bioinspired Assembliesfor Injectable Biomaterials 193Junji Watanabe, Mitsuru Akashi

6.1 Introduction 1936.1.1 Biominerals as Nanomaterials 1936.1.2 Nanomaterials for Biofunctions 1966.2 Fundamental Concept of Bioinspired Approach 1976.2.1 Bioinspired Approach to Materials 1976.2.2 Concrete Examples of the Bioinspired Approach 1986.3 Alternate Soaking Process for Biomineralization and their

Bio-functions 1996.3.1 Nanoassembly by Polyelectrolytes 1996.3.2 Alternate Soaking Process for Biomineralization 2006.3.3 Biomineralization of Hydrogels for Bio-functions 2016.4 Electrophoresis Process for Biomineralization 2036.4.1 Innovative Methodology of Electrophoresis Process

for Biomineralization 2036.4.2 Application for Injectable Materials 2046.5 Conclusions 206

References 206

7 Bioinspired Porous Hybrid Materials via Layer-by-LayerAssembly 209Yajun Wang , Frank Caruso

7.1 Introduction 2097.2 Porous Materials 2097.2.1 Microporous Materials 210

Contents VII

7.2.2 Mesoporous Material 2107.2.3 Macroporous Materials 2117.3 LbL Assembly 2137.4 LbL Assembly on MS Substrates 2147.4.1 Encapsulation of Biomolecules in MS Particles 2147.4.2 MS Spheres as Templates for the Preparation of Hollow Capsules 2187.4.3 Preparation of Protein Particles via MS Sphere Templating 2207.4.4 Template Synthesis of Nanoporous Polymeric Spheres 2217.5 LbL Assembly on Macroporous Substrates 2257.5.1 LbL Assembly on Tubular Substrates 2267.5.2 LbL Assembly on 3DOM Materials 2297.5.3 LbL Assembly on Naturally Occurring Porous Substrates 2317.6 Summary and Outlook 232

References 233

8 Bio-inorganic Nanohybrids Based on Organoclay Self-assembly 239Avinash J. Patil, Stephen Mann

8.1 Introduction 2398.2 Synthesis and Characterization of Organically Functionalized 2:1

Magnesium Phyllosilicates 2408.3 MagnesiumOrganophyllosilicateswithHigher-orderOrganization 2438.4 Intercalation of Biomolecules within Organically Modified

Magnesium Phyllosilicates 2468.4.1 Protein–Organoclay Lamellar Nanocomposites 2478.4.2 DNA–Organoclay Lamellar Nanostructures 2528.4.3 Drug–Organoclay Layered Nanocomposites 2538.5 Hybrid Nanostructures Based on Organoclay Wrapping of Single

Biomolecules 2548.5.1 Organoclay-wrapped Proteins and Enzymes 2548.5.2 Organoclay-wrapped DNA 2588.6 Functional Mesolamellar Bio-inorganic Nanocomposite Films 2608.7 Summary 262

References 262

9 Biodegradable Polymer-based Nanocomposites:Nanostructure Control and Nanocomposite Foamingwith the Aim of Producing Nano-cellular Plastics 271Masami Okamoto

9.1 Introduction 2719.2 Nano-structure Development 2729.2.1 Melt Intercalation 2729.2.2 Interlayer Structure of OMLFs and Intercalation 2739.2.2.1 Nano-fillers 2739.2.2.2 Molecular Dimensions and Interlayer Structure 2749.2.2.3 Correlation of Intercalant Structure and Interlayer Opening 277

VIII Contents

9.2.2.4 Nanocomposite Structure 2789.3 Control of Nanostructure Properties 2829.3.1 Flocculation Control and Modulus Enhancement 2829.3.2 Linear Viscoelastic Properties 2849.3.3 Elongational Flow and Strain-induced Hardening 2889.4 Physicochemical Phenomena 2909.4.1 Biodegradability 2909.4.2 Photodegradation 2959.5 Foam Processing using Supercritical CO2 2969.5.1 PLA-based Nanocomposite 2969.5.2 Temperature Dependence of Cellular Structure 2989.5.3 CO2 Pressure Dependence 3019.5.4 TEM Observation 3059.5.5 Mechanical Properties of Nanocomposite Foams 3079.6 Porous Ceramic Materials via Nanocomposites 3079.7 Future Prospects 309

References 310

10 Biomimetic and Bioinspired Hybrid Membrane Nanomaterials 313Mihail Barboiu

10.1 Introduction 31310.2 Molecular Recognition-based Hybrid Membranes 31410.2.1 Multiple Molecular Recognition Principles 31410.3 Self-organized Hybrid Membrane Materials 31810.3.1 Ionic-conduction Pathways in Hybrid Membrane Materials 31810.3.1.1 Ionic-conduction Pathways in Macrocyclic Hybrid Materials 31910.3.1.2 Ionic-conduction Pathways in Peptido-mimetic Hybrid Materials 31910.3.2 Self-organization in Hybrid Supramolecular Polymers 32410.3.2.1 Self-organization by Base Pairing in Hybrid

Supramolecular Polymers 32510.3.2.2 Self-Organization of the Guanine Quadruplex in Hybrid

Supramolecular Polymers 32810.4 Dynamic Site Complexant Membranes 33010.5 Conclusions 333

References 334

11 Design of Bioactive Nano-hybrids for Bone TissueRegeneration 339Masanobu Kamitakahara, Toshiki Miyazaki, Chikara Ohtsuki

11.1 Introduction 33911.2 Composite of Bioactive Ceramic Particles and Polymers 34011.3 Bone-bonding Mechanism of Bioactive Materials 34111.3.1 Interface between Bone and Bioactive Material 34111.3.2 Simulated Body Fluid 34211.3.3 Hydroxyapatite Formation on Bioactive Materials 343

Contents IX

11.4 Sol–Gel-derived Bioactive Nano-hybrids 34511.4.1 Silicate-based Nano-hybrids 34511.4.2 Nano-hybrids Starting from Methacryloxy Compounds 34711.4.3 Nano-hybrids Based on Other than Silicate 34911.4.4 Nano-hybrids Combined with Calcium Phosphates 35311.5 Nano-hybrid Consisting of Bone-like Hydroxyapatite and

Polymer 35411.5.1 Biomimetic Process 35411.5.2 Hydroxyapatite Deposition on Polymers Modified with

Silanol Groups 35611.5.3 Hydroxyapatite Deposition on Natural Polymers 35711.5.4 Hydroxyapatite Deposition on Synthetic Polymers 35811.5.5 Control of the Structure of Hydroxyapatite 35911.6 Nano-hybrid Consisting of Hydroxyapatite and Protein 36011.7 Conclusion 361

References 361

12 Nanostructured Hybrid Materials for Bone ImplantsFabrication 367María Vallet-Regí, Daniel Arcos

12.1 Introduction 36712.2 Bone: A Biological Hybrid Nanostructured Material 36912.3 Biomimetic Materials for Bone Repair. The Hybrid

Approach 37212.3.1 The Hybrid Approach 37412.4 Synthesis and Properties of Organic–Inorganic Hybrid

Materials for Bone and Dental Applications 37512.4.1 Class I Hybrid Materials 37512.4.1.1 BG–Poly(vinyl Alcohol) 37512.4.1.2 Silica Particles–pHEMA 37812.4.2 Class II Hybrid Materials 37812.4.2.1 PMMA–SiO2 Ormosils 38012.4.2.2 PEG–SiO2 Ormosils 38012.4.2.3 PDMS–CaO–SiO2–TiO2 Ormosils 38012.4.2.4 PTMO–CaO–SiO2–TiO2 Hybrid Materials 38312.4.2.5 MPS–HEMA Ormosils 38312.4.2.6 Gelatine–SiO2 Systems 38412.4.2.7 Poly(e-Caprolactone)–Silica Ormosils 38512.4.2.8 Bioactive Star Gels 38712.4.2.9 The Synthesis of Bioactive Star Gels 38812.4.2.10 How to Characterize Bioactive Star Gels? 38912.4.2.11 The Bioactivity of the Star Gels 38912.4.2.12 The Mechanical Properties of Bioactive Star Gels 39112.5 Conclusion 392

References 393

X Contents

13 Bio-inorganic Conjugates for Drug and Gene Delivery 401Jin-Ho Choy, Jae-Min Oh, Soo-Jin Choi

13.1 Introduction 40113.2 Synthesis of Bio-inorganic Conjugates 40313.3 Bio-inorganic Conjugate for Efficient Gene Delivery 40713.3.1 Cellular Uptake Kinetics of LDH–FITC Into Cells 40713.3.2 Effect of As-myc–LDHHybrid on the Suppression of Cancer Cells 40813.4 Bio-inorganic Conjugate for Efficient Drug Delivery 40913.4.1 Cellular Uptake of MTX–LDH Hybrid 40913.4.2 Effect of MTX–LDH on Cell Proliferation and Viability 40913.4.3 Effect of MTX–LDH Hybrid on the Cell Cycle 41013.4.4 Potential of Bio-inorganic Conjugates for Gene and Drug Delivery 41113.5 Cellular Uptake Mechanism of LDH 41213.5.1 Endocytosis of LDH 41213.5.2 Endocytic Pathway of LDH 41313.6 Conclusion 415

References 415

14 Halloysite Nanotubules, a Novel Substrate for the ControlledDelivery of Bioactive Molecules 419Yuri M. Lvov, Ronald R. Price

14.1 Halloysite Structural Characterization 41914.2 Macromolecule Loading and Sustained Release 42214.2.1 Nanotubule Loading Procedure 42214.2.2 Drugs and Biocides 42314.2.3 Globular Proteins 42714.3 Nanoassembly on Tubules and at the Lumen Opening 42814.4 Catalysis in a Nanoconstrained Volume of the Tubule Lumen 43114.5 Multilayer Halloysite Assembly for Organized Nanofilms.

Forming Low Density Tubule Nanoporous Materials 43614.5.1 Tubule–Polycation Multilayer 43614.5.2 Assembly of Tubule/Sphere Multilayer Nanocomposites 43714.6 Applications: Current and Potential 438

References 439

15 Enzyme-based Bioinorganic Materials 443Claude Forano, Vanessa Prévot

15.1 Introduction 44315.2 Enzymes versus Inorganic Host Properties 44515.2.1 Enzyme Properties 44515.2.2 Inorganic Host Structures 44615.3 Immobilization Strategy 44615.3.1 Adsorption Process 44815.3.2 Encapsulation Processes 44915.3.3 Nanostructuring of Enzyme-based Films 450

Contents XI

15.3.4 Covalent Grafting 45215.4 Bioinorganic Nanohybrids 45415.4.1 Immobilization of Enzymes in 2-D Inorganic Hosts 45415.4.1.1 Immobilization in Clay Minerals and Related Materials 45415.4.1.2 Immobilization in Layered Double Hydroxides 45715.4.1.3 Immobilization in Layered Metal Oxides 46015.4.1.4 Immobilization in Layered Zirconium Phosphate

and Phosphonate 46115.4.2 Immobilization of Enzymes in 3-D Inorganic Hosts 46415.4.2.1 Immobilization in SiO2 46415.4.2.2 Immobilization on Alumina 46715.4.2.3 Immobilization in Zeolite 46915.4.2.4 Immobilization in Hydroxyapatite and Tricalciumphosphate 47115.5 Enzyme–Host Structure Interactions 471

References 476

Index 485

XII Contents

Preface

Materials from living matter and inorganic materials are apparently on oppositesides in the materials' world. In this context, biological materials including poly-saccharides, proteins, nucleic acids, and lipids, have soft and flexible natures, andoften show incredible functions with high specificity and efficiency, which cannot beeasily re-generated or replicated by combination of man-made materials. Therefore,direct use of such a class of biological materials sounds like a rational way toconstruct highly sophisticated functional systems.The best way to cope with the high functionality and stability of bio-related

materials for practical applications is to create hybrids consisting of materials ofbiological origin and inorganic materials. However, simply mixing these materialstogether into a messy slurry is not a wise strategy. In biologically derived materialsboth components have their own well-organized, meaningful nanostructures, andtherefore, hybridization of inorganic and biological elements in controlled struc-tures with nanometer-scale precision is the most desirable strategy. The obtainedmaterials can be called bio-inorganic nanohybrids. They are well-blessed childrenfrom both worlds, and should succeed in providing essences and advantages of bothbiological and inorganic materials. Research in bio-inorganic nanohybrids can bedefined as an interdisciplinary field resulting from the interfaces between biotech-nology, materials science, and nanotechnology. Such a new field is closely related tosignificant topics such as biomineralization processes, bioinspired materials andbiomimetic systems. The incoming development of novel bio-nanocompositesintroducing multifunctionality and taking profit from the characteristics of bothtypes of constituents is nowadays an amazing research line taking advantage fromthe synergistic assembling of biopolymers with inorganic nanosized solids.Mother of pearl and marine shells, corals, teeth, bones, and microbe inclusions

(such as sulfur or iron nanocrystals) are examples of bio-inorganic nanocomposites.Inmany cases, these composites have biopolymers and inorganic parts organized onthe nanoscale, such as the regular alternation of proteins and calcium carbonatenanolayers in nacre. When struck, the layers glide over one another absorbing theshock. If cracks develop, plates simply grow back together. Such natural nanocom-posite materials often combine unique mechanical properties based on the nano-scale organization of hard and soft materials and have the ability for regeneration

XIII

and self-reproduction. However, these nanocomposites have a fatal drawback intheir application. In most cases, their functions are optimized only at ambientconditions and their structural stability is maintained in a limited environment.In contrast, inorganic materials usually have incredible stability and stiffness, evenin extreme conditions. In addition, they sometimes offer us the opportunity toprepare precise structures by both top-down fabrications and bottom-up assembly.Of course, superior aspects in electronic, photonic, magnetic, and mechanicalproperties can be expected in many inorganic materials. Nevertheless, no one canbelieve that the highly sophisticated functions seen in living systems may beconstructed by assembly and fabrication of materials of exclusively inorganic nature.The focus of this Bio-Inorganic Hybrid Nanomaterials book is to cover the wide

spectrum of recent developments in natural and artificial bio-hybrid materials,which is the result of the successful assembly of 15 chapters by world-wide expertsin their corresponding fields. Fundamental aspects on the preparation of bio-inorganic nanohybrids using various nanostructures including mesoporous materi-als, nanoparticles, gels, organoclays, membranes, and nanotubules with advancedmethodologies such as the sol–gel process, self-assembly, intercalation, templatesynthesis and layer-by-layer adsorption upon the concept of supramolecular chem-istry, biomimetics, and biomineralization are thoroughly described. Not limited tobasic sciences, several chapters introduce practical applications of bio-inorganicnanohybrids, as exemplified in biodegradation, bone tissue re-generation, controlleddelivery, and enzymatic activity. Readers can enjoy independent chapters and alsofeel the good harmony of the balanced assembly of the chapters. We hope that everyreader can find potential possibilities of bio-inorganic nanohybrids with certainwonder, surprise, and impression, just after closing these pages.

Eduardo Ruiz-HitzkyKatsuhiko ArigaYuri Lvov

XIV Preface

Contributors

Mitsuru AkashiOsaka UniversityDepartment of Applied ChemistryGraduate School of Engineering2-1, Yamada-oka,andThe 21st Century COE Programfor Center for Integrated Cell andTissue Regulation2-2, Yamada-oka, SuitaOsaka 565-0871Japan

Pilar ArandaInstituto de Ciencia de Materialesde MadridConsejo Superior de InvestigacionesCientíficas (CSIC)Cantoblanco28049-MadridSpain

Daniel ArcosUniversidad ComplutenseDepartamento de Quimica Inorgánicay BioinorganicaFacultad de FarmaciaPlaza Ramón y Cajal s/n28040 MadridSpain

Katsuhiko ArigaNational Institute for MaterialsScience (NIMS)Supermolecules Group1-1 NamikiTsukuba 305-0044Japan

Mihail BarboiuInstitut Européen des MembranesAdaptative SupramolecularNanosystems GroupUMR CNRS 5635Place Eugène Bataillon CC 04734095 MontpellierFrance

Roberta BraynerUniversité Pierre et Marie CurieUniversité Denis DiderotInterfaces, Traitements, Organisationet Dynamique des Systèmes (ITODYS)UMR-CNRS 70862 Place Jussieu75252 Paris Cedex 05France

XV

Frank CarusoThe University of MelbourneCentre for Nanoscience andNanotechnologyDepartment of Chemical andBiomolecular EngineeringVictoria 3010Australia

Soo-Jin ChoiEwha Womans UniversityCenter for Intelligent Nano-BioMaterialsDivision of Nanoscience andDepartment of ChemistrySeoul 120-750Korea

Jin-Ho ChoyEwha Womans UniversityCenter for Intelligent Nano-BioMaterialsDivision of Nanoscience andDepartment of ChemistrySeoul 120-750Korea

Thibaud CoradinUniversité Pierre et Marie CurieChimie de la Matière Coudenséede Paris (CMCP)UMR-CNRS 75744 Place Jussieu75252 Paris Cedex 05France

Margarita DarderInstituto de Ciencia de Materialesde MadridConsejo Superior de InvestigacionesCientíficas (CSIC)Cantoblanco28049-MadridSpain

Fernand FiévetUniversité Denis DiderotInterfaces, Traitements, Organisationet Dynamique des Systemès (ITODYS)UMR-CNRS 70862 Place Jussieu75251 Paris Cedex 05France

Claude ForanoUniversité Blaise PascalLaboratoire des MatériauxInorganiquesCNRS UMR 600263177 Aubiere CedexFrance

Kazuko FujiiNational Institute for Materials Science(NIMS)Special Subjects Group1-1 NamikiTsukuba 305-0044Japan

Narasimhan GokulakrishnanNational Institute for Materials Science(NIMS)Supermolecules Group1-1 NamikiTsukuba 305-0044Japan

Jonathan P. HillNational Institute for Materials Science(NIMS)Supermolecules Group1-1 NamikiTsukuba 305-0044Japan

XVI Contributors

Masanobu KamitakaharaTohoku UniversityGraduate School of EnvironmentalStudies6-6-20, AobaAramaki, Aoba-kuSendai 980-8579Japan

Jacques LivageUniversité Pierre et Marie CurieChimie de la Matière Condenséede Paris (CMCP)UMR-CNRS 75744 Place Jussieu75252 Paris Cedex 05France

Yuri M. LvovLouisiana Tech UniversityInstitute for Micromanufacturing911 Hergot Ave.Ruston, LA 71272USA

Stephen MannUniversity of BristolCentre for Organized Matter ChemistrySchool of ChemistryBristol, BS8 1TSUnited Kingdom

Toshiki MiyazakiKyushu Institute of TechnologyGraduate School of Life Science andSystems Engineering2-4, HibikinoWakamatsu-ku, Kitakyushu-shiFukuoka 808-0196Japan

Toshiyuki MoriNational Institute for Materials Science(NIMS)Nano Ionics Materials GroupFuel Cell Materials Center1-1 NamikiTsukuba 305-0044Japan

Jae-Min OhEwha Womans UniversityCenter for Intelligent Nano-BioMaterialsDivision of Nanoscience andDepartment of ChemistrySeoul 120-750Korea

Chikara OhtsukiNagoya UniversityDepartment of Crystalline MaterialsScienceGraduate School of EngineeringFuro-cho, Chikusa-kuNagoya 464-8603Japan

Masami OkamotoToyota Technological InstituteAdvanced Polymeric NanostructuredMaterials EngineeringGraduate School of Engineering2-12-1 Hisakata, TempakuNagoya 468 8511Japan

Contributors XVII

Avinash J. PatilUniversity of BristolCentre for Organized MatterChemistrySchool of ChemistryBristol, BS8 1TSUnited Kingdom

Vanessa PrévotUniversité Blaise PascalLaboratoire des MatériauxInorganiquesCNRS UMR 600263177 Aubiere CedexFrance

Ronald R. PriceAtlas Mining CorporationNanoclay and Technology Division1200 Silver City RoadEureka, UT 84628USA

Eduardo Ruiz-HitzkyInstituto de Ciencia de Materialesde MadridConsejo Superior de InvestigacionesCientíficas (CSIC)Cantoblanco28049-MadridSpain

Yury A. ShchipunovRussian Academy of SciencesInstitute of ChemistryFar East Department690022 VladivostokRussia

María Vallet-RegíUniversidad ComplutenseFacultad de FarmaciaDepartamento de Quimica Inorgánicay BioinorganicaPlaza Ramón y Cajal s/n28040 MadridSpain

Ajayan VinuNational Institute for Materials Science(NIMS)Nano Ionics Materials GroupFuel Cell Materials Center1-1 NamikiTsukuba 305-0044Japan

Yajun WangThe University of MelbourneCentre for Nanoscience andNanotechnologyDepartment of Chemical andBiomolecular EngineeringVictoria 3010Australia

Junji WatanabeOsaka UniversityDepartment of Applied ChemistryGraduate School of Engineering2-1, Yamada-oka,andThe 21st Century COE Programfor Center for Integrated Celland Tissue Regulation2-2, Yamada-oka, SuitaOsaka 565-0871Japan

XVIII Contributors

1

An Introduction to Bio-nanohybrid Materials

Eduardo Ruiz-Hitzky, Margarita Darder, Pilar Aranda

1.1

Introduction: The Assembly of Biological Species to Inorganic Solids

The assembly of molecular or polymeric species of biological origin and inorganicsubstrates through interactions on the nanometric scale constitutes the basis forthe preparation of bio-nanohybrid materials (Figure 1.1). The development of thesematerials represents an emerging and interdisciplinary topic at the border of LifeSciences, Material Sciences and Nanotechnology. They are of great interest due totheir versatile applications in important areas as diverse as regenerativemedicine andnew materials with improved functional and structural properties [1–5]. It must beremarked that the development of bio-nanocomposites also represents an ecologicalalternative to conventional polymer nanocomposites, as the properties of thebiodegradable polymers used ensure that the materials produced are environmen-tally friendly and renewable. Typical examples of this type of bio-nanocompositesresult from the combination of polysaccharides such as starch, cellulose or polylacticacid (PLA) with microparticulated solids, which are usually called green nanocom-posites or bioplastics [6,7].Recently, special attention has been paid to strategies for synthetic approaches to

bio-nanohybrids. One of these approaches is related to the preparation of bioinspiredor biomimetic materials following the examples found in Nature, as for instance,bone [8], ivory [9] and nacre [10–13]. These materials show excellent structuralproperties due to the special arrangement at the nanometric level of their assembledcomponents, that is biopolymers and inorganic counterparts. For instance, nacrerepresents a good example of a natural bio-nanocomposite, also known as native bio-mineral, formed by the stacking of highly oriented calcium carbonate (aragonite)platelets cemented by a fibrous protein (lustrin A). The resulting supra-architecturesshow exceptional mechanical properties compared to monolithic calcium carbonate[11,12].Nowadays, bio-nanocomposites mimicking these natural materials have been

prepared with the aim being to develop new biohybrids with improved mechanicalproperties together with biocompatibility and, in some cases, other interesting

j1

features such as functional behavior [14–18]. In this context, the development ofbiohybrid systems based on biomimetic building [18,19], that is following biomin-eralization processes similar to those that take place in the cell wall of diatoms, wherenanostructured silica nanospheres are assembled by the participation of cationicpolypeptides called silaffins (SILica AFFINity) [20,21] appears to be of great signifi-cance. In relation to these natural systems, the mechanisms of interaction betweencolloidal silica and peptides have been particularly studied with the aim being tounderstand the biomineralization processes and consequently to develop, in acontrolled manner, new improved synthetic bio-nanocomposites [22–24].Inorganic solids assembled with biological species are of diverse nature with

different chemical compositions, structures and textures, which determine theproperties of the resulting bio-nanohybrids. In this way, single elements such astransition metals and carbon particles, metal oxides and hydroxides, silica, silicates,carbonates and phosphates, are typical inorganic components of bio-nanohybrids(Table 1.1). The affinity between the inorganic and the bio-organic counterparts,which determines the stability of the resulting bio-composites, depends on theinteraction mechanisms governing the assembly processes.As indicated above, the development of bio-nanohybrids by mimicking biomin-

eralization represents an extraordinarily useful approach. This is, for instance, thecase for those bio-nanocomposites based on bone biomimetic approaches, whichshow excellent structural properties and biocompatibility. They are prepared by

Fig. 1.1 Number of publications per year related to bio-

nanohybrid materials. Data collected from the ISI Web of

Knowledge [v3.0]-Web of Science. Keywords for search:

(biopolymer*ANDnanocomposite*)OR (natural polymer*AND

nanocomposite*) OR (bio-nanohybrid*) OR (biohybrid* AND

nano*).

2j 1 An Introduction to Bio-nanohybrid Materials

assembling hydroxyapatite (HAP), which is the main mineral constituent of bonesand teeth, with biopolymers, for example collagen [25–27]. The coating of the micro-or nano-particulated solids with biopolymers often occurs through hydrogen-bonding or metal-complexing mechanisms. In this way, the assembly of magneticiron oxide nanoparticles (e.g., magnetite) with biopolymers (e.g., dextran) allows thepreparation of magnetic bio-nanocomposites applied in NMR imaging, hyperther-mia treatments or bio-carriers as drug delivery systems (DDS) [28,29].The assembly of biopolymers with inorganic layered solids can lead to bio-

nanocomposites in which the biopolymer becomes intercalated between the layersof the inorganic hosts [3]. The intercalation is a complex process that may simulta-neously involve several mechanisms. Thus, in addition to hydrogen bonding, it hasbeen invoked that certain biopolymers interact with the inorganic layers through

Tab. 1.1 Selected Examples of Bio-Nanohybrid Materials

Involving Different Types of Inorganic Solids.

Inorganic moiety Biological species Bio-nanohybrid features Authors/References

silica nanoparticles poly-L-lysine (PLL) biomimetic nanocompositeswith controlled morphology

Patwardhanet al. [39]

siloxane networks living bacteria encapsulation by sol-gel Fennouhet al. [62]

calcium carbonate chitosan andpoly(aspartate)

biomimetic preparationtowards artificial nacre

Sugawaraand Kato [88]

hydroxyapatite (HAP) collagen biomimetic porous scaffoldsfor bone regeneration

Yokoyamaet al. [94]

layered clay minerals(montmorillonite)

chitosan functional bio-nanocompositefor ion-sensing applications

Darder et al.[129]

fibrous clay minerals(sepiolite)

caramel bio-nanocomposite asprecursor of multifunctionalcarbon–clay nanostructuredmaterials

Gómez-Aviléset al. [153]

organoclays PLA green nanocomposites asbiodegradable bioplastics

Paul et al. [144]

layered doublehydroxides (LDHs)

deoxyribonucleicacid (DNA)

bio-nanocomposite asnon-viral vector for genetransfection

Choy et al. [159]

gold nanoparticles chitosan bio-nanohybrid processableas self-supporting filmsfor biosensor applications

dos Santoset al. [164]

magnetitenanoparticles

phosphatidylcholine magnetocerasomesfor targeteddrug delivery

Burgos-Asperillaet al. [73]

carbon nanotubes(CNTs)

galactose modified CNTs able to capturepathogens by protein binding

Gu et al. [194]

layered perovskites(CsCa2Nb3O10)

gelatin bio-nanocomposite thin filmswith dielectric properties

Ruiz et al. [220]

1.1 Introduction: The Assembly of Biological Species to Inorganic Solids j3

ionic bonds. This is the case for polysaccharides, proteins and nucleic acids that canact as polyelectrolytes intercalating, via ion-exchange reaction, solids provided withpositively or negatively charged layers, such as layered double hydroxides (LDHs) orsmectite clay minerals (see below).Microfibrous crystalline silicates such as sepiolite, similarly to amorphous silica,

contain silanol groups (Si�OH) covering the external mineral surface. These groupscan be effectively involved in hydrogen bonding by their association to OH, NHand other polar groups belonging to the biopolymers used. Silica generated by thesol–gel method from tetraethyl orthosilicate (TEOS) in the presence of chitosan,gives biopolymer-silica nanocomposites whose morphology can be determinedby the experimental conditions adopted for the preparation [30]. Chitosan andcollagen can also be assembled with sepiolite to give the corresponding biopolymer-sepiolite nanocomposites, which exhibit good mechanical properties resultingfrom the combination of the fibrous inorganic substrate with the biopolymer[31–34]. The interaction mechanisms governing the formation of sepiolite-basedbio-nanocomposites are mainly ascribed to hydrogen bonding, but it must be takeninto account that sepiolite exhibits cationic exchange capacity (CEC �15meq/100 g).Thus this silicate could also interact with positively charged polymers, such aschitosan, through electrostatic bonds.Although to only aminor extent, othermechanisms can be invoked, as for instance

covalent bonding (grafting) between hydroxy groups on the surface of the inorganicsubstrates and functional groups of the biopolymers [35].The aimof this chapter is to provide a general overviewof the preparation andmain

characteristics of bio-nanohybrids, with emphasis on the different types of inorganicsolids that can be involved in the formation of this class ofmaterials. Special attentionwill be devoted to the diverse mechanisms that govern the interaction between thecomponents of biohybrids, illustrating them with selected examples. Relevant fea-tures and potential or actual applications of recently developed bio-nanocompositeswill be discussed on the basis of their structure–property relationships.

1.2

Bio-nanohybrids Based on Silica Particles and Siloxane Networks

Biominerals are produced by living organisms following a set of processes known asbiomineralization, which results in a wide variety of biological materials includingshells, bones, teeth, ivory and magnetic nanoparticles in magnetotactic bacteria.Biomolecules secreted by living organisms control the nucleation and growth ofinorganic minerals (carbonates, phosphates, silica and iron oxide) leading to such adiversity of biological-inorganic hybridmaterials, which usually exhibit a hierarchicalarrangement of their components from the nanoscale to the macroscopic scale. Theskeletons of diatoms and radiolarians are astonishing examples of biosilicificationgiving rise to amorphous hydrated SiO2 (biosilica), also formed in sponges andmanyhigher plants [24]. As mentioned in Section 1, polycationic peptides, called silaffins,are involved in this process, controlling the assembly of silica nanoparticles to form


these siliceous structures [20,21,36]. Similarly, silica needles in the skeleton ofmarine sponges involve a central filament containing silicatein, an enzyme thatcatalyses the synthesis of biosilica [37]. Materials scientists try to understand andreproduce these biosilicification processes taking place in nature, with the aim beingto develop bioinspired or biomimetic hybrid nanostructured materials with con-trolledmorphologies and structural properties similar to those of biosilica [24,38–42],as shown in Figure 1.2. As recently reviewed by Coradin et al. [43], proteins (collagen,gelatin, and silk) and polysaccharides (alginate, carrageenans, chitosan, as well ascellulose and its derivatives) are the main biomacromolecules involved in thesynthesis of biopolymer/silica nanocomposites, while silicic acid, sodium silicateand different silicon alkoxides are employed as precursors of the silica or thepolysiloxane networks assembled with the biopolymer chains. Following biomimeticprocesses, lysozyme and bovine serum albumin (BSA) promote the precipitationof silica particles from sodium silicate solutions, leading to entrapment of the pro-tein [44]. Similarly, polysaccharides such as cationic and hydrophobic derivativesof cellulose also promote silica precipitation, acting as efficient templates to

Fig. 1.2 Scanning electron micrographs of (A)

the silica wall of the diatom Stephanopyxis turris

(reproduced from [21] by permission of Wiley-

VCH) and (B—D) singular morphologies of silica

synthesized using poly-L-lysine and pre-hydro-

lyzed tetramethyl orthosilicate (TMOS) under

different experimental conditions:

(B) unperturbed solution, (C) flowed through

a 1/800 I.D. tube and (D) stirred for 25min.Reproduced from [39] by permission of The

Royal Society of Chemistry.

1.2 Bio-nanohybrids Based on Silica Particles and Siloxane Networks j5

develop organic–inorganic hybrid nanocomposites in combination with tetrakis(2-hydroxyethyl)orthosilicate (THEOS) [45]. Chitosan is another natural polysaccharideinvolved in this type of silica-based hybrid material prepared by the sol–gel method.For instance, it has been assembled with siloxane networks derived from amino-propylsiloxane (APS) [46] or TEOS [30]. A similar chitosan–polysiloxane biohybridmaterial has been recently prepared from chitosan and 3-isocyanatopropyltriethox-ysilane, where chitosan is bound to the polysiloxane network by covalent bridges.This new functional material offers photoluminiscent features and bioactive behavior,since itpromotesapatite formation insimulatedbodyfluid [47].Chitosan–silicahybridspresent as microparticulate materials showing different shapes have been preparedby the sol–gel method using TEOS or polyethoxysiloxane oligomers in the presenceof the biopolymer. These materials can be used as a stationary phase in HPLC [48].In these examples as well as in analogousmaterials, the interaction of the biological

and the inorganic components has synergetic effects leading to hybrid materials withimproved mechanical resistance, higher thermal and chemical stability and biocom-patibility, and, in some cases, with functional properties. Biopolymer/silica nanocom-posites are suitable for the design of membranes and coatings, drug delivery systemsand also for the encapsulation of bioactive molecules such as enzymes, antibodies,yeast and plant cells or even bacteria, resulting in functional biomaterials for differentbiotechnological applications, including biosensors and bioreactors [43,49].Silica-based bio-nanocomposites for drug delivery purposes have been processed

as nanospheres by means of spray-drying or CO2 supercritical drying techniques.Hybrid nanoparticles based on algal polysaccharides such as alginate and carrageen-an are potential carriers for the targeted delivery of drugs due to their ability to go intothe intracellular space of cells and to their lack of cytotoxicity [50,51]. In other cases,silica nanoparticles serve as a support of biocide molecules and their dispersion inhydroxypropylcellulose allows the preparation of coatings and films with fungicideand pesticide activity [52]. Following a similar approach, Zhang and Dong [53] havedeveloped functional materials based on the dispersion of Ru(bpy)3

2þ-doped silicananoparticles in the biopolymer chitosan. The resulting hybridmaterial can be easilyspread onto the surface of electrodes as a stable electroactive coating, allowing thedevelopment of chemiluminiscence sensors.Similarly to the above-mentioned entrapment of proteins by biomimetic routes,

the sol–gel procedure is a useful method for the encapsulation of enzymes and otherbiological material due to the mild conditions required for the preparation of thesilica networks [54,55]. The confinement of the enzyme in the pores of the silicamatrix preserves its catalytic activity, since it prevents irreversible structural defor-mations in the biomolecule. The silica matrix may exert a protective effect againstenzyme denaturation even under harsh conditions, as recently reported by Frenkel-Mullerad and Avnir [56] for physically trapped phosphatase enzymes within silicamatrices (Figure 1.3). A wide number of organoalkoxy- and alkoxy-silanes have beenemployed for this purpose, as extensively reviewed by Gill and Ballesteros [57],and the resulting materials have been applied in the construction of optical andelectrochemical biosensor devices. Optimization of the sol–gel process is requiredto prevent denaturation of encapsulated enzymes. Alcohol released during the


hydrolysis process can be harmful for the entrapped biologicals and, thus, severalmethods propose its removal by evaporation under vacuum [58] or the use of polyol-based silanes that generate biocompatible alcohols [59]. Catalytic activity is alsopreserved when silica-polysaccharide bio-nanocomposites are used as immobiliza-tion hosts. This is the case for three-dimensional hybrid matrices resulting from thecombination of THEOS with xanthan, locust bean gum or a cationic derivative ofhydroxyethylcellulose, which have been reported as excellent networks for the long-term immobilization of 1! 3-b-D-glucanase and a-D-galactosidase [60].In addition to enzymes and antibodies, silica-based hybrid nanocomposites with a

suitable porosity can successfully entrap more complex systems including yeasts,algae, lichens, plant cells and bacteria [49]. The huge volume of biological tissues, incomparison to enzymes, may hinder the polymerization processes resulting infractures in the silica matrices. To overcome this drawback, lichen particles wereembedded in a flexible network, derived from 3-(trimethoxysilyl)propyl methacrylate(MAPTS) and tetramethoxysilane (TMOS), that offers improvedmechanical features(Figure 1.4A). This lichen-modified material was used to develop electrochemicalsensors for the determination of heavy metal ions by anodic stripping voltammetry[61]. Similarly, algal tissue can be immobilized in sol–gel derived matrices based onTMOS and methyltrimethoxysilane (MTMOS) (Figure 1.4B).

Fig. 1.3 Schematic representation of the entrapped enzyme in

a silica matrix (left side). Enzymatic activity, under extreme

alkaline conditions, of acid phosphatase (A) immobilized in silica

sol—gel matrices with or without CTAB, or (B) in solution.

Reprinted with permission from [56]. Copyright 2005, American

Chemical Society.

1.2 Bio-nanohybrids Based on Silica Particles and Siloxane Networks j7

One of the first works reporting the entrapment of E. coli proposed its incorpo-ration in a TMOS-derived silica network in which the water content was kept at about70wt% in order to guarantee the cells viability, but when silica gel was dried thebacterial activity decreased [62]. In order to overcome this drawback, the authorsexplored other possibilities such as the incorporation of glycerol in the silicamatrix toincrease bacteria viability (Figure 1.4C), leading to almost 50%of viable bacteria afteronemonth of ageing [63], or the addition of quorum sensingmolecules involved inintercellular communication, which increase the cells viability to 100% after onemonth [64]. Similar results have been achieved recently by Ferrer et al. [65], whoshowed that gluconolactone-bearing organopolysiloxane matrices are more efficientthan pure silica in extending E. coli the cells viability due to their increasedbiocompatibility (Figure 1.4D).New materials that mimic liposomes have been recently reported as a new family

of organic–inorganic hybrid compounds generated by a coupled process of sol–geland self-assembly of long-chain containing organoalkoxysilanes [18,66–71]. Thesenanohybrids essentially refer to biomimeticmaterials derived from the assembly of asurfactant covalently bonded to a silica-based network. The name cerasomes wasintroduced by Ariga and coworkers [66] combining the terms liposome andceramic, this last making reference to the silica network. As ceramic is derived

Fig. 1.4 Scanning electron micrographs of (A)

the lichen Pseudocyphellaria hirsuta, (B) the alga

Anabaena, and (D) the bacteria E. coli entrapped

in sol—gel generated organopolysiloxane matri-

ces (reprinted with permission from [65].

Copyright 2006, American Chemical Society).

(C) Transmission electron micrograph of the

same bacteria, E. coli, embedded in a silica

matrix containing 10% glycerol (reproduced

from [63] by permission of The Royal Society of

Chemistry).


from the Greek word keramikóz (keramikos) making reference to inorganic non-metallic materials whose formation is due to the action of heat [72], the termcerasome can be confusing as they are usually formed in soft conditions. Ruiz-Hitzkysuggests the use of HOILs (Hybrid Organic–Inorganic Liposomes) for this class ofcompounds [18]. Anyway, cerasome is actually the most popular term for thesehybrid materials. Interestingly, the bilayers formed by the surfactant tails are able toincorporate different organophyllic species [71] making these materials potentiallyapplicable as Drug Delivery Systems (DDS). More recently, these types of bilayershave been grafted onto magnetic nanoparticles giving rise to the so-calledmagnetocerasomes [73], which are nanohybrids simultaneously having lipophiliccharacter and magnetic properties (see below, Section 5).

1.3

Calcium Phosphates and Carbonates in Bioinspired and Biomimetic Materials

Aspointed out in Section 2, a widenumber of biominerals are synthesized innature byliving organisms using organic templates. Some well-known examples include boneand ivory, where the collagen matrix controls the growth of hydroxyapatite (HAP)mineral [8,9,74], or nacre in pearls and shells, showing a brick-like structureof aragonite layers cemented by proteins [11,12]. This assortment of biological–inorganic hybrid materials, showing a hierarchical arrangement from the nano- tothe macroscale, serves as a model for the development of new biomimetic andbioinspired materials. In vitro studies have demonstrated the controlled nucleationand growth of carbonates and phosphates by soluble proteins and peptides combinedwith insoluble polysaccharide matrices (cellulose, chitin, collagen), leading to biomi-metic materials that reproduce the exceptional features of native biominerals [1,75].Besides nacre in pearls and shells, calcium carbonate is also present in sea urchin

spines, coral skeleton, eggshell, and the exoskeleton of arthropods, forming organic–inorganic hybrid structures by assembly with biomacromolecules (soluble proteinsand insoluble matrices) [76–78]. Calcite and aragonite are the calcium carbonatepolymorphs that constitute the biominerals found innature, since they show ahigherstability than vaterite. However, in vitro studies have confirmed that the presence offunctionalised macromolecules as soluble proteins and insoluble matrices have aconsiderable effect on calcium carbonate crystallization, allowing the formation ofthe less stable polymorphs and even of amorphous CaCO3 [79,80]. Regarding solublematrices, living organisms secrete biomacromolecules with a high content ofglutamic and aspartic acids, bearing carboxyl groups that can interact with calciumions. A similar effect has been found using polymers providedwith sulfonic, hydroxyand even ether groups. Many of these studies have been carried out using the samebiopolymers that act as insoluble matrices for CaCO3 crystallization in nature, suchas collagen and chitin [79,81]. Calcium carbonate polymorphs are also formed onother natural and synthetic polymers including elastin that controls the formation ofcalcite [82], poly(ethylene glycol) that forces the selective formation of aragonite [83],poly(a-L-aspartate) that promotes vaterite formationwith a helicalmorphology [84], or

1.3 Calcium Phosphates and Carbonates in Bioinspired and Biomimetic Materials j9

poly(acrylate)-grafted chitosan giving rise to CaCO3 crystals of unusual morphology[85] similar to those created using poly(N-isopropylacrylamide-co-(4-vinylpyridine))as the platform for mineralization [86]. As confirmed by these reports, materialsscientists are able to produce calcium carbonate organic hybrid materials withdefined morphologies (Figure 1.5) by tuning the polymers and biomacromoleculesthat control the nucleation and growth of calcium carbonate crystals. Many studiesare currently centered on the crystallization of CaCO3 as thin films, as illustrated inFigure 1.5C, trying to mimic the nacre of shells [87–89]. The reason is that molluskshells, where the organic matrix constitutes only 1% of the total weight, present afracture toughness about 3000 times higher than that of pristine calcium carbonateand offer a good example of ultra-lightweight hybrid materials with exceptionalmechanical strength and interesting optical properties.Calcium phosphate minerals are present in living organisms as the most im-

portant constituents of biological hard tissues (bones, teeth, tendons, and cartilage)to provide them with stability, hardness and function [74,90]. Among the dif-ferent calcium phosphates, hydroxyapatite (HAP), with a chemical compositionCa10(PO4)6(OH)2 and Ca/P ratio of 1.67, is the most widely studied due to its hugeincidence in the field of regenerative medicine. Bone can be regarded as a naturalnanocomposite containing HAP nanocrystallites in a collagen-rich matrix alsoenclosing non-collagenous proteins. Besides providing structural support, boneserves as a reservoir of calcium and phosphate ions involved in numerous metabolicfunctions. Due to the significant role of bone in humans,most of the synthetic hybridmaterials that mimic its structure and composition are currently devoted to biomed-ical applications for regeneration of injured bone and this fact has led to a widenumber of scientific publications on this topic in the recent years.Among synthetic materials for bone grafting, nanocomposites are replacing

metals, alloys, ceramics, polymers, and composites, due to their advantageousproperties: large surface area, high surface reactivity, relatively strong interfacialbonding, flexibility, and enhanced mechanical consistency. It has been provedthat nanocrystalline HAP offers better results than microcrystalline HAP withrespect to osteoblast cells adhesion, differentiation and proliferation, as well as

Fig. 1.5 SEM micrographs of (A) donut-shaped

CaCO3 crystals grown on polyacrylic acid grafted

chitosan, (B) CaCO3 hollow helix, fractured by

micro-manipulation, formed on poly(a-L-aspar-tate), and (C) double layered aragonite thin films

grown on a chitosan matrix in the presence of

poly(aspartate) and MgCl2 by alternate spin

coating and crystallization. (A) and (B) adapted

from [84] and [85] with permission fromElsevier,

and (C) from [88] (reproduced by permission of

The Royal Society of Chemistry).


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