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Relevance of the Polymer-Induced Liquid-Precursor (PILP) Process to Biomineralization and Development of Biomimetic Materials

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Title:
Relevance of the Polymer-Induced Liquid-Precursor (PILP) Process to Biomineralization and Development of Biomimetic Materials
Creator:
CHENG, XINGGUO ( Author, Primary )
Copyright Date:
2008

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Subjects / Keywords:
Biomineralogy ( jstor )
Calcite ( jstor )
Calcium ( jstor )
Carbonates ( jstor )
Crystallization ( jstor )
Crystals ( jstor )
Hydrogels ( jstor )
Minerals ( jstor )
Polymers ( jstor )
Thin films ( jstor )

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Source Institution:
University of Florida
Holding Location:
University of Florida
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Copyright Xingguo Cheng. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
11/30/2005
Resource Identifier:
436098754 ( OCLC )

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RELEVANCE OF THE POLYMER-INDUCED LIQUID-PRECURSOR (PILP) PROCESS TO BIOMINERALIZATION AND DEVELOPMENT OF BIOMIMETIC MATERIALS By XINGGUO CHENG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by XingGuo Cheng

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To my dear wife, Rongfang Gu

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ACKNOWLEDGMENTS I would like to thank my parents who brought me up and have always been there when I met any difficulties. To get a better education and future, I have been in school for about 21 years without any significant contributions to the family in terms of social and economic activities, and they always showed complete faith and understanding. I thank my parent and my brother’s family from the bottom of my heart. Secondly, a great many thanks should be given to Dr. Laurie Gower for providing me direction and guidance throughout my education in the United States. As an international student, I saw in her the great generosity, humor, and uplifting spirit of people in the US. She helped me to get the F-1 visa to study in US. She picked me up at the Jacksonville airport on my first day of arrival. She secured financial support for my studies. She taught me the importance of strict, careful attention to my presentations, research reports, and manuscripts. She sent me to several conferences to broaden my view of the academic world. I also appreciate her friendship and understanding through many of the tough times in graduate school. I would also like to thank my supervisory committee (Dr. Jack Mecholsky, Dr. Hassan El-Shall, Dr. Wolfgang Sigmund, and Dr. David Hodell) for their advice, time and understanding. I also enjoyed talks and communications with Dr. Dempere, Dr. Douglas, Dr. Hummel, Dr. Dehoff, Dr. Ebrahimi, and Dr. Brennan, who are also very helpful and kind to me. I thank all of them. iv

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Many people at UF have influenced my personal life, and here I can only name a few. Lijun Dai and Meng Chen, my former roommates, were always kind and helpful to me whenever we were together. Fairland Amos, RajenDr.a Kumar, Yi-Yeoun Kim, Mattew Olsztza, Sangsoo Jee, Sara Jensen, Jim Mellman, Vishal Petal, Phil, Iris, Anika, Debra Lush and Barry Miller (good friends and lab mates) were always there to listen and lend a helping hand. Several fun things I did in the US are worth mentioning. Raj and I went to a casino and Burbon Street in New Orleans. In summer 2003, all of us in Dr. Gower’s group had a great time tubing the Ichetucknee river. Anika invited my wife and me to her house for thanksgiving meal in 2003 for which I thank her, because I cannot cook a good turkey to return the favor. I experienced the passion of sports in the Swamp. I spent a memorable 2004 Christmas eve with a lot of friends at MGM studios. I will never forget the fun of many Poker nights with Guojing Zhang, Nan Jiang, Minghan Chen, and many other people. None of these wonderful experiences could have happened without my true friends. I must stop here because I cannot include many other people and their stories here. They all are in my heart and in my memory. I wish everybody good luck and thank them all with the greatest sincerity. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................xi LIST OF FIGURES.........................................................................................................xiii ABSTRACT.....................................................................................................................xix CHAPTER 1 INTRODUCTION........................................................................................................1 2 AN OVERVIEW OF BIOMINERALIZATION AND BIOMIMETICS.....................4 Several General Observations in Biomineralization.....................................................6 Comparison of Biominerals with Inorganically Forming Minerals...........................11 Mechanisms of Biomineralization..............................................................................11 Classic Model of Nucleation...............................................................................11 Amorphous Precursor Process: a Kinetically Driven Crystallization Process....12 Purpose of Our Study..........................................................................................13 Biomimetics and Materials Science............................................................................14 3 NON-EQUILIBRIUM CRYSTAL MORPHOLOGY AND POLYMER-MINERAL ASSOCIATION......................................................................................21 Introduction.................................................................................................................21 Materials and Methods...............................................................................................23 Polymer-Induced Liquid-Precursor (PILP) Crystallization.................................23 Fluorescence Labeling of Polymer......................................................................24 Polarized Optical Microscope (POM) and Fluorescence Microscopy................25 Scanning Election Microscopy (SEM) and X-ray Energy Dispersive Spectroscopy (EDS).........................................................................................25 Results.........................................................................................................................26 Equilibrium Morphology of Calcite....................................................................26 In-situ Observation of PILP Process...................................................................26 Polymer-Mineral Association in the PILP Process by Fluorescence Study........28 Other Non-equilibrium Morphologies Produced by the PILP Process...............30 Discussion...................................................................................................................31 vi

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Polymer Exclusion in the PILP process......................................................................31 Conclusion..................................................................................................................34 4 MAGNESIUM ION IMPURITY INCORPORATION BY THE PILP PROCESS...47 Introduction.................................................................................................................47 Materials and Methods...............................................................................................49 Experimental Condition.......................................................................................49 Characterization...................................................................................................50 Polar optical microscopy (POM) and crystallinity analysis.........................50 X-Ray diffraction (XRD) analysis...............................................................51 Fourier transform infrared spectroscopy (FTIR)..........................................51 SEM and X-ray energy dispersive spectroscopy (EDS)..............................51 Inductively-coupled-plasma (ICP)-atomic emission spectroscopy (AES) analysis.....................................................................................................52 Results.........................................................................................................................52 Influence of Mg...................................................................................................52 Morphology of deposits w/o and w/ polymer..............................................52 Influence of Mg/Ca ratio in solution on crystallinity of film.......................55 Crystal structure verification........................................................................56 Mg incorporation in film determined by EDS, XRD and ICP.....................56 Non-equilibrium Composition in the PILP Process............................................58 Using FTIR for Characterization of CaCO3........................................................59 Discussion...................................................................................................................61 Influence of Temperature....................................................................................65 Influence of Polymer Concentration...................................................................66 POM characterization...................................................................................66 Morphology characterization by SEM.........................................................67 Influence of polymer Concentration on Mg incorporation..........................67 Assessing the Reliability of Magnesium as a Temperature Proxy......................68 Conclusion..................................................................................................................70 5 IMPURITY INCORPORATION BY THE PILP PROCESS-A STATISTICAL APPROACH...............................................................................................................85 Introduction.................................................................................................................85 Experiment and Method.............................................................................................86 Experimental Condition.......................................................................................86 Polarized Optical Microscopy (POM) and Crystallinity Analysis......................87 X-Ray Diffraction and Analysis..........................................................................87 SEM and X-ray Energy Dispersive Spectroscopy (EDS)...................................87 Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES).........87 Results and Discussion...............................................................................................88 Amorphous to Crystalline Transformation in the PILP Process.........................88 Influence of Mg in starting solution.............................................................88 Influence of Sr..............................................................................................89 vii

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Influence of polymer....................................................................................89 Influence of drying time: transformation rate..............................................89 Morphology of the Films Deposited by the PILP process..................................91 Crystal Structure Verification..............................................................................91 Mg and Sr incorporation in CaCO3 by the PILP process....................................92 Influence of Molecular Weight of Polymer.........................................................96 Distribution Coefficient.......................................................................................96 Discussion...................................................................................................................97 Incorporation of Mg in Calcite............................................................................97 Incorporation of Sr in CaCO3..............................................................................98 Role of Polymer in Impurity Incorporation.........................................................99 Conclusion................................................................................................................100 6 MOLDING CaCO3 MINERALS BY THE PILP PROCESS...................................112 Introduction...............................................................................................................112 Industrial Methods to Prepare Inorganic-Organic Composites.........................112 Natural Methods of Making Organic-inorganic Composites............................113 Biomimetic Methods of Making Organic-Inorganic Composites.....................114 Materials and Methods.............................................................................................115 Preparation of PHEMA Sheets and Micro-porous PHEMA Scaffold...............115 Mineralization of PHEMA Sheet and Porous PHEMA Scaffold......................116 Characterization.................................................................................................117 TGA/DTA analysis............................................................................................117 Results and Discussion.............................................................................................118 Mineralization of CaCO3 Thin Films on PHEMA Substrate............................118 Molding CaCO3 Minerals within Compartment by the PILP Process..............121 Structure and composition of sea urchin spine...........................................121 Structure and characterization of micro-porous PHEMA hydrogel...........122 Molding CaCO3 inside a microporous hydrogel by the PILP process.......123 Conclusion................................................................................................................126 7 INDUCTION OF THE PILP PROCESS USING NACRE PROTEINS..................142 Introduction...............................................................................................................142 Materials and Methods.............................................................................................144 Characterization of Nacre..................................................................................144 Protein Extraction Procedure.............................................................................144 Crystallization Study.........................................................................................145 Results and Discussions............................................................................................146 Structural Characterization of Nacre.................................................................146 Nacre Protein Characterization..........................................................................147 Influence of Nacre Protein on CaCO3 Crystal Growth.....................................148 On glass slides............................................................................................148 On PHEMA substrate.................................................................................149 CaCO3 crystal growth in the presence of both Mg and nacre protein........150 Conclusions...............................................................................................................153 viii

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8 PATTERNING HYDROXYAPATITE IN THE PRESENCE OF SOLUBLE POLYMER...............................................................................................................170 Introduction...............................................................................................................170 Experiments and Methods........................................................................................172 Crystallization System.......................................................................................172 BSP Peptide Synthesis.......................................................................................172 Micropatterning of Hydroxyapatite on Patterned SAMs...................................172 XRD...................................................................................................................173 Results and Discussion.............................................................................................173 Hydroxyapatite Formation on Glass Slides w/ and w/o Pasp or BSP-25..........173 Hydroxyapatite Micropatterning Formed on SAMs w/ and w/o Polymer........175 Micropatterning HA without polymer addition.........................................176 Micropatterning HA in the presence of Pasp or BSP-25...........................179 Conclusions...............................................................................................................181 9 HYDROXYAPATITE FORMATION ON ORGANIC SUBSTRATES.................199 Introduction...............................................................................................................199 Experiments and Methods........................................................................................202 Mineralization....................................................................................................202 Preparation of PHEMA and Carboxylmethylation of PHEMA........................202 Results and Discussion.............................................................................................203 Chemical Modification of Nonporous and Microporous PHEMA...................203 Mineralization of CaP on PHEMA and Carboxyl-PHEMA Disc.....................204 Without polymer addition..........................................................................204 With polymer addition...............................................................................205 Mineralization of Micro-porous PHEMA Hydrogel.........................................208 Without polymer addition..........................................................................208 With polymer addition...............................................................................208 Conclusions...............................................................................................................209 10 COLLAGEN-HYDROXYAPATITE NANOCOMPOSITES.................................227 Introduction...............................................................................................................227 Materials and Methods.............................................................................................229 Mineralization....................................................................................................229 Characterization.................................................................................................229 Scanning Electron Microscopy (SEM) analysis.........................................229 Transmission Electron Microscopy (TEM) analysis..................................229 X-ray Diffraction (XRD) analysis..............................................................230 Results and Discussion.............................................................................................230 Without Polymer...............................................................................................230 With Polymer.....................................................................................................231 Conclusion................................................................................................................233 11 CONCLUSION.........................................................................................................246 ix

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APPENDIX SCHEMATIC REACTION OF PHEMA AND CARBOXYMETHYLATED PHEMA SYNTHESIS..............................................250 LIST OF REFERENCES.................................................................................................251 BIOGRAPHICAL SKETCH...........................................................................................259 x

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LIST OF TABLES Table page 2-1 Three mechanical properties of various bones measured in tension and their functions for adaptational advantage..........................................................................4 2-2 Variety of biomineral types and biological systems..................................................5 2-3 Functions of cells in biomineralization......................................................................9 2-4 Comparison between biominerals and their inorganic counterparts........................10 2-5 A summary of biomineralization and biomimetics-related research.......................15 4-1 Experimental condition of the current study (two temperatures: 23C, 5C)..........50 4-2 Crystallinity of the films prepared at different Mg/Ca ratios at room temperature by PILP process........................................................................................................55 4-3 Mg content (molar percentage) in CaCO3 films prepared at different Mg/Ca ratios by PILP process determined by EDS (films prepared at two temperatures: 23C and 5C)..........................................................................................................57 4-4 Mg content (molar percentage) in CaCO3 films prepared at different Mg/Ca ratios by the PILP process determined by XRD (films prepared at 5C)................57 4-5 Mg content in CaCO3 films prepared at different Mg/Ca ratios by the PILP process, as determined by XRD (films prepared at 23C).......................................57 4-6 Mg content (molar percentage) in CaCO3 films prepared at different Mg/Ca ratios by the PILP process determined by ICP-AES ...............................................58 4-7 Influence of Polymer on Mg incorporation in PILP formed films (Mg/Ca=3.5)......68 5-1 Experimental condition of 223 factorial statistical design.......................................88 5-2 Mg and Sr incorporation in CaCO3 film in the PILP process using 223 factorial design (Pasp, Mw=35,400, room temperature)..........................................92 5-3 Mg and Sr incorporation in CaCO3 using 223 factorial design (Pasp, Mw=8,600, room temperature)................................................................................93 xi

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5-4 Analysis of variance table [Partial sum of squares] for Ca mol%...........................93 5-5 Analysis of variance table [Partial sum of squares] for Mg mol%..........................94 5-6 Analysis of variance table [Partial sum of squares] for Sr mol%............................95 6-1 Equilibrium water content (EWC%) determination of PHEMA sheets and porous PHEMA scaffolds made from replication of a sea urchin spine................116 6-2 Experimental condition of mineralization of CaCO3 onto PHEMA substrate. (Condition: room temperature, 3 days of vapor diffusion, Poly-, , d, L-aspartic acid, sodium salt, Mw=6200)...................................................................117 7-1 Eperimental condition of in-vitro crystallization of CaCO3 by nacre proteins......145 7-2 Condition of in-vitro crystallization using nacre proteins on PHEMA substrate..145 7-3 Crystallization of CaCO3 at different Mg/Ca ratio and constant low protein concentration (3 g/ml)..........................................................................................145 7-4 Crystallization of CaCO3 at different Mg/Ca ratio and constant protein concentration (3 g/ml)..........................................................................................146 7-5 Crystallization of CaCO3 at constant Mg/Ca ratio and at different protein concentrations.........................................................................................................146 9-1 EQW determination of chemical modified PHEMA hydrogel substrate...............203 9-2 EQW determination of chemical modified micro-porous PHEMA hydrogel substrate..................................................................................................................203 xii

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LIST OF FIGURES Figure page 2-1 Structure of nacre.....................................................................................................17 2-2 Seven levels of hierarchical structure of mammalian bone......................................18 2-3 Structure of sea urchin spine....................................................................................19 2-4 Crystallization pathways under thermodynamic and kinetic control.......................20 3-1 Comparison of morphology of typical mineral from inorganic origin and biological origin.......................................................................................................36 3-2 CaCO3 crystals grown in the presence of FITC.......................................................37 3-3 Polarized optical image and fluorescence image of the PILP process observed in the bottom of the in-situ crystallization chamber taken at different times of crystallization...........................................................................................................38 3-4 Polarized optical micrographs of crystals on the top slide of the in-situ crystallization chamber............................................................................................39 3-5 Optical image (left) and fluorescence image (right) of crystallites observed from the in-situ chamber ..................................................................................................40 3-6 Image of typical CaCO3 films formed by the PILP process ...................................41 3-7 Optical images of thin films showing the polymer-exclusion phenomenon in the PILP process.............................................................................................................42 3-8 CaCO3 crystals grown at high polymer concentration.............................................44 3-9 Scanning electron micrographs of some of the unusual CaCO3 morphologies.......45 4-1 POM micrographs (with 1st-order red -plate) of Mg-Ca carbonates formed at different Mg/Ca ratio ...............................................................................................71 4-2 SEM micrographs of CaCO3 crystals and corresponding EDS spectra ..................72 4-3 Film crystallinity six days after removal from the reaction chamber......................73 xiii

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4-4 Typical XRD spectra of the CaCO3 crystals............................................................74 4-5 Influence of Mg/Ca ratio in solution on the MgCO3 content in the films ...............75 4-6 FTIR spectrum of typical calcite crystals from the MCP0 control..........................76 4-7 FTIR of the magnesium-bearing CaCO3 films.........................................................76 4-8 A plot of the 4 peak maximum versus the Mg/Ca ratio in the parent solution for the films MCP2-MCP5.............................................................................................77 4-9 Mg content in the films from MCP0-MCP5 increases linearly as the Mg/Ca ratio in the parent solution increases (Mg content measured by ICP)..............................77 4-10 A comparison of using 4 shift in wave-numbers to correlate the Mg content in the Mg-CaCO3 films prepared by the PILP process to that from inorganic and biogenic magnesian calcite determined by another group.......................................78 4-11 Polarized optical micrographs of thin films.............................................................79 4-12 SEM micrographs of films deposited by the PILP process at low temperature.......80 4-13 Influence of temperature on the Mg incorporation in thin films prepared by the PILP process at different Mg/Ca ratios....................................................................81 4-14 Influence of polymer concentration and time of drying on the amorphous-crystalline transformation in the PILP process........................................................82 4-15 SEM and EDS of typical thin CaCO3 films formed at a higher polymer concentration............................................................................................................83 4-16 Effect of polymer concentration in the crystallization solution on the amount of Mg incorporation in thin films deposited by the PILP process at both temperatures.............................................................................................................84 5-1 Polarized optical micrographs of samples 1-16.....................................................102 5-2 POM pictures show amorphous-crystalline transformation of films after 7 days (Pasp Mw = 35,400, RT, samples 1-12, as shown in Table 5-1.)..........................103 5-3 POM pictures of each film after 50 days transformation ......................................104 5-4 POM pictures of each film after 10 days transformation ......................................105 5-5 Typical SEM images of thin CaxMgySr(1-x-y)CO3 films prepared by the PILP process....................................................................................................................106 5-6 Typical SEM pictures of the films deposited by the PILP process at higher magnification, along withtheir corresponding EDS spectra...................................107 xiv

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5-7 Typical XRD spectra of CaxMgySr(1-x-y)CO3 thin films deposited by the PILP process ...................................................................................................................108 5-8 Response of Ca content in the film versus variables..............................................109 5-9 Response of Mg content in the film versus variables.............................................110 5-10 Response of Sr content in the film versus variables...............................................111 6-1 POM pictures of calcitic crystals/films deposited on PHEMA substrate...............127 6-2 SEM micrographs of CaCO3 crystal/films formed under conditions described in Table 6-1................................................................................................................129 6-3 XRD spectra of mineralized PHEMA sheet shows typical calcite peaks..............131 6-4 SEM pictures of a sea urchin (Arbacia tribuloides) spine.....................................132 6-5 Comparison of the composition of commercial CaCO3, a sea urchin spine, and the products of the PILP process, by TGA and DTA.............................................133 6-6 XRD analysis of the reaction progress during the spine mineralization................135 6-7 SEM picture of the microporous PHEMA hydrogel scaffold made by replicating the structure of a sea urchin spine..........................................................................136 6-8 Comparison of Equilibrium Water Content (EWC%) of a non-microporous and micro-porous PHEMA hydrogel............................................................................137 6-9 TEM and ED of the amorphous nano-particles collected at one time from the mineralizing PHEMA hydrogel replica..................................................................138 6-10 Amorphous calcium carbonate particles collected from the solution after 1 day of mineralization....................................................................................................139 6-11 Comparison of hydrogel before and after mineralization, and sea urchin spine....140 6-12 Comparison of mineralized hydrogels prepared with and without polymer process-directing agent...........................................................................................141 7-1 Image of nacre from Atrina shells..........................................................................155 7-2 SEM images of nacre from Atrina shell.................................................................156 7-3 XRD spectra of nacre.............................................................................................157 7-4 SDS-PAGE of protein extracted from nacre by different extraction methods.......158 7-5 FTIR spectrum of soluble protein extracted from nacre........................................159 xv

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7-6 SEM pictures of the morphology of the modified crystals grown in the presence of crude nacre proteins...........................................................................................160 7-7 Polarized optical micrographs of thin films grown on the organic PHEMA substrate..................................................................................................................161 7-8 Comparison of polarized optical images of films grown on PHEMA substrate in the presence of nacre proteins for comparison to aragonite thin film from literature.................................................................................................................162 7-9 TEM and SAED of thin films produced by nacre proteins grown on PHEMA substrate..................................................................................................................163 7-10 Polarized optical micrographs of crystals formed at different Mg/Ca ratios in solution, at low protein concentration....................................................................164 7-11 Thin films formed in the presence of both Mg and crude nacre protein................165 7-12 Thin film formation at a constant Mg/Ca ratio of 3.5, at different protein concentrations.........................................................................................................166 7-13 Typical SEM images of thin film in the presence of both Mg and nacre protein..167 7-14 Typical SEM images of thin films at high Mg/Ca ratio and low nacre protein concentration..........................................................................................................168 7-15 XRD spectra of the thin films formed in the presence of both Mg and nacre protein verifies the crystalline phase is calcite.......................................................169 8-1 Schematic representation of micropatterning of Hydroxyapatite on Self-Assembled Monolayers (SAMs) using soft lithography technique.......................183 8-2 Polarized optical micrographs of CaP minerals grown on glass slides at different Polyaspartic acid concentration .............................................................................184 8-3 SEM and TEM of Hydroxyapatite spherulites grown on glass slides without any Pasp addition..........................................................................................................185 8-4 SEM image of thin CaP films formed in the presence of 5 g/ml Pasp................186 8-5 SEM images of CaP films formed in the presence of 15 ug/ml Pasp....................187 8-6 A comparison of CaP films formed in the presence of Pasp and BSP protein......188 8-7 Elemental mapping of hydroxyapatite micro-patterning on the self-assembled monolayer of COOH-(CH2)11SH...........................................................................190 8-8 Higher magnification SEM of the HA platy crystals grown on SAMs..................191 xvi

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8-9 TEM and ED of isolated platy crystals scraped from the SAMs...........................192 8-10 A comparison of XRD spectra of crystals of different origin................................193 8-11 Short scan (2 from 24 to 34) of the XRD spectra of HA crystals grown on mixed SAMs...........................................................................................................194 8-12 Hydroxyapatite micro-patterning on self-assembled monolayers of COOH-(CH2)11SH in the presence of 15 g/ml Pasp.........................................................195 8-13 Micropatterning of hydroxyapatite on self-assembled monolayers of COOH-(CH2)11SH in the presence of 15 g/ml BSP proteins...........................................196 8-14 HAP pattern formation on the same monolayer as in 8-13 in the presence of 75 g/ml BSP protein..................................................................................................197 8-15 XRD comparison of hydroxyapatite of different origins.......................................198 9-1 Digital images of microporous PHEMA hydrogels...............................................211 9-2 A comparison of Equilibrium Water Content (EWC%) of nonporous and microporous carboxylmethylated PHEMA............................................................212 9-3 Hydroxyapatite crystals formed on pure PHEMA substrate without any polymer addition...................................................................................................................213 9-4 Hydroxyapatite crystals formed on 0.25M BrCH2COOH modified PHEMA without any polymer addition................................................................................214 9-5 Hydroxyapatite formation on 1 M BrCH2COOH modified PHEMA substrate without any addition of polymer............................................................................215 9-6 Hydroxyapatite thin films formed on PHEMA substrate in the presence of 15 g/ml Pasp..............................................................................................................216 9-7 Hydroxyapatite thin films formed on 0.25 BrCH2COOH modified PHEMA substrate in the presence of 15 g/ml Pasp............................................................218 9-8 Hydroxyapatite thin films formed on 1M BrCH2COOH modified PHEMA substrate in the presence of 15 g/ml Pasp............................................................220 9-9 XRD spectra of CaP thin film formed in the presence of 15 g/ml Pasp..............222 9-10 Mineralization of microporous and chemically modified PHEMA without any polymer addition....................................................................................................223 9-11 SEM images of CaP mineralized microporous PHEMA hydrogel in the presence of 15 g/ml Pasp....................................................................................................225 xvii

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9-12 SEM images of CaP mineralized, microporous, 1M BrCH2COOH modified PHEMA hydrogel...................................................................................................226 10-1 SEM image of hydroxyapatite mineralized cellagen sponge without any polymer addition (control).....................................................................................235 10-2 SEM image of mineralized cellagen sponge in the presence of 15 g/ml Pasp (unbleached)...........................................................................................................236 10-3 Field emission images mineralized cellagen sponge in the presence of 15 g/ml Pasp (unbleached)........................................................................................237 10-4 Field emission images mineralized cellagen sponge in the presence of 15 g/ml Pasp (bleached ) ..........................................................................................238 10-5 SEM image of mineralized cellagen sponge in the presence of 75 g/ml Pasp (unbleached)...........................................................................................................240 10-6 SEM image of mineralized cellagen sponge in the presence of 75 g/ml Pasp. (bleached )..............................................................................................................242 10-7 X-ray diffraction (XRD) spectra of calcium phosphates crystallized in the absence and presence of polyaspartic acid.............................................................243 10-8 TEM image of extracted hydroxyapatite nanocrystals from mineralized cellagen sponge.....................................................................................................................244 10-9 Comparison of XRD spectra of mineralized cellagen sponge with commercial hydroxyapatite........................................................................................................245 xviii

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy RELEVANCE OF THE POLYMER-INDUCED LIQUID-PRECURSOR (PILP) PROCESS TO BIOMINERALIZATION AND DEVELOPMENT OF BIOMIMETIC MATERIALS By XingGuo Cheng May 2005 Chair: Laurie B. Gower Major Department: Materials Science and Engineering Natural biominerals often contain small amount of acidic macromolecules. These soluble macromolecules are thought to play a very important role in regulating the biomineralization process. By using synthetic acidic biopolymer and natural proteins extracted from biominerals to mimic these acidic macromolecules, the Polymer-Induced Liquid-Precursor (PILP) process is proposed to have great relevance to biomineralization. Like some biominerals, the minerals formed by the PILP process have both non-equilibrium morphology (e.g., “molded” crystal morphologies, films, rods, and tablets) and non-equilibrium composition (e.g., high magnesian calcite). Our goal was to study the formation of calcium carbonate and calcium phosphate formation by the PILP process. By using fluorescence labeling, in-situ observation, and TEM study, we examined the formation of liquid-precursor and polymer-mineral association (exclusion, occlusion). The cooperation of Mg and polymer leads to pronounced amorphous-crystalline transition and formation of thin films incorporating xix

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high amount of impurity comparable to biominerals. By taking advantage of amorphous-crystalline transition in the PILP process, calcium carbonate amorphous liquid precursors are molded inside a porous hydrogel and transform to crystalline calcite. After removal of the organic mold, a calcite scaffold with complex morphology is formed. Natural soluble proteins are extracted from nacre. Our in-vitro crystallization studies using these proteins show similar amorphous-crystalline transition and thin film morphology in the presence of Mg. The combination of organic substrate and nacre proteins leads to thin aragonite films. Finally we studied the formation of calcium phosphate using our in-vitro crystallization model. As in the calcium carbonate system, thin films were formed on organic substrate and glass slides in the presence of polymer. We also successfully prepared PHEMA-CaP and collagen-HA organic inorganic composites for tissue-engineering applications. By the above studies, we hope to unveil the role of the acidic proteins in biomineralization. We hope that our in vitro model system can show how different substrates and impurities influence the deposition and transformation of amorphous phases generated with the soluble polymeric process-directing agent. We believe that this new approach to mimicking biomineralization will allow fabrication of novel biomimetic materials from mild conditions. xx

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CHAPTER 1 INTRODUCTION From a materials science perspective, nature can make materials with such superior hierarchical structure, complex morphology, and prejudicial choice and placement of organic and inorganic components that mankind can hardly duplicate them. This is well demonstrated in biominerals such as the bone of vertebrates, the spines of sea urchins, and the nacre of mollusks. An overview of biomineralization and biomimetics in materials science is described in Chapter 2. Recently researchers found that biominerals such as the spine of sea urchin or nacre of mollusk larvae are formed by transformation of an amorphous precursor (1-8). It has also long been suspected that Hydroxyapatite (HAP) in bone may also be formed by transformation of an amorphous calcium phosphate precursor. Our recent discovery of the Polymer-Induced Liquid-Precursor (PILP) process may have great relevance to the biomineralization process associated with amorphous precursor phases. Crystallization of calcium carbonate or calcium phosphate in the presence of small amounts of acidic biomimetic polymer leads to the formation of droplets of a liquid phase mineral precursor that grows from the nanoscopic scale and coalesces to larger structures. Solidification and transformation then occurs, resulting in the formation of mosaic single-crystalline film patches of calcite (9). The central goal of our study was to examine this PILP process generated in the crystallization systems of calcium carbonate (CaCO3) and calcium phosphate (CaP), and to compare this novel crystallization model to traditional crystallization model with 1

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2 respect to gaining insight into biomineralization. We were also interested in learning from nature and developing novel biomimetic materials. By combining organic substrates with different functionalities (PHEMA and carboxylmethylated PHEMA); or self-assembled monolayers (alkylthiol on Au); or organic compartments (Microporous PHEMA and fibrous collagen sponges) with the PILP process, we formed different organic-inorganic composites or minerals with complex morphology that may be useful for tissue engineering or biomedical materials. By using the fluorescence labeling techniques and in-situ observations, we expected to learn how the polymer is associated with mineral phase at the early stage, and how it is excluded during solidification. Exclusion of this organic polymer greatly affects the composition of the precursor phase, which in turn leads to formation of “transition bars”, which can be seen in images showing both polymer-rich and polymer-poor domains (Chapter 3). By introducing Mg and/or Sr ion into the above crystallization system, we hoped to use impurities to stabilize the precursor phase and investigate its transformation. We found that the cooperative action of Mg-ion and polymer led to the formation of CaCO3 with both non-equilibrium morphology and non-equilibrium composition, with some features greatly resembling biominerals (Chapter 4 and 5). Both the organic matrix and soluble acidic macromolecules play very important roles in biomineralization. Chapter 6 shows the synergistic effect of a polymeric process-directing agent (acidic protein from nacre) and template (a polymeric hydrogel) in mimicking aragonite tablet formation in mollusk nacre.

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3 Chapter 7 shows the formation of crystals with complex morphology by mimicking the compartmentalization strategy found in nature and combining with the amorphous-crystalline transition in our in vitro model. In the presence of acidic polymer and combined with a porous hydrogel, the liquid precursor nanoparticles are molded inside, and transform to crystalline calcite, whose morphology is dictated by the moldthe microporous hydrogel. Chapter 8 describes the formation of amorphous calcium phosphate and crystalline hydroxyapatite. By comparing crystallization of CaP with and without polymer, we hope to observe features which may give some insight to CaP biomineralization. We also successfully micro-patterned nano-crystals using self-assembled monolayer systems combined with the soft lithography technique of microcontact printing. Chapter 9 describes the formation of CaP on an organic substrate and mineralization of CaP inside a porous hydrogel. The goal of our study was to progress towards novel organic-inorganic composites with potential applications for hard-tissue engineering. Chapter 10 describes the preparation and characterization of collagen-Hydroxyapatite nanocomposites using an amorphous precursor process. We developed a heavily mineralized collagen nanocomposite and the orientation of HAP nanocrystals inside the collagen matches that of bone, as proved by XRD, FEM, and TEM-ED. Such composites have great potential for application as bone-graft substitutes and tissue-engineering applications.

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CHAPTER 2 AN OVERVIEW OF BIOMINERALIZATION AND BIOMIMETICS Biomineralization refers to the processes by which organisms form inorganic minerals for functional applications. It is a process very common in the vast majority of organisms. The reason that organisms form such a variety of biominerals is mainly for adaptational advantage (10). For example, red deer’s antler is less mineralized than standard bone because its high toughness and anti-fracture properties enable the males to compete to fight each other to lead a herd; while the ear bone of a whale is heavily mineralized, its brittleness does not matter because it is hidden away and protected in the skull cavity (Table 2-1). Table 2-1. Three mechanical properties of various bones measured in tension and their functions for adaptational advantage E(GPa) (MPa) Function Red deer, mature antler 7.2 158 0.114 High toughness, good impact properties for fighting in rutting season Cow, femur 26.1 148 0.004 Fairly stiff and strong, standard bone Fin whale, ear bone 34.1 27 0.002 High brittleness is OK because it is inside the skull Reprinted with permission from Currey, J. D. Journal of Experimental Biology, 1999, 202, 3285-3294. (Table 1, Page 3287). There are over 60 types of biominerals, ranging from the widely observed calcium carbonate (CaCO3), which is found mainly in invertebrate structural components, to calcium phosphate, which is commonly found in vertebrate skeletal parts such as bone, the enamel, and dentin of teeth. These minerals exist in different location and perform different functions for organisms (Table 2-2). 4

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5 Table 2-2. Variety of biomineral types and biological systems Biogenic Minerals Formula Organism Biological Location Biological Function Calcium carbonates: (Calcite, Vaterite, Aragonite, Mg-Calcite, Amorphous) CaCO3 (Mg,Ca)CO3 CaCO3nH2O Many marine organisms, Aves, plants, mammals Shell, test, eye lens, crab cuticle, eggshells, leaves inner ear Exoskeleton, optical, mechanical strength, protection, gravity receptor, buoyancy device, Ca store, precursor Calcium phosphates: (Hydroxyapatite, Dahllite, Octacalcium Phosphate) Ca10(PO4)6(OH)2 Ca5(PO4,CO3)3(OH) Ca8H2(PO4)6 Vertebrates, mammals, fish, bivalves Bone, teeth, scales, gizzard plates, gills mitochondria Endoskeleton, ion store, cutting/grinding, protection, precursor Calcium oxylates: (Whewellite, Wheddellite) CaC2O4 H2O CaC2O4 2H2O Plants, fungi, mammals Leaves, hyphae, renal stones Protection/deterrent, Ca storage/removal, pathological Iron oxides: (Magnetite, Goethite, Lepidocrocite, Ferrihydrite) Fe3O4 a-FeOOH, g -FeOOH 5Fe2O3 9H2O Bacteria, chitons, tuna/salmon, mammals Intracellular, teeth, head, filaments, Ferritin protein Magnetotaxis, magnetic orientation, mechanical strength, iron storage Sulfates : (Gypsum, Celestite, barite) CaSO4 2H2O SrSO4 BaSO4 Jellyfish, acantharia, loxodes, chara Statoconia, cellular, intracellular, statoliths Gravity receptor, skeleton, gravity device/receptor Halides: (Flourite, Hieratite) CaF2 Mollusc, crustacean Gizzard plate, statocyst Crushing, gravity perception Sulfides: (Prite, Sphalerite, Wrtzite, Galena, Geigite) FeS2 ZnS, PbS Fe3S4 Thiopneutes Cell wall Sulfate reduction, ion removal Silicon oxides: (Slica) SiO2nH2O Diatoms, radiolaria, plants, etc. Cell wall, cellular, leaves Exoskeleton, skeleton, protection From http://gower.mse.ufl.edu/research.html , last accessed on Apr. 8, 2005.

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6 Several General Observations in Biomineralization The biomineralization field is a true multidisciplinary field and researchers can be found from many disciplines other than materials science, such as cell biology, orthopedics and dentistry, and structural biology. Years of study produced several observations or rules. Observation 1: Soluble acidic macromolecules are often associated with biominerals. Biominerals are actually composites. Besides the inorganic minerals, there are also two common kinds of macromolecules. The first are soluble acidic macromolecules, which are often rich in aspartic acid or glutamic acids, or phosphorylated serine and threonine residues, or polysaccharides rich in carboxylate groups or sulfated groups (10). These acidic macromolecules are found in almost all biologically-controlled mineralization systems, but not generally found in some species (such as the calcareous algae that forms aragonite crystals) under poorly controlled conditions, which is referred to as biologically-induced mineralization. One classic example of biologically-controlled mineralization is the nacre tablets of mollusk shells, which consist of aragonite tablets closely associated with aspartic acid-rich macromolecules (11). Although it is self-evident that these highly charged macromolecules may play an important role in biomineralization, little is known about their function and how they regulate biomineral formation. The main focus of our study is to use synthetic or natural proteins to mimic the role of these macromolecules with an in-vitro crystallization model that uses the Polymer-Induced Liquid-Precursor Process (explained more in detail later in section 1 of Chapter 3).

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7 Observation 2: A preformed organic matrix framework exists prior to mineralization. Before the mineralization process occurs in most skeletal parts, cells often synthesize organic extracellular matrix macromolecules. For example, in dentin biomineralization of rat incisors, type I collagen (a proteinaceous elastic hydrogel) is first secreted and self-assembled into fibrillar structures at some time within 24 hours; then acidic proteins are secreted by a different pathway, through the odontoblast process (a long cellular tube) directly into the site of mineralization. The preformed organic matrix framework is formed just ahead of the mineralization front, enabling both spatial and temporal control of mineral deposition (10). In abalone shells, the core of the organic matrix is composed of a layer of -chitin layered between “silk-like” glycineand alanine-rich proteins. The outer surfaces of the matrix are coated with hydrophilic acidic macromolecules (11). Nacre has a fracture toughness reportedly 3,000 times higher than its pure inorganic counterpart, aragonite. The key to this fracture toughness is the interlayering of organic matrix (which disrupts crack propagation), and the high degree of order over long distances (Figure 2-1A-D) (12). Similarly, in bone, which has a complex hierarchical structure, the basic level of structure is formed by mineralization of hydroxyapatite in a preformed matrix of collagen fibrils and other proteins (Figure 2-2) (13). In a broader view of the organic matrix, it can also include membrane-bound vesicles which act as a compartment for constraining mineral growth. Some biominerals are formed within intracellular vesicles (particularly the CaCO3 biominerals), while others are deposited on the extracellular surface, such as S-layers of bacteria. For the case of intracellular deposition vesicles, the mineralization of magnetite (Fe3O4) crystals takes

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8 place in special phospholipid vesicles of magnetotactic bacteria (noteprokaryotes normally do not have membrane-bound intracellular organelles). Similarly, the biomineralization of eukaryotes such as coccoliths and diatoms also takes place in vesicles, which compartmentalize the formation of CaCO3 and silica, respectively (14). It is traditionally believed that the organic matrix provides initial structural information for the inorganic mineral to nucleate on and grow outward in the desired manner, often referred to as an epitaxial template (although not strictly following the rules governing epitaxy as defined by traditional ceramic processing). However, in recent observations, it has been found that some biominerals result from transformation of an amorphous precursor. Because of the difficulty in discerning an amorphous phase in biominerals, it was only recently discovered that the amorphous precursor process is quite common in echinoderms, mollusks shells and so on (1,3,6). These observations support the concept that an organic matrix serves as a “mold” to direct mineral morphology, although interactions with the matrix will also influence the nucleation and growth of minerals via transformation of the precursor in contact with the organic matrix. One focus of our study has been to combine organic matrices with biomimetic polymers in our in-vitro crystallization model to examine the formation of CaCO3 and CaP crystals which form via an amorphous precursor phase. We believe that interactions between the two different types of biomacromolecular constituents can lead to different crystallization behaviors, and therefore give insight into the role of both organic matrix macromolecules and soluble acidic macromolecules in biomineralization.

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9 Observation 3: Cells direct the process of biomineralization. Almost all cases of biomineralization are the direct consequences of cellular activities. Cells control almost every stage of biomineralization (Table 2-3). In cases with certain dysfunctions of cells, disease happens. For example, the elderly tend to have brittle bones because their osteoblasts cannot produce enough bone to compensate for the osteoclast resorption of bone. Blood cells and bone-forming cells (osteoblasts) are made from the bone marrow by stem cells. The bone marrow is the soft spongy material in the center of bones. Large flat bones such as the pelvis and breast-bone (sternum) contain the most bone marrow. If bone marrow is not healthy, people may suffer leukemia or bone diseases. Health-related problems such as dental caries, over-calcification of cartilage, kidney stones, and osteoporosis are all direct results of unhealthy cell function. Table 2-3. Functions of cells in biomineralization Identified functions of cells in biomineralization Regulate ion transport Secrete proteins to modulate mineral nucleation and/or growth Secrete organic matrix to template or compartmentalize the mineral phase Produce enzymes to temporary modify the function of proteins Coordinate the rate of mineralization with the organisms’ growth rate Control location and type of mineral Adapted from source: 1) Boskey, A. L. Connective Tissue Research, 2003, 44, 5-9. 2) Lowenstam, H. A.; Weiner, S. On biomineralization, Oxford University Press: New York, Oxford 1989. As previously mentioned, biomineralization is a multidisciplinary field, and it is difficult to directly study how cells control all of these multiple functions. However, it is possible to examine the types of molecules (proteins, organic matrix, enzymes, growth hormone) that cells secrete to affect the mineralization process using an in-vitro crystallization model. This is why biomimetic crystallization systems can provide some understanding of biomineralization, as well as contribute to the development of

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10 biomimetic materials. By combining the organic matrix and acidic macromolecules in our mineralization model, we found several examples of how to make biomimetic materials (making laminating composites by mimicking nacre, making calcite crystals with complex morphology by mimicking sea urchin spine, and making collagen-hydroxyapatite nanocomposites by mimicking the nanostructure of bone). Table 2-4. Comparison between biominerals and their inorganic counterparts Biominerals Corresponding mineral formed inorganically Magnetite (in bacteria, vertebrates) Ferrihydrite precursor (transforms to magnetite in Chitin teeth) Elevated temperatures and pressures in igneous and metamorphic rocks Fluoride (mollusca, opossum, shrimps, gastropods) Amorphous ? fluoride (gastropods) Formed at high temperatures in hydrothermal systems and sedimentary rock from diagenesis Celestite (marine protoctists) Sea water is greatly undersaturated with respect to SrSO4 Opal (diatoms, silicoflagellates and radiolarians) Sea water is greatly undersaturated with opal Biogenic aragonite and calcite (in all depths and temperatures in the oceans, even in the deepest trenches) Only the upper portion of sea water is saturated sufficiently for aragonite and calcite Sr and Mg contents in CaCO3 of most mollusk shells (higher incorporation than equilibrium) Only low content of Mg and Sr is thermodynamically stable Stable C and O isotopic compositions in coral skeletons are out of equilibrium Equilibrium Amorphous calcium carbonate precursor (Echinoderms, mollusks) Quickly dissolves or transforms in water Amorphous calcium phosphate precursor (blue crab) Quickly dissolves or transforms in water Compiled from source: 1) Lowenstam, H. A.; Weiner, S. On biomineralization, Oxford University Press: New York, Oxford 1989. 2) Lowenstam, H. A. Minerals Formed by Organisms, Science, 1981, 211, 1126-1131.

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11 Comparison of Biominerals with Inorganically Forming Minerals Biominerals differ strikingly from their inorganic counterparts (Table 2-4). Many organisms can form biominerals (magnetite, fluoride, SiO2) from mild reaction conditions while their inorganic counterparts are synthesized at higher temperature or pressures (Table 2-4). Organisms also can synthesize biominerals from environments where the ionic constituents of the minerals are highly undersaturated in the surrounding water. Biominerals often contain impurities (e.g., Mg , Sr) and isotopic compositions (C and O) out of equilibrium with their environment. The differences between biogenic minerals and inorganically formed (geological) minerals have great significance. For example, determining the mineral origin of meteorites would make it possible to detect the existence of past extraterrestrial life. Funding of this work comes from the NASA exobiology program, because NASA is interested in developing biomarkers for extraterrestrial life. To do that, one must first have a good understanding of biomineralization mechanisms, which in turn can be used to identify different features of the mineral that can be definitively assigned to biological origin. Mechanisms of Biomineralization Classic Model of Nucleation Biominerals are produced in a heterogeneous environment at ambient temperature and pressure, and most often at neutral pH. There are around 60 types of minerals number, but they include both thermodynamically stable and kinetically driven phases. Traditional classical nucleation theory is based on a thermodynamic treatment of the interplay between energy gain through bulk crystallization and energy loss through formation of a surface. The organic matrix is thought to provide nucleation sites via

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12 molecular recognition (geometric, electrostatic and stereochemical matching) with the crystal faces of specific nuclei. When the organic matrix alone cannot control the crystal polymorph or orientation by in-vitro crystallization studies, it is explained that the acidic macromolecules are absorbed onto the organic matrix to provide nucleation sites within the biological system (15-18), which is missing within the model system. Aizenberg et al. demonstrate that by controlling the nucleation sites, the density and crystal orientation can be templated using a self-assembled-monolayer model patterned via a soft lithography technique (19-23). Amorphous Precursor Process: a Kinetically Driven Crystallization Process In recent years, amorphous precursors have been found in biominerals such as mollusk larvae, sea urchin spicule and spine (Figure 2-3), and ascidian (sponge) spicule. A review of the known distribution of biogenic amorphous calcium carbonate is well done by Addadi et al. (6). The amorphous precursor phase can transform to a crystalline phase, which is either thermodynamically stable or unstable. For example, the amorphous CaCO3 precursor can transform to single crystalline aragonite in the mollusk larvae, which is not the most stable phase, but is far more stable than ACC. In this case, the amorphous precursor forms first, then after compositional and structural modifications, it transforms to more stable phase. How this amorphous phase transforms to the crystalline phase is still enigmatic (i.e., by dissolution and recrystallization, or by a solid-state transformation). How many phases are crossed during this process depends on the solubility of the minerals and the free energy of activation of their inter-conversions. As shown in Figure 2-4, this kinetically driven crystallization cannot be readily explained by the classic model of nucleation and growth which often presumes an organized nucleus.

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13 Researchers have found that these amorphous biominerals often contain a large amount of acidic macromolecules, as well as certain amounts of Mg or PO43ions. So amorphous precursor phases can possibly be stabilized by such ion impurities as Mg2+, PO43-. In the case of the sea urchin spicule, the precursor phase has been shown to be stabilized by enclosing the amorphous phase in an impermeable sheath of organic macromolecules, such as polysaccharides, and proteins rich in glutamic acid, theronine and serine (7,24-26). Purpose of Our Study Our recently discovery of the Polymer-Induced Liquid-Precursor (PILP) process may have great relevance to the biomineralization mechanisms associated with amorphous precursor phases. We find that the crystallization of calcium carbonate or calcium phosphate in the presence of a small amount of acidic biomimetic polymer leads to the formation of a liquid-phase mineral precursor, which grows from nanoscopic scale and coalesces to larger structures. Solidification and transformation then occurs, resulting in formation of mosaic single-crystalline film patches (9) and other non-equilibrium crystal morphologies. In this dissertation, we hope to study how polymer (acidic biomimetic polymer, natural proteins from nacre) and impurities (Mg and Sr) lead to the formation of an amorphous liquid-phase precursor, and how they influence the transformation to the crystalline phases. CaCO3 and CaP are two main minerals studied. By combining organic substrates (e.g., PHEMA, patterned self-assembled monolayers), or compartments (e.g, microporous hydrogel, collagen nano-fibrous sponges), we are able to form novel biomimetic mineral structure or organic-inorganic composites for biomedical or structural applications.

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14 We hope to establish the relevance the PILP process to biomineralization, and further optimize this process to develop novel biomimetic materials. Biomimetics and Materials Science Biomineralization is a process very common in vast majority of organisms since many organisms use hard tissues for skeletal or protective functions. From years of studies with different models such as bone, sea shells, enamel, and so on, some of the chemistry, structure, properties, functions and controlling mechanisms in biomineralization have gradually and continually been revealed. Man can learn from such processes for the design and synthesis of novel materials, which leads to either biomimetic processing or fabrication of biomimetic materials. Table 2-5 summarizes some popular biomimetic topics in materials science which result from learning the chemistry, structure, function, properties and control mechanisms in biomineralization. As seen from the Table, biomimetics driven research is truly multi-disciplinary and holds great promise to synthesize novel materials for a variety ranges of applications. Several advances have already been made. For example, by considering the chemical composition of bone, Hench invented P2O5-containing, silica based bioactive glass for bone repair (27,28). In our group (as discussed in Chapter 10 of this dissertation), we developed a intrafibrillar mineralized collagen-HA bone analogue material by using the PILP process to mimic the amorphous-crystalline controlling mechanism which we hypothesize is active in bone formation. Aizenberg et al. try to learn from the natural example of the lenses of brittlestars to build large artificial lenses based on CaCO3 by using a molecular recognition strategy (29). Several groups are trying to synthesize strong and tough organic-inorganic composites by mimicking the nanostructures from bone and sea shells for applications such as ceramic armor. The

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15 whole biomimetics strategy can also be extended to form other inorganic minerals/crystals such as semiconductor crystals such as CdSe, ZnS, CdS (known as AII-BVI compounds), and possibly to many cationic-anionic systems. Table 2-5. A summary of biomineralization and biomimetics-related research Model Biomineralization concepts, chemistry, structure, properties, functions, control mechanisms Examples of biomimetics in materials science All Molecular recognition in biomineralization system Biosensors: Organic surface to recognize bacteria, viruses, or even drugs; Crystal engineering; Organizing inorganic crystals using monolayers All Enzymes, growth factors New catalyst for synthesis of material at mild conditions All Protein synthesis, Gene control Man-made gene transfer into “bacteria factory” to produce biopolymer with monomolecular weight and super properties; Gene therapy All Cell control Stem cell to grow artificial organs; "Living composites" for bone graft substitutes Diatom, bacteria, Cocolith etc Compartmentalization strategy Supramolecular protein cages or template (e.g.,ferritin, S-layer) in the synthesis of nano-phase inorganic materials; Reverse micelles synthesis, Phospholipid vesicles synthesis Bone Stiffness and toughness, Composition (collagen-HA-protein), Hierarchical structure, Design bone analogue material Nacre, Enamel, sea urchin teeth Strong and tough Hierarchical structure extended to long distance and judicial placement of organic, laminar organic-inorganic structure, Plywood structure Sea urchin spine Strong and tough Occluding protein to toughen inorganic Fish scale Tough Multi-oriented lamellar structure Ossified tendon Anisotropic mechanical properties Strong elastic Individual fibers bundles Sea urchin spine, mollusk shell Amorphous-crystalline transition mechanism PILP process to make biomimetic materials Several key advantages of biomimetic synthesis can be generalized as follows: Mild conditions

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16 Biomimetic processing generally takes place from aqueous solution and temperatures below 100C. No toxic or harsh solvents or chemicals are used. Such processes are in line with what is called “green chemistry” today. Nano-scale processing Biomineralization is known to build materials from a “bottom-up” approach. Self-assembly and material synthesis occur from nano-scale and are then extended to larger dimensions through hierarchical assembly. This technique holds great promise for synthesizing materials with novel mechanical, optical, magnetic and electronic properties Multi-functional or multi-plastic, tailored properties A known example is the three kinds of bone (mature antler of red deer, femur of cow, ear bone of fin whale) mentioned earlier. By controlling the different degree of mineralization, the material-bone can have different strength and toughness. The abundance of chemistry (functional groups, impurities, protein sequence and structure, catalysts), structure, composition and strategically control can leads to a variety of structures with tailored properties (30).

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17 A B C D Figure 2-1. Structure of nacre. A) Optical image of nacre’s inner layers (Atrina). B) SEM image of the cross-section showing the layering structure. C) Optical image of nacre’s inner layers (Geukensia). D) Corresponding SEM images showing layering structure. Bar=1 m.

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18 Figure 2-2. Seven levels of hierarchical structure of mammalian bone. Level 1: Major components of bone, type-I collagen and hydroxyapatite (HAP). Note the native 64 nm banding pattern of type-I collagen. Bars = 100 nm (right). Level 2: Intrafibrillar mineralization of type-I collagen with nanoscopic platy hydroxyapatite crystals. Bar = 200 nm. Level 3: Fibrillar array of intrafibrillarly mineralized collagen. Note how the banding pattern of the collagen fibrils aligns across adjacent fibrils, suggesting that the collagen is highly aligned before mineralization. Bar = 200 nm. Level 4:Fibrillar array patterns of mineralized collagen, oriented to account for mechanical forces within bone (e.g., oriented tangentially around holes to account for increased stress concentration.) Level 5: Cylindrical motifs, osteons, which serve as structural members of bone. Note the Haversian canal in the middle of the osteon, used to supply blood and cells to bone tissue. Level 6: Spongy vs compact bone. Spongy bone is a continuous network of mineralized trabeculae, resembling a sponge, observed at the ends of bones to take up shock. Compact bone is comprised of osteons packed tightly to provide mechanical strength, and is observed on the outer case of bone and in the shafts of long bones. Level 7: This level consists of a range of all the bones in the body (e.g., flat and long bones). Reprinted, with permission, from Weiner S and Wagner H., The Annual Review of Materials Science 28 , 1998, by Annual Reviews www.annualreviews.org . (Figure 1, Page 273).

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19 A B 1m m C Figure 2-3. Structure of sea urchin spine. A) Image of sea urchin spines. B) SEM showing the cross-section of sea urchin (Arabacia Punctulata) spine. C) SEM showing the microporous structure and the conchoidal fracture surface (inserted).

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20 Figure 2-4. Crystallization pathways under thermodynamic and kinetic control. Whether a system follows a one-step route to the final mineral phase (pathway A) or proceeds by sequential precipitation (pathway B), depends on the free energy of activation (G) associated with nucleation (n), growth (g), and phase transformation (t). Amorphous phases are common under kinetic conditions. Reprinted, with permission, from Colfen, H. and Mann, S., Angewandte Chemie-International Edition, 42, 2003, Higher-order organization by mesoscale self-assembly and transformation of hybrid nanostructures. (Figure 1, Page 2353).

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CHAPTER 3 NON-EQUILIBRIUM CRYSTAL MORPHOLOGY AND POLYMER-MINERAL ASSOCIATION Introduction Biominerals formed in nature are often found to have non-equilibrium composition and morphologies. In the system of CaCO3, three kinds of polymorphs are common: vaterite, aragonite and calcite, with decreasing order of solubility, and calcite being the most thermodynamically stable phase at ambient conditions. CaCO3 biominerals are very different from CaCO3 crystals formed from inorganic origin (e.g., geological minerals). First, CaCO3 biominerals often include kinetically favorable but thermodynamically unfavorable phases. For example, the nacreous layer of mollusk shells consists of aragonite, yet aragonite is not the most thermodynamically stable phase of the three CaCO3 polymorphs. Geological CaCO3 favors the thermodynamically stable phase-calcite, when precipitated under ambient conditions. Aragonite is only common in many modern carbonate sediments as shells and crystals that precipitated from sea water. Second, biominerals formed in nature often form complex non-equilibrium morphologies. For example, geological calcite often shows the typical rhombohedral habit of calcite (Figure 3-1A), while the tooth of a sea urchin is composed of calcite rods (Figure 3-1B). Inorganic precipitated aragonite often is in the shape of needles-comprising spherulitic dumbbells (Figure 3-1C). However, the aragonite in mollusk nacre has adopted the morphology of thin plates (Figure 3-1D). 21

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22 The remarkable difference between biominerals and mineral formed from inorganic origin intrigues researchers who seek to find the mechanisms and principles of biomineralization. In particular, materials scientists wish to use these mechanisms to form bio-inspired materials with novel optical, mechanical, and magnetic properties. On the other hand, some researchers are interested in understanding biomineralization because different features of biominerals can be used to establish biomarkers to distinguish the origin of a mineral (i.e., whether it is of geological or biogenic origin). For example, scientists have argued that the Martian meteorite ALH 84001 may reveal information about past microbial life on mars (31), but to fully make this claim, they need to establish features specifically produced in biominerals which can be used as potential biomarkers to identify whether it is of inorganic-origin or biological-origin (32). For example, they can examine the morphology (texture, habit, crystal size), chemical composition (especially impurity incorporation), organization and structural perfection of the minerals within the meteorite and compare it to minerals (carbonaceous or iron oxides) formed from biological and inorganic origin. The ability to establish biomarkers for biologically-produced minerals requires an understanding of the processes involved in biomineralization. Our recent discovery of the Polymer-Induced Liquid-Precursor (PILP) process may have important implications in this regard. Polymer-Induced Liquid-Precursor (PILP) Process Polyaspartic acid is used in our studies to mimic the acidic proteins typically associated with calcium carbonate growth in biominerals (10,33,34). We have shown that this soluble acidic polymer can induce an amorphous liquid-phase mineral precursor,

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23 which then gradually transforms into mineral crystals while retaining the non-equilibrium morphology of the precursor phase. This process is induced at room or low temperature from aqueous conditions similar to the physiological environment for biominerals. We propose that the acidic polymer acts similar to the soluble acidic macromolecules in biominerals since crystals with non-equilibrium morphology are formed, which is the hallmark of biominerals. Thus, it is proposed that the PILP process may be relevant to biomineralization at its most fundamental level, enabling crystals to be formed with a wide variety of complex morphologies. . A detailed review of this process is described by Gower (9,35), in which the first non-equilibrium morphology that was discovered and examined in detail was the deposition of thin ( 500 nm) films of CaCO3. In the next few sections of this chapter, several more non-equilibrium morphologies of CaCO3 are described which have been produced and studied more recently, and the association of CaCO3 with polymer is discussed. Materials and Methods Polymer-Induced Liquid-Precursor (PILP) Crystallization The PILP process was performed similarly as that described earlier by our group (9), which consists of a modified version of the vapor diffusion technique by Addadi et al. (36). The polymer used was poly-(, )-D,L –aspartic acid sodium salt (Mw=8,600 unless specified, Sigma), at concentrations ranging from 2-667 g/ml. Calcium chloride dihydrate (CaCl2H2O, 99.99%, Aldrich) stock solutions were prepared as 36 mM using double-distilled deionized water. The crystallization dishes used for the reaction were filled to make up to 3 ml each with double-distilled deionized water. The resultant

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24 solutions contained 12 mM or 9 mM Calcium chloride by diluting the stock CaCl2 solution and different polymer concentrations (2-100 g/ml). Each solution was allowed to crystallize at room temperature (20C) for three days in the presence of vaporized ammonium carbonate (which slowly decomposes to CO2 and NH3). The films or precipitates formed on glass microscope coverslips were taken out from each solution, washed with deionized water and alcohol, and then dried for later examination. Fluorescence Labeling of Polymer FITC, fluorescein isothiocyanate (C21H11NO5S, Mw=389.4, Sigma) was used to label the Polyaspartic acid, the detailed procedure is as follows: Preparation of PBS buffer pH = 7 ~ 9, 10 ~ 20 mM For the preparation of PBS buffer, 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g of KH2PO4 were dissolved in 800 ml distilled water. The pH of the solution was then adjusted to 7.4 with diluted HCl. Ultra-pure H2O was then added to the above solution to bring the volume up to 1 liter. Preparation of 0.1 M sodium carbonate buffer For the preparation of 0.1 M sodium carbonate buffer, First 0.2 M sodium carbonate solution was prepared by dissolving 21.2 g Na2CO3 in 1 liter H2O, then 0.2 M sodium bicarbonate was prepared by dissolving 16.8 g NaHCO3 in 1 liter H2O. Finally 8 ml of 0.2 M sodium carbonate, 17 ml of 0.2M sodium bicarbonate (NaHCO3), and 25 ml of water were mixed. The pH of this buffer solution should be around 9.6. Then pH of this solution was adjusted between 9.1-8.3 by adding drops of 0.01 N HCl solution. Labeling reaction

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25 Two solutions were prepared by dissolving 20 mg of Polymer in 2 ml 0.1 M Sodium carbonate buffer and by dissolving 10 mg of FITC in 1 ml DMSO. After that, 200 l of the above FITC solution was slowly added to the above polymer solution and incubated in the dark at 5C for 8hr. In order to remove the unbound FITC, a Centricon YM-3 centrifuge tube (Mw cutoff=3,000) was used to filter the labeled solution. The retentate conjugate containing the FITC-labeled polymer was stored in a lightproof container in the fridge at 4C. Polarized Optical Microscope (POM) and Fluorescence Microscopy The cover slip from each sample was examined using an Olympus BX60 polarized optical microscope in transmission mode, using crossed polars and an optional first-order red (gypsum) wave plate. When being used for fluorescence study, a mercury arc lamp was used as a light source and a fluorescence mode NIB filter was chosen. All images from the microscope were acquired digitally (MTI 3CCD camera) by Scion image software (Scion Corporation). The first-order gypsum plate was used primarily to distinguish amorphous and crystalline phases, in which amorphous phases will appear magenta (the same color as the background) while crystalline phases will exhibit birefringence and extinction patterns. A calibrated scale bar was put on the digital images using Photoshop software. Scanning Election Microscopy (SEM) and X-ray Energy Dispersive Spectroscopy (EDS) Selected samples were gold coated for SEM microscopy on a JEOL 6400 SEM instrument equipped with an EDS detector system. The EDS detector includes an ultra-thin window, which permits analysis of elements from Boron to Uranium.

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26 Results Equilibrium Morphology of Calcite As mentioned earlier, the major goal of this chapter is to compare the morphology of mineral products from the PILP process to those formed without any presence of polymer. Another goal is to investigate the polymer-mineral association throughout the reaction using in situ fluorescence-labeling techniques. Figure 3-2A shows the typical equilibrium morphology of calcite formed without any presence of polymer additive, but with the addition of a fluorescence-labeling agent (FITC). The calcite as formed still adopts the typical rhombohedral habit found in calcite of inorganic origin. Figure 3-2B is a fluorescence image of the calcite formed in the presence of FITC, which clearly shows the FITC alone does not affect the crystal growth of calcite, establishing that the effect of polymer will be clearly distinguished in the subsequent fluorescence study. In-situ Observation of PILP Process Direct observation of the PILP process was done by in-situ examination through a thin glass chamber similar to that designed by Gower (9), which allows the crystallizing dish to fit under the ultra-long-working-distance objectives of an optical microscope. A previous study by the group had shown that the liquid-precursor droplets grew from nano size to micrometer size (37,38). Because nano-size droplets are beyond the resolution of an optical microscope, the pictures shown in Figure 3-3 only reveal micrometer-size amorphous phase droplets that had grown during in-situ observation after 1 day (Figure 3-3A). Depending on the setup of the in-situ chamber, the droplets are quite stable (i.e., do not dissolve) and grow in size through aggregation and coalescence (Figure 3-3B & C). The precursor droplets gradually solidify (Figure 3-3C & D), and some of them

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27 transform into crystalline film patches so that both amorphous phase and crystalline phase coexists (Figure 3-3E & F). Figure 3-4 shows all the typical film morphologies formed by the PILP process within the in-situ chamber, which clearly differ from the rhombohedral habit of crystals formed without any polymer addition. Figure 3-4A shows a sector-pattern texture of a distorted but single-crystalline patch of film which was examined with cross-polarized light using a gypsum wave-plate (Notethe single-crystalline nature is determined from the uniform birefringence color and extinction upon rotation of the sample). The dark lines have been referred to as transition bars in Gower’s prior work because they are only seen during the amorphous to crystalline transformation, and ultimately the dark transition bars becomes birefringent as the crystallization proceeds. Using only cross-polarized light, the transition bars are seen as white and black striations (Figure 3-4B). The periodic texture of the film could either result from “wrinkles” caused by shrinkage of the film during solidification (due to water loss), or from smooth films which have regions of delayed crystallizability, which might result from the exclusion of polymer to form a polymer-rich CaCO3 phase and a polymer-poor CaCO3 phase. Crystalline films with spherulitic texture also form (Figure 3-4C). This polycrystalline growth of CaCO3 by the PILP process indicates that a dynamically heterogeneous environment is present in the crystallizing media. The small precursor droplets/clusters and the solidified phase apparently have different mobility and interfacial energy, so that single crystal growth is switched to polycrystalline growth, which is often caused by introducing foreign particles or by reducing the orientational mobility of molecular constituents as they join with the crystal (39). Figure 3-4D shows the coexistence of amorphous film and single-crystalline

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28 film. Figure 3-4E shows transition bar-patterns in some single-crystalline films that exhibit a more uniform rhombohedral symmetry, as seen by the well-defined transition bars, than the distorted tablet as shown in Figure 3-4A. After 15 days, this transition bars were diminished (Figure 3-4F). In general, in-situ observation of the PILP process gives very informative and interesting morphology of crystals. The thin film morphology is distinctly different from the rhombohedral calcite formed without the PILP process. The single crystalline films typically contain transition bars seen only during the amorphous to crystalline transformation, which indicates the heterogeneity in the crystallinity during the solidification and transformation of PILP product. Since not all films containing transition bars appear to contain physical corrugations (wrinkles), it was considered that the bars may be due to the exclusion of polymer into diffusion-limited zones during the solidification and crystallization process, leading to crystallographically defined polymer enriched zones within the final deposited film. To address this issue, I examined the reaction in situ with fluorescein-labeled polymer. Polymer-Mineral Association in the PILP Process by Fluorescence Study In order to study how polymer is distributed in the PILP product, the polyaspartic acid was coupled with a fluorescence dye (FITC) so that the polymer will appear bright yellow-green wherever present when being observed under fluorescence microscope. The dye is bound to the amine functional groups of polyaspartate, but does not appear to disturb the crystallization reaction since the mineral products appear similar. Figure 3-5 shows polarized microscope images compared with corresponding fluorescence microscope images of the same regions. In the spherulitic film (Figure 3-5A), the film appears to originate from a core which is thicker than the surrounding film,

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29 almost to the extent of starting to form a 3-dimensional spherulite. The core region of the film is clearly enriched in polymer as shown in Figure 3-5B, and seemingly more so than just the simple additivity of fluorescence in the thicker region, suggesting that thicker regions also may have more difficulty in eliminating the polymer during crystallization. Non-perfect spherulitic film also forms as shown in Figure 3-5C, and the corresponding fluorescence image again shows polymer enrichment in the core region (Figure 3-5D). On the spherulitic film, there are also some condensed particles, which appear to be polymer enriched (Figure 3-5E & F). One apparent conclusion from this study is that polymer is closely associated with the CaCO3 phase, and not just adsorbing to all surfaces, because all the mineral films appear bright (and particularly in the thicker regions), while the other region of the glass, without CaCO3 film, appear dark in fluorescence microscope. As the polymer concentration was raised to 50 g/ml in the starting crystallization solution, more films formed on the glass slides (relative to crystal aggregates). Figure 3-6A show the typical film morphology under the polarized microscope with a gypsum wavelength plate. A mosaic film formed on the glass slides with some particles embedded in the film. These particles follow the same crystallographic orientation as the background film because they exhibit the same interference color (and extinction direction) using the wavelength plate. The mosaic film contains both amorphous and crystalline regions (Figure 3-6B). The polymer is quite uniformly distributed in the background film while the embedded particles are much more bright under the fluorescence microscope (Figure 3-6 D). It seems that polymer is more enriched in the particles than in the films. Again, comparison of the optical image and fluorescence

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30 image indicates the polymer is closely associated with CaCO3 phase, particularly in the more solidified droplets that didn’t coalesce with the films as well, while the dark region in the fluorescence image corresponds to the area containing no CaCO3 (blank in Figure 3-6C). Figure 3-7 gives a similar comparison of polarized images and fluorescence images taken in situ midway in the reaction during the transformation. One noticeable feature is the concentric ring pattern in the optical image (Figure 3-7A & B) that also was shown clearly in the fluorescence image (Figure 3-7C), indicating that polymer is excluded from the crystalline region during the amorphous to crystalline transition in the PILP process. Similar features of polymer-exclusion are also shown by comparison of the optical and fluorescence images in Figure 3-7D-G). A striking polymer-exclusion phenomena occurs clearly at higher polymer concentration when the CaCO3 phase crystallizes into core-shell particles (Figure 3-8A), where the polymer is enriched in the shell region (Figure 3-8B). Apparently the crystallization attempts to exclude the polymeric impurity, but as the material solidifies, the diffusion becomes more limited so that it becomes entrapped in a shell region. Other Non-equilibrium Morphologies Produced by the PILP Process When using higher molecular weight Poly(aspartic acid), several unusual morphology are also observed. Cones composed of assembled nanofibers are formed (Figure 3-9A & B), which resemble the BaSO4/BaCrO4 cone structure formed in the presence of Polyacrylate and block copolymers (40-42). The cone structures are explained by Colfen et al. to originate from single sheets of nanofilaments during the very early stage by edge-to-edge attraction and fusion, followed by splaying as a result of lateral packing forces (43). It is worth doing more research in this area to determine how

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31 the precursor nanoparticles of PILP phase can be fused and lead to such defect-free filaments, and then packed into such hierarchal structures. Similar fibers of CaCO3 also formed in the length of 50 m (Figure 3-9C). Strands of beads of CaCO3 are also formed (Figure 3-9D), which probably results from the coalesced PILP droplets. Prickly fibrous extensions from the thin CaCO3 films may result from the secondary particle fusion via Solution-Precursor-Solid (SPS) mechanism described by colleague Olszta (Figure 3-9E & F) (44). Figure 3-9G shows clearly the “molten morphology” of PILP products, where PILP droplets are clearly seen in the curved mineral fibers emanating from the CaCO3 aggregates. EDS was performed to confirm the composition of these unusual structures (Figure 3-9H). Discussion Polymer Exclusion in the PILP process Our fluorescence study gives very interesting results about polymer-mineral interaction in the PILP process. At the very early stage of the PILP process, the polymer is mainly bound with the Ca2+ in the solution (A rough calculation shows that there are still enough free Ca2+ in the solution when CaCl2 concentration is 12 mM and Pasp (Mw = 8,000) concentration is 20 g/ml. At least 2808 g/ml of Pasp is need to fully chelate with the Ca2+, even if we assume that all of the Pasp is deproteinated, and two carboxylate groups will bind with one Ca2+ cation). The polymer-Ca2+ complex is very stable if there is no CO32in the solution. However, as more CO32diffuses into the solution and builds up the supersaturation, the polymer-Ca2+ complex presumably will interact with the newly formed CaCO3 colloidal particles and induces the liquid-like precursor particles. As the supersaturation becomes higher, more precursor particles

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32 form, which then aggregate and coalesce together. This coalescence and growth of precursor phase is also favorable through reduction in surface energy. The polymer also interacts within the CaCO3 colloidal phase by inhibiting it from forming classical nuclei for the rhombohedral calcite crystals. As the amorphous liquid-like precursor phase coalesces into films, the film will gradually transform (crystallize) as time goes on due to the metastability of the precursor phase. However, the inhibitory effect of the polymer is lessened as it is gradually excluded from the precursor phase during this transformation. The polymer is excluded into certain regions to become intracrystalline phase as well as excluded out of the crystals and surrounding precursor phase to become intercrystalline. Sometimes the polymer may be excluded onto the outer surface of the crystals so that the crystalline film may appear bright under fluorescence microscope (Figure 3-3F). During the amorphous-crystalline transition, transition bars are observed due to polymer exclusion into diffusion-limited zones, as can be seen by the polymer-enriched and polymer-poor zones in the fluorescence image. The fact that similarly spaced transition bars appear in both spherulitic and single-crystalline mineral films suggests that the spacing of these bars are due to diffusional limitations, in which polymer ultimately gets entrapped when the film becomes more solidified. It also suggests that there may be a kinetically favorable surface upon which the polymer will be excluded, which is a result of fast growth directions of the crystal (such as the radial direction in spherulites, or specific crystallographic directions in the single-crystalline rhombs), rather than specific (e.g., stereoselective) interactions. In each case, the polymer is closely associated with the mineral phase and actually becomes occluded within the mineral phase.

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33 These studies may shed some light on reports in the literature regarding selective protein occlusion in biominerals. In-vitro crystallization studies using sea urchin spine proteins suggests that the proteins specifically bind to different crystal planes by the observation that new (10l) faces formed. Proteins were present within the final crystals as determined by quantitative amino acid analysis (45). In support of this claim, synchrotron x-ray diffraction studies of biological specimens showed anisotropic defect textures of the urchin spine suggesting an anisotropic distribution of proteins within the biomineral. We think this anisotropic protein incorporation within biominerals may actually result from the anisotropic polymer exclusion during the precursor transformation stage. Before this stage, the polymer is already occluded within the amorphous precursor. Then during this precursor transformation stage, the polymer is gradually excluded onto the newly formed crystalline plane, grain boundaries, and amorphous-crystalline interface, as the crystallization tries to exclude organic impurities to form more perfect crystals. During this restructuring period, the polymer exclusion may be affected by the kinetics of crystal growth. For example the fast growth plane may end up with less amount of polymer exclusion along those specific crystallographic planes. Polymer exclusion may also be affected by the difference in fluidity (diffusion kinetics) between the precursor and crystalline phase, so finally the polymer distribution will be different on each crystalline plane. We believe this difference of occluded polymer on each crystalline plane maynot result form the selective adsorption of macromolecules on crystal planes during the crystallization process, as suggested by Aizenberg et al. (45). This concept has been at the foundation of biomineral and biomimetics research, but with the recent evidence of

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34 the spine and other biominerals being formed via a transient amorphous precursor, this stereoselective adsorption hypothesis seems less relevant. It rather results from the kinetic transformation and crystal growth and fluidity of the precursor phase. This polymer-exclusion phenomenon might be expected for any crystallization process, but the polymer-occlusion within minerals, and in particular the specific zonal occlusion patterns as seen in the final products produced by the PILP process, are highly relevant to the mechanism(s) of biomineral formation. An example is sea urchin spine, whose shape and crystallographic texture cannot readily be explained by classic nucleation and growth mechanism, but exhibits features to suggest that the PILP process may lie at the foundation of such biomineralization processes. Conclusion The above studies demonstrate distinct features of the Polymer-Induced Liquid-Precursor process as compared to the traditional crystallization of CaCO3 (without polymer additive) in terms of formation mechanism, morphology and hierarchical structure of crystals. Several conclusions can be drawn: At some point of supersaturation level, the precursor phase separates from the crystallizing media in the forms of nano-meter sized droplets. These droplets gradually grow in size, coalescing to larger structures until they become more “viscous” and subsequently vitrified. The polymer plays a very important role in stabilizing this droplet-like precursor phase, and yet becomes excluded as the phase tries to crystallize and finally become occluded in the CaCO3 phase. The acidic biomimetic polymer induced a liquid-like amorphous precursor which contains polymer, inorganic ions, Ca and CO3 ions, and H2O. While this has been demonstrated by prior work, the simultaneous observation of the amorphous to crystalline transformation in the in-situ chamber set up shows phase segregation of the polymer into polymer-rich and polymer-poor regions during the transformation. The amorphous precursor gradually transforms to crystalline CaCO3 product. Depending upon the concentration of the polymer and crystallization kinetics, the phase transformation products can yield different forms: mosaic single crystalline films, polycrystalline spherulitic films, hierarchical structures such as cones, fibers,

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35 and condensed “molten morphology” aggregates. At the higher polymer concentration, large particles that transform into spherulites consisting of inorganic cores and organic enriched shells are produced, which we believe are a result of strong attractive interactions between droplets leading to a more pronounced aggregation tendency. This liquid precursor transformation mechanism shown by the PILP process resembles several biomineral formation mechanisms such as sea urchin spine, mollusk shell formation, cocolith etc.; in all these cases they share similar features: an amorphous precursor, occluded macromolecules that are anisotopically located on specific crystallographic planes, transformation to crystalline phase, and non-equilibrium morphology.

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36 A B C D Figure 3-1. Comparison of morphology of typical mineral from inorganic origin and biological origin. A) Geological calcite ( http://www.themysticeye.com/info/calcite.htm , last accessed Feb 06, 2005). B) The keel region a sea urchin tooth (Arbacia tribuloides) contains calcite “rods”, 5-7 m in diameter, embedded in an amorphous CaCO3 matrix. Bar = 20 m. C) Inorganically precipitated aragonite (Aragonite was prepared by growth from minute amounts of SrCO3 nuclei at a Mg/Ca = 5, room temperature). Bar = 50 m. D) Thin plates of aragonite in Atrina nacre. Bar = 10 m.

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37 A B Figure 3-2. CaCO3 crystals grown in the presence of FITC. A) Polarized optical micrograph of the typical equilibrium morphology of calcite without any addition of polymer, but only FITC as additive (CaCl2 20mM, [FITC] ~ 7 mg/ml, 3 days, vapor diffusion). B) Fluorescence micrograph of same crystals demonstrates that the calcite as formed has almost no fluorescence emission, indicating FITC alone does not adsorb strongly to calcite nor does it affect the CaCO3 crystal growth.

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38 A B C D E F Figure 3-3. Polarized optical image and fluorescence image of the PILP process observed in the bottom of the in-situ crystallization chamber taken at different times of crystallization. (Condition: CaCl2 20mM, Pasp-FITC ~ 20 mg/ml, room temperature) A) Droplets of micro-meter size can be seen after one day of reaction. B) C) Amorphous liquid-precursor droplets were still stable after three days. D) Fluorescence image of the droplets as shown in C), indicate that polymer is enriched in droplets and can be distinguished from the background. E) The amorphous liquid-phase precursor coalesces and transforms into crystalline film by four days. F) Fluorescence image as shown in E) at same magnification, showing excess polymer in the crystalline patches, which may be thicker and trap the polymer. (all bars = 100 m).

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39 A B C D E F Figure 3-4. Polarized optical micrographs of crystals on the top slide of the in-situ crystallization chamber. (Condition: 20mM CaCl2 , 20 g/ml Polyapartic acid (Mw=6,200), room temperature). A) A single crystal patch which is likely a distorted calcite crystals (3 days crystallization) can be distinguished as single-crystalline due to the uniform extinction pattern upon rotation of sample stage. B) Optical image of A) but without the red-1 wavelength plate shows a variable thickness in this tablet as varying shades of gray. C) A uniform film with contiguous spherulitic patches and one faceted patch of single-crystalline calcite. D) Transformation of a holey amorphous film into a single-crystalline patch. Note how the crystallization path follows the outline of the precursor phase. E) A well faceted calcite tablet showing pronounced transition bars (without polarized light) F) The same patch of film as in E has expanded in crystallinity, while the transition bars are diminishing after 15 days of growth.

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40 A B C D E F Figure 3-5. Optical image (left) and fluorescence image (right) of crystallites observed from the in-situ chamber (Condition: 20 mM CaCl2, FITC-labeled Pasp 6 l (final concentration~20 g/ml), 2 days at room temperature). A) Polarized optical image with red-1 wavelength plate. B) Fluorescence image as shown in A). C) Polarized optical image without red-1 wavelength plate. D) Corresponding fluorescence image. E) Polarized optical image without red-1 wavelength plate. F) Corresponding fluorescence image.

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41 A B C D Figure 3-6. Image of typical CaCO3 films formed by the PILP process (condition: 20 mM CaCl2, FITC-labeled Pasp = 60 l (Final [Pasp] ~ 50 g/ml in 3 ml total solution, 3 days). A) With the red-1 wavelength plate, the magenta color indicates amorphous film (or crystals oriented in the extinct direction or along their isotropic axis) while other retardation colors indicate crystalline film. B) Without the red-1 wavelength plate, the black areas correspond to the magenta areas with the red-1 waveplateC) Aon-polarized optical image showing that the film is quite continuous but bumpy due to poor coalescence of partially solidified droplets. D) Fluorescence image showing that polymer is closely associated with CaCO3, but isparticularly enriched in the less fluidiclate-stage PILP droplets that didn’t coalesce well with the film.

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42 A D B E C F Figure 3-7. Optical images of thin films showing the polymer-exclusion phenomenon in the PILP process. (Condition: CaCl2 20mM, FITC-Pasp ~=111 g/ml, 2 days crystallization, room temperature). A) and D) POM image with red-1 plate. B and E) POM image w/o red-1 plate. C) and F) Fluorescence images of same films A)-C): scale bars = 20 m; D)-F) scale bars = 50m. G) Fluorescence images image of F) a higher magnification. Scale bar =20 m.

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43 G Figure 3-7. continued

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44 A B Figure 3-8. CaCO3 crystals grown at high polymer concentration. (Condition: CaCl2, 20mM; FITC-Pasp = 666 g/ml, 2 days, room temperature, vapor diffusion). A) POM image of core-shell particles. B) Fluorescence image of the core-shell particles showing pronounced polymer enrichment in the shell.

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45 B A 20 m 5 m D C 50 m 50 m F E 100 m 20 m Figure 3-9. Scanning electron micrographs of some of the unusual CaCO3 morphologies induced by a higher molecular weight poly(aspartic acid) (Mw=35,400) under the following conditions: A -B) Nanofibrous cones of vaterite. C) fibrous CaCO3, produced with 9mM CaCl2, 28 g/ml PolyAsp; room temperature (23C). D) Strands of beads of CaCO3 (presumably coalesced PILP droplets); E –F) “prickly” fibrous extensions emanating from CaCO3 films, produced at 9 mM CaCl2, 24 g/ml PolyAsp; low temperature (4C); G) Fibrous extruded from aggregates of CaCO3 crystals. H) EDS spectrum shows the Ca and O in CaCO3.

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46 20 m G H Figure 3-9. continued.

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CHAPTER 4 MAGNESIUM -ION IMPURITY INCORPORATION BY THE PILP PROCESS Following the discussion of the non-equilibrium morphology of the biominerals and crystals from the PILP process, this chapter will give a brief overview of the non-equilibrium composition of biominerals, especially the formation of high Mg-calcite in nature. Then we investigate impurity (Mg) incorporation in CaCO3 deposited by the PILP process, and compare the amount of impurity incorporation in CaCO3 to that in biominerals. Finally we hope to establish impurity incorporation as a means for defining biomarkers, and tie the PILP process to biomineralization by this comparison. Introduction Calcium carbonate minerals comprise a large majority of marine exoskeletons and tissues in organisms ranging from simple bacteria and algae, to crustaceans, mollusks, sponges, and even in human otoliths (26,46-49). Most of these biological calcium carbonate phases characteristically contain magnesium at higher values than observed in Mg-calcite cement inorganically precipitated from seawater (50), as well as synthetically produced calcite. They also contain different ensembles of intra-crystalline and/or inter-crystalline acidic matrix proteins on the order of 0.1 wt% or less (51,52), which are thought to play an important role in regulating the CaCO3 biomineral formation (8,10). In geology, high-magnesium calcite (HMC) is classified as that containing more than 5% Mg ion in the calcite lattice. HMC is considered thermodynamically less stable than low-magnesium calcite (LMC) and loses its excess magnesium early in diagenesis 47

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48 (The physical, chemical, and biological processes by which a sediment becomes a sedimentary rock). The biomineralization process of calcium carbonates most often occurs in environments that contain relatively high levels of magnesium ion, such as sea water, which has a typical concentration up to 51 mM Mg-ion. Mg-ion has a pronounced influence on the growth of calcite in vitro, yet the mechanism by which magnesium and macromolecules cooperate in the formation of biological calcium carbonate still eludes researchers. Several studies have examined the influence of Mg-ion on calcite grown by the traditional solution crystallization process (53-57). At low concentration (<24 mM), Mg modifies the habit to form calcite with a prismatic shape; while at higher concentrations, approaching that of sea water concentration (51 mM), the calcite is more inhibited, thus enabling the formation of the metastable CaCO3 phase, aragonite, which is typically in the form of small dumbbell-shaped spherulites. At the concentrations which form calcite, only around 8% Mg-ion is found to be incorporated into the calcite lattice, which is significantly lower than the high Mg-bearing calcite found in many biominerals (e.g., red coralline algae secrete a high-magnesian calcite of up to 30% of Mg-ion). Therefore, we believe that the incorporation of Mg-ion in biomineral crystals could be due to a precursor process, which we suggest is regulated by the presence of acidic proteins. In particular, we have proposed that the Polymer-Induced Liquid-Precursor (PILP) process may play a fundamental role in biomineralization (9), and therefore are interested in the influence of Mg-ion on this “biomimetic” process. In this chapter, the influence of magnesium ion on the deposition of the PILP phase has been examined. The interplay of magnesium ions and polymer in the PILP process

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49 leads to formation of calcitic films with varying magnesium content, which further influence the percent crystallinity and the rate of the amorphous-crystalline transformation. The very high levels of Mg which are incorporated within the calcitic films matches those found in a variety of biominerals, and reinforces our hypothesis regarding the relevance of the PILP process to biomineralization. Duplication of biomineralization mechanisms holds promise for innovative materials processing strategies, e.g., low temperature fabrication of ceramic films, biomimetic coatings, and synthesis of organic-inorganic composite materials (58-60). Materials and Methods Experimental Condition The polymer used was poly-(, )-D,L –aspartic acid sodium salt (Mw=8600, Sigma) . Stock solutions of 36 mM of calcium chloride dihydrate (CaCl2H2O, 99.99%, Aldrich) were combined with a range of quantities of 180 mM MgCl2H2O (Sigma) solution to produce the desired Mg/Ca ratios. The solutions used for the PILP process were placed in small polystyrene petri dishes and filled up to a total volume of 3 ml each with ultra pure water, and a glass microscope cover slip (cleaned with Nochromix-acid solution) was placed in the bottom of each dish. The resultant solution contained 12 mM CaCl2 combined with MgCl2 solution of varying Mg/Ca ratios (0, 2, 3, 3.5, 4 and 5) and polymer concentrations (0, 2-30 g/ml). Each solution was allowed to crystallize at room temperature (20C) for three days, or in the fridge (5C) for seven days, in the presence of vaporized ammonium carbonate by placing vials of the powder (which slowly decomposes to CO2 and NH3) in a desiccator. The coverslips with precipitated films and/or crystal aggregates (magnesium-bearing carbonates) were taken out from each

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50 solution, gently rinsed with deionized water and ethanol, and then dried for later examination. Table 4-1. Experimental condition of the current study (two temperatures: 23C, 5C) Experiment Mg/Ca ratio [CaCl2] (mM) [MgCl2] (mM) [PolyAsp] (g/ml) MC0 0 12 0 0 MC1 2 12 24 0 MC2 3 12 36 0 MC3.5 3.5 12 42 0 MC4 4 12 48 0 MC5 5 12 69 0 MCP0 0 12 0 2 MCP2 2 12 24 2 MCP2 3 12 36 2 MCP3.5 3.5 12 42 2 MCP4 4 12 48 2 MCP5 5 12 69 2 P0 (same as MC3.5) 3.5 12 42 0 P1 (same as MCP3.5) 3.5 12 42 2 P2 3.5 12 42 6 P3 3.5 12 42 10 P4 3.5 12 42 15 P5 3.5 12 42 20 P6 3.5 12 42 25 P7 3.5 12 42 30 Characterization Polar optical microscopy (POM) and crystallinity analysis. The cover slip from each sample was examined using an Olympus BX60 polarized optical microscope in transmission mode, using crossed polars and an optional first-order red wave plate. The wave plate enables one to simultaneously view both amorphous (isotropic) and crystalline (birefringent) regions of the samples. The % crystallinity of each sample was determined by measuring the birefringent area of the films using image Pro software to isolate and quantify those regions. Averaging of at least five random sections of each film was used to reduce scatter due to the inhomogeneous nature of the

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51 films. Notegiven the spherulitic nature of the crystalline films, this method yields an underestimate of the % crystallinity since the Maltese cross region is isotropic, even though crystalline. X-Ray diffraction (XRD) analysis X-Ray powder diffraction spectrums were recorded using a Philips PW1710 diffractometer, operating with a Cu source (Cu K1, =1.54056 ). The amount of magnesium incorporated into the calcite lattice (XMg) was calculated by measuring the shift of the (202) peak of calcite (61), following the procedure of Goldsmith et al. (62), with the (202) peak being chosen due to its higher sensitivity to magnesium incorporation (56). XMg=(d202-d'202) /(d202-d"202), where XMg is molar fraction of Mg in the structure of magnesian calcite (Ca(1-x)MgxCO3), XMg%=(d202-d'202) 100/(d202-d"202), where d202, d'202 and d"202 are the lattice spacings of pure calcite, magnesium-bearing calcite, and pure magnesite, respectively. From the data of the Joint Committee on Powder Diffraction Standards (JCPDS), d202 and d"202 are 2.095 and1.939 , respectively. Fourier transform infrared spectroscopy (FTIR). Films deposited on the glass slides were scraped off and dispersed in KBr pellets. Infrared absorption spectra were obtained using a Nicolet MAGNA 760 Fourier transform infrared (FTIR) spectrometer with a resolution of 0.1cm-1. The data presented here have had the corresponding background subtracted out. SEM and X-ray energy dispersive spectroscopy (EDS) Selected samples were gold coated for SEM microscopy on a JEOL 6400 SEM instrument equipped with an EDS system. The Mg/Ca ratio of the samples was

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52 determined using the EDS software, which calculates the relative counts of Mg and Ca in the EDS peaks from at least five average areas within the samples. Different levels of Mg are expected, as compared to the ratios obtained by XRD (which arise from ions incorporated into the lattice), since the Mg/Ca ratios were calculated using the magnesium content in the whole sample area scanned, which includes Mg ions excluded to grain boundaries and defect sites. Inductively-coupled-plasma (ICP)-atomic emission spectroscopy (AES) analysis Selected films were dissolved in 0.1 N HCl and the Ca, Mg, and Sr concentration was measured by a Perkin-Elmer Plasma 3200 Inductively Coupled Plasma Spectroscopy (ICP) system. A calibration curve was established firstly by using an ICP standard solution before measuring the impurity amount in the sample. Results The experiments denoted in Table 4-1 were done at both room temperature (20C) and low temperature (5C). This section first shows the results from room temperature, then gives a brief description of the results from low temperature and compare the results from the two temperatures, Finally the influence of polymer concentration at constant Mg/Ca ratio is discussed. Influence of Mg Morphology of deposits w/o and w/ polymer At constant calcium chloride and polymer (polyaspartic acid, sodium salt) concentrations, a pronounced influence of the Mg-ion addition is observed. Our experiments reveal an increasing inhibitory effect of Mg2+ on calcite growth with the increase of Mg/Ca ratio in the parent solution, but this effect is manifest in different ways, depending on the absence or presence of polymer, as shown in Figure 4-1.

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53 Figure 4-1A-D shows representative crystals grown in solution in the absence of polymer at room temperature, with Mg2+ to Ca2+ ratios ranging from zero to 5:1 (denoted as MC0 to MC5); and Figure 1E-H shows representative crystals grown in the presence of polymer (2 g/mL), with Mg2+ to Ca2+ ratios ranging from zero to 5:1 (denoted as MCP0 to MCP5). CaCO3 crystals grown in the absence of acidic polymer have a distinctly different morphological structure than those grown in the presence of polymeric additives due to the PILP process, which typically leads to films/coatings morphology. As expected in the control MC0, without polymer or Mg-ion, classical rhombohedral calcite crystals are nucleated on the glass slides. In samples MC2, MC3.5 and MC5, aragonitic dumbbell structures and spherulitic calcitic crystals were observed to nucleate on the glass slides. This result is consistent with prior work that shows that Mg2+ ions added to a supersaturated calcium carbonate solution will impede the growth of calcite, while allowing the aragonite phase to form. This is thought to be due to the surface poisoning of calcite nuclei by absorption of firmly hydrated Mg2+(56). The polymer control sample (with no Mg2+) produced a mixture of 30-40 mm diameter calcite rhombohedrals and vaterite spherulites (Figure 4-1E). Also present in the control are minute amounts of small PILP droplets, but not enough to produce films (a higher level of polymer is required without Mg2+, as shown in the previous chapter). When Mg2+ ions are added in the presence of acidic polymer additive, a prominent change in the morphology of the CaCO3 occurs due to the PILP process. At Mg/Ca ratios between 2 and 5, while in the presence of this same small amount of polyaspartic acid as in Figure 4-1F-H, thin CaCO3 films were produced. Thin films were not formed at a ratio of Mg/Ca=1 using this low polymer concentration, but will form with higher polymer

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54 concentrations (results not shown). Unlike the mosaic single-crystalline patches of film described in our previous chapter and reported in previous work (9), these films are all spherulitic and more continuous, covering the entire glass coverslip. Also with the presence of magnesium, the critical concentration of polyaspartic acid needed to produce these pure precursor films is much lower (only 2 g/ml). By pure, we mean only PILP deposited mineral (i.e. films), with elimination of the large crystal aggregates formed by the traditional solution growth process that is prevalent in our prior report (9). As the concentration of Mg2+ ions is increased, precursor films are still deposited, but the transformation stage of the PILP process is also influenced by the Mg. After the slides were removed from the reaction chamber and dried, each precursor film shows an amorphous to crystalline transformation. (Noteif the films are left in solution, the metastable amorphous phase will frequently re-dissolve if it has not crystallized to the more stable state quickly enough; therefore, the samples are usually removed prior to the transformation). The crystalline patches of film are round with spherulitic texture, as seen by the Maltese cross under polarized microscopy. At the lower Mg/Ca ratio of 2 in the parent solution, the resultant precursor film has almost completely transformed into spherulitic crystals which impinge upon one another to form polygonal shapes, as seen in MCP2 of Figure 4-1F. As the Mg/Ca ratio is increased, the precursor films did not completely transform into a fully crystallized film, and only isolated spherulites are observed sporadically throughout the film (Figure 4-1H), indicating inhibition of either nucleation or growth of calcium carbonate at higher magnesium content. While the inhibitory effect of Mg is also true for the solution grown crystals (without polymer), the result there is to form aragonite, while the result for the PILP system is to still form

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55 calcite, as verified by XRD and FTIR studies, but with a reduction in the transformation rate. A typical SEM image of the film is shown in Figure 4-2C & D, from which it can be seen that the films are continuous at the macroscopic scale. On the surface of the film there are small spherical particles of ~ 2 m diameter, which are post-precipitate PILP droplets that did not coalesce with the film. Cracking of the film occurs due to rapid drying during vacuum preparation for SEM examination. EDS analysis shown in Figure 4-2E & F shows Ca and O peaks in CaCO3. Influence of Mg/Ca ratio in solution on crystallinity of film The kinetics of crystallization as related to the Mg2+ content in the solution was quantified using image analysis at a time of six days after the deposited PILP film was removed from the reaction chamber (Figure 4-3). The % crystallinity of the films from MCP2-MCP5 decreased as the Mg/Ca ratio in the PILP process was increased from 2 to 5 (Table 4-2). Table 4-2. Crystallinity of the films prepared at different Mg/Ca ratios at room temperature by PILP process Mg/Ca ratio Crystallinity (%) Standard error 2 94.627 7.409 3 65.586 19.416 3.5 32.338 8.447 4 12.880 3.448 5 1.097 0.666 As seen also from Figures 4-1 and Figure 4-3, at the lowest Mg level (Mg/Ca=2) in the crystallization solution, the resultant film is composed mainly of spherulites after six days and has highest crystallinity of around 95% (noteit maybe actually fully crystalline, but the maltese cross is isotropic and contributes to the non-crystalline amount); At the highest Mg level (Mg/Ca = 5), the film is mostly amorphous and shows

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56 only a little crystallinity of around 1% after being dried for six days for transformation; while at the intermediate Mg levels (Mg/Ca = 3, 3.5, 4), the amorphous calcium carbonate (ACC) and spherulitic crystals coexist within the deposited film during this time period. Crystal structure verification Figure 4-4A shows an XRD of the crystals in MCP0 (without the addition of Mg, and only a small amount of polymer), the crystals are mainly calcite. Figure 4-4B shows the XRD spectrum of the thin film sample in MCP2 and MCP4, (formed in the presence of both Mg and polymer), which corresponds to the films shown in Figure 4-1E-H, which have both amorphous calcium carbonate (ACC) and calcite. Mg incorporation in film determined by EDS, XRD and ICP The inhibiting effect of Mg2+ on the film transformation suggests that high levels of Mg2+ ion might be entrapped in the precursor films since Mg2+ ions are known to be an inhibitor of calcite growth (63). Indeed, EDS analysis of the films shows that both Ca2+ ions and Mg2+ ions coexist in the film (Figure 4-2E & F). Due to the phase boundaries delineating the PILP phase, impurities that also interact with the charged polymer (e.g., Mg2+) can be entrapped within the amorphous precursor during solidification, and upon crystallization, yield impurity incorporation at levels far removed from equilibrium conditions. The results of Mg content in the deposited films determined by EDS are shown in Table 4-3 and Table 4-4. At each temperature, the Mg incorporated in the film increases near linearly with the Mg/Ca ratio in the starting crystallization solution. On the other hand, Mg content determined by XRD only reflects the amount of Mg incorporated in the calcite lattice according to Vegard’s Law, which states that unit cell parameters should

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57 change linearly with the degree of foreign ion substitution (64). The result of Mg content in the calcite lattice of MCP2-MCP5 prepared at low temperature and room temperature is shown in Table 4-4 and Table 4-5, respectively. Finally, the films of MCP2-MCP5 were dissolved and the actual Mg incorporation in the total film was determined using ICP-AES, and the results are shown in Table 4-6. Table 4-3. Mg content (molar percentage) in CaCO3 films prepared at different Mg/Ca ratios by PILP process determined by EDS (films prepared at two temperatures: 23C and 5C) Mg/Ca ratio MgCO3 %, Room Temp Standard error MgCO3 %, Low Temp. Standard error 2 22.9 0.19 18.8 1.5 3 25.4 1.05 22.5 0.4 3.5 30.7 1.79 24.5 0.8 4 34.6 2.96 25.3 1.7 5 37.3 3.80 29.2 2.7 Table 4-4. Mg content (molar percentage) in CaCO3 films prepared at different Mg/Ca ratios by the PILP process determined by XRD (films prepared at 5C) Mg/Ca ratio 2 theta d 202 Mg content (mole fraction) Percent MgCO3 % 2 43.385 2.0840 0.0705 7.05 3 43.520 2.0778 0.1103 11.03 3.5 43.555 2.0762 0.1205 12.05 4 43.730 2.0683 0.1712 17.12 5 43.890 2.0611 0.2173 21.73 Table 4-5. Mg content in CaCO3 films prepared at different Mg/Ca ratios by the PILP process, as determined by XRD (films prepared at 23C) Mg/Ca ratio 2 theta d 202 Mg content Percent MgCO3 % 2 43.4250 2.0821 0.0827 8.27 3 43.6475 2.0720 0.1474 14.74 3.5 43.7600 2.0670 0.1795 17.95 4 43.8350 2.0636 0.2013 20.13 5 44.0600 2.0536 0.2650 26.54

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58 Table 4-6. Mg content (molar percentage) in CaCO3 films prepared at different Mg/Ca ratios by the PILP process determined by ICP-AES at two different temperatures Room Temp. Low Temp. Mg/Ca ratio MgCO3 (%) MgCO3 (%) 2 15.53 7.07 3 21.89 9.50 3.5 23.74 10.44 4 25.36 11.36 5 31.56 13.88 Figure 4-5 shows how the MgCO3 content in films, determined by EDS, XRD and ICP measurements, increases nearly linearly with the initial Mg/Ca ratio in crystallizing solution. The measured concentrations of Mg2+ were consistently higher using EDS than XRD. This is because EDS measures the total Mg content (mol%) in both ACC phase and calcite phase in film, including the magnesium impurities located at defects and grain boundaries, while XRD measures the reduction in calcite lattice spacing (d202) caused by the incorporation of Mg ion into the calcite lattice only. ICP measures the average amount of Mg amount in the film after the films were totally dissolved. Non-equilibrium Composition in the PILP Process Apparently there is a compositional heterogeneity in the film deposited by the PILP process, as deduced from the above results: We know all the films contain both the ACC and calcite crystalline phase, and the calcite is transformed from the amorphous precursor film. EDS and ICP show higher Mg content in CaCO3 than XRD results, indicating that the amorphous precursor phase originally has a high Mg entrapment (probably close to that determined by ICP, which detects all chemical species, regardless of structure). After transformation of the precursor phase into ACC and calcite, a smaller amount of Mg was incorporated in the calcite lattice while the excess Mg was excluded into the surrounding

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59 ACC phase, ultimately ending up concentrated at the phase boundaries, which would lead to a compositional heterogeneity in the deposited film. Most importantly, the above measurements indicate that the PILP process can lead to much higher magnesium incorporation into calcium carbonate (up to 37% by EDS and 27% by XRD) than calcite precipitated inorganically (8%), or precipitated from standard sea water (at a Mg/Ca ratio of 5) where the magnesium content is less than 10%, as reported from different studies (53,54,57,65). Our results indicate that Mg and a small amount of polymer act together to produce CaCO3 with both non-equilibrium morphology (film) and non-equilibrium composition (composition heterogeneity and high Mg incorporation) by the PILP process. Using FTIR for Characterization of CaCO3 In order to further identify the crystal structure of the films prepared at higher Mg/Ca ratio (e.g., MCP5), we allowed the films to transform to be fully crystalline after three and a half months. Then the films were characterized by FTIR. In the range of 1800 and 600 cm-1, each spectrum of the films contains three sharp peaks and one broad peak. The assignments of peaks essentially follow from various articles in the literature (26,66,67). In the infrared spectrum of calcium carbonate, the 2 (out-of-plane-bending), 3 (asymmetric stretching), and 4 (in-plane-bending) vibration modes of carbonate anion are active. The symmetric stretching mode 1 is forbidden in the Mg-calcite spectra and is generally observed as a weak absorption band (67). It should be pointed out that the distinctive peaks in calcium carbonate are 4, 3. For example, vaterite has distinctive peaks at 744 cm-1 and 877 cm-1, and calcite has distinctive peaks at both 876 and 713 cm-1, while aragonite has distinctive peaks at 713 cm-1 and 860 cm-1.

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60 As seen from Figure 4-6, the IR spectrum of the control sample shows peaks at both 713 and 876 cm-1, which indicate it is calcite. The 1 absorption of calcite around 1080 is barely seen. Figure 4-7 shows IR spectra of samples MCP2-MCP5 at room temperature. The infrared spectra of all the films show similar peaks as those of calcite. However, due to the incorporation of Mg into the calcium carbonate, there is a significant shift of 4 towards higher wavenumbers. This is also characteristic of inorganic or biogenic magnesian calcite (67). A plot of the peak position of the 4 band versus the Mg/Ca ratio in solution, shown in Figure 4-8, was found to be quite linear (R=0.9909), which indicates the magnesium content in the film increases linearly with the Mg/Ca ratio in solution, according to Eq. 4-1 through 4-3: 4 (cm-1) = 712.32 + 2.683 (Mg/Ca ratio in solution). (4-1) In order to establish a relationship between 4 and Mg content in the film, ICP data in Table 4-5 was used to determine the magnesium content in the film. As seen from Figure 4-9, the ICP data also shows a linear relationship between the MgCO3 content in the films and the Mg/Ca ratio in solution, according to the following: XMg = 0.015 + 0.062 (Mg/Ca ratio in solution). (4-2) Where XMg is the molar fraction of Mg in the film. Substituting the Mg/Ca ratio in (1) by XMg from Eq. 4-2, a relationship between 4 and XMg is established as follows: 4 (cm-1) = 711.67 + 43.2 x XMg (4-3) Eq. 4-3 is relationship of 4 with XMg for Mg-calcite thin films synthesized by the PILP process. The relationship of 4 with the molar fraction of Mg in the calcium

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61 carbonate films deposited by the PILP process is similar to that in inorganic and natural high magnesian calcites reported by Bottcher et al. (67). Their result is 4(cm-1) = 712.20 + 39.4 XMg. (4-4) Eq. 4-4 is relationship 4 with XMg for Mg-calcite from inorganic and biogenic origin. A comparison of Eq. 4-3 and Eq. 4-4 is shown in Figure 4-10. From the above comparison it can be verified that our FTIR characterization is accurate and consistent with the FTIR characterization for both inorganic and biogenic calcium carbonate. One important aspect of this study is that the magnesian calcite is caused by an amorphous precursor phase in the PILP process, and in nature, recent evidence has shown that several biominerals are formed from a transient amorphous phase. For example, Beniash et al. found that amorphous calcium carbonate was present in the early stage of sea urchin spicule growth, which transforms into magnesian calcite with time (68). Aizenberg et al. also found that amorphous calcium carbonate in ascidian skeletons serves as a precursor for magnesian calcite (25,26). In this sense, our synthesis of Mg bearing calcite by the PILP process may offer an explanation for the non-equilibrium compositions, such as magnesian calcite, found in biominerals. The amount of Mg incorporation in the resulting film by the PILP process is conveniently controlled by varying the Mg/Ca ratio in solution, and the high magnesian calcite is presumably stabilized due to the presence of polymer. Discussion Based on previous investigations of the Gower group (9), there are two main stages in the PILP process. The first stage is deposition of the precursor phase, which often

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62 occurs as film formation. As a critical concentration is reached during the infusion of carbonate species, isotropic droplets (~2-5 m in diameter) phase-separate from the solution and accumulate on the glass coverslip. These liquid-like droplets coalesce to form a continuous isotropic precursor film. The second stage is transformation of the precursor phase, in which the amorphous precursor film gradually crystallizes as the waters of hydration are driven off. Our present study further investigates this process using magnesium as an impurity to see how it affects both the deposition and transformation stages of the PILP process. The incorporation of magnesium in the crystallizing solution caused a pronounced change in the deposition stage of the PILP process. Magnesium lowers the concentration of polymer needed to produce the precursor films. As described earlier, without magnesium in the solution, a concentration of 20 g/ml polyspartic acid or above is needed in order to produce films by the PILP process, and even then, the films are not fully continuous unless produced at lower temperature. When magnesium is added into the solution at a ratio greater than 2, a concentration of only 2 g/ml PolyAspartic acid is sufficient to produce films. Although Mg impurity alone is a potent inhibitor, it does appear that at least a small amount of polymer is required to generate the PILP process (at least at this moderate level of calcium, which is biologically relevant). This enhanced PILP formation by the addition of Mg might be explained by the following possible mechanisms: As magnesium is incorporated into the precursor phase, surface poisoning of calcite nuclei is caused by adsorbed hydrated magnesium. The relatively large dehydration barrier of Mg ion acts as an inhibitor of crystal growth in addition to the polymer, thus the critical concentration of polymer needed for PILP formation is

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63 significantly lowered. In addition, although the inhibitory action of the polymer is deemed important for the formation of an amorphous phase, the degree of hydration is likely enhanced with the hydrated Mg-ions. These hydration waters are the important component of the PILP process because they impart the amorphous phase with fluidic properties, distinguishing it from the more typical description of amorphous minerals. The second stage of the PILP process, the transformation stage, is also strongly effected by the Mg-ion. We have found that the addition of magnesium in the crystallization solution leads to the incorporation of magnesium in the calcium carbonate precursor film. As Mg2+ is entrapped in the precursor phase, which then gradually transforms (i.e., solidifies and crystallizes), a significant amount of the magnesium becomes incorporated into the calcite lattice and results in the formation of high magnesium-bearing calcite. Somewhat surprisingly, the precursor films transform into calcite rather than aragonite. In the traditional solution crystallization, aragonite results from the inhibitory action produced at high magnesium concentrations, as shown in the control reaction. However, our results are consistent with the results of Kitano and Kanomori (69,70). They found that some organic compounds such as citrate, pyruvate and maleate of sodium lead to enhanced incorporation of magnesium into calcites and suppress the formation of aragonite, which is the dominant phase without the presence of these organics. In their study, The MgCO3 content of magnesian calcite ranged from 8 to 12 percent when precipitated from calcium carbonate solution at the same Mg/Ca ratio of 5, depending on the concentration of organic concentration. As the concentration of citrate ions was higher, the Mg content was higher too. Even if high-magnesian calcites were formed in the presence of these small organic molecules, the Mg content in their

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64 study is much lower than that in our present study. As shown in Figure 4-5, under similar conditions of Mg/Ca ratio of 5, the Mg content in calcite by the PILP process can reach up to 27% as determined by XRD. The final magnesium content in the films increases linearly with Mg/Ca ratio in the solution, as one might expect since the precursor process arises from the ion-binding affinity of the polyanionic additive, which will also bind to a certain amount of Mg-ion. It seems likely that the amount of Mg entrapped within the precursor phase (and ultimately the crystals) could be adjusted by choosing polymers with different ion-binding affinities. The highest magnesium content incorporated by the PILP process is ~37mol% at the highest Mg/Ca ratio tested, which was 5, the concentration of seawater. Notably, inorganic precipitation of carbonates from sea water or solutions with similar Mg/Ca ratios have been shown to give a molar percentage of MgCO3 below 10%. On the other hand, high magnesium levels are typical in biogenic calcium carbonate minerals formed by coralline algae and various invertebrates. Red algae and echinoderms form high magnesium calcite that contains up to 25% magnesium. Some organisms, most notably red algae, secrets a high-magnesian calcite of up to 37% (71).The magnesium content obtained in the PILP process (10%-37%) encompasses the range found in biologically formed Mg-bearing calcite (12%-30%) (72,73). In addition, the formation of high magnesium calcite (HMC) in our PILP process is similar to corals, red algae, echinoderms, bryozoans and some benthic foraminifera (74-76), in that the composition of the deposited calcium carbonates are not in equilibrium with their host water environment (Mg/Ca solution ratio). Such measurements are not only of interest to the biogeochemist, since high-magnesian calcite is unstable and difficult to synthesize at low

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65 temperatures and normal pressures, but also shed a light on the influence of biopolymers (especially the acidic proteins mimicked by polyaspartic acid) on the morphology, phase transformation, and composition of CaCO3 minerals in nature, helping to unravel the mysterious mechanisms of biomineralization. We believe the PILP process can be considered as an in vitro model of biomineralization, where the acidic polyaspartic acid serves as a process directing agent, which we suggest is a primary function of some of the acidic proteins in biomineralization. In this report, a Mg/Ca ratio of 2 to 5 is used to simulate the conditions of sea water (although the Mg concentration within the organism’s mineralization compartment may differ since it is regulated by cellular processes); a slow vapor diffusion of ammonium carbonate is used to gradually raise the supersaturation, mimicking the transportation of carbonate species across a cellular membrane (or via enzymatic production). We are currently examining various organic substrates instead of glass slides to mimic the function of insoluble matrix in biomineralization, which can influence both the deposition and transformation of PILP phase, thus playing a role in controlling the location (77), orientation (44), and phase of the crystals (60). A combination of all these conditions may allow the synthesis of inorganic crystals with different impurity incorporation and controlled crystal properties. All these characteristics are typical of biomineral formation, suggesting that the PILP process may conceivably lie at the foundation of the biomineralization process. Influence of Temperature The above section gives a detailed results and discussion of experiments prepared at room temperature. The same set of experiments in MC0-MC5, and MCP0-MCP5 was

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66 also done at low temperature, and similar trends regarding the crystal structure, morphology and influence of Mg/Ca ratio were obtained. Here a brief comparison of film morphology at the same condition (Mg/Ca ratio, and Pasp concentration) except temperature is shown in Figure 4-11. Generally film prepared at lower temperature has higher crystallinity than that at higher temperature, despite the same Mg/Ca ratio and polymer concentration. For example, Figure 4-11A has lower crystallinity compared to Figure 4-11B, and Figure 4-11C has lower crystallinity than Figure 4-11D. Figure 4-12 is an SEM picture of film deposited from low temperature by the PILP process, demonstrating that the morphology is similar to that of films formed at room temperature, as shown in Figure 4-2C & D. A comparison of the Mg incorporation in the films from two temperatures is shown in Figure 4-13. The film prepared at room temperature has much higher magnesium incorporation than that prepared at low temperature by the PILP process. For example at a Mg/Ca of 5, which is close to the seawater condition, EDS measurement indicates that the Mg content is 37%, as compared to 29% at the lower temperature. A similar result is also obtained by XRD measurement. Influence of Polymer Concentration The experiment from P0 to P7 in Table 4-1 investigates the influence of Pasp concentration on CaCO3 crystal growth at constant Mg/Ca ratio of 3.5. POM characterization Figure 4-14 shows optical images of samples P1, P3, and P7 at two periods of drying time. From top down the influence of polymer concentration on crystallinity of the film can be seen. Without any polymer at a Mg/Ca ratio of 3.5, only single crystals of calcite and dumbbells of aragonite formed. Similar to the results described earlier (data

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67 not shown), a small amount of polymer dramatically modified the CaCO3 into a film morphology due to the PILP process. Polymer has a general inhibitory effect by not only inducing the precursor, but it also stabilizes the amorphous phase after solidification. In other words, the polymer concentration affects the kinetics of the amorphous to crystalline transformation. For example, P1 (Figure 4-14A) has more crystallinity than P7 (Figure 4-14E) at 1 day’s drying. This effect is more pronounced after 13 days’ drying at room temperature, when more of the amorphous phase has transformed to crystalline phase. For example, sample formed from lower polymer concentration (Figure 4-14B, 2 g/ml Pasp) shows much higher crystallinity than sample formed from higher polymer concentration (Figure 4-14D, 10 g/ml Pasp, and Figure 4-14F, 30 g/ml Pasp). Morphology characterization by SEM Figure 4-15 shows a typical SEM image of sample P6, which contained 25 g/ml Pasp in the starting crystallizing solution. At lower magnification (Figure 4-15A), the film morphology is not obvious since it is very smooth except for the large non-coalesced droplets, but can more clearly be seen by comparison to a dark scratch line, where the glass slide is exposed. The film at higher magnification (Figure 4-15B) shows a very smooth background with embedded and exposed particles (possibly due to the post-precipitated PILP droplets). An EDS spectrum shows the presence of Mg and Ca (Figure 4-15C). Influence of polymer Concentration on Mg incorporation Table 4-7 shows the influence of polymer concentration on the amount of Mg incorporation in the deposited films at two different temperatures. As seen from Table 4

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68 7 and Figure 4-16, the influence of polymer concentration is a minor factor as compared to temperature. Even if the Polymer changes from 2 g/ml to 30 g/ml, the amount of Mg incorporation is about the same at each temperature. Table 4-7. Influence of Polymer on Mg incorporation in PILP formed films (Mg/Ca=3.5) Low temperature Room Temp `No. Polymer (g/ml) d202 XMg MgCO3% d202 XMg MgCO3 % P1 2 2.0730 0.1410 14.10 2.0627 0.2071 20.71 P2 6 2.0792 0.1013 10.13 2.0681 0.1724 17.24 P3 10 2.0724 0.1449 14.49 2.0602 0.2231 22.31 P4 15 2.0758 0.1231 12.31 2.0601 0.2237 22.37 P5 20 2.0769 0.1160 11.60 2.0596 0.2269 22.69 P6 25 2.0780 0.1090 10.90 2.0602 0.2231 22.31 P7 30 2.0753 0.1263 12.63 2.0565 0.2468 24.68 Assessing the Reliability of Magnesium as a Temperature Proxy The incorporation of minor or trace elements in biogenic carbonates has been used to trace the relationships with the chemical environment (e.g., Mg/Ca ratio) and physical environment (e.g., Temperature), resulting in their use as geological biomarkers (a biological-monitoring material). Given the potential relevance of the PILP process to biomineralization, these studies can provide important information in this regard. First, the magnesium content in the deposited film is proportional to the Mg/Ca ratio in parent solution. This means the water chemistry directly influenced the resultant magnesium content, in a well-defined linear fashion, but at values which differed dramatically from those seen for the traditional solution precipitates. As is suggested by the geological community, the long oceanic residence times for Ca and Mg, the Mg/Ca ratio in sea water is thought to be of little variance for several million years (78-80). So it is assumed that for foraminiferal calcite, coral skeletons, ostracodes and marine cement, the magnesium content (or Mg/Ca ratio) may not have been influenced much by the seawater

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69 for several million years. However, beyond this time range, or in the speleothem, karst, lacustrine environments, there are possible variations in the Mg/Ca ratio. Many marine cements form in microenvironments (e.g., cells in organisms, peloids, foram chambers, algal bores, etc. (81)) or semi-enclosed environments where Mg/Ca ratio may differ significantly from sea water (82). So in all these cases, magnesium as a temperature proxy should be used with caution, particularly in light of our studies, which show that compositions far removed from equilibrium may be expected to result from biomineralization that utilizes a precursor mechanism. Second, a very small amount of organic matter, and especially in polymeric form (e.g., polyaspartic acid in our case) can greatly change the amount magnesium incorporation in mineral deposited by the PILP process. However, an increased polymer concentration does not lead to an increase in magnesium incorporation, at least not with this particular polymer. Our results show that the magnesium content does not change significantly after a critical concentration of polymer is reached (the concentration which leads to the PILP process). Although the influence of organic matter in organisms is much more complex than that in our simple model system, our results show that acid polypeptide, which mimics the acidic proteins commonly associated with calcium carbonate biominerals, does not affect much the resultant magnesium content despite its concentration change. Further work is needed to determine if this is true for other biologically relevant macromolecules. Finally, our results show that at constant Mg/Ca ratio, temperature does influence the magnesium content in the deposited film. The higher is the temperature, the higher will be amount of magnesium content in the film. In the case where the Mg/Ca ratio does

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70 not change much (such as seawater conditions), the magnesium can still serve as a reliable temperature proxy after careful calibration of the particular species biomineralization conditions. Conclusion This chapter investigates the influence of Mg impurity on the stages of the PILP process, as well as its incorporation into PILP formed mineral films. High-magnesian calcite films are formed by the PILP process. The influence of Mg/Ca ratio, polymer concentr4ation, temperature, and time of drying, on the film crystallinity and Mg incorporation are discussed. The main conclusions from these studies are listed below: Mg shows an inhibitory effect on CaCO3 crystal growth, which affects both stages of the PILP process: Mg-ion enhances the ability of the polymer to induce the PILP process, producing pure precursor phase (eliminating solution crystal products), while also becoming entrapped in the precursor phase, stabilizing the amorphous phase and delaying the formation of crystalline calcite. The Mg/Ca ratio in the starting solution greatly affects the amount of Mg incorporation in CaCO3, and further affects the kinetics of the amorphous-crystalline transformation in the PILP process Temperature has a primary effect on the amount of Mg incorporation, as determined by EDS, XRD and ICP, in which these difference techniques allow for comparison between the total Mg and the Mg discriminately incorporated into the crystal lattice. Polymer induces the precursor phase and tends to stabilize the amorphous phase, reducing the kinetics of the amorphous to crystalline transformation, as the concentration of polymer goes to higher values at constant Mg/Ca ratio. Mg may be a useful a temperature proxy due to the sensitivity of impurity incorporation with temperature; the constant sea water composition over long periods of time; and the insignificant effect of organic polymer. The PILP process not only leads to the formation of CaCO3 with non-equilibrium morphology, but also leads to the formation of CaCO3 with non-equilibrium composition. High-magnesian calcite films are formed by the PILP process and the amount of Mg incorporation covers the whole range of composition in biogenic magnesian calcite.

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71 A E B F C G D H Figure 4-1. POM micrographs (with 1st-order red -plate) of Mg-Ca carbonates formed at different Mg/Ca ratio without (left) and with (right) polyaspartic acid. A)-D) MC0, MC2, MC3.5, and MC5 show all solution crystal growths without polymer addition E)-H) MCP0, MCP2, MCP3.5, and MCP5 show mineral deposited in the presence of Pasp.

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72 20 m A 20 m B C E 50 m D F 20 m Figure 4-2. SEM micrographs of CaCO3 crystals and corresponding EDS spectra of the films. A) Calcite rhomb, as in MCP0. B) Possible vaterite spherulite, as in MCP0. C) MCP2 (Mg/Ca=2, Pasp=2 g/ml, room temperature). D) MCP4 (Mg/Ca=4, Pasp=2 g/ml) E) EDS shows the film has Mg incorporation in the calcium carbonate film of MCP2. F) EDS of the film of MCP4. Notethe Si and K peaks are due to the glass slides, and the Au and Pd peaks, are due to the gold coating used for SEM.

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73 0 20 40 60 80 100 1 2 3 4 5 Mg/Ca ratio in solution Six day Crystallinity % Figure 4-3. Film crystallinity six days after removal from the reaction chamber. (These films were prepared at room temperature at different Mg/Ca ratios with Pasp at 2 g/ml, as shown in Table 4-1 from MCP2 to MCP5). The % crystallinity was determined by measuring the coverage area of birefringence versus isotropic regions of the films using Image Pro software. The crystallinity is underestimated because the Maltese-cross pattern in the spherulitic films contributes to isotropic area even though it is actually crystalline (just oriented perpendicular to one of the polars).

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74 0200400600800100012001400160020304050602 thetaintensity 01002003004005006007008000102030405060 MCP4 MCP2 104 A 024 006 018 113 202 B 104 006 110 202 113 Figure 4-4. Typical XRD spectra of the CaCO3 crystals. A) XRD spectrum of crystals in MCP0 without Mg and only small amount of Polymer (2 g/ml). Notethe weak broad background peak at around 27 due to the glass coverslip. B) XRD of the thin films synthesized by the PILP process in MCP2 and MCP4, in the presence of both Mg and polymer. Notethe inhibition of Mg results in both amorphous calcium carbonate (large broad peak) and calcite coexisting in the film due to its slow transformation. The peak widths of the calcite are also somewhat broadened, suggesting either small crystallites (which is not observed optically), poor crystallinity and/or lattice strain (from Mg and/or polymer incorporation in the lattice).

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75 0102030401.522.533.544.555.5Mg/Ca ratio in solutionMgCO3 Content in Film Figure 4-5. Influence of Mg/Ca ratio in solution on the MgCO3 content in the films prepared at different Mg/Ca ratios (MCP2-MCP5) by the PILP process at room temperature (the Mg contents were determined separately by EDS, XRD and ICP).

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76 Figure 4-6. FTIR spectrum of typical calcite crystals from the MCP0 control. Figure 4-7. FTIR of the magnesium-bearing CaCO3 films. Both ACC and calcite coexist in the deposited films. Films were prepared under at room temperature under the following conditions: A) Mg/Ca=2; B) Mg/Ca=3; C) Mg/Ca=4; D) Mg/Ca=5 in parent solution, while polyaspartic acid was kept at a constant concentration of 2 g/ml. Note the peak shift of the 4 band, which arises from the in-plane bending mode of the carbonates, which ranges from 718cm-1 to 726 cm-1, according to the amount of Mg incorporation.

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77 712714716718720722724726728012345Mg/Ca ratio in solutionv4 of Carbonate R = 0.9909 Figure 4-8. A plot of the 4 peak maximum versus the Mg/Ca ratio in the parent solution for the films MCP2-MCP5. Mg/Ca ratio in solutionMgCO3 content in film R = 0.9846 Figure 4-9. Mg content in the films from MCP0-MCP5 increases linearly as the Mg/Ca ratio in the parent solution increases (Mg content measured by ICP).

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78 0.000.050.100.150.200.250.300.35710712714716718720722724726728 v4(cm-1)XMg Mg calcium carbonate by PILP Process Inorganic and biogenic magnesian calcite Figure 4-10. A comparison of using 4 shift in wave-numbers to correlate the Mg content in the Mg-CaCO3 films prepared by the PILP process to that from inorganic and biogenic magnesian calcite determined by another group.

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79 A B C D Figure 4-11. Polarized optical micrographs of thin films formed at two different temperatures. A) Mg/Ca=3.5, room temperature, 23C. B) Mg/Ca=3.5, low temperature, 5C. C) Mg/Ca=4, room temperature, 23C. D) Mg/Ca=4, low temperature, 5C. Notethe films prepared at low temperatures have higher crystallinity than films prepared under the same conditions except at room temperature (as judging from the amount of isotropic (magenta colored) material).

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80 10 m 20 m A B A Figure 4-12. SEM micrographs of films deposited by the PILP process at low temperature. A) Mg/Ca = 3.5, Pasp = 2 g/ml. B) Mg/Ca = 5, Pasp = 2 g/ml. The thickness of the film appears to be about 0.5m, similar to those measured previously. The cracks in the film either resulted from vacuum coating procedure, or under the beam of the SEM, suggesting that these regions were still highly hydrated, causing stress fractures.

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81 05101520253035404522.533.544.55Mg-Ca Ratio in Crystallizing SolutionMgCO3 content in film (mol%) Room Temp(20) EDS Low Temp (5) EDS Low Temp(5) XRD RoomTemp(20)XRD Figure 4-13. Influence of temperature on the Mg incorporation in thin films prepared by the PILP process at different Mg/Ca ratios.(Samples MCP2-MCP5). The Mg content was determined separately by EDS and XRD.

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82 A B C D E F Figure 4-14. Influence of polymer concentration and time of drying on the amorphous-crystalline transformation in the PILP process. A) P1-Pasp = 2 g/ml. 1 day drying time. B) P1-Pasp=2 g/ml. 10 days drying time. C) P3Pasp = 10 g/ml. 1 day drying time. D) P3Pasp = 10 g/ml. 10 days drying time. E) P7Pasp = 30 g/ml. 1 day drying time. F) P7Pasp = 30 g/ml. 10 days drying time. (Other condition is shown in samples P1 to P7 in Table 4-1, Mg/Ca=3.5, low temperature, seven days, vapor diffusion)). G H

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83 50 m 20 m A B C Figure 4-15. SEM and EDS of typical thin CaCO3 films formed at a higher polymer concentration. A)-B) SEM image P6 (Mg/Ca = 3.5, Pasp = 25 g/ml, low temperature) at low and high magnification, respectively. C) An EDS spectrum of P6 shows Ca and Mg present in the film.

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84 Effect of polymer on MgCO3 content in film05101520253035404550051015202530Polymer concentration(ug/ml)MgCO3 content (mol%) Low temp, Mg/Ca=3.5 room temp, Mg/Ca=3.5 Figure 4-16. Effect of polymer concentration in the crystallization solution on the amount of Mg incorporation in thin films deposited by the PILP process at both temperatures.

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CHAPTER 5 IMPURITY INCORPORATION BY THE PILP PROCESS-A STATISTICAL APPROACH Introduction CaCO3 is the most common constituent of the mineralized tissues of many organisms in marine and natural waters. The growth of CaCO3 is invariably from an environment where there are many other cations available besides Ca. Among them, the most important ones are Mg and Sr. These two elements are in the same elemental group as Ca in the Periodic table, and they have close atomic radius and same charge, so that both Mg and Sr can be easily substituted into the CaCO3 crystal lattice (Mg much easier into calcite, while Sr much easier to aragonite. ionic radius: Mg2+, 0.65 ; Ca2+, 0.99 ; Sr2+, 1.13 ) (50). In recent years, the Mg/Ca ratio and/or Sr/Ca ratios in CaCO3 from foraminiferal shells (83-87), corals skeletons (88-91), and ostracod (tiny marine and freshwater crustaceans with a shrimp-like body enclosed in a bivalve shell)(92-94) have been used as a paleothermometer or temperature proxy for sea surface temperatures (SST) or bottom water temperatures (BWT). For example, geologists rely on the fossilized carapaces of ostracods to date sediments. Therefore, understanding CaCO3 crystallization has important implications for biogeochemistry, paleo-oceanography, and biomineralization (95). One important aspect is that there is a large disparity in impurity incorporation in biogenic and inorganic phases, thus implicating the accuracy of use of Mg/Ca and Sr/Ca ratio as a temperature proxy. In this chapter, the impurity incorporation of Mg and Sr in CaCO3 formed by the PILP process are investigated using a statistical approach. By varying the starting 85

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86 Mg/Ca, Sr/Ca and polymer levels, we hope to investigate the impurity incorporation mechanism in CaCO3 by the PILP process, thus giving insights and recommendations to the application of Mg/Ca and Sr/Ca ratio as a temperature proxy from mineral deposits of biogenic origin. Finally, by comparing the disparity of impurity incorporation in biogenic and inorganic CaCO3 phases, we hope to establish impurity incorporation (especially Mg and Sr) in CaCO3 as a mineralogical signature of biological origin, which can be of value to NASA, for example (who funded this research), towards determining biomarkers for microbial life, such as from meteorite samples. Experiment and Method Experimental Condition The polymer used was Poly-L-aspartic acid, sodium salt, at two different molecular weights, (Mw=8600, P5387, Lot 10K5906, Sigma, and Mw=35400, Lot 10K5905). The calcium chloride dihydrate (CaCl2H2O, 99.99%, Aldrich) solutions were combined with a range of quantities of 180 mM MgCl2H2O (Sigma), and 36 mM SrCl2 solution to produce the desired Mg/Ca and Sr/Ca ratios. The solution used to generate the PILP process was made up to 3 ml each with ultra-pure deionized water. Here, we adopt a standard three factorial statistical design (223) to investigate the MgCl2, SrCl2 and Polymer concentration influence in the PILP Process. The experimental conditions are described in Table 5-1. In each experiment No., the calcium concentration is kept constant; while the Mg concentration is at three levels: 24, 42, and 60 mM; and the Sr concentration is at two levels 0.12 mM and 1.2 mM. (In sea water composition, the Sr/Ca ratio is close to 0.01, and the Mg/Ca ratio is close to 5, with a Ca concentration close to 12 mM). Finally, the poly-L-aspartic acid concentration is at two levels, 2 and 20 g/ml. Each solution (3 ml in volume) was

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87 kept at room temperature (20C) for three days in a closed desiccator in the presence of vaporized ammonium carbonate (ACS, Aldrich). Then the films/crystals that formed on the cover slips were taken out from each solution and washed with deionized water and ethanol, and then dried for later examination. Polarized Optical Microscopy (POM) and Crystallinity Analysis The cover slip from each sample was examined using an Olympus BX60 polarized optical microscope in transmission mode, using crossed polars and an optional first-order red -plate. X-Ray Diffraction and Analysis. X-Ray powder diffraction patterns were recorded using a Philips PW1710 diffractometer, operating with a Cu source (Cu K1, =1.54056 ). The diffraction angle varied from 20 to 60 degrees, with a step size of 0.02/sec. SEM and X-ray Energy Dispersive Spectroscopy (EDS) Selected samples were gold coated for SEM imaging on a JEOL 6400 SEM instrument equipped with a EDS tractor system. The EDS detector includes an ultra-thin window which permits analysis of elements from Boron to Uranium. Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) Each film was dissolved in 0.1 N HCl and the Ca, Mg, and Sr concentrations were measured by a Perkin-Elmer Plasma 3200 Inductively-Coupled-Plasma Spectroscopy (ICP) system. The partition coefficient (D) was determined by the following equation: solutionCaMcrystalCaMD])[:]([])[:]([ , (5-1) Where M is Mg2+ or Sr2+, and [M] symbolizes the molar concentration of these ions.

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88 Table 5-1. Experimental condition of 223 factorial statistical design No. CaCl2 (mM) MgCl2 (mM) SrCl2 (mM) PAsp (g/ml) 1 12 24 0.12 2 2 12 42 0.12 2 3 12 60 0.12 2 4 12 24 1.2 2 5 12 42 1.2 2 6 12 60 1.2 2 7 12 24 0.12 20 8 12 42 0.12 20 9 12 60 0.12 20 10 12 24 1.2 20 11 12 42 1.2 20 12 12 60 1.2 20 13* 12 0 0 0 14* 12 0 0.12 2 15* 12 42 0 2 16* 12 42 0.12 0 * Note: experiment No. 13, 14, 15, and 16 are experimental controls without Mg, or Sr, or Polymer. Results and Discussion Amorphous to Crystalline Transformation in the PILP Process Figure 5-1A, Figure 5-2, and Figure 5-3, clearly show the influence of various factors (Mg, Sr and Polyaspartic acid) on the crystallography of the films deposited via the PILP process, at different times of drying, respectively. Each figure shows the inhibitory effect of Mg, Sr and Pasp on the crystallization of CaCO3. Influence of Mg in starting solution From top to bottom in each column, as the Mg concentration in the starting solution increases, the resultant films become less crystalline. For example, in Figure 5-1A, sample 1 (24 mM Mg in starting solution) has more spherulitic crystals than sample 3, which contained 52 mM Mg in the starting crystallization solution, while the levels of Sr and Pasp in the solution remain the same. As discussed in chapter 4, Mg can be entrapped in the precursor phase, and when solidified, it tends to stabilize

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89 the amorphous calcium carbonate phase, thus delaying the amorphous to crystalline transformation and making sample 3 less crystalline than sample 1. A similar inhibitory effect can be seen from each column from 4, 5 to 6; from 7, 8 to 9; and from 10, 11 to 12; in Figure 5-1A, Figure 5-2 and Figure 5-3. Influence of Sr While the influence of Mg is pronounced in the inhibition of the amorphous to crystalline transformation in the films deposited by the PILP process, the influence of Sr is not so evident. However, the inhibitory effect of Sr can still be seen when the Mg level in the starting solution is low. For example, when the Mg level is 24mM, sample 4 appears to be less crystalline than sample 1 in Figure 5-1A, and the same is true when comparing sample 7 to sample 10. When the Mg level goes higher, to 42 mM, this inhibitory effect of Sr can only be seen when comparing sample 2 to sample 5, at a low level of Pasp in Figure 5-1A. Influence of polymer The Polyaspartic acid at two levels plays a key role in precursor film formation since the control experiments. Figure 5-1B, No. 13, 14 and 16, show that no film can be deposited without polymer addition. Also, it can be shown that the higher polymer concentration tends to stabilize the amorphous films more than lower concentration. For example, when comparing 1 to 7, 2 to 8, and 4 to 10, in Figure 5-1A, the inhibitory effect of polymer on the amorphous to crystalline transformation can be clearly seen. Influence of drying time: transformation rate After the precursor films are deposited on the glass slides, which take about 3 days at room temperature, each film is washed with H2O and dried with ethanol and kept at room temperature. We find each precursor film tends to transform gradually to

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90 the crystalline phase (notewithout Mg impurity, the transformation is usually already complete before removal of the films from the solution). The mechanism of this transformation at the molecular level is not known, but the important overall feature is that the shape delineated by the phase boundaries of the precursor is retained, leading to crystals with non-equilibrium morphologies. The mobility of the ions within the liquid-precursor phase, loss of waters of hydration, stress due to impurity incorporation and water loss, and polymer-CaCO3 association, probably play very important roles in this transformation during this structural re-organization. As demonstrated in this study, the impurity levels entrapped in the precursor phase greatly influence this transformation rate. For example, when the starting Mg, Sr level is very low, as shown in sample 1, the precursor film quickly transforms to be fully crystalline after 1 day. More spherulitic crystals are shown after 7 days in in No.1 of Figure 5-2, and the spherulitic crystals cover the whole slide after 50 days. On the other hand, when the Mg and Sr level is very high, as shown in sample 6, the transformation rate is so slow that it remains almost all amorphous, even after 50 days, as shown in No. 12 of Figure 5-3. Another important factor that affects this transformation is polymer concentration. The fluorescence study in chapter 1 already shows that polymer is closely associated with the precursor CaCO3 phase. The incorporation of polymer in the precursor phase is seen to have a strong influence on the transformation rate. For example, Sample 5 and 11 both remain amorphous at 1 day; however, after 50 days, sample 5 is quite crystalline while sample 11 remains almost entirely amorphous. The only difference between these two is that the latter has a much higher polymer concentration in the starting solution, thus more amount of polymer associated in the precursor phase.

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91 An extreme example is sample 12, which contains the highest level of Mg, Sr and polymer in the starting solution, such that the deposited film remains amorphous even after 50 days due to the high level of impurity and polymer association in the precursor film. Morphology of the Films Deposited by the PILP process Figure 5-5 shows the typical morphology of the deposited films from sample 1 to 12, as formed from the conditions shown in Table 5-1. All the films appear similar under SEM, being quite continuous films embedded with many particles, probably post-precipitated PILP droplets with dimensions around 2 m in diameter. Figure 5-6 shows typical SEM features seen at higher magnification, and the corresponding EDS spectra of the films. Figure 5-6A is an SEM image of sample 2, as described in Table 5-1 (Mg/Ca ratio = 3.5, Sr/Ca = 0.01, Pasp = 2 g/ml). The smooth nature of the film and coalescence of particles can be clearly seen from this picture. Corresponding EDS spectra show the presence of Ca and Mg in the deposited films (due to the low amount of Sr incorporated in the film, it can not be detected by EDS). Figure 5-6C shows an SEM picture of sample 3 (Mg/Ca = 5, Sr/Ca =0.01, Pasp = 2 g/ml). The film has an appearance similar to the previous one. Once again, the EDS spectrum shows the presence of Mg and Ca in the deposited film (Figure 5-6D), and it clearly shows a higher Mg peak in sample 3 compared to sample 2 due to the higher Mg incorporation. Crystal Structure Verification After 6 months, XRD was performed on all samples from 1 to 12 to verify the crystal phase. Figure 5-7 shows typical XRD spectra of sample 4, 5 and 6. All the samples show typical calcite peaks with a very broad background. By comparison with the synthetic calcite XRD spectrum, which is characterized by very sharp and

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92 high intensity 104 peaks, it can be inferred that there is still some amorphous calcium carbonate phase coexisting in each of our samples. Mg and Sr incorporation in CaCO3 by the PILP process Table 5-2 shows the results of impurity incorporation in CaCO3 determined from ICP measurements, when using higher molecular weight polymer (Mw=35400, determined by LALLS), and Table 5-3 shows impurity incorporation with polymer at lower Mw (8600). Table 5-2. Mg and Sr incorporation in CaCO3 film in the PILP process using 223 factorial design (Pasp, Mw=35,400, room temperature). Variable Response Distribution coefficient Exp No. Ca (mM) MgCl2 ( mM) SrCl2 (mM) Pasp (g/ml) Ca mol% Mg mol Sr mol% DMg DSr 1 12 24 0.12 2 81.75 17.50 0.75 0.11 0.92 2 12 42 0.12 2 75.20 24.12 0.68 0.09 0.90 3 12 60 0.12 2 64.23 35.20 0.57 0.11 0.88 4 12 24 1.2 2 77.55 13.96 8.49 0.09 1.09 5 12 42 1.2 2 69.52 22.41 8.07 0.09 1.16 6 12 60 1.2 2 62.06 31.17 6.77 0.10 1.09 7 12 24 0.12 20 82.20 17.02 0.78 0.10 0.94 8 12 42 0.12 20 75.11 24.23 0.66 0.09 0.88 9 12 60 0.12 20 67.96 31.46 0.58 0.09 0.86 10 12 24 1.2 20 78.99 12.86 8.15 0.08 1.03 11 12 42 1.2 20 71.89 21.64 6.48 0.09 0.90 12 12 60 1.2 20 63.53 29.72 6.75 0.09 1.06 Three variables, Pasp, SrCl2, and MgCl2 concentration, in the starting crystallization solution were studied at 2, 2 and 3 levels, respectively, and for each set of experimental variables, the thin film morphology was deposited via the PILP process. The impurity (Ca mol%, Mg mol% and Sr mol%) in each film was set as the response. Then this table of values was put in Minitab software for statistical analysis of the relationship between variables and responses.

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93 Table 5-3. Mg and Sr incorporation in CaCO3 using 223 factorial design (Pasp, Mw=8,600, room temperature). Variable Response Distribution coefficient Exp No. Ca (mM) MgCl2 ( mM) SrCl2 (mM) Pasp (g/ml) Ca mol% Mg mol% Sr mol% DMg DSr 1 12 24 0.12 2 83.71 15.51 0.78 0.09 0.93 2 12 42 0.12 2 74.41 24.95 0.64 0.10 0.86 3 12 60 0.12 2 65.88 33.63 0.49 0.10 0.75 4 12 24 1.2 2 78.74 13.99 7.28 0.09 0.92 5 12 42 1.2 2 74.93 21.25 3.83 0.08 0.51 6 12 60 1.2 2 68.64 28.21 3.15 0.08 0.46 7 12 24 0.12 20 85.69 13.86 0.46 0.08 0.53 8 12 42 0.12 20 78.05 21.56 0.39 0.08 0.50 9 12 60 0.12 20 66.53 33.14 0.33 0.10 0.50 10 12 24 1.2 20 81.41 14.17 4.42 0.09 0.54 11 12 42 1.2 20 71.70 24.42 3.87 0.10 0.54 12 12 60 1.2 20 62.90 33.87 3.23 0.11 0.51 Factor A: MgCl2 , Factor B: SrCl2 ; Factor C: Polyasp (35400) Response 1: Ca mol%, ANOVA for selected factorial mode Table 5-4. Analysis of variance table [Partial sum of squares] for Ca mol%. Source Sum of squares DF Mean Square F value Prob > F Model 546.25 9 60.69 41.62 0.0237 A 492.75 2 246.38 168.95 0.0059 B 3.74 1 43.74 30 0.0318 C 7.28 1 7.28 4.99 0.1550 AB 0.68 2 0.34 0.23 0.8103 AC 1.65 2 0.82 0.56 0.6391 BC 0.12 1 0.12 0.081 0.8027 Residual 2.92 2 1.46 Corr. Total* 549.14 11 * Correction for total invariance As shown in Table 5-4, the Model F-value of 41.62 implies the model is significant. There is only a 2.37% chance that a "Model F-Value" this large could occur due to noise.Values of "Prob > F" less than 0.0500 indicate model terms are significant. In this case A, B are significant model terms. Values greater than 0.1000 indicate the model terms C, AB, AC and BC are not significant. So C (polymer level) is not a significant term to influence the Ca mol%.

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94 From the above analysis, we know that both MgCl2 and SrCl2 are significant factors to influence the total amount of Ca in the final deposited film. The higher the levels of MgCl2 and SrCl2 in the starting solution, the lower is the amount of Ca percentage in the final film due to the incorporation of Mg and Sr in the CaCO3 lattice. On the other hand, factor C, the Polymer concentration, is not a statisticaly significant factor to influence the Ca percentage in the final film. This response of Ca (mol%) versus different levels of MgCl2, SrCl2 and Pasp levels is plotted in Figure 5-8. It clearly shows that there is an inverse relationship between the Ca (mol%) in the film and the concentrations of MgCl2 and SrCl2 in the starting solution, while polymer level does not hardly modify the amount of Ca (mol%) in the film. Response 2: Mg mol%, ANOVA for selected factorial mode Table 5-5. Analysis of variance table [Partial sum of squares] for Mg mol% Source Sum of squares DF Mean Square F value Prob > F Model 583.95 9 64.88 82.70 0.0120 A 548.64 2 274.32 349.64 0.0029 B 26.33 1 26.33 33.56 0.0285 C 4.59 1 4.59 5.86 0.1366 AB 1.45 2 0.73 0.93 0.5189 AC 2.87 2 1.44 1.83 0.3531 BC 0.051 1 0.051 0.065 0.8225 Residual 1.57 2 0.780 Cor. Total* 585.52 11 * Correction for total invariance As shown in Table 5-5, the Model F-value of 82.70 implies the model is significant. There is only a 1.20% chance that a "Model F-Value" this large could occur due to noise. Values of "Prob > F" less than 0.0500 indicate model terms are significant. In this case A, B are significant model terms. Values greater than 0.1000 indicate the model terms C, AB, AC and BC are not significant. There is no interactions between A, B and C.

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95 From the above statistical analysis it can be shown that both MgCl2 and SrCl2 in the starting crystallization solution are significant factors to influence the final Mg percentage in the deposited film. Similarly, polymer levels does not contribute much to the amount of impurity incorporation in CaCO3 (as long as the concentration is sufficient to induce the amorphous precursor, which then allows for large amounts of Mg and Sr incorporation by the PILP process). The response of Mg mol% to these three variables is plotted in Figure 5-9. It clearly shows that as the MgCl2 in the solution increases, the amount of Mg incorporation in the film also increases dramatically. However, as the SrCl2 in the solution increases, the Mg incorporation in the film decreases, which is rational because Sr competes with Mg to be incorporated into the CaCO3 phases. Response 3: Sr mol%, ANOVA for selected factorial mode Table 5-6. Analysis of variance table [Partial sum of squares] for Sr mol% Source Sum of squares DF Mean Square F value Prob > F Model 141.51 9 15.72 96.31 .0103 A 1.57 2 0.79 4.82 .1610 B 137.96 1 137.96 845.07 0.0012 C 0.31 1 0.31 1.89 0.3213 AB 0.98 2 0.49 3.02 0.2142 AC 0.36 2 0.18 1.11 0.4230 BC 0.32 1 0.32 1.99 0.3021 Residual 0.33 2 0.16 Cor Total* 141.84 11 *Means correction for total invariance. As shown in Table 5-6, the Model F-value of 96.31 implies the model is significant. There is only a 1.03% chance that a "Model F-Value" this large could occur due to noise. Values of "Prob > F" less than 0.0500 indicate model terms are significant. In this case B are significant model terms. Values greater than 0.1000 indicate the model terms A, C, AB, AC and BC are not significant.

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96 The above statistical analysis indicates that the Sr incorporation in CaCO3 is only influenced by SrCl2 concentration in the starting solution. Surprisingly, it is not influence much by the MgCl2 concentration despite the fact that Mg and Sr compete against each other to be incorporated into CaCO3. Figure 5-10 shows the amount of Sr percentage in the film versus MgCl2, SrCl2 and polymer levels. It clearly shows that SrCl2 has a primary influence on Sr incorporation in CaCO3. Even with a MgCl2 change from 24 to 60mM, there is not much difference in Sr incorporation. Similarly, polymer also shows little influence on Sr incorporation. These results suggest that impurity incorporation in the amorphous calcium carbonate state will be independent of polymer. Polymer only appears to affect the kinetics of precursor formation and transformation, and after the precursor film is formed, the impurity incorporation is only determined by this distribution effect. Influence of Molecular Weight of Polymer It should be mentioned that the above results and discussion are from experiments with high molecular weight polymer (Mw=35,400). Similar results have been observed for the influence of Mg, Sr and polymer levels in the starting crystallization solution on the amorphous to crystalline transformation in the PILP process using lower polymer Mw (8,600). For example, comparison of Figure 5-3 and Figure 5-4 show that polymer at higher molecular weight (Mw=35,400) is more inhibitory than that at lower molecular weight (Mw=8,600). Distribution Coefficient The Distribution coefficient of Mg, DMg, was found to equal ~0.1 in all 12 samples (DMg = Mg/Ca ratio in film/Mg/Ca ratio in solution), as shown in Table 5-2 and Table 5-3. The DSr values range from 0.88 to 1.06 at Mw = 35400 (Table 5-2), while Dsr ranges from 0.46 ~ 0.93 for Mw= 8600 (Table 5-3). This is close to the DSr

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97 = 1 in coral and DSr = 0.4 ~ 0.6 in some foraminifera shells grown at room temperature. The reason for this difference is unknown. It seems polymer of higher molecular weight tends to lead to more Sr incorporation than that of lower molecular weight. Due to the large radius of Sr as compared to Ca, it may be more sensitive than Mg to the influence of polymer, which plays a key role to induce the liquid precursor phase. On the other hand, Mg has a much smaller radius compared to Ca, so it is much easier than Sr for incorporation, and was found to not be sensitive to the change of molecular weight of polymer. Discussion Incorporation of Mg in Calcite Our result shows that the amount of Mg incorporated in CaCO3 by the PILP process is influenced by the Mg/Ca ratio in solution. By varying the Mg/Ca ratio in the starting solution from 2 to 5, the amount of Mg incorporation ranges from 12.8 mol% to 35.2 mol%. This is much higher than the Mg-ion incorporation from inorganic precipitation methods. Mucci et al. precipitated inorganic magnesian calcite from sea water at room temperature, and found a Mg content of 8.1% at a Mg/Ca ratio of 5.14 (standard sea water), and a distribution coefficient DMg of 0.017, which is significantly lower than the value of 0.1 in the PILP process. Katz determined the effect of temperature on the partition coefficient for Mg in inorganically precipitated calcite to be DMg = 0.0009(TC) + 0.035. At 23C used in our experimental study, the DMg would be expected to be 0.0557, which is much lower than the measured value of 0.1 in our experiments. Both the high Mg incorporation and distribution coefficients support the premise that polymer plays an active and direct role in the precipitation of calcium carbonates in biogenic minerals.

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98 It can also be inferred that not only the PILP process obtains a high amount of Mg incorporation. Any process involving an amorphous calcium carbonate transformation process may also lead to a high amount of Mg incorporation in calcium carbonate (e.g., high super-saturation ratio, high Mg/Ca ratio to induce ACC phase, low temperature). In this case, although high Mg incorporation in the ACC phase can be achieved initially, the impurity can be excluded and re-dissolved during the formation of aragonite, which contains a very low amount of Mg. A recent study by Meldrum et al. demonstrated that even small organic additives, such as citric acid and maleic acid, will inhibit aragonite growth and form polycrystalline calcite aggregates with Mg contents of up to 22mol% at a Mg/Ca ratio of 6.25 in the parent solution (51). As mentioned earlier, the small molecule organics can yield similar behavior, albeit much less pronounced than with polymeric additives. Additionally, our PILP process can control the stability of the ACC phase when varying the Mg/Ca and Sr/Ca ratios, and Polymer concentration in the starting solution, which leads to formation of CaCO3 with both non-equilibrium morphology and non-equilibrium composition. It is the combination of both of these two features that makes the PILP process relevant to biomineralization processes. For example, nature can make high-magnesian calcite with a Mg incorporation of up to 35mol%, where such high MgCO3 percentages in biogenic HMC (29-35%) have been reported from red algae (96-98). One feature that still remains a challenge to us is in determining how nature manages to build single-crystalline structures out of high magnesian calcite. The addition of Mg-ion tends to lead to spherulitic films in our system. Incorporation of Sr in CaCO3 Strontium has been found to be favorably incorporated into aragonite due to the similar radius of the Sr ion to that of the larger cation sites in the aragonite crystal

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99 lattice as compared to calcite (56). Sr has also been found to be incorporated in biogenic calcite as well as inorganic calcite(93). Morse and Bender observed partition coefficients of Sr in inorganic calcite to vary from 0.03 to 0.12 (71). A large discrepancy in partition coefficients between inorganically precipitated calcite and biogenic calcite is observed in planktic foraminifers, where DSr = 0.16. Recently, Deckker et al. derived an equation of Dsr related to temperature in ostracod calcite, where Dsr = 0.223 + 0.0086*(TC). This equation was obtained from in-vitro culturing of juveniles of the euryhaline ostracod Cyprideis australiensis grown to adulthood in waters of ranging salinities, Mg/Ca ratio, Sr/Ca ratios, and under two different temperature regimes, 20C and 25C (93). At 23C, the equation will give a value of 0.42, which is close to our observed Dsr of 0.46 at a Mg/Ca ratio of 5, using the low molecular weight polymer (Mw=8600) to induce the PILP process. In red algae, Dsr equals 0.326, which falls within the range of 0.26-0.43 for HMC from diverse invertebrates (96). On the other hand, in coral the Dsr has a high value almost equal to unity, but in this case, the Sr is incorporated into aragonite (56). In the PILP process, in accord with the Mg experiments, a high amount of Sr can be incorporated into CaCO3. The DSr values obtained, which range from 0.46 ~ 1.16, are much higher than that found in inorganically precipitated calcite, and are much closer to that found in biogenic calcium carbonate. For example, our value is close to DSr = 1 in coral and DSr = 0.4 ~ 0.6 in certain foraminifera shells (96). Role of Polymer in Impurity Incorporation Polyaspartic acid at the two levels tested had no significant influence on impurity incorporation. This suggests that impurity incorporation in the amorphous calcium carbonate state will be independent of polymer conc., which could be useful

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100 information with respect to biogeochemical studies obtained from biogenic sediments. Preliminary studies show polymer concentration primarily affects the induction time of precursor formation, and has some influence on the amorphous to crystalline transformation time once the precursor film is formed, while the impurity incorporation seems to be primarily determined by distribution effects of constituent ions. Conclusion From the above results and discussion of impurity incorporation in CaCO3 by the PILP process, several conclusions can be drawn: High magnesian calcite (HMC), ranging from 12 mol% to 35 mol% MgCO3, can be obtained by the PILP process. The HMCs are presumably formed from entrapment in the polymer-induced amorphous precursor phase. The final amount of Mg incorporation in CaCO3 is much influenced by the MgCl2 and SrCl2 levels in the starting solution, but shows no dependence on the level and molecular weight of the Pasp, as determined from a factorial statistical analysis. High amounts of Sr (0.33~8.49 mol% of SrCO3) can be incorporated into CaCO3 by the PILP process. Again, the Sr is first incorporated into an amorphous precursor, which later gradually transforms into calcite. The final amount of Sr incorporation in CaCO3 is only influenced by the SrCl2 level in the starting solution, showing little dependence on the levels of MgCl2 and Pasp, as determined from statistical analysis. However, unlike the Mg additive, the Sr incorporation is also strongly influenced by the molecular weight of Pasp, with a higher molecular weight polymer leading to higher Sr incorporation. The amount of Mg, Sr and polymer incorporation in the precursor CaCO3 phase greatly affects the amorphous-crystalline transformation in the PILP process. Both impurity and polymer show inhibitory effects on this transformation, but such effects are much more pronounced for Mg than Sr. Polymer plays a critical role by inducing the amorphous precursor, which not only allows for the large amount of impurity incorporation, but also stabilizes the amorphous phase and produce thin films with both non-equilibrium morphology and non-equilibrium composition. The distribution coefficient of Mg, DMg, is independent of SrCl2, MgCl2 and polymer levels in the solution, while it is strongly influenced by temperature (results from previous chapter). This result strengthens the suggestion that Mg/Ca ratio in biominerals can be used as a temperature proxy. Both Mg and Sr can be incorporated in CaCO3 at levels substantially higher than impurity incorporation from inorganically precipitated calcite, with levels much closer to those found in natural biominerals. The formation of minerals with both non-equilibrium morphology and non-equilibrium composition suggests the relevance of the PILP process to biomineralization. Due to the disparity of impurity incorporation in

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101 biogenic minerals to those from inorganic origin, impurity incorporation in minerals becomes an important feature of potential biomarkers for a biological formation mechanism.

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102 A 1 4 7 10 2 5 8 11 3 6 9 12 B 13 14 15 16 Figure 5-1. Polarized optical micrographs of samples 1-16, prepared as described in Table 5-1. A) POM pictures of samples 1 to 12 show films with different crystallinity after 1 day of drying time (Pasp Mw = 35,400, RT). Most of the films are amorphous since they are just taken out from the solution and dried. Bar = 100 m). B) POM pictures of control samples from No. 13 to No.16, as described in Table 5-1. Note: in 13 and 16, without Polyaspartic acid, there is no precursor film deposition. Previous reports showed that only a very small amount of Polyaspartic acid is needed to induce precursor films in the presence of Mg, which is shown in sample 15. (All pictures taken at same magnification; bar = 100 m)

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4 7 10 1 11 2 5 8 12 6 3 9 12 Figure 5-2. POM pictures show amorphous-crystalline transformation of films after 7 days (Pasp Mw = 35,400, RT, samples 1-12, as shown in Table 5-1.)

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104 1 4 7 10 10 2 5 8 11 11 3 9 12 6 12 Figure 5-3. POM pictures of each film after 50 days transformation (Pasp Mw=35,400, RT, samples 1-12 as shown in Table 5-1).

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105 1 4 7 10 2 5 8 11 3 6 9 12 Figure 5-4. POM pictures of each film after 10 days transformation (Pasp Mw=8,600, RT, Sample 1-12 as shown in Table 5-1.). Note that the low molecular weight polymer produces films that take less time (10 days) to transform to similar crystallinity than those formed with higher molecular weight polymer (50 days as shown in Figure 5-3; all pictures are same magnification.).

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106 1 4 7 10 2 5 8 11 3 6 9 12 Figure 5-5. Typical SEM images of thin CaxMgySr(1-x-y)CO3 films prepared by the PILP process. (Sample No. 1-12 as described in Table 5-1. Pasp Mw=35,400, RT). (All bars = 20 m).

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107 A B C D Figure 5-6. Typical SEM pictures of the films deposited by the PILP process at higher magnification, along withtheir corresponding EDS spectra. A) Higher magnification of SEM pictures of sample 2 (Mg/Ca = 3.5, Sr/Ca = 0.01, Pasp = 2 g/ml, Mw = 35,400, RT). B) EDS spectrumof the sample shown in A). C) Higher magnification SEM pictures of sample 3 (Mg/Ca = 5, Sr/Ca = 0.01, Mw=35,400, Pasp = 2 g/ml). D) EDS spectrum of the sample shown in C). Bars = 10 m.

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108 05010015020025030020304050602 thetaintensity 6 5 4 012 104 113 202 116 Figure 5-7. Typical XRD spectra of CaxMgySr(1-x-y)CO3 thin films deposited by the PILP process (Samples No 4, 5 and 6 are shown in Table 5-1. Pasp Mw=35,400) XRD was done after 6 months transformation, and all samples showed the typical calcite peaks and some amount of amorphous calcium carbonate.

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109 2025303540455055606560626466687072747678808284 Ca (mol%)MgCl2(mM) Sr/Ca=0.01, Pasp=20 Sr/Ca=0.01, Pasp=2 Sr/Ca=0.1, Pasp=20 Sr/Ca=0.1, Pasp=2 Figure 5-8. Response of Ca content in the film versus variables. Statistical treatment of the data shown in the above plot reveals that the calcium content in the deposited film is influence by both Mg and Sr. As Mg or Sr concentration increase, there will be a decrease in the calcium content. This phenomena is due to the incorporation of Mg and Sr in calcium carbonate lattice.

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110 2025303540455055606512141618202224262830323436 Sr/Ca=0.01, Pasp=2 Sr/Ca=0.01, Pasp=20 Sr/Ca=0.1, Pasp=2 Sr/Ca=0.1, Pasp=20Mg(mol%)MgCl2 (mM) Figure 5-9. Response of Mg content in the film versus variables. The above plot means the Mg content in the film is influence by both Mg and Sr; the higher the Mg concentration in solution, the higher the Mg content in film. However, the higher the Sr concentration in film, the smaller Mg content in film, because the Sr and Mg compete to be incorporated into the calcium carbonate.

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111 202530354045505560650123456789 Sr/Ca=0.1, Pasp=2 Sr/Ca=0.1, Pasp=20 Sr/Ca=0.01, Pasp=20 Sr/Ca=0.01, Pasp=2Sr (mol%)MgCl2 (mM) Figure 5-10. Response of Sr content in the film versus variables. Statistical treatment of data shown in the above plot reveals that the Sr content in the film is only influenced by the Sr concentration in parent solution, and not by polymer concentration or Mg content. 1 4 7 10 2 5 8 11 3 6 9 12

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CHAPTER 6 MOLDING CaCO3 MINERALS BY THE PILP PROCESS This chapter is mostly concerned with potential applications of the PILP process. First it shows that the PILP process can be used to produce biomimetic laminated organic-inorganic (Polymer-CaCO3) composites by deposition of the precursor phase on a polymer hydrogel substrate. Then, by adopting a compartmentalization strategy, the PILP process can enable the to formation of molded CaCO3 with complex morphology. Introduction Industrial Methods to Prepare Inorganic-Organic Composites There are many ways to make organic-inorganic (hybrid) composites on the market today. However, the most usual way in industry is to mix the organic (usually plastic) and inorganic mineral phase, and then mold, extrude, or blow it into the final product. This has to be performed at high temperatures, at least above the melting point of the polymer. Another common way to make organic-inorganic composites is by solution casting, where the organics are dissolved in a solvent and inorganic particles are dispersed in the solution. But this process needs to remove a large amount of solvent. The solvents used are usually toxic and expensive. In addition to issues relating to “green” chemistry, it should also be mentioned that high mineral loading cannot easily be achieved in synthetic composites, which in turn leads to unique mechanical properties, particularly materials with both high strength and toughness, which is of keen interest to the biomimetics community. In addition, the interaction between mineral phase and organic phase may be very weak in the above synthetic methods, often requiring surface modification of the 112

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113 particles, so the organic-inorganic interface in biomineralized tissues has been another active area of research. Solid-free-form fabrication is another method to make inorganic-organic hybrid composites, where thin layers of materials are repetitively added to build large structures by a machine (e.g., inkjet printer or a system like a pen-plotter). This process is similar to the sol-gel process, where solidification and shrinkage is very difficult to control. Of all these methods to prepare organic-inorganic composites, none successfully mimics the natural biomineralization process to make organic-inorganic composites, where inorganic phase are usually molded inside the organic compartment or matrix at mild conditions. Natural Methods of Making Organic-inorganic Composites Biominerals often have exquisite morphologies and exhibit a high degree of crystallographic controls, such as crystal size and shape (morphology), as well as crystal orientation, texture, location, and assembly. One important example is the structure of the sea urchin spine. Recent study has shown that spine regeneration of the adult sea urchin proceeds via the initial deposition of amorphous calcium carbonate granules which are deposited on the outer surface of the fracture spine, and meld into the single-crystalline calcite base as it is regrown, ultimately being shaped into a three-dimensional microporous calcium carbonate structure with interconnected pores of around 10 m in diameter. The authors suggested that this is likely a generalized strategy in biomineralization, in which these transient amorphous phases transform to produce single crystals of calcite with complex morphology, the hallmark of biominerals. Macromolecules associated with the mineral phase play a very important role in not only regulating this amorphous-crystalline transformation, but also in controlling the

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114 mechanical properties of the biominerals. The single-crystal Mg-calcite spine of adult sea urchin spines contain 0.1wt% of protein. Such a small amount of protein is sufficient to modify their mechanical properties (99). The material design strategy learned from this classic biomineralization model (there are also many other examples of how occluded macromolecules affect mechanical properties of biominerals such as nacre, conch shell) is that materials can be isotropic in macroscopic scale (e.g., fracture behavior) while they are highly anisotropic at the nanoscopic scale due to occlusion of polymers into the crystal texture. Biomimetic Methods of Making Organic-Inorganic Composites Meldrum et al. have proven a synthetic approach can be successful in producing a single-crystalline calcium carbonate replica by using double-diffusion approach, in which counter ions (calcium and carbonate) are diffused across a gel into a synthetic sea urchin spine replica placed in the middle of a U-tube. However, the role of macromolecules associated with the sea urchin spine was ignored in their study, and an amorphous phase was not observed or used to form this structure. The formation of a complex calcite morphology without an amorphous calcium carbonate (ACC) precursor does not reflect the true mechanisms of biomineralization in the sea urchin spine (100). We are trying to develop a biomimetic material synthesis method to make an artificial material by learning two widespread strategies to produce inorganic-organic composites in nature: using transformation of amorphous precursor phases, and using occluded polymer inside the crystals to form interpenetrating composites which at the nanoscopic scale influence the mechanical properties. Our method of molding minerals inside or onto a compartment involves the use of a polymer-induced liquid precursor phase that is formed from aqueous solution at low

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115 temperature. This biomimetic process is environmentally benign by not adopting the use of harsh solvents. The mineral phase is formed from the aqueous solution and molded in-situ within/onto the compartment. This feature allows the mineral phase to interact with the compartment at the nanoscopic scale in-situ so that large structures can be built up from this bottom-up approach. This feature also allows for high mineral loading and better mechanical properties of the resultant material due to the occlusion of polymers inside the crystals. Due to the fluidity of the PILP phase, large amounts of impurities or active agents can be entrapped inside the mineral phase so that a hybrid organic-inorganic can be achieved for multi-functional applications. Materials and Methods Preparation of PHEMA Sheets and Micro-porous PHEMA Scaffold PHEMA (poly-2-hydroxyethyl-methacrylate) hydrogels were made by a thermo-initiated free radical polymerization. Briefly, 8 g of HEMA monomer was mixed with 0.022 g AIBN into a transparent solution. Then 5.333 g of H2O was added into the above solution. After flushing with N2 for 2 min., the solution was poured into the mold and put in an oven (67C) for 2 hr. Thin sheets of PHEMA were prepared inside a glass-mold created with Teflon spacers (The spacers were put between two pieces of glasses, which were held together by clips). Microporous PHEMA was prepared by using a “sea urchin spine” as a mold, and after polymerization, the calcium carbonate of the sea urchin spine was dissolved away to generate the PHEMA hydrogel replica. These micro-porous hydrogel replicas were then rinsed in large amounts of ultra-pure water to remove any remaining organic and salts associated with original sea urchin plate. The dry PHEMA replica had the same structure as the sea urchin plate, with pore diameters of about 10 ms.

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116 The equilibrium water content of the porous hydrogel replicas and the non-microporous hydrogel was determined as follows: Equilibrium water content (EWC %) = (Wwet-Wdry) /W0, 100, where W0, Wwet, Wdry are: W0: original weight Wwet: weight after swelling for 24 hr. Wdry: weight after drying in vacuum for 24 hr. The data of EWC determination is shown in Table 6-1. Table 6-1. Equilibrium water content (EWC%) determination of PHEMA sheets and porous PHEMA scaffolds made from replication of a sea urchin spine. Pure PHEMA W0 Wwet Wdry EWC% Standard error 1 0.1314 0.2085 0.1294 60.2 2 0.1242 0.1835 0.1153 54.9 3 0.1320 0.2040 0.1286 57.1 4 0.0803 0.1231 0.0769 57.5 Average EWC of PHEMA sheet 57.4 2.17 Porous PHEMA W0 Wwet Wdry EWC% Standard error 1 0.0073 0.0191 0.0074 160.3 2 0.0056 0.0129 0.0059 125.0 3 0.0159 0.0405 0.0161 153.5 4 0.0101 0.0251 0.0103 146.5 5 0.0157 0.0472 0.0161 198.1 6 0.0107 0.0306 0.0108 185.0 7 0.0091 0.0242 0.0092 164.8 8 0.0289 0.0716 0.0292 146.7 Average EWC of Sea urchin replica 160.0 23.1 Mineralization of PHEMA Sheet and Porous PHEMA Scaffold The mineralization is similar as that described in the previous chapter. The experimental conditions of mineralization of PHEMA sheets is shown in Table 6-2 .

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117 Table 6-2. Experimental condition of mineralization of CaCO3 onto PHEMA substrate. (Condition: room temperature, 3 days of vapor diffusion, Poly-, , d, L-aspartic acid, sodium salt, Mw=6200). Exp. No. 1 2 3 4 5 PHEMA substrate Yes yes yes yes yes CaCl2 (mM) 12 12 12 12 12 Pasp (g/ml) 0 2 20 100 500 The mineralization study of the porous PHEMA hydrogel was done by placing these replicas into 0.56M CaCl2 solutions containing 1000 g/ml Pasp, and allowing for slow diffusion of (NH4)2CO3 into the solution. After a certain time, these replicas, as well as the mineral from the solution and surface of the solution, were collected, washed with water and dried. Characterization The material or mineral collected was characterized by using POM, SEM (JEOL-SEM 6400), TEM (JEOL-TEM 200 CX) and XRD analysis, similar as described in the previous chapter. XRD was carried out by scanning with Cu-K X-ray radiation from a Philips XRD 3720 at 40 KV and 20 mA, using a step size of 0.01 with a time of 2 sec/step, over a 2 range of 20-60. TGA/DTA analysis Thermo Gravimetric Analysis (TGA) and Differential Thermo Analysis (DTA) of commercial CaCO3, sea urchin spine and amorphous calcium carbonate precursor phase was carried out on a Shimadzu DT 30 thermal analyzer at a heating rate of 10C/min under an air atmosphere with about 5-10 mg samples.

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118 Results and Discussion Mineralization of CaCO3 Thin Films on PHEMA Substrate Figure 6-1 shows polarized optical micrographs of samples 1 through 5 (conditions specified in Table 6-1. In the control, without any polymer addition, typical rhombohedral calcite single-crystals formed on the PHEMA thin sheet (Figure 6-1A). When 2 g/ml of polymer was introduced into the solution, calcite single crystals with modified morphology were formed (Figure 6-1B). As the Pasp concentration in the solution increased to 20 g/ml, thin calcitic film patches began to form on the PHEMA sheet. Figure 6-1C is the image using a gypsum accessory plate. The PHEMA sheet is magenta in color (1st-order red) due to its amorphous nature, and is not easily seen, while the film patches are quite crystalline and birefringent. On the film patches there are also some crystal aggregates, which appear brown since very little light passes through the thicker aggregates. The corresponding polarized image without the gypsum wavelength plate is shown in Figure 6-1D. The amorphous PHEMA sheet became dark and the mineral film is bright due to its crystalline nature. When the concentration of Pasp in the solution was increased to 100 g/ml, the film formed on the PHEMA sheet became more continuous and almost covered the whole sheet. The POM image with gypsum wavelength plate is shown in Figure 6-1E, and the corresponding image without the gypsum plate is shown in Figure 6-1F. As the concentration of Pasp goes up to 500 g/ml, the film formed become more continuous and covered the whole PHEMA sheet (Figure 6-1G). The film is spherulitic as determined by the presence of a “Maltese cross“ pattern (Figure 6-1H).

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119 SEM images of the mineral deposited on the PHEMA substrates is shown in Figure 6-2. Again, the control sample 1 only shows rhombohedral calcite (Figure 6-2A). Modified calcite single crystals were formed on PHEMA substrates when the polymer concentration is only 2 g/ml (Figure 6-2B). Film patches along with crystal aggregates were deposited when the polymer in the solution went up 20 g/ml (Figure 6-2C). The film became more continuous on the PHEMA substrate at a polymer concentration of 100 g/ml in the solution. However, they tend to crack under the beam of the SEM (Figure 6-2D). Figure 6-2E is a lower magnification image of a film formed on the PHEMA substrate when the polymer concentration went up to 500 g/ml. It clearly shows that film covers almost the whole sheet. The higher magnification image shown in Figure 6-2F indicates that there are also rounded particles deposited on the CaCO3 films, which is very similar to the morphology of the films deposited on glass slides by the PILP process. A cross-section of the mineralized PHEMA is shown in Figure 6-2G), showing the thickness of the film is around 3 m. This clearly shows an organic-inorganic composite structure is achieved by the PILP process by using PHEMA as a substrate. A typical EDS spectrum of the thin film shows the presence of Ca, C, and O in CaCO3 composite (Figure 6-2H). An XRD spectrum of a sample film verifies that they are mineralized with the calcite crystal structure (Figure 6-3). The above results clearly show that the PILP process can offer a new strategy to form biomimetic thin ceramic films at room temperature from aqueous solutions. It should also be pointed out that PHEMA is a hydrophilic polymer (similar to PEG, which is an anti-fouling polymer), which does not show a tendency to absorb protein or biopolymer. The mechanism of mineral film formation should be similar to that formed

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120 on glass slides. Mainly, at certain polymer concentration, the polymer induces a liquid-like precursor phase, which adsorbs onto the PHEMA sheet. Then the precursor phase transforms to the crystalline structure, presumably retaining by occlusion some of the inducing polymer, and possibly interpenetration into the PHEMA hydrogel as well. Polymer additive at higher concentration tends to result in more continuous films. There is evidence that the PHEMA substrate has some favorable interaction with the liquid-precursor phase during the deposition stage (either through preferred adsorption, or preferred nucleation of the amorphous phase), because even at highest polymer concentration, there are no films observed in the solution or on the polystyrene dish. Another interesting feature is that the films formed on PHEMA are all of spherulitic texture, which is different from the single-crystalline mosaic films deposited on glass slide at normal non-scale-up condition (Figure 3-6A & B in chapter 3), as described earlier. This is puzzling since biominerals, which presumably are in close association with the organic matrix, are often single-crystalline, such as the sea urchin spine. On the other hand, these single-crystalline biominerals are usually formed within phospholipids vesicles (referred to as mineral deposition vesicles), while there are many other biominerals which have spherulitic textures, and these seem to be formed in association with polymeric matrices. Although these types of biominerals are not typically referred to as having a spherulitic texture, since the overall shape is not spherical, they do have polycrystalline radial outgrowths, with a texture that resembles that found in spherulites. From the appearance of various micrographs in the literature, the overall shape is not spherical, but rather appears constrained, and it usually elongated in some direction, apparently directed by the organization of the surrounding organic matrix.

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121 Molding CaCO3 Minerals within Compartment by the PILP Process Structure and composition of sea urchin spine SEM images of the cross-section of a fractured adult sea urchin spine (Arbacia tribuloides) is shown in Figure 6-4. Figure 6-4A indicates the spine has a skin-core structure. The skin region is about 100 thick with micro-channels, possibly for transportation functions since the spine motion is hydraulically driven (Figure 6-4B). The core region is composed of high-magnesian calcite with interconnected pores of pore size around 6-8 m in diameter (Figure 6-4C). An EDS spectrum shows the presence of Ca, C, O, and Mg peak (Figure 6-4D). A comparison of the composition of commercial CaCO3, a sea urchin spine, and an amorphous precursor phase generated by the PILP process, is shown in Figure 6-5, as determined by TGA/DTA analysis. Commercial calcium carbonate only shows one large endothermic peak in DTA, which corresponds to the dissociation of CaCO3 into CaO and CO2. TGA indicates the weight loss from this dissociation is about 44% (Figure 6-5A). The sea urchin spine shows an additional exothermic peak in the DTA at around 440C, which corresponds to heat emission due to the burning reaction of organics in the sea urchin spine. TGA determines that the organic phase is about 2.6% by weight of the spine (Figure 6-5B). Figure 6-5 C shows TGA/DTA of the mineral phase formed by the PILP process (condition: [MgCl2]/[CaCl2] = 3, [CaCl2] = 12 mM, Pasp = 1000 g/ml, vapor diffusion, 1 day, Room temperature). This intermediate ACC phase, between PILP precursor to CaCO3 thin film, distinctively has 3 peaks in DTA: one heat adsorption peak due to water loss, one heat emission peak due to burning of polymer, and another heat absorption peak due to the dissociation of MgCO3 and CaCO3 in the air/N2 atmosphere

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122 (101). TGA determines the water content is about 4.4% and the polymer content is about 1.7%. XRD of sea urchin spine is shown in Figure 6-6E. There is a shift of the peak to the right due to the inclusion of Mg in the calcite lattice. For example, the (202) peak shifts from 43.15 in pure calcite to 43.41 in the spine, and the d202 changes to 2.0826. According to the procedure described in Chapter 4, the Mg content in the sea urchin spine which causes this peak shift is: (2.095 2.0826) 100(2.095 1.939) = 7.9 mol%, which corresponds to about 6.8 wt% MgCO3 in calcium carbonate. Structure and characterization of micro-porous PHEMA hydrogel Figure 6-7 shows the structure of the microporous PHEMA hydrogel made by replicating the structure of sea urchin spine. This 3D microporous scaffold has a similar structure as that of the original sea urchin spine (Figure 6-7A), with interconnected pores and a pore size of around 6-8 m (Figure 6-7B). Notethe terminology “microporous” is used here to distinguish the fact that it has microporosity from replication of the spine, in addition to the typical nanoporosity of a PHEMA hydrogel. The EDS spectrum shows only C and O peaks, and no Ca peak, indicating the mineral from the original sea urchin spine has been totally leached out (Figure 6-7C). The equilibrium water content (EWC%) was determined and it shows that the microporous hydrogel absorbs almost 3 times more water than the non-microporous PHEMA hydrogel (Figure 6-8). This is an important point, as will be shown shortly, because the microporous hydrogel swells in water and leads to decrease of the pore size. In other words, the polymerization reaction of the PHEMA was performed under only

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123 40% water conditions, so when such a hydrogel is immersed in water, it will swell and the replicated shape will not be identical to the original shape of the spine. Molding CaCO3 inside a microporous hydrogel by the PILP process The microporous PHEMA hydrogels were put inside the crystallization solution. After 1 day of mineralization, particles were collected inside the hydrogel and dispersed and dried on a TEM grid. Figure 6-9 shows the TEM Bright-field (BF) image and corresponding electron diffraction (ED) pattern. As seen from the Figure, the mineral nanoparticles have a wide range of diameters. Figure 6-9A shows nanoparticles with a size of around 10 nm in diameter. Figure 6-9B shows that coalescence of the nanoparticles leads to larger structures, with diameters of around 60 nm. Absorption of small nanoparticles onto a large particle, 400 nm in diameter, is clearly shown in Figure 6-9C. Figure 6-9D shows particles that have grown as large as 1 m in diameter (approaching of the typical plateau size limit we have observed in the optical microscope). All particles are non-diffracting, as shown by ED, and presumed to be amorphous. This TEM and ED analysis indirectly shows ex situ evidence of the liquid-like nature of the amorphous precursor which is induced by the acidic polymer in the PILP process. The XRD spectrum of mineralized hydrogel after only 1 day shows a broad amorphous peak and a small crystalline calcite peak, indicating that the amorphous calcium carbonate begins to gradually transform to crystalline calcite after 1 day in solution (Figure 6-6B). At the same time frame, minerals were also collected from the solution after 1 day of mineralization. Figure 6-10A & B demonstrate that these amorphous particles are quite fluidic as they appear to have coalesced into complex structures. XRD also confirms that

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124 all these particles are amorphous (Figure 6-6A). At the air-water interface of the mineralization solution, amorphous and partially fluidic droplets are also observed (Figure 6-10C). These amorphous droplets transform to crystalline calcium carbonate after 7 day’s mineralization (Figure 6-10D). In this case, however, the transformation likely involved dissolution and recrystallization of the metastable precursor into the larger spherulites, as is sometimes observed in these reactions, rather than crystallizing via the pseudo-solid-state transformation which leads to crystals that retain the shape of the precursor phase. After 7 days of mineralization, the mineralized PHEMA hydrogel was taken out of the solution and dried. It is immediately apparent that the mineralization reaction has occurred because the mineralized PHEMA has become rigid, even while in the aqueous solution. The appearance of CaCO3 mineralized PHEMA hydrogels is shown in Figure 6-10. The non-mineralized PHEMA hydrogel is shown in Figure 6-11A; it is translucent due to the scattering of light inside the micro-pores. After 7 days, the mineralized PHEMA hydrogel appears solid white in color, indicating a heavily mineralized composite has been achieved (Figure 6-11B). The amorphous calcium carbonate phase has totally transformed to crystalline calcite after 7 days of mineralization (Figure 6-6C). A section of the original sea urchin spine is also shown in Figure 6-10C for comparison, although the coloration is purplish-brown due to biological organics. In order to examine the structure of the mineralized component within the hydrogel, it was burned (calcined) at 500C to remove the PHEMA phase. XRD was used to confirm that this temperature does not alter the calcite crystal structure in the composite (Figure 6-6D). Figure 6-12 shows SEM images of the remaining mineral structure after

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125 calcination. Figure 6-12A shows a microporous structure that closely resembles that of sea urchin spine was achieved. EDS shows the presence of C, O and Ca from the CaCO3 phase. In some areas, the structure collapsed and fell apart due to the heating, and perhaps being not as fully mineralized. In other regions, an interesting strutted network of mineral was formed, bearing a rather striking resemblance to some biominerals. We believe this change in morphology is due to the swelling of the microporous hydrogel, as mentioned earlier, which leads to a change in the dimensions and shape of replicated structure when the PHEMA is immersed in the aqueous solution. Overall, all structures were found to be composed of calcium carbonate structured into a 3-D porous network, with interconnected pores (Figure 6-12B & C). A higher magnification picture of this molded crystal structure shows pore diameters of around 6-8 m (Figure 6-12D). The EDS spectrum indicates the presence of Ca, C and O. The XRD spectrum (Figure 6-12D) of this calcined PHEMA replica shows calcite peaks similar to original sea urchin spine (Figure 6-12E), except for the small peak displacements from Mg incorporation in the spine, which was not included in our replication experiment. A control experiment was also done without the addition of any polymer. Figure 6-12F shows that only large rhombohedral calcite crystals attached to the surface of the hydrogel (which is not burnt out in this sample), and the mineral could not infiltrate inside the hydrogel. Although Meldrum was also using a mineralization reaction without polymer, and in that case, did replicate the microporous structure, her technique differed in that calcium carbonate was formed inside a microscopy resin (a flexible polymer) replica inside a U-tube by double-diffusion of Ca and CO32ions. We do not know if our microporous structure is single-crystalline, as was the case for Meldrum, but this does not

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126 appear to be the case, as judging by POM. Most likely a single nucleation site needs to be prescribed, perhaps by placing a calcite seed somewhere in the scaffold. Conclusion The above results show a mineralization study using PHEMA sheets and micoporous PHEMA scaffolds, with and without the PILP process. Several conclusions can be drawn: Mineralization of PHEMA sheets by the PILP process leads to the formation of thin CaCO3 films deposited on the PHEMA substrate. The films become more continuous as the polymer concentration increases. By sequential deposition of organic layers and sequential mineralization using the PILP process, a laminated composite can conceivably be fabricated from aqueous solution at low temperature. The precursor phase can be molded inside a compartmentin this case, a microporous scaffold, which capitalizes on the fluidity and mobility of the liquid-like precursor phase. The infiltration and subsequent solidification of precursor phase inside a porous compartment leads to the formation a rigid organic-inorganic composite. After the organic hydrogel is removed, a porous ceramic scaffold is formed. We first demonstrate that the presence of a liquid-like precursor phase grows from nanometer droplets to the microscopic scale. TEM also demonstrates the coalescence of precursor droplets. XRD, POM and TEM analysis indicates that the amorphous precursor phase transforms to crystalline calcite gradually with time. Further application of the PILP process and compartmental confinement can conceivably lead to the development of novel hybrid organic-inorganic composites. By in-situ precursor phase transformation and solidification, the PILP process can enable processing of mineralized composites which are structured at the nanoscopic scale. And more, due to the fluidity of the precursor phase, large amounts of impurities, which could be active agents, aqueous-based medicines, etc., can be molded within compartments in situ, leading to the formation of multi-functional hybrid composites. I will also demonstrate the formation of nano-structured composite by using nano-sized compartments in Chapter 8. Pasp = 2 ug/m l

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127 A B C D E F Figure 6-1. POM pictures of calcitic crystals/films deposited on PHEMA substrate. A) No. 1, control with no polymer. B) No. 2, Pasp = 2 g/ml. C) No. 3, Pasp = 20 g/ml, pictures with gypsum wavelength plate. D) No. 3, Pasp = 20 g/ml, pictures without gypsum wavelength plate. E) No. 4, Pasp = 100 g/ml, pictures with gypsum wavelength plate. F) No. 4, Pasp = 100 g/ml, pictures without gypsum wavelength plate. G) No.5, Pasp = 500 g/ml, pictures with gypsum wavelength plate. H) No.5, Pasp = 500 g/ml, pictures without gypsum wavelength plate. (All bars = 100 m)

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128 G H Figure 6-1. continued

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129 C D 1 mm 500 m E F 1m m 50 m Figure 6-2. SEM micrographs of CaCO3 crystal/films formed under conditions described in Table 6-1. A) No.1only calcite single crystals grow on the PHEMA substrate without the polymer process-directing agent. B) No. 2mainly calcite single crystals with distorted rhombohedral morphology are produced at a low polymer concentration of Pasp = 2 g/ml. C) No.3CaCO3 film patches are deposited on the PHEMA substrate at Pasp = 20g/ml. D) No.4more films are deposited on PHEMA at Pasp=100g/ml. E-F) No. 5at higher polymer concentrations of 500 g/ml, the film becomes more continuous and thicker. G) No.5A cross-sectional view of the PHEMA-CaCO3 composite shows that the thickness of the CaCO3 film produced in the presence of 500g/ml Pasp is about 3 m. H) An EDS spectrum shows the Ca, C, and O peaks of CaCO3.

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130 G H 50 m PHEMA CaCO3 3 m Figure 6-2. continued

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131 02004006008001000120014002025303540455055602 thetaintensity 104 202 018 113 006 110 116 012 122 Figure 6-3. XRD spectra of mineralized PHEMA sheet shows typical calcite peaks. (No. 5 in Table 6-1, Pasp = 500 g/ml.)

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132 A B 1mm 100 m C D 100 m 100 m Figure 6-4. SEM pictures of a sea urchin (Arbacia tribuloides) spine. A) Cross-section view of a fractured spine showing the skin-core structure. B) The skin region is composed of micro-channels, possibly for transportation function. C) The core region of the spine is composed of single-crystalline high-magnesian calcite, with interconnected pore size of around 10 m in diameter. This sea urchin spine also shows a conchoidal fracture, as has been described in the literature. D) EDS shows the presence of Ca, Mg, C and O. The Mg content in the CaCO3 spine is about 8 %, as determined by ICP, and confirmed by XRD, which is about 6.9%.

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133 A 020040060080010005060708090100110 TGA DTATemp(oC)TGA(%)-10-8-6-4-20246810 793.5 oC, 59%DTA (uv) 020040060080010005060708090100 785.5oC, 57.1% TGA DTATemp (oC)TGA (%)-25-20-15-10-505101520 583.6 oC, 95.1%408.5oC, 97.7%DTA (uv) B Figure 6-5. Comparison of the composition of commercial CaCO3, a sea urchin spine, and the products of the PILP process, by TGA and DTA. A) Commercial calcium carbonate (Sigma). There is only one heat absorption peak observed in DTA, which corresponds to the chemical dissociation reaction of CaCO3 into CaO around 720C. The TGA measures the corresponding weight loss, which is about 41% due to emission of CO2. B) Sea urchin spine. There are two apparent peaks in DTA; one corresponds to a heat emission peak due to the burning reaction of organics in sea urchin spine, and the other corresponds to the dissociation of CaCO3. TGA determines that the organic phase is about 2.6% by weight of the spine.C) Mineral phase formed by the PILP process (condition: [MgCl2]/[CaCl2] = 3, [CaCl2] = 12 mM, Pasp = 1000 g/ml, vapor diffusion, 1 day). This intermediate phase, between liquid precursor to CaCO3 thin film, distinctively has 3 peaks in the DTA: one heat adsorption peak due to water loss, one heat emission peak due to burning of polymer, and another heat absorption peak due to continuous dissociation of MgCO3 and CaCO3 in the N2 atmosphere (101). TGA determines the water content is about 4.4% and the polymer content is about 1.7%.

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134 020040060080010005060708090100 132.9 oC, 95.6% 373.9 oC, 93.9% 590.1 oC, 92.21%777.6 oC, 53.25% TGA DTATemp (oC)TGA (%)-4-202468 DTA (u v) (u v) C Figure 6-5. continued Figure 6-5. continued

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135 10203040506020040060080010001200140016001800200022002400260028003000320034003600 Intensity2 Theta E D C B A 014 202 110 018 012 012 116 122 006 1010 Figure 6-6. XRD analysis of the reaction progress during the spine mineralization. A) Amorphous calcium carbonate from the surrounding solution, at 1 day. B) CaCO3 mineralized PHEMA hydrogel after 1 day, showing both amorphous calcium carbonate and a weakly crystalline calcite phase (the relatively broad peak at 29). C) CaCO3 mineralized PHEMA hydrogel after 7 days. Noteit is totally transformed into crystalline calcite. D) CaCO3 scaffold after calcining the mineralized PHEMA sample at 500C for 2hr. E) Sea urchin spine. Notethe mineralized PHEMA hydrogel shows a similar XRD spectrum to the sea urchin spine after 7 days, and it is formed by a process that we believe mimics that of the biomineralization mechanisms, via an amorphous precursor phase.

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136 A B 200 m 20 m C Figure 6-7. SEM picture of the microporous PHEMA hydrogel scaffold made by replicating the structure of a sea urchin spine. A) Low magnification shows the 3-D interconnected porous structure. B) Higher magnification shows that the pore size is about 10 m. C) The EDS spectrum shows the absence of a Ca peak in the replica, and the presence of a high intensity C peak, indicating no CaCO3 debris, with a high purity PHEMA replica.

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137 0.020.040.060.080.0100.0120.0140.0160.0180.0200.01(EWC%) Microporous hydrogel Non-microporous hydrogel Figure 6-8. Comparison of Equilibrium Water Content (EWC%) of a non-microporous and micro-porous PHEMA hydrogel. The micro-porous PHEM hydrogel is the replica of the sea urchin spine, and it holds 3 times more water than the pure non-microporous PHEMA hydrogel.

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138 A B C D Figure 6-9. TEM and ED of the amorphous nano-particles collected at one time from the mineralizing PHEMA hydrogel replica. A) Particles from tens of nanometers in size, with the corresponding ED pattern showing that they are all amorphous. B) Particles from 50 nm to 150 nm in size. (note the coalesce of particles into larger structures.) C) A single particle with a diameter of around 400 nm, and adsorption of small particles of around 5 nm. ED shows a faint ring pattern which is weakly crystalline. D) Particles have grown to around 1 m meter size.

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139 A B C D Figure 6-10. Amorphous calcium carbonate particles collected from the solution after 1 day of mineralization. A) TEM image of coalesced structures of amorphous precursor particles, and the corresponding ED patterns. B) TEM image of coalesced particles and a “helical rope” structure, supporting the premise of the fluidic nature of the amorphous calcium carbonate precursor phase. ED is consistent with XRD in that it is totally amorphous. C) Polarized optical micrograph of mineral at the air-water interface showing that it is totally non-birefringement (and therefore amorphous) after only 1 day of mineralization. D) The amorphous phase recrystallized into polycrystalline spherulites after 7 days.

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140 Figure 6-11. Comparison of hydrogel before and after mineralization, and sea urchin spine. A) The pure non-mineralized microporous PHEMA hydrogel is translucent due to the scattering of light inside the micro-pores. Note: a nonporous PHEMA is totally transparent. B) The mineralized microporous PHEMA hydrogel becomes solid white due to heavy mineralization. C) A section of the sea urchin spine used as a mold for replica fabrication. The brown color is due to the organics associated with biological CaCO3 phase.

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141 100 m A 200 m B 100 m C 10 m D E F 200 m Figure 6-12. Comparison of mineralized hydrogels prepared with and without polymer process-directing agent. A)-D) are samples that were calcined at 500C to remove the organic phase. A) One section of the calcined composite showing a very similar structure to the original sea urchin spine. B) One section of the collapsed structure after calcinations, shown at lower magnification. C) Same as of B) except at higher magnification, shows an interesting strutted network of mineral. D) Higher magnification pictures of the pore structure of shows it is a 3D microporous structure with interconnected pore sizes close to that of original sea urchin spine. E) EDS spectrum shows the presence of Ca, C and O from the CaCO3 (Note: the intensity of the C peak after removal of PHEMA is significantly lower than that in the EDS spectrum of pure PHEMA). F) A control sample without polymer. Only large calcite crystals grew on the surface of the PHEMA hydrogel, and after 7 days, the hydrogel remained soft while in solution.

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CHAPTER 7 INDUCTION OF THE PILP PROCESS USING NACRE PROTEINS The last few chapters discuss the use of synthetic acidic polymers to induce the PILP process. In this chapter, natural proteins from nacre are extracted and used to induce precursor film formation. A comparative study shows that the natural proteins work similarly to the synthetic acidic polymer, but under modified conditions, to induce single-crystalline as well as spherulitic films. Introduction In the classic book “On Biomineralization”, Lowenstam and Weiner (10) discussed the relationship between minerals and macromolecules. They found that “ In almost all cases, macromolecules are found in solution (after demineralization) and, on further examination, many of them are found to have one particular characteristic: they are highly acidic”. These proteins are rich in aspartic acid and/or glutamic acids, and in addition, some may have phosphorylated serine and theronine residues. They often have covalently bound polysaccharides that are also acidic, being rich in carboxylate groups and sometimes sulfate as well. All these macromolecules are commonly referred to as acidic macromolecules. The Polymer-Induced Liquid-Precursor (PILP) process uses synthetic biopolymers (e.g., Polyaspartic acid) to mimic the natural acidic proteins associated with biominerals, and is considered an in vitro model system for studying the role of acidic biopolymers in biomineralization and biomimetic materials. Thus far, we have demonstrated that synthetic biomimetic polymers can lead to the formation of minerals with both non142

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143 equilibrium morphology (e.g., molten morphology, film, rods) and non-equilibrium composition (e.g., high magnesian calcite), reproducing many of the features found in biominerals. We also found that the polymeric impurity is excluded during the transformation process into specific zones delineated by transition bars, where in biomineralization the occlusion of organic polymer along crystallographically defined zones has been reported. Here, we go further to investigate the relevance of the PILP process to biomineralization by determining if natural proteins can also induce this process in vitro. The proteins are extracted from nacre and tested under similar conditions to see if there is evidence of such biological polymers inducing the PILP process. We found that protein alone can induce single-crystalline thin aragonite films on organic substrates, which is similar to the mosaic single crystalline calcitic films generated using polyaspartic acid. In the presence of both Mg impurities and proteins, thin calcitic films formed resulting from transformation of an amorphous precursor phase. The morphology, transformation and composition of thin films formed by nacre proteins greatly resemble those films generated with the synthetic biomimetic polymerpolyaspartic acid. We hope to unveil the role of the acidic biopolymers in biomineralization by this comparative study, and establish the relevance of the PILP process to biomineralization. In addition, this research will also establish a new approach towards making biomimetic materials from mild conditions by this novel process.

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144 Materials and Methods Characterization of Nacre Nacre layers from Atrina shells collected from the Gulf of Mexico were peeled off by hand. Selected layers were etched with commercial bleach and observed by POM and SEM. Protein Extraction Procedure Several demineralization methods were tried, such as using dilute acid (6% acetic acid at pH 4; and 6% acetic acid at pH 5.2) and chelating agent (10% EDTA at pH 5.2; and 10% EDTA at pH 7.4). We find that in order to get pure and well-defined Mw (separable bands in SDS-PAGE) proteins, 10% EDTA at pH 7.4 should be used according to the following procedure. 1. Powder preparation: We crushed nacre into fine powder, then put 26.775 g fine powder of nacre into 200ml 10% Ethylene Diamine Tetra Acetic acid (EDTA) solution (22.2 g EDTA, di-sodium salt in 200 ml H2O) solution. 2. Demineralization: Put the above solution in nacre powder and stir continuously, control pH at 7.4, using drops of diluted HCl to continuously control the pH. 3. Centrifuge: After three days, centrifuge the solution (5000 rpm at 4C for 15 min.) to remove insoluble proteins (e.g., chitin) and collect the clear supernatant, which contains the soluble acidic proteins. 4. Dialysis: Put the above solution in a dialysis bag (Spectra/Por Regenerated Cellular Membrane tubes, MWCO 3500) and dialyses against 10-fold deionized water; change the water 2 times a day and continue for 2 days. 5. Concentration: Put the solution was in a Centricon YM-3 or YM-10 tube (MWCO:3,000 and 10,000) to remove the lower Mw molecules and concentrate the solution. It should be noted this method is only valid for a small amount of sample; for each filter device, the initial volume of solution should not exceed 2 ml. 6. Lyophilization: Freeze dry the concentrated or dialyzed solution (at C) to remove all the water, leaving a powder of crude protein extract 7. SDS-PAGE: Examine a small amount of protein extract using SDS-PAGE to characterize the molecular weight.

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145 Crystallization Study Five kinds of experiments were done to investigate the role of nacre proteins. The first one was an in-vitro crystallization of CaCO3 on glass slides using extracted nacre proteins. The experimental conditions are shown in Table 7-1. Table 7-1. Eperimental condition of in-vitro crystallization of CaCO3 by nacre proteins. No. 1 2 3 4 CaCl2 (mM) 12 12 12 12 Protein (g/ml) 0 2 100 500 The second experiment was an in-vitro crystallization using a PHEMA substrate. The conditions are shown in Table 7-2. Table 7-2. Condition of in-vitro crystallization using nacre proteins on PHEMA substrate No. 1 2 3 4 5 7 8 PHEMA substrate No Yes Yes Yes Yes Yes Yes CaCl2 (mM) 12 12 12 12 12 12 12 Protein (g/ml) 2700 0 6 60 300 1000 1500 The third experiment was crystallization of CaCO3 at different Mg/Ca ratio in solution. while keeping protein concentration constant at a very low concentration (3 g/ml, Table 7-3) in order to compare to the results using polyaspartic acid (in Chapter 4). Table 7-3. Crystallization of CaCO3 at different Mg/Ca ratio and constant low protein concentration (3 g/ml). No. 1 2 3 4 5 6 7 8 CaCl2 (mM) 12 12 12 12 12 12 12 12 Protein (g/ml) 0 3 3 3 3 3 3 3 MgCl2 (mM) 0 0 12 24 36 42 48 60 The fourth experiment was crystallization of CaCO3 at different Mg/Ca ratios in solution, while keeping protein concentration constant at a higher concentration (100 g/ml, Table 7-4).

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146 Table 7-4. Crystallization of CaCO3 at different Mg/Ca ratio and constant protein concentration (3 g/ml). No. 1 2 3 4 CaCl2 (mM) 12 12 12 12 Protein (g/ml) 100 100 100 100 MgCl2 (mM) 12 24 42 60 The fifth experiment was crystallization of CaCO3 at a constant Mg/Ca ratio of 3.5, while changing the protein concentration (Table 7-5). Table 7-5. Crystallization of CaCO3 at constant Mg/Ca ratio and at different protein concentrations. No. 1 2 3 4 5 6 7 CaCl2(mM) 12 12 12 12 12 12 12 Protein((g/ml) 42 42 42 42 42 42 42 MgCl2(mM) 0 3 20 50 100 500 1200 Results and Discussions Structural Characterization of Nacre When looking at the inner surface, nacre shows iridescent colors (Figure 7-1A), which is caused by both the diffraction and interference of light with the fine-scale diffraction grating structure, and stacks of thin crystalline nacreous layers or platelets below the surface (102). After etching with bleach, the inner surface shows a layered structure under the polarized optical microscope (Figure 7-1B). Figure 7-2A is an SEM image of the inner surface of nacre. It is composed of very thin and smooth plates/tablets of aragonite. The layered structure is clearly shown in Figure 7-2B. These layers stack together and are bridged by organic matrix composed of acidic macromolecules, silk fibroin like proteins and -chitin (5% by weight). The cross-section of nacre can be clearly seen in Figure 7-2C & D, showing that the tablets are around 400 nm thick.

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147 An XRD spectrum of the powder of nacre verifies that it has an aragonite crystal structure (Figure 7-3A). XRD was also done on the demineralized sample (Figure 7-3B). All aragonite peaks are lost after demineralization, indicating that demineralization of aragonite is complete. Nacre Protein Characterization SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) was done to characterize the proteins extracted by the different methods (Figure 7-4). Basically the diluted acidic demineralization method gave the largest amount of proteins from nacre (Figure 7-4A & B), but these proteins were hardly separable by gel electrophoresis (Mr difficult to resolve), possibly due to dissociation or denaturing of the protein at acidic conditions. Therefore, we decided to use EDTA at relatively neutral pH so that proteins are extracted with a relatively preserved state without any other additives (Figure 7-4D). It should be noted that it is very difficult to stain these highly acidic proteins. They have a pronounced tendency to diffuse out of the gel due to their highly acidic nature (8). Future improvements should be done to adopt new fixation conditions using Formaldehyde and Glutaraldehyde (8). According to the literature, the whole protein is 0.01% in weight to that of mineral, and 45 mol% of the proteins are highly acidic (contain high levels of Asp, Glu and Gln residues). FTIR was done on the protein powder. As seen from Figure 7-5, the characteristic carbonate peaks around wavenumbers of 713 and 700(4), 864 and 844(2), 1090(1), and 1490 (3) are all lost, indicating there is no mineral left in the protein extract. Instead, a broad peak around 3436cm-1 corresponds to the N-H stretch or O-H stretch. A broad peak around 1585 cm-1 is a mixture of amide I (1700-1600 cm-1, primarily

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148 governed by the stretching vibrations of the C=O (70-85%) and C-N groups (10-20%)), and amide II (1580-1510 cm-1, derived mainly from in-plane N-H bending). The other regions are very complex bands dependent on the details of the chemical environment, the nature of the side chains and hydrogen bonding. Therefore, these bands are only of limited use for the extraction of structural information. Influence of Nacre Protein on CaCO3 Crystal Growth On glass slides Possibly due to the quality and purity of the crude protein extracts, the protein hardly modifies the CaCO3 at low crude protein concentration (i.e., the extract contains a mixture of several proteins, not all of which are highly acidic). Figure 7-6 shows a modified calcium carbonate morphology when crystals are grown on glass slides in the presence of 500 g/ml proteins. The crystals no longer have a rhombohedral habit (Figure 7-6A & B). Although some of the crystal faces appear smooth, the (104) faces that are usually expressed, higher magnification images show that some surfaces of the crystals are not smooth. Figure 7-6C shows plate-like micro-facets with well-defined edges. Figure 7-6D shows tile-like structures. Figure 7-6E shows well-defined platy crystals forming a foliated fabric structure. The platy crystals are large in two-dimensions, while they are very small in the other dimension (~95 nm thickness determined from Figure 7-6F. These plates resemble the layered structure of aragonite, but are not thought to be related mechanistically. The results here suggest nacre protein may adsorb onto specific crystallographic faces and lead to pronounced defect textures, such as stacking faults.

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149 On PHEMA substrate Since no clear films grew at crude protein concentrations of up to 500 g/ml, we decided to use an organic substrate instead of glass slides to investigate the CaCO3 crystal growth in the presence of nacre proteins. Figure 7-7 shows thin CaCO3 film growth on the PHEMA substrate at a crude protein concentration of 1500 g/ml, after 3 days crystal growth. Figure 7-7A shows single-crystalline film patches along with some crystal aggregates. Figure 7-7B shows both single-crystalline film patches and a spherulitic patch on the PHEMA substrate. At higher magnification, the film patches are seen to have striations (possibly fibers, as suggested by TEM) (Figure 7-7C & D), which may be transitions bars due to the exclusion of organic polymer, as shown previously, but given the pronounced appearance, even at the completion of the crystallization, we believe they may be a result of shrinkage of the substrate film during the crystallization of the mineral precursor. When the samples were left in solution to continue to grow, more films were deposited on the PHEMA substrate. Figure 7-8A shows films on PHEMA after 36 days of crystal growth. Figure 7-8B shows both single-crystalline films and spherulitic films form on the PHEMA substrate. The morphology of these films resembles that of the thin aragonite films reported earlier in literature (103), using nacre extracts. However, here the thin films are grown on organic substrate instead of on purified organic sheets. In order to determine the structure of the thin films in Figure 7-8, they were peeled off the glass slide and transferred directly to a TEM grid. Figure 7-9A is BF-TEM image of a film patch showing a rod-like texture (~64 nm in diameter). A SAED pattern shown in Figure 7-9B shows this large tablet has a single-crystalline spot pattern. Further

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150 analysis of the d-spacings found that the crystal structure matches that of aragonite. Figure 7-9C shows another smooth film with an ED pattern (Figure 7-9D) that also matches the aragonite structure. CaCO3 crystal growth in the presence of both Mg and nacre protein As shown in Chapter 4, at a Mg/Ca ratio higher than 2 in solution, only 2 g/ml of polyaspartic acid is needed to induce thin CaCO3 films. Here we will compare the results of using nacre proteins to those of using polyaspartic acid. Figure 7-10 shows polarized optical micrographs of the crystals grown in the presence of only 3 g/ml nacre protein at different Mg/Ca ratio in the solution. As seen from the Figure 7-10A & B, no films were formed at Mg/Ca ratio of 1 and 2. Thin film begins to form when the Mg/Ca ratio goes higher, up to 3 (Figure 7-10C & D), however, there are also some large crystal aggregates deposited on the films. Thin and smooth spherulitic films are formed at higher Mg/Ca ratios in the presence of this amount of protein (Figure 7-10E & F). These results are a little bit different from those using the synthetic polymer, Polyaspartic acid. It seems that crude nacre protein does not act as potent as polyaspartic acid to induce film formation, because no film is formed at 3g/ml protein at a Mg/Ca ratio of 2, while film is formed at 2 g/ml polyaspartic acid at same Mg/Ca ratio. In order to induce film formation at a Mg/Ca ratio of 2, a higher concentration of protein is needed. Figure 7-11 shows polarized optical images of thin films formed at 100 g/ml protein at different Mg/Ca ratios. Figure 7-11A, B & C shows that thin film formed at a Mg/Ca ratio of 2, 3.5, and 5 after 3 days’ crystallization and one day’s drying time. A few spherulitic patches were observed at day 1 for the solution with a Mg/Ca

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151 ratio of 2, while the film was still amorphous at day 1 for the Mg/Ca ratios of 3.5 and 5. As time goes on, these films gradually transform from amorphous to crystalline, and this transition is dictated by the amount of Mg incorporated in the films. Figure 7-11D shows that most of the amorphous phase has transformed to crystalline after 4 days at Mg/Ca ratio of 2, while the amorphous phase shows little transformation for the Mg/Ca ratio of 3.5 (Figure 7-11E), and no transformation at a Mg/Ca ratio of 5 (Figure 7-11F). This clearly indicates the inhibitory effect of Mg on the amorphous to crystalline transition in the precursor films induced by nacre proteins. After 52 days, almost all the film produced at Mg/Ca ratio of 2 has transformed into spherulitic crystallinity (Figure 7-11G), and more spherulitic crystal growth is seen at the Mg/Ca ratio of 3.5 (Figure 7-11H). Some spherulitic crystals are also seen in the film produced at Mg/Ca ratio of 5 after 52 days of transformation (Figure 7-11I). The inhibitory effect of nacre proteins and their influence on amorphous to crystalline transformation is similar to that observed when using synthetic polymerPolyaspartic acid or Polyacrylic acid, to induce the amorphous precursor phase. At a constant Mg/Ca ratio of 3.5, the influence of protein concentration on thin film growth was also examined. Figure 7-12A & B shows polarized optical micrographs of a thin film formed at a protein concentration of 2 g/ml, which shows on the spherulitic films that there are also many crystalline particles (post-precipitate PILP droplets). Figure 7-12C & D shows image of the films grown at a higher protein concentration of 1200 g/ml. Compared to films formed at lower protein concentration, there are less crystal aggregates deposited at the higher concentration.

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152 Figure 7-13A-C shows the corresponding SEM images of the thin films formed at the high protein concentration (Condition: MgCl2: CaCl2=3.5, room temperature, protein = 1200 g/ml, 3 days vapor diffusion.). At lower magnification, the film looks quite smooth (Figure 7-13A); however, higher magnification shows that the film that appears “smooth” under the optical microscope is covered with a layer of very fine particles (Figure 7-13B), which apparently did not fully coalesce, similar to the large particles.. Figure 7-13C shows that the large particles on the films are around 2 m in diameter, similar to prior observations. Differently though, the large particles appear to be either covered with, or composed of the nano particles. We believe that as the polymer gets excluded from the precursor phase during solidification, that it can induce a second round of droplets (often seen as the 2 – 4 micron particles); or in this case, perhaps even a third round of these finer precipitates, given that a much higher polymer concentration was used for this experiment. Figure 7-13D) is an EDS spectrum of the film showing Ca, Mg, C and O peaks, typical of magnesian calcium carbonate. Figure 7-14A-C shows SEM images of a thin film formed at low protein concentration (3 g/ml) while at a high Mg/Ca ratio of 5. The morphology of this thin film resembles that formed using polyaspartic acid. On the smooth films, there are also particles around 2 m in diameter (Figure 7-14B). This film cracked under the electron beam, revealing even smaller particles of around 64 nm in diameter within the film (Figure 7-14C). EDS analysis (Figure 7-14D) shows the presence of Ca, Mg, C and O in the film, and the amount of Mg in the film formed at a Mg/Ca ratio of 5 is much higher than that in the filmed formed a Mg/Ca ratio of 3.5 (Figure 7-13D).

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153 After 2 years transformation time, most of the amorphous phase has transformed to crystalline phase. Figure 7-15A is the XRD spectrum of the film formed at a Mg/Ca ratio of 3.5 and a protein concentration of 1200 g/ml, and Figure 7-15B is the XRD spectrum of the film formed at a Mg/Ca ratio of 5 and protein concentration of 2 g/ml. Both XRD spectra verify that the crystalline phase is calcite. Conclusions In this chapter, proteins were extracted from nacre and investigated for their ability to induce precursor films. A comparative study of crystal morphology induced by nacre proteins and polyaspartic acid was also done, and the conclusions are as follows: Crude extracts of nacre protein alone (without Mg) modifies the crystal growth of calcium carbonate when added at high concentrations to the in-vitro crystallization assay. Thin plates, tiles, and foliated fabric type morphologies were observed on the surface of the crystals, which appear to be micro-facets produced by surface reorganization of the unstable new faces that were produced. In combination with an organic substrate, PHEMA, thin aragonite films were formed in the presence of extracted nacre proteins. The organic substrate PHEMA alone does not induce aragonite thin film formation without the polymeric process-directing agent. It requires the synergic effect of PHEMA and nacre protein to induce the formation thin aragonite film. As shown in the previous chapter, a combination of PHEMA and Pasp leads to spherulitic calcitic films, while a combination of PHEMA and nacre protein leads to both single crystalline aragonite films and spherulitic films. This suggests that the nacre proteins contain some active component for controlling the polymorphs of calcium carbonate. At low crude protein concentration (3 g/ml), addition of Mg at a Mg/Ca ratio of 3 in the solution results in amorphous precursor film deposition, which gradually transforms to calcite as time goes on. This study also suggests the crude protein does not act as potent as polyaspartic acid does towards inducing thin films, since Pasp can do this at a level of only 2 g/ml at a Mg/Ca ratio of 2. At high crude protein concentration (100 g/ml), addition of Mg at a Mg/Ca ratio of 2 is enough to induce thin film formation. The morphology, amorphous-crystalline transformation, composition and influence of Mg on the films formed using nacre protein greatly resemble those features formed using a synthetic acidic biopolymer

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154 such as Pasp. This similarity probably results from the non-specificity of the PILP process, which is primarily a result of their highly acidic nature. At a constant Mg/Ca ratio of 3.5 in the solution, higher protein concentration leads to film formation with less crystal aggregates than lower protein concentration. Also, the film formed at high protein concentration tends to be more stable in the electron beam than film formed at low protein concentration, but this may simply be due to the much longer drying time, which has allowed full removal of hydration waters. Both natural acidic proteins and synthetic acidic polymers have the ability to induce precursor film formation. This suggests that the PILP process plays a very important role in biomineralization, and also this study provides new tools towards making organic-inorganic biomimetic composites similar to nacre.

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155 A B Figure 7-1. Image of nacre from Atrina shells. A) Iridescent appearance of nacre. B) Side view of surface viewed under POM showing layered structure.

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156 10 m 10 m A B C D 20 m Figure 7-2. SEM images of nacre from Atrina shell. A) Planar view of the aragonite tablets forming on the surface of nacre. B) Side-view of the plates showing the layer-by-layer structure. C) Cross-section of nacre showing the nano-laminated layer-by-layer structure. D) Higher magnification pictures of nacre plates show the “brick and mortar” structure (D is from http://web.mit.edu/cortiz/www/ , last accessed Feb 17,2005).

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157 0100200300400500600700202530354045505560 A B Figure 7-3. XRD spectra of nacre. A) Before demineralization. B) After demineralization in 0.5M EDTA solution for three days. (Note: all peaks of nacre disappear after demineralization, the peak around 28 is not due to the calcite, and maybe from the Ca-EDTA complex. Other peaks should also be seen if it is calcite).

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158 A B C D E 32.5 Kda 25 16.5 6.5 Figure 7-4. SDS-PAGE of protein extracted from nacre by different extraction methods. Dilute acid extraction leads to a higher amount of protein, but less resolution in SDS-PAGE (Acidic proteins are notorious for anomalous migration in PAGE), possibly also due to partial degradation of proteins under acidic condition. A) Acetic acid, pH = 4. B) Acetic acid, pH = 5.2 C) EDTA at pH = 5.2. D) EDTA, pH = 7.4. E) Standard M.W. markers.

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159 40003500300025002000150010005007654321 856931717112012701340141315853436IntensityWavenumber (cm-1) Figure 7-5. FTIR spectrum of soluble protein extracted from nacre. Notethe free carboxylate group around 1700cm-1 disappears. The broad peak around 3436cm-1 is from N-H stretch, or O-H stretch vibrations. The broad peak around 1585cm-1 is a mixture of amide I (1700-1600cm-1, primarily governed by the stretching vibrations of the C=O (70-85%) and C-N groups (10-20%)), and amide II (1580-1510 cm-1, which derives mainly from in-plane N-H bending). The other regions are very complex and difficult to extract information. C

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160 200m A 50 m B C D 10 m 10m E F 5 m 2 m Figure 7-6. SEM pictures of the morphology of the modified crystals grown in the presence of crude nacre proteins. (Condition: 10 mM CaCl2, 500 g/ml Protein, vapor diffusion for three days). A) This low magnification SEM picture shows that the calcite has a modified morphology and loses its typical rhombohedral habit. B)–F) Higher magnification SEM of the modified crystal faces shows structures similar to the layer-by-layer seen in nacre, which arise from microfacets that stabilize the modified surface.

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161 A B C D Figure 7-7. Polarized optical micrographs of thin films grown on the organic PHEMA substrate. (Condition: 12 mM CaCl2=, 1500 g/ml nacre protein, vapor diffusion for three days.) A) Lower mag. image shows thin single-crystalline film patches along with centralized crystal aggregates. B) Single-crystalline film patches along with a spherulitic film patch (top right). C) Image of “transition bars”, possibly due to exclusion of protein during crystal growth. D) Higher mag. image shows the striped lines of transition bar.

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162 A B C Figure 7-8. Comparison of polarized optical images of films grown on PHEMA substrate in the presence of nacre proteins for comparison to aragonite thin film from literature. A) Lower mag. image shows patchy thin formation. B). Higher mag. image shows both single-crystalline and spherulitic crystalline patches. C) Image of aragonite films in the presence of nacre proteins grown on organic from literature (103). In particular, this article describes a “windmill” extinction pattern upon rotation of the microscope stage, and this same extinction patter is seen in our films, and apparently results from a coherent radial growth pattern of the patches around the central core aggregate (like a course-grained spherulite). Reprinted, with permission, from Belcher, A. M.; Wu, X. H.; Christensen, R. J.; Hansma, P. K.; Stucky, G. D.; Morse, D. E. Nature, 1996, 381, 56-58.

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163 A B C D Figure 7-9. TEM and SAED of thin films produced by nacre proteins grown on PHEMA substrate. A) The TEM image shows rod-like features within the single-crystalline patch of film. The patch is laying across some holey carbon film. The diameter of the rods are about ~67 nm. B) The corresponding ED pattern matches the aragonite crystal structure. C) Another TEM image of a different single-crystalline film patche. D) Another similar SEED pattern matching the aragonite crystal structure. The d-spacings for the planes are as follows: 121 (2.78), 200 (2.46), 321 (1.474); with corresponding inter-planar angles: (121)(321) = 29.7. (321)(200) = 26.8, (200)(12) = 56.6. Angles were calculated according to the following equation for Orthorhombic crystals: 1 ) )222()111(212121(222222222222222clbkahclbkahcllbkkahhACos , where for aragonite, a = 4.9623 , b = 7.968 , c = 5.7439 , respectively.

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164 A B C D E F Figure 7-10. Polarized optical micrographs of crystals formed at different Mg/Ca ratios in solution, at low protein concentration. (Condition: 12 mM CaCl2, 3 g/ml protein, 3 days vapor diffusion at RT). Thin films formed only at Mg/Ca > 3. Note: thin films formed at Mg/Ca = 2 when in the presence of 2 g/ml Pasp, which means the crude protein extract is not as potent as Pasp towards inducing films. A) Mg/Ca = 1. B) Mg/Ca = 2. C) Mg/Ca = 3 with red-1 wavelength plate D) Mg/Ca = 3, without red-1 wavelength plate. E) Mg/Ca = 5, with red-1 wavelength plate. F) Mg/Ca = 5, without red-1 wavelength plate. (This image was taken after 2 years of drying)

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165 A B C D E F G H I Figure 7-11. Thin films formed in the presence of both Mg and crude nacre protein. (Condition: 100 g/ml protein, RT, vapor diffusion, 3 days in solution for crystal growth). At a constant Mg/Ca ratio in the solution, each resultant film transformed to crystalline film as time progressed from 1 day, 4 days, to 52 days, after removal from solution (top to bottom). At constant time and protein concentration, the films tends to stay more amorphous at the higher Mg/Ca ratio in the starting solution (left to right). A) MgCl2:CaCl2 = 2 (24 mM:12 mM), 1 day dried. B) Mg/Ca = 3.5, 1day. C) Mg/Ca = 5, 1day. D) Mg/Ca = 2, 4 days. E) Mg/Ca= 3.5, 4 days. F) Mg/Ca = 5, 4 days. G) Mg/Ca = 2, 52 days. H) Mg/Ca= 3.5, 52 days. I) Mg/Ca = 5, 52 days.

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166 A B C D Figure 7-12. Thin film formation at a constant Mg/Ca ratio of 3.5, at different protein concentrations. The higher protein concentration tends to lead to smoother films and less aggregates. A) Protein = 3 g/ml, image taken with red-1 wavelength plate. B) Protein = 3 g/ml, image taken without red-1 wavelength plate. C) Protein = 1200 g/ml, image taken with red-1 wavelength plate. D) Protein = 1200 g/ml, image taken with red-1 wavelength plate. (Condition: MgCl2: CaCl2 = 3.5, room temperature, 3 days, vapor diffusion, Pictures were taken after 2 years drying time.)

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167 A B 5 m 20 m C D 2 m Figure 7-13. Typical SEM images of thin film in the presence of both Mg and nacre protein. (Condition: MgCl2: CaCl2 = 3.5, room temperature, 1200 g/ml protein, 3 days of vapor diffusion.) A) Lower Mag. B) Higher Mag. picture shows the “smooth” film under optical microscope is not really smooth, and consists of an overlayer of fine precipitates. C) Higher Mag image shows the large particles are around 2 m in diameter. The particles are either composed of smaller particles, or have a layer adsorbed on their surface. D) The EDS spectrum shows Ca, Mg, C and O peaks, typical of magnesian calcium carbonate.

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168 A B 2m m 20 m C D 5 m Figure 7-14. Typical SEM images of thin films at high Mg/Ca ratio and low nacre protein concentration. (Condition: MgCl2: CaCl2 = 5, room temperature, 3 g/ml protein, 3 days of vapor diffusion.). A) Lower mag. B) Higher Mag image show smooth films and embedded particles. This film morphology resembles the films formed in the presence of both Mg and Pasp under similar conditions (2 g/ml Pasp, Mg/Ca = 5). C) The film is quite smooth and cracks under the electron beam, revealing very fine particles of around 120 nm in diameter. These particles are significantly different in size to the particles on films (~2 m in diameter). D) EDS shows the presence of Mg, Ca, C and O peaks of magnesian calcium carbonate.

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169 02004006008001000120014001600202530354045505560 104 202 110 116 A 113 B Figure 7-15. XRD spectra of the thin films formed in the presence of both Mg and nacre protein verifies the crystalline phase is calcite. In both cases there are still amorphous calcium carbonate phases existing, even after 2 years of transformation time. A) MgCl2: CaCl2= 3.5, 1200 g/ml protein. B) MgCl2: CaCl2 = 5, 3 g/ml protein. (Condition: room temperature, vapor diffusion, 3 days.)

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CHAPTER 8 PATTERNING HYDROXYAPATITE IN THE PRESENCE OF SOLUBLE POLYMER Introduction The previous chapters describe the formation of CaCO3 minerals and related composites by the PILP process. This chapter mainly describes the formation of another mineral common to skeletal parts of vertebrates, apatite. We further investigate how the PILP process can be utilized in the formation of hydroxyapatite thin films. The inorganic constitutes of biological hard tissues, such as bone, teeth and tendons, are comprised mainly of hydroxyapatite (Ca10(PO4)6(OH)2), although it is more accurately identified as non-stoichiometric carbonated apatite. Biologically formed calcium phosphates are often in the form of nano-crystals that are precipitated under mild conditions (ambient pressure and near room temperature), modulated in size and shape by the presence of biological macromolecules. There have been numerous studies of calcium phosphate growth in-vitro. Dorozhkin and Epple have reviewed the biological and medical significance of calcium phosphates thoroughly (104). Traditionally, biological calcium phosphate nucleation is thought to be initiated by a soluble negatively charged polymer (105-107). It has also been suggested that calcium binding onto negatively charged sites of insoluble organic matrix also promotes heterogeneous nucleation (108,109). In the current study, we challenge the traditional view of calcium phosphate formed in biological systems. We think that the negatively charged polymer first will bind to the calcium ions, and similar to the formation of calcium carbonate by the PILP process, this 170

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171 polymer will induce a liquid-like precursor phase which consists of calcium and phosphate ions, water and binding polymer, and ultimately transform to crystalline hydroxyapatite. We first developed a calcium phosphate crystallization system that is very reliable, stable and close to physiological conditions (pH, temperature, ionic strength and concentration). In this system, hydroxyapatite spherulites with well-defined platy aggregates are formed without any polymer addition. We then investigate calcium phosphate formation with polymer (Polyaspartic acid and Bone sialoprotein) addition. Thin films of calcium phosphate are formed in the presence of very small amounts of these polymers. Both amorphous and crystalline phase exist in the films, indicating that it may be formed by the PILP process. In the second part of this chapter, we investigate calcium phosphate formation in the presence of a templatea negative charged surface formed by Self-Assembled-Monolayers (SAMs). Without any polymer addition, this negatively charged surface does greatly promote nucleation and growth of calcium phosphate, and has the ability to modulate the orientation of well-defined platy crystals. In this case, there is no true film formation; instead the “film” consists of compact crystal aggregates. However, in the presence of a small amount of polymer, thin films formed instead of the well-defined platy crystals. Upon crystallization of the films, the crystal plate habit became curved and much smaller in size compared to those formed without any polymer addition. We also find that BSP protein domains greatly promote calcium phosphate formation in the presence of this negatively charged template, which is contrary to the traditional view that this protein is inhibitory for calcium phosphate.

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172 Experiments and Methods Crystallization System A supersaturated solution of calcium phosphate is formed by addition of equal volumes of 9 mM CaCl2 and 4.2 mM of K2HPO4 in tris-buffer (final concentration: 4.5 mM CaCl2 and 2.1M of K2HPO4). The solution is mixed at room temperature and the crystallization study is done at 37C and at a pH of 7.4. BSP Peptide Synthesis BSP-25, BSP65-89 (Mw=2792) is a 25 amino acid peptide whose sequence corresponds to residues 65 to 89 of bone sialoprotein II (BSP). The primary sequence of BSP65-89 is DSSEENGDDSSEEEEEEEETSNEGE. BSP65-89 was synthesized using the protein core facility at the University of Florida. For the synthesis, standard automated Fmoc couplings were used with the exception of manual couplings and a different deprotection protocol for the three aspartic acid residues. After synthesis, BSP65-89 was deprotected and cleaved from the resin in the laboratory of Dr. Joanna R. Long and purified to >97% purity via HPLC. A 250 mole synthesis yielded over 150 mg of purified BSP65-89. The purity of the BSP65-89 was verified via MALDI mass spectrometry and analytical HPLC. Micropatterning of Hydroxyapatite on Patterned SAMs A review of patterning of SAMs using soft lithography techniques can be found in Yi-Yeoun Kim’s dissertation (77). The schematic of the procedure is shown in Figure 8-1. Briefly, a micro-channel molded PDMS is used as a stamp, for patterning an “ink” of alkane thiols. The width of the projected channel and concaved channel on the stamp is about 90 m and 30m (Figure 8-1A), respectively. The 11-mecaptoundecanoic acid

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173 (COOH-(CH2)11SH) and 11-mercapto-1-undecanol (HO-(CH2)11SH ) were dissolved in high quality ethanol at a concentration of 2 mM. The mixed monolayer was prepared by mixing different volumes of the above solution at ratio of (2:2, 1:3 and 0.5:3.5 in ml) for use as the SAM inks. The patterned SAMs were stamped on a Au substrate as described elsewhere (110). Each patterned SAM substrate was placed upside-down in the above supersaturated solution of calcium phosphate w/ or w/o polymer additive. The patterned HA was taken out after 1 day, and washed with nanopure water and ethanol twice, and dried for SEM, TEM and XRD analysis. XRD XRD was done directly on the Au substrate containing the mineralized SAMs, and samples were scanned by Cu-K X-ray radiation from a Philips XRD 3720 at 40 KV and 20 mA, using a step size of 0.01 with a time of 2 sec/step, over a 2 range of 10-60, or for short scan over a 2 range of 25-35. Results and Discussion Hydroxyapatite Formation on Glass Slides w/ and w/o Pasp or BSP-25 Figure 8-2 shows optical micrographs of calcium phosphates formed without and with polyaspartic acid addition, after 4 days of crystallization. Without polymer addition, calcium phosphate spherulites formed on the glass slides (Figure 8-2A). However, with only 5 g/ml polyaspartic acid added to the solution, thin films formed on the glass slides (Figure 8-2B). The thin films are bumpy, as seen by the magenta color dotted with thicker black particles. Without the red-1 wavelength plate, the film shows both black (amorphous phase) and white (crystalline phase) regions (Figure 8-2C). It appears that the particles on the films are contributing to the birefringent regions, and therefore are

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174 more crystalline than the film underneath. Similar features of the films can still be seen at higher Pasp concentration (Figure 8-2D-F), but the dark particles seem larger or more aggregated. SEM images of the above spherulites in the control sample are shown in Figure 8-3 A-C. These spherulites are composed of tiny protruding plates. The EDS spectrum shows the Ca and P elements comprising these crystals (Figure 8-3D). A bright field TEM image of the tiny plates isolated by crushing the spherulites is shown in Figure 8-2 E. The ED pattern matches that of hydroxyapatite (Figure 8-3F). Figure 8-4A-D shows SEM images of the thin film formed in the presence of 5 g/ml Pasp. The thin film shows curved platy features, and scattered on the surface of the polycrystalline film, there are larger clusters, possibly due to post precipitated calcium phosphate particles. The EDS spectrum shows Ca and P present in the film (Figure 8-4E). A bright field TEM image of the thin film is shown in Figure 8-4F, and the corresponding ED pattern shows this film is weakly polycrystalline. Figure 8-5A-C shows SEM images of the thin film formed in the presence of 15 g/ml Pasp. The film looks similar to that formed at 5 g/ml Pasp, except that the surface clusters appear larger, and one can even see that presence of clusters embedded within the films, suggesting that the films are formed by coalescence of precursor particles that did not fully coalesce. The EDS spectrum shows Ca and P in the film (Figure 8-5D). At higher Pasp concentration (100 g/ml), the film looks rather different (Figure 8-6A-C). The film is not quite continuous but porous (Figure 8-6C). The curved platy features are not always seen, and instead replaced by many particulate agglomerates. EDS, however, shows similar Ca and P peaks in the film (Figure 8-6D). These results

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175 appear to show a trend with polymer concentration, such that the primary PILP particles do not coalesce into smooth films as well at higher polymer concentration. It is not clear if the polymer causes the particles to solidify more rapidly, or if the polymer blocks the coalescence of the particles (perhaps by electrostatic or steric repulsion). Figure 8-6E & F show a thin film formed in the presence of 75 g/ml BSP-25. This is not a full protein, but rather a synthetic peptide mimicking the acidic domains, with sequence DSSEENGDDSSEEEEEEEETSNEGE. As seen from Figure 8-6F, the film has crystals with similar curved features as those formed at low concentrations of Pasp (5, 15 g/ml). Figure 8-6G & H shows the film formed at a much higher BSP concentration (300 g/ml). This film has similar particle agglomerate features as those formed at high Pasp concentration (100 g/ml) as shown in Figure 8-6A-C. The above results indicate that addition of acidic polymer (Pasp or BSP-25) changes the hydroxyapatite crystal morphology and modifies its growth. Thin films are formed in the presence of these polymers, and on these films, both amorphous and crystalline phases coexist, which suggest that hydroxyapatite is formed possibly by transformation of an amorphous precursor induced by the polymer. In particular, the apparent coalescence of particles which comprise the films suggests that the films are formed by deposition of a “fluidic” amorphous precursor (and not heterogeneous nucleation and growth of an amorphous phase). Hydroxyapatite Micropatterning Formed on SAMs w/ and w/o Polymer In recent years, methods for organizing inorganic nano-crystals have achieved great success (111). The formation of inorganic crystals which are found in natural biominerals, such as calcium carbonate (22,23,29), iron oxide (112), and silica (113,114),

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176 is an important focus of the biomimetics field. The ability to pattern synthetic minerals not only helps to elucidate different parameters involved in the biomineralization process, but also leads to the development of novel materials having unique optical, magnetic and mechanical properties. Hydroxyapatite is the main mineral in the skeletal parts of vertebrates. Patterning of hydroxyapatite, especially at a nanoscopic scale, is of great value in developing novel bioactive templates for biomedical and tissue engineering applications. Because Soft Lithography can be conveniently used to introduce organic functional groups on a Au substrate, pattering of hydroxyapatite using soft lithography combined with a biomimetic crystallization process provides valuable information on the role of these surface functional groups in combination with polymer additive on HA formation. Micropatterning HA without polymer addition We find carboxyl-terminated SAMs greatly promote the nucleation of hydroxyapatite crystals. On all of the patterned surfaces containing carboxylate groups (even at a COOH-(CH2)11SH: HO-(CH2)11SH ratio of 0.5:3.5 in the monolayer solution), a uniform HA pattern can be achieved in less than 24 hours, while monolayers terminated with only hydroxyl group (at a COOH-(CH2)11SH: HO-(CH2)11SH ratio of 0:4 in the monolayer solution) take longer (3 days) to form a uniform hydroxyapatite pattern. Figure 8-7 shows typical SEM images of the micropatterned hydroxyapatite on patterned SAMs (COOH-(CH2)11SH). Crystal growth only appears on the area where the carboxyl terminated SAM has been stamped (Figure 8-7A). The x-ray element mapping of Ca and P is consistent with the SEM images for calcium phosphate micro-pattern formation (Figure 8-7B & C). On the patterned substrate, the HA distribution is very uniform except in the boundary area where the surface monolayer disappears (Figure 8-7D). The width of

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177 a single channel of micro-patterned HA is about 90 m, which is consistent with the projected area of the PDMS stamp used for patterning the monolayer template. A higher SEM magnification shows that most of the platy crystals are densely packed and have grown normal to the Au substrate. The thickness of each plate is around 80 nm (Figure 8-8A). In order to investigate the fine structure of the platy HA crystals, they were ground in liquid-N2 and transferred onto a TEM grid. Figure 8-9A shows a typical bright field TEM image of one plate, and Figure 8-9 B shows the corresponding selected area diffraction pattern. The ED pattern shows the plate is single crystalline, with d-spacings of 3.44, 3.11, 2.84, 2.79 and 1.71 respectively, which are consistent with the d-spacings of the (002), (210), (211), (112) and (004) planes of hydroxyapatite, respectively. For the hexagonal lattice of hydroxyapatite, the angle between the (002) and (112) plane should be 36.2 (and should match the angle between diffraction spots), and the angle between the (112) and (211) planes should be 30.8. For triclinic octacalcium phosphate (OCP), the angle between the (112) and (211) plane is only 14.3. Angle calculations, together with d-spacing measurements, confirm that hydroxyapatite, and not OCP, has formed, despite similar d-spacings. The literature has suggested that OCP may serve as a precursor phase to HAP (115), but there is no evidence of that here. When the electron beam direction is oriented parallel to the [010] direction of the crystal, each plate essentially shows two dominant planes, (002) and (300), which are normal to each other (Figure 8-9C & D), as expected for a platelet of the hexagonal unit cell that is lying on it’s flat (010) face. This electron diffraction pattern is confirmed by the XRD data showing a strong intensity of the (002) and (300) peaks in crystals grown on pure carboxyl-terminated SAMs (Figure 8-10A). As seen from SEM picture of Figure 8-8A

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178 and the top spectra of Figure 8-10, these HA crystals are predominantly oriented in the [002] direction, or in other words, the c-axis of apatite plates are normal to the Au substrate. The preferential growth of plates in the perpendicular direction relative to other direction can be seen in Figure 8-8A. On mixed SAMs of different ratios of carboxyl and hydroxyl functional groups (3:1, 2:2, 1:3 and 0.5:3.5 in volume ratio of COOH-(CH2)11SH and HO-(CH2)11SH ), uniform micropatternings of nano-crystalline hydroxyapatite, similar to those shown in Figure 8-7, were also observed. Interestingly, the morphology and size of the hydroxyapatite crystals are different, as shown in the higher magnification SEM pictures in Figure 8-8. When the concentration of hydroxyl groups is low in the SAMs (3:1), the size of the crystals is reduced, and the edge of the HA crystals looks more curved (Figure 8-8B). When the volume ratio of the two surfactants reaches to 1:1 in the starting “ink”, the size of HA crystals formed on the SAMs become even smaller (Figure 8-8C). This ratio appears to give a maximum of nucleation sites, as deduced by the SEM image. However, as the concentration of hydroxyl groups increases to 1:3 and 0.5:3.5, the size of the HA crystals increases again, and the edges of the plates become sharper (Figure 8-8D, E, and F). The preferred orientation of HA plates on mixed monolayers also changes as the composition of monolayer changes. On pure carboxyl or hydroxyl terminated SAMs, the HA formed has a preferred [002] orientation, despite different formation times (Figure 8-10). Conversely, on the mixed SAMs, as more hydroxyl groups substitute for the carboxylate groups, there is a decrease in the degree of orientation of the (002) plane. This is proved by XRD data showing the intensity of the (002) peak of HA decreases

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179 relative to the normalized (300) peak as more hydroxyl groups substitute for carboxyl groups in the SAMs (Figure 8-11). Micropatterning HA in the presence of Pasp or BSP-25 Figure 8-12A shows a polarized optical micrograph (POM) of micropatterned CaP formed in the presence of 15 g/ml Pasp on a patterned monolayer of COOH-(CH2)11SH. The pattern is very thin and smooth, with some scattered crystal clusters/aggregates (which appear black under POM). The SEM image shows the patterned channels to be around 90 m wide (Figure 8-12B & C). Higher Mag. SEM shows that there are many asymmetric particles or aggregates that are around 0.8 m long (Figure 8-12D). Further experiments and characterization needs to be done to get more information on optimizing the film thickness and time of formation. Figure 8-13A-E shows SEM images of the micropatterned HA formed in the presence of 15 g/ml BSP protein (BSP-25) on the same patterned monolayer COOH-(CH2)11SH. The Ca and P mapping shows the mineral pattern formation (Figure 8-12B & C). The width of a single patterned HA channel is also around 90 m (Figure 8-13D). However, at higher magnification (Figure 8-13E), the crystallites within the patterned HA films have a different appearance compared to the film formed without any polymer addition, shown in Figure 8-8A. The HA plates are more curved and smaller in size as compared to the flat plates formed without any polymer. The curved morphology resembles the thin films formed on glass slides in the presence of BSP protein shown earlier in this chapter, suggesting that the curvature is due to the polymer additive, and not the SAM. Some of the crystals were scraped off and crushed for TEM observation.

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180 Figure 8-12F shows a TEM image of isolated platy crystals and the corresponding Selected-Area-Electron-Diffraction (SAED) confirms that the crystals are hydroxyapatite. At higher BSP protein concentration (75 g/ml), some micropatterning was accomplished (Figure 8-14A); however, many large crystals aggregates had overgrown into the non-“inked” bare Au area (Figure 8-14B), so the patterns were not composed of empty channels, but rather differently textured mineral. On the patterned SAM region, the hydroxyapatite that formed shows similar curved feature as before (Figure 8-14C). In the regions of bare Au, rounded clusters formed, suggestive of poor “wetting” of the mineral in these more hydrophobic regions. An EDS spectrum shows the Ca and P elements present in the crystals (Figure 8-14D). In comparison with micropatterning formed without any polymer addition, these results clearly show that BSP protein greatly promotes hydroxyapatite formation, reducing its specificity for forming on preferential substrates. This patterning is formed faster (in less than 24 hours) as compared to that without any BSP, and the films formed appear thicker and even over-grown on to non-patterned areas. BSP also seems to reduce the size of the crystalline plates and imparts them with curvature. XRD was done on all patterned surfaces to confirm that the crystalline phase is hydroxyapatite. Figure 8-15A shows the XRD pattern of commercial hydroxyapatite. The (002) peak is significantly lower than the (211) peak because the commercial powder is randomly oriented. However, hydroxyapatite patterned on SAMs terminated with a COOH group, without or with polymer addition, shows a strong (002) peak relative to the (211) (Figure 8-15B-D), confirming that the crystals are predominantly oriented in the c-axis direction. The c-axis growth of hydroxyapatite is very common because it is the

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181 kinetically preferred growth direction for HA. The (001) plane of hydroxyapatite crystals has the highest atomic density so that the crystal grows favorably in this direction by lowering its surface energy. When the surface is enriched with nucleation sites by introduction charge density, each HA will nucleate and grow preferably in c-axis, thus giving highly c-axis oriented HA crystals in the control. Our results show that BSP protein does not alter the orientation of HA crystals very much, suggesting that the orientation effect may be the result of the charge density distribution in the self-assembled monolayers (supported by the mixed monolayer study), and not due to the adsorption of protein to the different crystal planes. On the other hand, the curved morphology of the crystals is more likely a result of protein adsorption. The peaks appear somewhat broader with polymer addition, which can be an indication of crystals of smaller dimensions. But given that the opposite is observed in the micrographs, the peak broadening is likely a consequence of lattice strain and crystal defects. Conclusions In this chapter, hydroxyapatite formation in the presence of patterned SAM templates, and w/ and w/o polyaspartic acid or BSP proteins, was investigated. The conclusions are as follows: A fast and reliable crystallization system for hydroxyapatite was developed, which is carried out under physiological conditions (pH = 7.4 and Temperature = 37C). Without any polymer addition, hydroxyapatite spherulites composed of platy crystals are formed. They most likely from in solution and settle onto the slides. When a negatively charged template (SAM) is introduced, well-defined hydroxyapatite plates are formed with a strongly preferred c-axis orientation. This orientation results from the charge density of the substrate, suggested by the mixed monolayer studies, where the orientation decreases when a mixed monolayer with decreasing charge density is introduced. With the addition of small amounts of Pasp or BSP proteins (both are highly acidic) in the solution, thin films are formed on the glass slides. The crystals comprising the

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182 films show a slightly curved morphology. When a negatively charged template (SAM) is introduced, patterned mineral thin films are formed which are composed of curved polycrystals of HA with preferred c-axis orientation. HA films formed in the presence of Pasp resemble films formed in the presence of BSP proteins (although at lower concentrations), suggesting it is the acidic nature of the polymer that induces the precursor particles that deposit and coalesce into films, prior to crystallization. This mechanism is also suggested by the presence of embedded non-coalesced particles on the films. The calcium phosphate system behaves differently than the calcium carbonate system. In the latter case, work by Kim has shown that the same polymer and templating system leads to smooth and fairly large single-crystalline patches of film due to a pseudo-solid-state transformation of the amorphous precursor film. Here, while the patterning behavior was fairly similar, the amorphous CaP films appear to transform by a solution-recrystallization process, leading to polycrsytals with the more typical faceted habit. But in both cases, addition of small amount of biomimetic polymer can change the crystal growth dramatically into non-equilibrium morphology, but the driving force for small and anisotropic crystals appears to be stronger in the CaP system. Future work is needed to further understand the role of the polymer as a process-directing agent, particularly towards determining the fluidity of the precursor phase, and towards elucidating the mechanisms of CaP biomineralization.

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200um 183 200m PDMS Stamp Figure 8-1. Schematic representation of micropatterning of Hydroxyapatite on Self-Assembled Monolayers (SAMs) using soft lithography technique. A) Optical micrograph of the PDMS stamp with a micro-channel pattern. B) Patterning of a SAM containing COOH and/or OH functional groups by micro-contact printing the SAM on the Au surface. C) Patterned surface being placed upside-down in a crystallization solution. D) Crystals of HA (or precursor phase) template exclusively on the patterned SAM surface.

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184 A B C D E F Figure 8-2. Polarized optical micrographs of CaP minerals grown on glass slides at different Polyaspartic acid concentration (4 days of crystallization). Spherulitic Hydroxyapatite aggregates grow in the control reaction without any Pasp (phase confirmed by XRD and ED). Thin CaP films are formed with polymer, with an increasing aggregation tendency observed for increasing Pasp concentration. A) 0 g/ml Pasp. B 5 g/ml Pasp, with red-1 wavelength plate. C) 5 g/ml Pasp, without red-1 wavelength plate, showing both amorphous and crystalline phase exists within the film. D) 15 g/ml Pasp. E) 50 g/ml Pasp. F) 100 g/ml Pasp.

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185 50 m A 5 m B C D 5 m E F Figure 8-3. SEM and TEM of Hydroxyapatite spherulites grown on glass slides without any Pasp addition. A) Lower Mag. B) Higher Mag. image shows every spherulite is composed of many tiny plates. C) Spherulite aggregates. D) EDS spectrum showing Ca and P peaks. E) TEM image of isolated plates from crushed spherulites. F) ED pattern verifying the plate’s apatite structure.

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186 A B 5 m 200m C D 2m 2 m E F Figure 8-4. SEM image of thin CaP films formed in the presence of 5 g/ml Pasp. A) Low magnification shows continuous film formation, where the dark streak is from film scratched off with a needle. B) The particles on the film are composed of small platy clusters. C) SEM image showing the curved platy morphology of the polycrystals. D) Higher magnification showing the particles deposited on the film E) EDS spectrum showing Ca and P peaks. The Si, K and Na peak are due to the glass underneath the thin film. The high intensity of glass elemental peak possibly indicates that the film is rather thin and porous. F) TEM image shows the platy features in the film and the inserted ED pattern suggests the film is weakly polycrystalline, but it is difficult to judge given the amorphous halo from the glass slide.

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187 A B 10 m 200 m C D 10 m Figure 8-5. SEM images of CaP films formed in the presence of 15 ug/ml Pasp. A) Lower Mag. Shows bumpy films with scattered clusters on the surface of the films B) At higher Mag. it can be seen that the aggregates are partially embedded in the film. C) The curved features of the crystals are also seen at higher Mag. D) EDS spectrum showing similar Ca and P peaks as before.

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188 200 m A 50 m B C D 5 m 2 mm E F 5 m Figure 8-6. A comparison of CaP films formed in the presence of Pasp and BSP protein. A) Low Mag. image of the film formed in the presence of higher concentration of Pasp (100 g/ml). B) SEM image showing the rough texture of the film. C) Higher Mag showing pores and particle aggregates which lead to this rough texture. D) EDS showing similar Ca and P peaks. E). SEM image of film formed in the presence of 75 g/ml BSP protein. F) Higher Mag. shows this film has similar features as that formed in the presence of low concentration of Pasp (Figure 8-3 or 8-4). G) SEM image of CaP formed in the presence of higher concentration of BSP protein (300 g/ml). H) The film has similar features as the film formed at 100 g/ml of Pasp.

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189 G H 10 m 20 m Figure 8-6. continued

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190 A B D C D 50um Figure 8-7. Elemental mapping of hydroxyapatite micro-patterning on the self-assembled monolayer of COOH-(CH2)11SH. A) Secondary electron image showing the HA patterned to form micro-channels. B) Phosphorous elemental x-ray map of sample shown in A). C) Calcium elemental x-ray map. D) Higher mag. SEM image of a HA pattern

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191 A B 5 m C D E F Figure 8-8. Higher magnification SEM of the HA platy crystals grown on SAMs. The SAMs are mixed with different volume ratios of 2mM COOH-(CH2)11SH and 2mM OH-(CH2)11SH. A) COOH-(CH2)11SH only, 1:0. B) 3:1. C) 1:1. D) 1:3. E) 0.5:3.5. F) OH-(CH2)12SH only 0:1. Note the changes in crystal size and morphology. All Bars = 5 m.

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192 A B 500 C D 200nm Figure 8-9. TEM and ED of isolated platy crystals scraped from the SAMs. A) TEM image of a single-crystalline hydroxyapatite plate grown on COOH terminated SAM. B) Corresponding electron diffraction pattern of the plate, indicating angles between the indexed planes. C) TEM image of the single-crystalline HA plates lying right at the zone axis of 0 10. D) Selected area diffraction pattern of C)

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193 010020030040050060070080020253035404550552 thetaIntensity 002 A Au 111 004 B C Figure 8-10. A comparison of XRD spectra of crystals of different origin. A) HA grown on pure COOH-(CH2)11SH SAMs. B) HA grown on pure OH-(CH2)12SH. C) Commercial HA.

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194 010020030040050060070080024.625.626.627.628.629.630.631.632.633.62 ThetaIntensity 002 300 A B C D E Figure 8-11. Short scan (2 from 24 to 34) of the XRD spectra of HA crystals grown on mixed SAMs. The SAMs are mixed with different volume ratios of 2mM COOH-(CH2)11SH and 2mM OH-(CH2)12SH. A) COOH-(CH2)11SH only. B) 3:1. C) 2:2. D) 1:3. E) 0.5:3.5. (All peaks are normalized to the 300 plane. All XRD were done at the same condition)

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195 500 m A B C D 50 m 5 m Figure 8-12. Hydroxyapatite micro-patterning on self-assembled monolayers of COOH-(CH2)11SH in the presence of 15 g/ml Pasp. This experiment shows a difference in morphology of the crystals grown with polymer addition, as compared to the control shown in Figure 8-7. A) Lower Mag optical and B) SEM micrographs show pattern formation, although at higher mag. C) it appears that a thinner layer of mineral may have deposited in the channels alongside the thicker film in the patterned regions. D) The patterned surface is very smooth (notethis image is at same the mag. as those shown for Figure 8-8), but marked by some scattered crystals and aggregates that appear anisotropic in shape. The polarized Optical micrographs suggests the mineral maybe amorphous..

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196 A B C 50 m D 5 m E F G Figure 8-13. Micropatterning of hydroxyapatite on self-assembled monolayers of COOH-(CH2)11SH in the presence of 15 g/ml BSP proteins. A) SEM showing micro-channel pattern formation. There is also overgrowth of HA with larger crystal size in the non-patterned bare Au area. B) Phosphorous x-ray mapping. C) Calcium x-ray mapping. D) SEM image of a single channel of patterned HA. E) Higher Mag. showing the crystals are bent and curved, and smaller in size as compared to crystals formed without any BSP protein addition (Figure 8-8 A). F) TEM image of isolated crystal plates. G) Single-crystalline ED pattern matches that of hydroxyapatite.

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197 A B C D Figure 8-14. HAP pattern formation on the same monolayer as in Figure 8-13 in the presence of 75 g/ml BSP protein. A) BSP protein greatly promotes hydroxyapatite formation such that more crystals grew and filled the non-patterned monolayer area. The film is very dense with around 37 m thick, as can be seen by the thick crack that formed under the beam. B) Crystals grown on non-patterned Au areas grew into round clusters, with polycrystals of larger size than those in the polycrystalline films on the patterned SAM area. C). Curved morphology of hydroxyapatite crystals on SAMs. D) EDS spectrum showing Ca and P.

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198 0200400600800100012001400160018002000202530354045505560 Au (111) D C B 002 211 112 330 213 A 310 222 004 Figure 8-15. XRD comparison of hydroxyapatite of different origins. A) Commercial powder of Hydroxyapatite. B) Hydroxyapatite grown on SAMs without any polymer addition. C) Hydroxyapatite grown on SAMs with 15 g/ml BSP protein. D). Hydroxyapatite grown on the same SAMs in the presence of 75 g/ml BSP protein.

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CHAPTER 9 HYDROXYAPATITE FORMATION ON ORGANIC SUBSTRATES Following the discussion of the influence of polymer on the crystal growth of hydroxyapatite in Chapter 8, this chapter will further investigate the formation of hydroxyapatite thin films on an organic substrate-Poly(HEMA), towards fabrication of laminated composites. Similar to Chapter 6, I have also tried mineralization of hydroxyapatite inside a microporous hydrogel to form an interpenetrating composite Introduction The formation of biomimetic hydroxyapatite on an organic substrate has attracted considerable attention because this can offers the new possibility of combining the distinctive properties of organic and inorganic components as a new material. The inorganic phase give the composite stiffness and strength, while the organic phase give toughness and flexibility. Natural bone is a nanostructured-composite, in which an assembly of small HAP particles effectively reinforces a matrix of collagen fibers (109,116). Collagen is a protein-based hydrogel which is believed to provide the scaffold for bone apatite growth on and within. The unusual combination of hydroxyapatite with an elastic collagen hydrogel enables the bone to have enhanced resistance to tensile and compressive forces, and higher fracture toughness than its inorganic constituent-hydroxyapatite. Duplication of the structure of bone to make bone-like materials is very difficult, and no great success has ever been achieved for a long time. Yet, patients with fractured bone or bone disease need bone implant materials to replace the lost bone or fill the bone 199

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200 defect. In the United States, over 2 million orthopedic procedures and over 10 million dental implant procedures are performed every year. For example, in 1999, more than 500,000 bone graft procedures were performed in the US, which means a huge market of around 2.5 billion /year (117). Obviously there is a great need to replace the structure and function of bone lost by trauma or disease. In order to meet the above demand, better synthetic bone graft substitute materials should be designed and synthesized. Among synthetic materials, most successful and widely used are bone cement and metallic alloys. Bone cement (E=2.55 GPa) has a modulus comparable to cortical bone (E=3-20 GPa) after setting (118). The problem with Polymethyl methacrylate (PMMA)-based bone cement is that the setting process is an exothermal reaction, and the temperature around the tissue can reach 80C. A conservative guideline used by authorities is that tissue damage will be expected at temperatures around 56C. PMMA is not biodegradable in body, which leads to long term inflammatory response(119). Bioactive materials such as bioglass or bioglass ceramics show good bonding behavior to living bone through a biologically active calcium phosphate layer formed on their surfaces in-vivo(27). However, their lower fracture toughness and higher elastic modulus than those of bone prevent their use at defect sites that are heavily loaded. Engineering alloys such as stainless steel, cobalt-chromium alloys, and titanium alloys have been widely used in orthopedic surgery (e.g., artificial hip replacement). However, these materials have a much higher modulus than that of cortical bone, which is the strongest kind of bone in human body. When such a stiff implant is placed in contact with bone, the bone will sustain less mechanical stress

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201 and gradually be resorbed, a phenomena known as bone stress shielding (118). Therefore, each currently used synthetic material has significant drawbacks. We adopt a biomimetic synthetic approach using natural bone formation as a guide. An inexpensive and biocompatible polymeric hydrogel, PHEMA, is used to mimic the hydrophilic nature of protein-based hydrogel in bone-collagen. Mineralization studies were also done on the hydrogel substrate w/o or w/ the presence of acidic biomimetic polymers. The acidic polymers are used to mimic the acidic bone proteins, such as bone sialoprotein (BSP), that exist during bone growth. The hydroxyapatite mineral is used to mimic the inorganic constituent of bone-apatite. The use of PHEMA as one component of our biomimetic composites has many advantages. PHEMA is well tolerated by biological tissues and has been used in a number of biomedical applications, such as soft contact lenses and intraocular lenses. Another advantage of PHEMA is that it can be tailored to meet different cell-PHEMA interactions. For example, cell attachment or proliferation on PHEMA can be changed by copolymerization with other monomers (120). Moreover, PHEMA has a mechanical strength related to equilibrium water content and concentration of cross-linking agent. The lower is the equilibrium water content (EWC), the higher is the mechanical strength. When PHEMA is polymerized without water addition, it has a modulus comparable to bone. The idea that a PHEMA hydrogel can be used as a scaffold for bone implant is further supported by the observations that the long term usage of PHEMA soft contact lenses suffers from calcification problems (121). Previously, Song et al. have investigated the mineralization of PHEMA substrate by a high-temperature urea-mediated method (122). Filmon et al. also investigated the

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202 effects of negatively charged groups on the calcification of carboxylate functionalized PHEMA (123), and their mineralization study was achieved in Simulated Body Fluids (SBFs). Here we mineralized PHEMA and negatively charged (carboxymethyl) PHEMA by the method described by the previous chapter. We believe our method is faster than mineralization in SBF, and avoids the high temperature of the urea-mediated method. Experiments and Methods Mineralization A supersaturated solution of calcium phosphate is formed by addition of equal volumes of 9 mM CaCl2 and 4.2 mM of K2HPO4 in tris-buffer (final concentration: 4.5 mM CaCl2 and 2.1M of K2HPO4). The solution is mixed at room temperature and the crystallization study is done at 37 C and at a pH of 7.4. A PHEMA substrate or porous PHEMA hydrogel was put in the above solution containing w/ or w/o polymer (15 g/ml Poly-L-aspartic acid, Mw = 6,200Da, Sigma). After 6 days, the sample was taken out and washed with H2O and ethanol twice and dried for later characterization. The characterization and preparation of PHEMA hydrogels (non-porous and microporous) have already been shown in the previous chapters. Preparation of PHEMA and Carboxylmethylation of PHEMA The polymerization reaction of HEMA and carboxylmethylation are shown in appendix. Carboxylmethylated-PHEMA was prepared following the procedure described in literature (123). Briefly, in order to get a different degree of carboxyl-PHEMA, each hydrogel was put in a different concentration of BrCH2COOH solution (0, 0.125, 0.25, 0.5 and 1M dissolved in 2M NaOH solution) overnight at room temperature, under gentle agitation. Each sample was washed three times (10min. each) in deionized water and

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203 dried in oven overnight at 37C. The equilibrium water content (EWC%) was determined in the same manner as described in Chapter 6, and the data is shown in Table 9-1 and Table 9-2. Table 9-1. EQW determination of chemical modified PHEMA hydrogel substrate. Non-porous Pure PHEMA Carboxylated PHEMA [BrCH2COOH] 0 0.1250 0.25 0.5 1 Wo PHEMA 0.3482 0.3881 0.4159 0.4039 0.4403 Wt PHEMA 0.5725 1.0815 1.5738 3.0267 5.0728 EQW(%) 64.42 178.67 278.41 649.37 1052.12 Table 9-2. EQW determination of chemical modified micro-porous PHEMA hydrogel substrate. Micro-porous Pure PHEMA Carboxyl-PHEMA [BrCH2COOH] 0 0.1250 0.2500 0.5000 1 Wo PHEMA(porous) 0.0191 0.0157 0.0142 0.0185 0.0157 Wt PHEMA(porous) 0.0479 0.0818 0.1717 0.4260 0.6893 EQW(%) 150.79 421.02 1109.15 2202.70 4290.45 Results and Discussion Chemical Modification of Nonporous and Microporous PHEMA There are two common ways to make a hydrogel hold more water: one is chemical modification and the other is introduction of porosity into the hydrogel. Table 9-1 shows the Equilibrium Water Content (100%) of PHEMA thin sheets with different degrees of Carboxylmethylation. As more hydroxyl groups are replaced by carboxyl groups by using higher BrCH2COOH concentration, the hydrogel becomes more hydrophilic. The pure PHEMA sheet EWC content is about 64%. However, after being treated with 1M BrCH2COOH, the EWC increases to 1052%, which correspond to 9 fold increase in water weight content relative to its original weight.

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204 Introduction of porosity into PHEMA also greatly increases the EWC of PHEMA. As seen from Table 9-2, the EWC of microporous is 151%, which is much higher than 64% of nonporous PHEMA. Table 9-2 also shows that the EWC of the microporous hydrogel is much higher after Carboxylmethylation. A digital image of these microporous hydrogels before and after chemical modification is shown in Figure 9-1. The higher the degree of carboxylmethylation, the more the hydrogel swells in water. The effect of porosity and carboxylmethylation on the EWC of hydrogel is clearly shown in Figure 9-2. It clearly indicates that the porous hydrogel holds more water than nonporous, and the carboxylmethylated hydrogel holds more water than the non-carboxylmethylated hydrogel. Mineralization of CaP on PHEMA and Carboxyl-PHEMA Disc Without polymer addition Figure 9-3 shows a pure PHEMA disc mineralized with CaP, without the use of a polymeric process-directing agent. As seen from the polarized optical micrograph (Figure9-3A), the mineral phase is crystalline. The appearance of mineral is similar to the hydroxyapatite spherulites formed on glass slides shown in the previous chapter. The spherulites composed of plates are growing and impinging on each other to form a compact coating (Figure 9-3B & C). However, there is also some area uncovered by the spherulitic crystals (Figure 9-3D). EDS analysis shows Ca and P peaks similar to hydroxyapatite (Figure 9-3E). Figure 9-4A-E shows the results of 0.25M BrCH2COOH modified PHEMA after mineralization. Basically the morphology and coverage of mineral on this slightly modified PHEMA looks similar to that shown in Figure 9-3. However, the size of the spherulitic crystals is smaller than that on pure PHEMA by comparison of Figure 9-4C to

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205 Figure 9-3C. This is due to enhanced nucleation of CaP due to Carboxylmethylation, which leads to more nucleation and less growth. Figure 9-5 shows the results of 1 M BrCH2COOH modified PHEMA after mineralization. As seen from Figure 9-5A, CaP spherulites formed on the substrate. The background is pink and the corresponding image without the red-1 wavelength plate in Figure 9-5B is dark, suggesting it is PHEMA or a thin amorphous calcium phosphate film. Indeed, SEM images in Figure 9-5C & D shows there is thin film formation underneath the spherulitic crystals. An EDS spectrum done on the film region shows Ca and P peaks. This result indicates that Carboxylmethylation of PHEMA greatly promotes the nucleation of calcium phosphate. The carboxyl groups in the modified PHEMA provide ion-binding sites for CaP nucleation. However, in this case, the nucleation event is for an amorphous phase due to the high localized concentration of ions. This result also indicates that a thin and amorphous CaP film can be formed with a highly localized surface charge, even without the presence of soluble polymer. After the thin amorphous film formed covered the PHEMA surface, normal CaP nucleation and growth apparently resulted in the spherulitic hydroxyapatite formation on the film surface. With polymer addition Figure 9-6, Figure 9-7, and Figure 9-8 show the mineralization of CaP on PHEMA discs in the presence of 15 g/ml Pasp. Similar to the previous mineralization results on glass slides, thin films formed on the substrate in the presence of a small amount of Pasp. Figure 9-6A & B shows polarized optical micrographs of the thin films deposited on a pure PHEMA disc. With the red-1 wavelength plate, the film shows a magenta color alongside higher interference colors, indicating both amorphous and crystalline phases

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206 are present (Figure 9-6A). It is also possible that the isotropic regions are crystalline but oriented along their isotropic optical axis. The colors change to black and white in without the red-1 wavelength plate (Figure 9-6B), showing a spherulitic texture in the crystalline regions. Indeed, XRD verifies that both amorphous calcium phosphate and a crystalline phase exists on the film (Figure 9-9A). XRD was run from 2 to 60, and there is no peak below 5 (data not shown), indicating that the crystalline phase is hydroxyapatite, and not octacalcium phosphate (OCP). Figure 9-6C & D shows SEM images of the thin film formed on the pure PHEMA disc. The film looks rough with radiating ridges or “wrinkles” that seem to emanate from a central point, which is likely the center of the spherulites. At higher magnification, a “fibrous” texture is seen from the polycrystals of the spherulites, which are around 100 nm in diameter (Figure 9-6E). EDS shows Ca and P elements present in the film (Figure 9-6F). A cross-section of the fracture surface of this sample is shown in Figure 9-6G. As seen from this picture, the mineral does not go inside the hydrogel because it is not porous. There is a thin layer of calcium phosphate (marked as red), and from edge on, the “wrinkled” topology can be seen on top of the PHEMA (marked as blue). EDS confirms that on the film there are Ca and P elements while on PHEMA there are not. The thickness of the film is determined to be about 1 m. Figure 9-7 shows CaP thin films on 0.25M BrCH2COOH modified PHEMA formed in the presence of 15 g/ml Pasp. Again, the thin film appears magenta, suggesting it is amorphous, (Figure 9-7A); but weakly birefringent regions can be seen without the red-1 wavelength plate, only under cross-polars. This suggests that the film is in fact crystalline, but with a crystallographic orientation that is perpendicular to its optic

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207 axis. The undulating birefringence appears to be brought out due to the waviness of the mineral film, which would cause a gradual shift in crystal orientation which is sufficiently removed from the isotropic c-axis to be weakly birefringent. The film is quite continuous and covers the whole PHEMA substrate (Figure 9-7C & D). In some areas, the film cracks under the beam (Figure 9-7E). The “smooth” film shows some pronounced “wavy” features under high magnification (Figure 9-7F). A side view of mineralized 0.25 M BrCH2COOH modified PHEMA disk is shown in Figure 9-7G. The film formed on this modified PHEMA is about 7 m in thickness. The EDS spectrum shows Ca and P in the film (Figure 9-7H). Figure 9-8 shows CaP mineralized PHEMA with the highest degree of carboxymethylation (1M BrCH2COOH modified) in the presence of 15 g/ml Pasp. Under polarized optical microscopy, it appears there is no film formation (Figure 9-8A & B), only some crystalline patches. However, SEM verifies that there is also a thin film formation on this sample. The film appearance can be clearly seen in Figure 9-8C & D. EDS confirms it is calcium phosphate not PHEMA (Figure 9-8E). The thickness of the thin film is around 600 nm (Figure 9-8F). A cross-section of the fracture surface of this sample is shown in Figure 9-8G. The thicker crystalline film patches shows Ca and P in EDS while the PHEMA shows no Ca and P in EDS. XRD of this sample shows a broad amorphous peak with a little (002) oriented hydroxyapatite crystal structure (Figure 9-9B). However, the broad amorphous peak could arise from the PHEMA, so it is possible that the mineral film is highly oriented (the XRD beam was perpendicular to the thin film, so the signal is very low for the mineral). Highly oriented HA has been observed

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208 for CaP grown on Langmuir monolayers in work by Aksay and coworkers, so it would not be unreasonable to suggest this possibility (124). Mineralization of Micro-porous PHEMA Hydrogel Without polymer addition Figure 9-10A shows the cross-section of a sea urchin spine. It was used directly for making a microporous PHEMA hydrogel (Figure 9-10B), which has a microporous structure similar to that shown in the central region of the spine shown Figure 9-10A. Figure 9-10C shows the appearance of a CaP mineralized microporous PHEMA hydrogel (without polymer additive). The whole surface is covered with large spherulitic clusters of HAP crystals. A cross-section of the hydrogel shows there are some spherulites which grew inside the pores of hydrogel (Figure 9-10D). Figure 9-10E shows the structure of a CaP mineralized, microporous, 0.25M BrCH2COOH modified PHEMA hydrogel. The SEM image shows that there are more crystals inside the pores of the hydrogel (Figure 9-10F). Figure 9-10 G shows the surface of CaP mineralized, microporous, 1M BrCH2COOH modified PHEMA hydrogel. It clearly shows there are some platy crystals within the pores. The spherulitic structure of the crystals is not as apparent. With polymer addition Figure 9-11 shows CaP mineralized microporous PHEMA in the presence of 15 g/ml Pasp. As seen from Figure 9-11A, the pore structure is no longer clear and highly filled with CaP mineral. Figure 9-11C shows Carbon elemental mapping, which corresponds to the PHEMA structure. Figure 9-11D-E shows the P and Ca mapping, which correspond to the CaP mineral phase. The pore structure in Figure 9-11D and E is

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209 a little bit smaller than that in Figure 9-11C, indicating that CaP mineral coated the walls of the PHEMA pores, but did not totally fill in the pores. Figure 9-12 shows CaP mineralized, microporous, and 1M BrCH2COOH modified PHEMA in the presence of 15 g/ml Pasp. As seen from Figure 9-12A & B, some CaP crystals filled in the pores and even emerged from the pores. Carbon mapping shows the porous PHEMA structure (Figure 9-12C), while Phosphorous mapping shows the pores are filled with CaP crystals (Figure 9-12D). Conclusions In this chapter, I demonstrate the preparation of nonporous and microporous PHEMA hydrogels, and then chemically modified them with carboxyl groups to enhance its calcification ability. Mineralization studies were done without and with polymer additive, which leads to a different crystal morphology and degree of mineralization. The conclusions are as follows: From swelling studies, the porous hydrogels are seen to hold more water than nonporous hydrogels, and carboxylmethylated PHEMA holds more water than pure PHEMA. The higher the degree of carboxylmethylation, the more water it holds. Without polymer addition, hydroxyapatite spherulitic crystals formed on the surface of pure PHEMA and 0.25 BrCH2COOH modified PHEMA. Due to the higher degree of carboxymethylation, amorphous calcium phosphate thin films first formed on the 1M BrCH2COOH modified PHEMA surface, and then many spherulitic hydroxyapatite crystals were formed on top of the film. This result indicates that carboxymethylation of PHEMA greatly enhances the nucleation of CaP and thus enhances its calcification ability, including the nucleation of an amorphous film. With polymer addition, thin films formed on both pure PHEMA and carboxylmethylated PHEMA surface. The thickness of the film differs, but the cross-section clearly indicates the formation of a laminated structure. On the surface of 1M BrCH2COOH modified PHEMA, a non-birefringent calcium phosphate film formed on the surface, upon which some crystalline hydroxyapatite film patches were formed. The non-birefringent film may either be amorphous, or highly oriented, with

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210 the latter suggested supported by the fact that some degree of c-axis orientation is seen by XRD. Without polymer addition, more HA spherulites infiltrated inside the pores of the PHEMA hydrogel as the degree of carboxymethylation was increased. With polymer addition, CaP minerals coated the walls of the pores of the microporous PHEMA hydrogel. However, CaP minerals can entirely fill in the pores of the 1M BrCH2COOH modified hydrogel, as confirmed by X-ray elemental mapping. This result also indicates that carboxymethylation of a porous hydrogel can leads to enhanced calcification through the precursor process as well. The above results indicate that with the addition of polymer, it is feasible to mineralize organic substrates or porous organic structures into organic-inorganic composites, which can have potential biomedical applications.

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211 A B 0 .125 .25 .5 1 Figure 9-1. Digital images of microporous PHEMA hydrogels. A) At drying state after different degree of carboxymethylation. B) At swelling state after different degree of carboxymethylation. The hydrogels swell more as the degree of carboxylmethylation become higher due to reaction with different concentrations of bromoacetic acid (0, 0.125,0.25, 0.5 and 1 M BrCH2COOH-modified).

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212 05001000150020002500300035004000450012345Concentration of bromoacetic acid used for carboxylation EQW of PHEMA (%) Non-microporous carboxylmethylatedPHEMA Microporous carboxylmethylatedPHEMA Figure 9-2. A comparison of Equilibrium Water Content (EWC%) of nonporous and microporous carboxylmethylated PHEMA. 1) 0, pure PHEMA. 2) 0.125 3) 0.25 M. 4) 0 .5M. 5) 1M BrCH2COOH-modified PHEMA.

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213 A B 1 mm C D 100 m 50 m E Figure 9-3. Hydroxyapatite crystals formed on pure PHEMA substrate without any polymer addition. A) Polarized optical micrograph with red-1 wavelength plate. B) Low mag. showing hydroxyapatite coated PHEMA. C) In some areas, the PHEMA is not fully covered with HA. D) The coating is a result of HA spherulites impinging upon each other. Each individual HA spherulite is clearly seen and reminiscent of the single HA spherulites grown on glass slides without any polymer addition. E) EDS shows Ca and P, and the morphology suggests the spherulites are HAP.

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214 A B 1 mm C D 100 m 50 m E Figure 9-4. Hydroxyapatite crystals formed on 0.25M BrCH2COOH modified PHEMA without any polymer addition. A) Polarized optical micrograph with red-1 wavelength plate. B) Lower Mag. showing that the whole substrate is nearly coated. C) In some areas, it is not fully covered with spherulites. D) In some areas, crystals aggregate and form a compact polycrystalline mesh. E) EDS spectrum shows Ca and P.

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215 B A C D 1 mm 50 m Ca E P Thin film formation under the spherulites! The thin film cracks under the beam Figure 9-5. Hydroxyapatite formation on 1 M BrCH2COOH modified PHEMA substrate without any addition of polymer. A) Polarized optical image with red-1 wavelength plate show large spherulites. B) Polarized optical image without red-1 wavelength plate. C) SEM image shows thin film underneath the spherulites. D) A crack ran right through a spherulite and thin film underneath. E) EDS spectrum shows Ca and P in hydroxyapatite.

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216 A B C D 1 mm 100 m E F 2 m Figure 9-6. Hydroxyapatite thin films formed on PHEMA substrate in the presence of 15 g/ml Pasp. A) Polarized optical image with the red-1 wavelength plate. B) Same sample as in A) without the red-1 wavelength plate. The film underneath is either amorphous, or highly c-oriented along the optic axis. C)-D SEM image shows the mineral films which formed on the PHEMA were wavy and wrinkled. E) Higher Mag shows a fibrous texture, with polycrystals around 100 nm in diameter, which may be due to a fibrous spherulitic structure. F) EDS shows Ca and P. G) A fracture surface of the cross-section of mineralized PHEMA showing a “laminated” structure. The thickness of the film is around 1 m. H) EDS shows no Ca and P on the PHEMA. I) EDS of the film shows Ca and P.

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217 G 50 m 1 m PHEMA Hydroxyapatite H I Figure 9-6. continued.

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218 A B C D 1 mm 100 m E F 2 m 50 m Figure 9-7. Hydroxyapatite thin films formed on 0.25 BrCH2COOH modified PHEMA substrate in the presence of 15 g/ml Pasp. A) Polarized optical image with red-1 wavelength plate. B) As in A) without the red-1 wavelength plate. The film underneath is weakly to non-birefringent. C)-E) SEM images show the smooth topology of the thin mineral films. F) High Mag shows the film has a fine-grained fibrous texture. G) A side-view of mineralized PHEMA substrate showing the film is quite “wavy”, and has a thickness of around 7 m, much thicker than that on unmodified PHEMA. H) EDS spectrum shows Ca and P element on the film.

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219 G H 7 m Figure 9-7. continued.

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220 A B 100 m C D 1m m 100 m E F 5 m Figure 9-8. Hydroxyapatite thin films formed on 1M BrCH2COOH modified PHEMA substrate in the presence of 15 g/ml Pasp. A) Polarized optical image with red-1 wavelength plate. B) As in A) without red-1 wavelength plate. C)-D) SEM shows thin film formation. E) EDS shows Ca and P in the film. F) The thin film cracked under SEM. The thickness of the film is around 600 nm. G) A cross-section of the fracture surface showing PHEMA substrate and CaP thin films. H) EDS spectra shows no Ca and P on the PHEMA section. I) EDS spectra shows Ca and P peak on mineral section.

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221 G H I Figure 9-8. continued.

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222 0200400600800100012001400202530354045505560 211,112,300 002 213 004 B A Figure 9-9. XRD spectra of CaP thin film formed in the presence of 15 g/ml Pasp. A) Pure HEMA mineralized with CaP. XRD shows broad peaks, which may arise from amorphous mineral and/or PHEMA, plus small crystalline peaks of hydroxyapatite. The XRD beam is placed perpendicular to the thin films, so the signal is quite small. B) 1M BrCH2COOH modified PHEMA. XRD shows amorphous peaks, from either calcium phosphate or PHEMA films, and 002-oriented hydroxyapatite film patches.

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A B C D E F Figure 9-10. Mineralization of microporous and chemically modified PHEMA without any polymer addition. A) Sea urchin spine used for making the PHEMA replica. B) The microporous PHEMA hydrogel. C) Appearance of CaP mineralized porous PHEMA, showing the surface is covered with large spherulites of HAP. D) SEM of cross-section shows some of the pores are infiltrated by hydroxyapatite spherulites. E) Low mag. view of CaP mineralized, porous, 0.25M BrCH2COOH modified PHEMA. F) Some hydroxyapatite spherulites formed inside the pores. G) Appearance of CaP mineralized, porous, 1M BrCH2COOH modified PHEMA. The pores are filled with crystals. H) SEM showing the appearance of crystal aggregates inside the pore. 223

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224 G H Figure 9-10. continued

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225 A B C D E Figure 9-11. SEM images of CaP mineralized microporous PHEMA hydrogel in the presence of 15 g/ml Pasp. A) Surface of mineralized hydrogel showing mineral infiltrated inside the pores. B)-E) Elemental mapping, with B) showing original SEM image. C) Carbon-mapping showing the porous PHEMA structure. D) Phosphorous mapping showing CaP coated the PHEMA area. E) Calcium mapping showing similar CaP coating structure.

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226 A B C D Figure 9-12. SEM images of CaP mineralized, microporous, 1M BrCH2COOH modified PHEMA hydrogel. A) SEM image shows there are some rounded crystals growing out of the pores. B) SEM image used for mapping. C) C mapping showing the hydrogel structure. D) P mapping shows the CaP filled in the pores.

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CHAPTER 10 COLLAGEN-HYDROXYAPATITE NANOCOMPOSITES Introduction Bone is a complex and highly organized biomineral. It is mainly composed of type-I collagen (70% dry weight of bone), mineral (~30% dry weight of bone) similar to hydroxyapatite, and H2O. In bone, hydroxyapatite formation is related to the cellular control of enzymes to release phosphate ions, with subsequent hydroxyapatite formation inside the pre-formed collagen matrix. The superior mechanical properties of bone rely on the interaction between the nano-sized hydroxyapatite crystals and collagen matrix at different hierarchical levels. In the second level of structure, the nanocrystals of Hydroxyapatite (20-40nm long) are mineralized and imbedded in the collagen fiber bundles, which is referred to as intrafibrillar mineralization, and is very important for the strength and stiffness of the fibers. Then these mineralized fibers are assembled into arrays, which further are organized into lamellae, approximately 4 m thick (125). In any lamellae, the c-axis of the HAP crystals are orientated parallel to the associated collagen fibrils. Then these lamellae are packed into even higher order structure, such as the concentric layers of osteons (13). The molecular structure of the collagens (there are around 20 types) indicates that they are macromolecular proteins of approximate molecular weight 300,000, composed of three helical polypeptide chains wound around each other to form a triple helix. The axial structure of a native collagen fibril shows a periodic banding pattern with a periodic 227

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228 repeat of about 67 nm. Collagen is believed to provide mechanical support to the connective tissue such as bone and dentin. The ductile collagen scaffold makes bone less brittle than other calcium-based materials such as eggshells, oyster shells and chalk. The collagen and other organic molecules provide bone with tensile strength, which is resistance to being stretched or torn apart, while the mineral phase provides stiffness for compressive loads. The noncollagenous proteins in bone usually contain a large amount of acidic amino acids, such as aspartate and glutamate. The role of these noncollagenous proteins in the formation of bone is still not known. Traditionally, such charged molecules are believed to bind to collagen, providing nucleation sites for hydroxyapatite to grow on and within the fibrils (126). The complex hierarchical structure, unique choice of material (collagen, HA), and synthesis from the nanoscale self-assembly of collagen, make bone a classic model of biomineralization. Presently, no synthetic bone analogue materials are comparable to bone in terms of structure and functional properties. This chapter will show the use of a biomimetic method to develop a material which is suitable as a bone graft substitute. Cellagen, reconstituted type-I collagen was chosen to mimic the native collagen in bone. Polyaspartic acid was chosen to mimic the acidic noncollagenous proteins in bone mineralization. The mineralization was carried out with a supersaturated CaP system, pH of 7.4, and temperature of 37 C, to mimic the physiological conditions in the human body.

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229 From this biomimetic approach, we hope to not only develop a novel biocompatible bone graft substitute material, but also to contribute to the understanding of bone formation in vivo. Materials and Methods Mineralization Supersaturated solution of calcium phosphate is formed by addition equal volume of 9 mM CaCl2 and 4.2 mM of K2HPO4 in tris-buffer (final concentration: 4.5 mM CaCl2 and 2.1M of K2HPO4). The solution is mixed at room temperature and the crystallization study is done at 37 C and at pH of 7.4. Cellagen, a hemostatic sponge (ICN Biomedicals), was put in the above solution containing w/ or w/o polymer (Poly-L-aspartic acid, Mw=6,200, Sigma). After certain amount of time (1 day, 2 day and 6 days), the sample was taken out and washed with H2O and ethanol twice and dried for later characterization. Characterization Scanning Electron Microscopy (SEM) analysis Samples were prepared for scanning electron microscopy (SEM) by allowing whole samples to dry in air, mounting the dried samples on an aluminum stub covered in double-sided copper tape, and then sputter coating the stub with either a Au/Pd or an amorphous carbon film. The samples were then analyzed using either a 6400 JEOL SEM or a 6330 JEOL FEGSEM at 15-20 kV. Elemental x-ray analysis (EDS) was performed on the mineralized sample with a Link-ISIS which was attached to both SEMs. Transmission Electron Microscopy (TEM) analysis Samples were prepared for transmission electron microscopy (TEM) following the protocols performed on bone and naturally mineralized tendon by Weiner and Traub

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230 (127). This included crushing the samples into a nanometer powder in a liquid nitrogen mortar and pestle. A few small drops of ethanol were then placed on the powder, followed by drawing the slurry into a micropipette. The slurry was transferred to a 3mm diameter carbon/Formvar coated copper TEM grid, followed by an optional stain with 1% phosphotungstic acid (PTA) in a PBS buffer. Before analysis, all samples were sputter coated with a thin layer of amorphous carbon for stability. The samples were analyzed using a 200cx JEOL TEM at 200kV in brightfield (BF), darkfield (DF) and selected area diffraction (SAD) modes. Normal brightfield images are produced using the transmitted spot to produce the brightest image possible. When a crystal phase is present, all of the planes that are parallel to the electron beam will diffract, thus creating diffraction patterns. X-ray Diffraction (XRD) analysis X-ray analysis was used to determine the crystal structures of samples mineralized in the absence and presence of polymeric additives, as well as HA and bone as standards. The samples were scanned with Cu-K x-ray radiation from a Philips XRD 2500 at 40 KV and 20 mA, using a step size of 0.02 mrad/s over a 2 range of 10-70. Results and Discussion Without Polymer Figure 10-1A shows SEM image of pure cellagen sponge. It is composed of a mesh of collagen fibers. The size of fibers is not quite uniform, ranging from 200nm-400nm. Figure 10-1B shows the appearance of hydroxyapatite mineralized cellagen without any polymer addition. The surface of cellagen sponge is covered with large hydroxyapatite spherulites (confirmed by XRD). At higher magnification (Figure 10-1C

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231 E, each spherulite is composed of assembly of plates. EDS spectra shows Ca and P in the spherulitic crystals. With Polymer With the polymer addition, the cellagen mineralized shows distinct difference to that without any polymer. Figure 10-2A-E shows SEM image of mineralized cellagen sponge in the presence of 15 g/ml Pasp (unbleached, 7 days’ mineralization). The cellagen mineralized shows the mineralized fibers morphology along with crystal aggregates. These aggregates do not show well-defined platy morphology, instead, they are more like particulate aggregates (Figure 10-2A). Some mineralized collagen fibers have different contrast under SEM, probably due to different degree of mineralization (Figure 10-2B). The hydroxyapatite minerals take the shape of collagen fibers and in some cases the minerals binding the fiber together (arrow, Figure 10-2C-E). EDS shows Ca and P on all the mineralized cellagen fibers, indicating at least the fibers are coated with the mineral. Field emission images of the mineralized cellagen sponge reveal greater details about the surface features (Figure 10-3A & B). The collagen fibers are heavily mineralized. In some area the minerals grow out of the fibril and form a “bamboo” like appearance (Figure 10-3C & D). The mineralized fibers are around 200 nm in diameter. In order to determine the degree of mineralization, the mineralized sample was bleached in 2% NaOCl solution to remove any un-mineralized collagen. After bleaching, some mineralized fiber broken indicating not all collagen fibers is mineralized (red arrow). There is also a thin hydroxyapatite coating on the mineralized collagen (white arrow in Figure 10-4A. The retain of fibrous shapes in this sample indicate that the

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232 collagen fibers are heavily mineralized to such a level that almost the entire surface of collagen is covered by the mineral so that the remaining hydroxyapatite form continuous fibers by itself (Figure 10-4B-E). EDS spectra showing Ca and P element in all the fibers (Figure 10-4F). Figure 10-5 shows the SEM image of mineralized cellagen sponge in the presence of 75 g/ml Pasp (unbleached). Figure 10-5A shows the overall appearance of mineralized collagen fibers. There are also some film patches on the mineralized collagen fibers. In some area, the mineral can hold different fibers together (Figure 10-5B-F). Figure 10-5G-H show the field emission images of unbleached collagen fibers. At higher Pasp concentration, the “bamboo” appearance disappears and less film patches form, indicating higher Pasp concentration may leads to more intrafibrillar mineralization. EDS shows Ca and P on the fibers (Figure 10-5I). Figure 10-6 shows the SEM image of mineralized cellagen sponge in the presence of 75 g/ml Pasp after being bleached in 2% NaOCl solution to remove unmineralized collagen). Figure 10-6A & B shows the appearance of mineralized cellagen after bleach. Again, field emission image of mineralized cellagen fibers indicating heavy mineralization (Figure 10-6C & D). Figure 10-6E & F show broken fibers after bleach, from which we can see the needle-shape hydroxyapatite crystals. A kinetic study of mineralization is shown in Figure 10-7 by XRD. XRD spectra is done directly on mineralized sample without any polymer addition and it matches hydroxyapatite (Figure 10-7A). Cellagen mineralized with calcium phosphate after 1 day shows broad amorphous peak (Figure 10-7B), and it gradually transform to crystalline after 2 day (Figure 10-7C). It transform to quite crystalline after 7 days’ mineralization

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233 and the spectra shows similar pattern as that of bone. This kinetic study clearly indicate the presence of amorphous calcium phosphate at early stage of mineralization and the amorphous phase transform to crystalline as time goes on. In order to determine the crystal size inside the mineralized cellagen in the presence of Pasp, the sample was sonicated in H2O for 15 min. to remove the mineral coatings. Then the sample was crushed in liquid-N2 into fine powders, and then adding 2% NaOCl to remove any collagen inside the sample. After washed with H2O and ethanol, the nanocrystals was transferred to TEM grid. Figure 10-8A-C shows the appearance of nanocrystals. The crystals are less than 200 nm long and very small in diameter and they tend to aggregate together. Figure 10-8D shows SAED pattern and it matches that of hydroxyapatite. XRD is also done on mineralized cellagen at different polymer concentration. As seen from Figure 10-9, at lower Pasp concentration, XRD pattern of the mineral in the cellagen sponge was almost the same as that in hydroxyapatite control (Figure 10-9A-C). However, at highest polymer concentration (200 g/ml), the mineralized cellagen shows both amorphous and crystalline phase, indicating polymer induce the amorphous phase and stabilize it. Conclusion The above results demonstrate the mineralization study of type-I reconstituted collagen (cellagen sponge) without and with the presence of Pasp. The conclusion is as follows: Mineralization of cellagen without Pasp leads to large hydroxyapatite spherulites crystals on the surface. The morphology of the HA crystals is similar to that formed on glass slides or PHEMA substrate shown in previous chapter without any polymer addition.

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234 Small amount of polymer can change the CaP mineralization via amorphous to crystalline transition mechanism. At early stage the mineral as formed is amorphous calcium phosphate, which transforms to hydroxyapatite as time goes on. Intrafibrillar mineralization is achieved in the collagen fibrils. The sample retains fibril shape even after bleaching. At higher Pasp concentration, the sample shows much smoother surface and less mineral coating compared to that at low Pasp concentration. Crystals extracted from the mineralized samples are nanometer in size. A heavily mineralized collagen-HA nanocomposites is prepared, which will be used for bone tissue engineering applications.

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235 A B 1 mm 50 m C D 50 m 50 m E F 5 m Figure 10-1. SEM image of hydroxyapatite mineralized cellagen sponge without any polymer addition (control). A) SEM of pure un-mineralized cellagen sponge showing a mesh of type-I collagen fibers. B) Overview showing many HAP spherulites covered the sponge. C)-D) These spherulites resemble the morphology of HAP formed on glass slides or PHEMA substrate without any polymer addition. The collagen underneath is hardly mineralized due to large crystal size. E) Each spherulite is composed of assembled plates. F) EDS spectra shows Ca and P in HAP.

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236 A B 5 m 50 m C D 2 m 5 m E F 2 m Figure 10-2. SEM image of mineralized cellagen sponge in the presence of 15 g/ml Pasp (unbleached). A) Overview showing the mineralized fibers and crystal aggregates. B) Some collagen fibers have different contrast under SEM, probably due to different degree of mineralization. C)-E) hydroxyapatite mineral take the shape of collagen fibers and in some cases the minerals binding the fiber together (arrow). F) EDS show Ca and P on the fibers.

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237 A B 1 m 1m C D 100 nm 1m Figure 10-3. Field emission images mineralized cellagen sponge in the presence of 15 g/ml Pasp (unbleached). A)-B) collagen fibers are heavily mineralized. C)-D) In some area the mineral grow out of the fibril and form a “bamboo” like appearance. The mineralized fibers are around 200 nm in diameter.

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238 A B C Figure 10-4. Field emission images mineralized cellagen sponge in the presence of 15 g/ml Pasp. (bleached in 2% NaOCl solution to remove unmineralized collagen.) A) After bleaching, some mineralized fiber broken indicating not all collagen fibers is mineralized (red arrow). There is also a thin hydroxyapatite coating on the collagen (white arrow). B)-C) Appearance of mineralized collagen after bleaching. D) Hydroxyapatite nanofibers formed after collagen being bleached. E) Appearance of heavily mineralized collagen. F) EDS spectra showing Ca and P element.

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239 D E F Figure 10-4. continued.

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240 A B 10 m 50 m C D 2 m 5 m E F 2 m 2 m Figure 10-5. SEM image of mineralized cellagen sponge in the presence of 75 g/ml Pasp (unbleached). A) Overview showing the mineralized fibers and film patches. B)-F) Morphology of mineralized collagen fibers. The mineral can hold different fibers together. G)-H) Field emission images of unbleached collagen fibers. At higher Pasp concentration, the “bamboo” appearance disappears and less film patches form, indicating higher Pasp concentration may leads to more intrafibrillar mineralization. I) EDS show Ca and P on the fibers.

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241 G I H Figure 10-5 continued.

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242 A B 20 m 10 m C D 100nm 1 m E F 100nm 100nm Figure 10-6. SEM image of mineralized cellagen sponge in the presence of 75 g/ml Pasp. (bleached in 2% NaOCl solution to remove unmineralized collagen). A)-B) Appearance of mineralized cellagen after bleach. C)-D) Field emission image of mineralized cellagen fibers indicating heavy mineralization. E-F) Broken fibers showing the needle-shape hydroxyapatite crystals.

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243 60 50 40 30 20 10 Normalized Intensit y ( a.u. ) 00000001015202530354045505560 F Bone (equine) E 15 g/ml Pasp, 6 day D 15 g/ml Pasp, 2 day C 15 g/ml Pasp, 1 day B 0 g/ml Pasp, 6 day A Commercial H A 202 210 310 222 213 004 300 211 002 112 2 (degrees) Figure 10-7. X-ray diffraction (XRD) spectra of calcium phosphates crystallized in the absence and presence of polyaspartic acid. The bottom spectrum A) is of a commercial hydroxyapatite (HA) standard showing the typical diffraction planes present in a randomly-oriented powder pattern of HA. Spectrum B) is of mineralized collagen in the absence of polyaspartic acid, which serves as a control sample that only produces HA clusters on the surface of the sponge (Fig. 2C). Therefore, the same planes are expressed as the standard HA due to the high degree of crystallinity and random orientation of the crystallites in the clusters. Spectra C)-E) show stages of the PILP mineralization process in which collagen samples were removed from the mineralizing solution at 1, 2, and 6 days respectively. As can be seen, the mineral phase was initially amorphous in the early stages, and gradually transformed into poorly crystalline HA. Note the similarity in spectrum F), which is of equine bone, to that of the fully mineralized sample (E), both of which exhibit relatively broad peaks, presumably due to the extremely small size of the intrafibrillar crystallites (or crystal lattice strain and/or paracrystallinity).

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244 A B C D Figure 10-8. TEM image of extracted hydroxyapatite nanocrystals from mineralized cellagen sponge. A)-C) Bright field image of the needle-like crystals. They tend to aggregate and resemble HA crystals in bone. D) SAED shows patterns of hydroxyapatite.

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245 0501001502002503003504004501015202530354045505560 Pasp200 Pasp 75 Pasp 15 HA D C B A Figure 10-9. Comparison of XRD spectra of mineralized cellagen sponge with commercial hydroxyapatite. A) Commercial hydroxyapatite. B) Mineralized cellagen in the presence of 15 g/ml Pasp. C) 75 g/ml Pasp. D) 200 g/ml Pasp.

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CHAPTER 11 CONCLUSION Natural minerals often contain both soluble acidic proteins and insoluble framework macromolecules. In this dissertation, we try to use an acidic polymer to mimic the acidic macromolecules, and form minerals onto different substrates/compartments (e.g., glass slides, PHEMA substrate, porous PHEMA mold, collagen fibrous sponges, self-assembled monolayers) to mimic the role of the insoluble framework macromolecules. We find the crystallization of calcium carbonate in the presence of a small amount of acidic biomimetic polymer leads to the formation of droplets of a liquid-phase mineral precursor, which grows from the nanoscopic scale and coalesces to form larger structures. Solidification and an amorphous-crystalline transformation then takes place and results in formation of the crystalline phase which exhibits a different morphology than the faceted habit of crystals grown from the traditional solution crystallization process, which can be modulated and even molded, depending on the substrate/compartment used. By using a fluorescence labeling technique, we were able to prove that polymer is closely associated with the precursor phase, even at the very early stage. During solidification and transformation, the polymer is partially excluded from the crystalline phase, so that “transition bars” are formed. These non-birefringent “bars” result from a slower crystallization rate in the regions where excess impurity (polymer) resides (some polymer is presumably also excluded out into the solution). This diffusion limited 246

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247 exclusion process results in compositional inhomogeneity of the polymer occlusion within the final crystal structure. Due to the fluidity of the precursor phase, large amount of impurities (Mg, Sr) can be entrapped in the precursor phase which is induced by the acidic polymer. We found that this impurities and polymer overall tend to stabilize the amorphous phase and have great effect on the transformation. The calcium carbonate as formed has both non-equilibrium morphology and non-equilibrium composition (e.g., high magnesian calcite), which are different from minerals formed from inorganic origin and with some features greatly resembling that of biominerals. Non-equilibrium morphology and non-equilibrium composition is the hallmark of biomineralization. By using acidic polymer to mimic the role of acidic macromolecules in biomineralization, PILP process have great relevance to kinetically-driven crystallization, such as the biomineralization of sea urchin spine, mollusk larvae shells and so on. Both the organic matrix and soluble acidic macromolecules play very important roles in biomineralization. We’ve demonstrated the synergistic effect of a polymeric process-directing agent (acidic protein from nacre) and template (a polymeric hydrogel) towards mimicking aragonite tablet formation in mollusk nacre. Because the precursor phase is growing from nano-size, it is possible for the precursor droplets to diffuse into a mold and crystals with complex morphology can be made via amorphous to crystalline transition of the precursor. In the presence of acidic polymer in combination with a porous hydrogel, the liquid precursor phase is molded inside a sea urchin replica before they transform to crystalline calcite, whose morphology is dictated by the moldthe microporous hydrogel. The mineralized hydrogel itself can be

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248 used for structural materials or for biomedical applications. Moreover, after the polymer phase is being removed, a porous 3-D scaffold is formed. So we’ve found a new method to make porous scaffold. This molding crystals method can be easily extended toward other mineral system (e.g., CaP, ZnS) or other compartment (e.g., fibrous, tubular, micelles, lipids), so that different minerals with different shapes can be achieved by combination of PILP process with compartment. Calcium phosphate is another important mineral for skeletal parts in organisms. We’ve describes the formation of amorphous calcium phosphate precursor and crystalline hydroxyapatite. By comparing crystallization of CaP with and without polymer, we find polymer can induce calcium phosphate with non-equilibrium morphology. We also successfully micro-patterned nano-crystals using self-assembled monolayer systems combined with the soft lithography technique of microcontact printing. Such micro-patterned nanocrystals have great potential for applications such as protein separation devices or for study of cell-mineral interactions. Current drawback of allografts and autografts call for synthetic biomaterials for bone graft substitutes. We successfully form CaP on an organic substrate and mineralization of CaP inside a porous hydrogel. The goal of this ongoing study is to generate novel organic-inorganic composites which have potential applications for hard tissue engineering. We’ve also describes the preparation and characterization of Collagen-Hydroxyapatite nanocomposites using an amorphous precursor process. We developed a heavily mineralized collagen nano-composite. The orientation of HAP nanocrystals inside the collagen matches that of bone, as proved by XRD, FEM and TEM-ED. Such

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249 composites have great potential for application as bone graft substitutes and tissue engineering applications. In a word, by combining different substrates with different functionality (e.g., PHEMA and carboxylmethylated PHEMA, glass slides), or self-assembled monolayers (alkanethiol on Au), or organic compartments such as fibrous collagen sponges, with the PILP process, different organic-inorganic composites or minerals with complex morphology are formed with applications towards tissue engineering or biomedical materials. Polymer-Induced-Liquid-Precursor process, which is using polymer as a process-directing agent, has not only relevant to biomineralization, but also can be applied towards formation of novel biomimetic materials.

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APPENDIX SCHEMATIC REACTION OF PHEMA AND CARBOXYMETHYLATED PHEMA SYNTHESIS H2CCH3OOCH2CH2OHCH2CH3OOCH2CH2OHnn AIBN 67 C R NaOH R-OH+BrCH2COOH R-OCH2COOH+NaBr 250

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LIST OF REFERENCES (1) Weiss, I. M.; Tuross, N.; Addadi, L.; Weiner, S. Mollusc larval shell formation: Amorphous calcium carbonate is a precursor phase for aragonite. Journal of Experimental Zoology 2002, 293, 478-491. (2) Politi, Y.; Arad, T.; Klein, E.; Weiner, S.; Addadi, L. Sea Urchin Spine Calcite Forms via a Transient Amorphous Calcium Carbonate Phase. Science 2004, 306, 1161-1164. (3) Raz, S.; Hamilton, P. C.; Wilt, F. H.; Weiner, S.; Addadi, L. The transient phase of amorphous calcium carbonate in sea urchin larval spicules: The involvement of proteins and magnesium ions in its formation and stabilization. Advanced Functional Materials 2003, 13, 480-486. (4) Raz, S.; Testeniere, O.; Hecker, A.; Weiner, S.; Luquet, G. Stable amorphous calcium carbonate is the main component of the calcium storage structures of the crustacean Orchestia cavimana. The Biological Bulletin 2002, 203, 269-274. (5) Weiner, S.; Levi-Kalisman, Y.; Raz, S.; Addadi, L. Biologically formed amorphous calcium carbonate. Connective Tissue Research 2003, 44, 214-218. (6) Addadi, L.; Raz, S.; Weiner, S. Taking advantage of disorder: Amorphous calcium carbonate and its roles in biomineralization. Advanced Materials 2003, 15, 959-970. (7) Aizenberg, J.; Weiner, S.; Addadi, L. Coexistence of amorphous and crystalline calcium carbonate in skeletal tissues. Connective Tissue Research 2003, 44, 20-25. (8) Gotliv, B. A.; Addadi, L.; Weiner, S. Mollusk shell acidic proteins: In search of individual functions. Chembiochem 2003, 4, 522-529. (9) Gower, L. B.; Odom, D. J. Deposition of calcium carbonate films by a polymer-induced liquid-precursor (PILP) process. Journal of Crystal Growth 2000, 210, 719-734. (10) Lowenstam, H. A.; Weiner, S. On biomineralization; Oxford University Press: New York, Oxford, 1989. (11) Naka, K.; Chujo, Y. Control of crystal nucleation and growth of calcium carbonate by synthetic substrates. Chemistry of Materials 2001, 13, 3245-3259. (12) Smith, B. L.; Schaffer, T. E.; Viani, M.; Thompson, J. B.; Frederick, N. A. et al. Molecular mechanistic origin of the toughness of natural adhesives, fibres and composites. Nature 1999, 399, 761-763. (13) Weiner, S.; Wagner, H. D. The material bone: Structure mechanical function relations. Annual Review of Materials Science 1998, 28, 271-298. (14) Bauerlein, E. Biomineralization of unicellular organisms: An unusual membrane biochemistry for the production of inorganic nanoand microstructures. Angewandte Chemie-International Edition 2003, 42, 614-641. 251

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BIOGRAPHICAL SKETCH Xingguo Cheng was born on November 11, 1973, to Xiantao Zhu in a small village called “Tu Ku Wan” in Xishui county, Hubei Province, China. He is kin to brother Xinhua Cheng. He had a happy childhood, attending primary school and middle school close to his home. After high school he entered the University of Science and Technology of China (USTC), a well-known University in Anhui Province in central China, to study for a bachelor’s degree in the Department of Materials Science and Engineering. After graduation from USTC, he worked for 2 years as an assistant engineer in the Xi’an Modern Chemistry Research Institute, Xi’an, ShanXi Province, China. He took his postgraduate entrance exam on a snowy winter day in 1997 and entered graduate school at the University of Science and Technology of China, Beijing in 1998. From 1991 to 2001 he did research on preparing microcellular polymer blends using supercritical carbon dioxide, in the State Key Lab of Engineering Plastics, Institute of Chemistry, Chinese Academy of Science (ICCAS). His advisor was Jiasong He. He was awarded his master’s degree in chemistry in June 2001. In August 2001, he flew halfway around the world to pursue a PhD degree in the Department of Materials Science and Engineering at the University of Florida, where he loved gator football and saw the ocean and gulf for the first time. He worked hard, studying and doing research under the supervision of Dr. Laurie Gower. He explored the Polymer-Induced Liquid-Precursor (PILP) process and made CaCO3 and CaP structures 259

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260 in different conditions. He successfully molded the liquid-like CaCO3 precursor into a polymeric hydrogel to form complex morphology, developed a new method of hydroxyapatite precipitation at 37C and made a heavily mineralized bone-like material (Collagen-HA nanocomposites). He also learned soft lithography technique and micro-patterned nano HA on self-assembled-monolayers (SAMs). He was awarded the PhD degree in Spring 2005.