A New Paradigm for Biomineral Formation: Mineralization via an Amorphous Liquid-Phase Precursor Process

Material Information

A New Paradigm for Biomineral Formation: Mineralization via an Amorphous Liquid-Phase Precursor Process
OLSZTA, MATTHEW JOHN ( Author, Primary )
Copyright Date:


Subjects / Keywords:
Biomineralogy ( jstor )
Bones ( jstor )
Calcite ( jstor )
Calcium ( jstor )
Carbonates ( jstor )
Collagens ( jstor )
Crystal morphology ( jstor )
Crystals ( jstor )
Minerals ( jstor )
Solar fibrils ( jstor )

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Matthew John Olszta. Permission granted to University of Florida to digitize and display 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:
Resource Identifier:
55893190 ( OCLC )


This item has the following downloads:

Full Text




Copyright 2004 by Matthew John Olszta


I dedicate this dissertation to my family and friends, for without their strength and support, I would not be where I am today.


ACKNOWLEDGMENTS I would be remiss if I did not first thank my mother and father, Cora and John Olszta, for all they have done for me in my pursuit of my doctorate. Sometimes it is hard justifying why it took 9 years to finish college, but they always showed complete faith and understanding and I thank them 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 tenure as a graduate student. The two things that I truly appreciate are her guidance in writing scientific papers, and her unwavering belief in my research abilities. Although it seemed like an eternity going back and forth with revision after revision in writing journal articles, when it came time to write this dissertation, everything seemed to flow much easier because of it. Additionally, I appreciate her friendship through many of the tough times in graduate school, as well as starting weekend roller hockey games, which I had not played since I was in high school. I would also like to thank my supervisory committee (Dr. Anthony Brennan, Dr. Elliot Douglas, Dr. Jack Mecholsky Jr. and Dr. Andrew Rapoff) for their advice, time and understanding. While he did not serve as a formal committee member, Dr. Kaufman provided numerous insights into crystallography and electron microscopy, and for that I am grateful. There have been many people at UF that have influenced my personal life, and it is a shame that I have but one paragraph to thank them all. Jake Mauldin, the first person I really met in Gainesville, always provided a constant source of camaraderie and iv


laughter. Vishal Patel, a good friend and lab mate for my first two years here, was always there to listen and lend a helping hand. Dan Urbaniak and I hashed out what it really meant to be a graduate student in our time here, concluding that it was all about Doing the extra. Vasana Maneeratana, a great friend and fellow student teacher, helped me to understand the best way to write my dissertation defense such that anyone could understand it. Lastly, Kristin Morgan, who shared with me an equal passion for pool and Guinness, was always a constant source of poignant intellectual conversation and will be a longtime friend. Throughout my time here, I had the pleasure of working with many colorful people in the Gower lab, who always made research interesting. I thank Charley, Yi-yeoun, Fairland, Debra, Barry, Xingguo, Lijun, Munisamy and Gajjeraman for their patience, time and understanding. While not directly associated with the Gower group, I would also like to thank Dr. Elaine DiMasi, who is an amazing research scientist, and Kerry Siebein, a wonderful electron microscopist, for sharing their time and experience. Lastly, I need to thank my brothers and friends, who always treated me as though I never left Illinois. In no particular order, I give thanks to Mary, Mark, Jim, Matt, Aaron, Andrew, Michael, Abigail, Dave, JJ, Carrie, The Swede, Marco and Steve. I could write a chapter on how each of the aforementioned people affected my life in the past four years, and it pains me not to be able to write more about each of them. I thank them all with the greatest sincerity. v


TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES.........................................................................................................viii ABSTRACT.........................................................................................................................x CHAPTER 1 INTRODUCTION........................................................................................................1 2 BACKGROUND..........................................................................................................6 Biomineralization.........................................................................................................7 Biomineralization Mechanisms.............................................................................9 Insoluble organic substrate.............................................................................9 Soluble acidic macromolecules....................................................................11 Vesicular compartments and ion transport...................................................12 Sea Urchins..........................................................................................................14 Vertebrate Teeth..................................................................................................16 Bone.....................................................................................................................19 Formation.....................................................................................................21 Mineral phase...............................................................................................22 Organic matrix..............................................................................................23 Mineralization mechanism...........................................................................25 Mineral precursor phases.............................................................................26 Bone Graft Substitutes................................................................................................27 Metallic substitutes.......................................................................................30 Polymeric substitutes....................................................................................31 Ceramic substitutes......................................................................................32 Biomimetic Mineralization.........................................................................................33 CaCO 3 mineralization..................................................................................35 HAp mineralization......................................................................................37 3 SYNTHESIS OF FIBROUS MINERAL VIA A SOLUTOIN-PRECURSOR-SOLID (SPS) MECHANISM.......................................49 Introduction.................................................................................................................49 Materials and Methods...............................................................................................52 Synthesis..............................................................................................................52 vi


Crystal substrates for fiber growth...............................................................52 Fiber growth.................................................................................................53 Analysis...............................................................................................................54 Polarized optical light microscopy (POM)...................................................54 Scanning electron microscopy (SEM)..........................................................55 Transmission electron microscopy (TEM)...................................................55 Results and Discussion...............................................................................................55 Fiber growth........................................................................................................55 SPS mechanism...................................................................................................57 SPS in relation to fibrous biominerals.................................................................60 SPS in relation to geological CaCO 3 ...................................................................63 4 MINERALIZATION OF TYPE-I COLLAGEN VIA A CALCIUM CARBONATE PILP PHASE..............................................................72 Introduction.................................................................................................................72 Materials and Methods...............................................................................................75 Synthesis..............................................................................................................75 Analysis...............................................................................................................77 Acid etch and bleach treatment....................................................................77 Scanning electron microscopy (SEM)..........................................................77 X-ray diffraction (XRD)...............................................................................78 Transmission electron microscopy (TEM)...................................................78 Results and Discussion...............................................................................................78 5 INTRAFIBRILLAR MINERALIZATION OF TYPE-I COLLAGEN VIA A CALCIUM PHOSPHATE PILP PHASE.......................................................96 Introduction.................................................................................................................96 Methods and Materials...............................................................................................99 Synthesis..............................................................................................................99 Analysis.............................................................................................................100 Scanning electron microscopy (SEM)........................................................100 Transmission electron microscopy (TEM).................................................100 Results and Discussion.............................................................................................101 6 CONCLUSIONS......................................................................................................114 LIST OF REFERENCES.................................................................................................122 BIOGRAPHICAL SKETCH...........................................................................................145 vii


LIST OF FIGURES Figure page 2-1 Scanning electron micrographs (SEM) of various highly organized biominerals observed in nature, composed of a range of inorganic material, from calcium carbonate (CaCO 3 ), to calcium phosphate (CaP), to silica (SiO 2 )...........................40 2-2 Electron micrographs illustrating various morphologies of calcite observed within the structure of a sea urchin......................................................................................41 2-3 Tri-radiate growth of sea urchin spicules from a single calcite seed crystal.........42 2-4 Seven levels of hierarchical structure of mammalian bone........................................43 2-5 Figures copied from a paper by Weiner and Traub showing the deck-of-cards arrangement of crystallites within a collagen fibril..................................................44 2-6 Schematic illustrating the prevailing theory on intrafibrillar mineralization of type-I collagen in hard tissues............................................................................................45 2-7 Examples of bone-like apatite nucleated on collagen fibrils when collagen substrates are introduced into simulated body fluid (SBF).......................................................46 2-8 Electron micrographs of mineral deposited on functionalized polymer and collagen surfaces.....................................................................................................................47 2-9 Mineralization of HAp in the presence of fibrillogenesis of type-I collagen.............48 3-1 SEM micrographs depicting the differences in morphology of synthetically grown calcite (A and B) versus that of biologically formed calcite (C and D)...................66 3-2 SEM micrographs of calcium carbonate deposited onto rhombohedral substrate crystals in the presence and absence of micromolar amounts of acidic polymer.....67 3-3 SEM micrographs demonstrating mechanistic aspects of the SPS mechanism. .......68 3-4 Single crystalline analysis of calcite fibers.................................................................69 3-5 Electron microscope analysis of the fibers.................................................................70 3-6 Schematic depicting the proposed solution-precursor-solid (SPS) mechanism.........71 viii


4-1 SEM micrograph of native bone at the surface of a canine femur diaphysis............89 4-2 EM micrographs of Cellagen sponge samples.......................................................90 4-3 SEM micrographs representing the sequential PILP mineralization process of Cellagen sponges in the presence of polyacrylic acid...........................................91 4-4 SEM micrographs of mineralized samples subjected to etching, for determination of the extent of mineral infiltration...............................................................................92 4-5 SEM micrographs of mineralized collagen bundles that had been treated with a dilute bleach solution for 15 minutes.......................................................................93 4-6 XRD spectra of the different conditions of Cellagen mineralization.....................94 4-7 Transmission electron micrographs of mineralized Cellagen................................95 5-1 Schematic illustrating the mineralization of type I collagen via a polymer-induced liquid-precursor (PILP) process.............................................................................108 5-2 Electron micrographs illustrating various stages of mineralization using the PILP process....................................................................................................................109 5-3 SEM micrographs of mineralized samples subjected to etching, for determination of the extent of mineral infiltration.............................................................................111 5-4 Electron micrographs of mineralized collagen fibrils..............................................112 ix


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 A NEW PARADIGM FOR BIOMINERAL FORMATION: MINERALIZATION VIA AN AMORPHOUS LIQUID-PHASE PRECURSOR PROCESS By Matthew John Olszta May 2004 Chair: Laurie Gower Cochair: Elliot Douglas Major Department: Materials Science and Engineering Biominerals, in both invertebrate and vertebrate systems, are well recognized for their unusual crystal morphologies and hierarchically organized composite structures. Soluble acidic macromolecules associated with biominerals are thought to play an important role in modulating the mineral morphology. Using acidic polypeptides to modify classical crystallization of synthetic, calcium-based minerals, it has been demonstrated that crystallization that proceeds via a liquid-phase mineral precursor, denoted as a polymer-induced liquid-precursor (PILP) process, leads to non-equilibrium morphologies. The goal of this dissertation is to demonstrate that the PILP phase can be used to recreate specific inorganic/organic biocomposite structures observed in Nature, thereby supporting the hypothesis that an amorphous liquid-phase mineral precursor plays a fundamental role in biomineralization. By depositing a calcium carbonate PILP phase on calcite rhombohedral seed crystals, calcite fibers with diameters ranging from 100-800 nm have been produced. x


These fibers closely resemble structures observed in sea urchins, including regions of the teeth and during the early stages of spiculogenesis. A solution-precursor-solid mechanism, which has features similar to both the vapor-liquid-solid and solution-liquid-solid mechanisms that are used to produce fibrous semiconductor materials, is proposed as the growth mechanism. This discovery suggests an interesting link between two seemingly unrelated processes, high-temperature semiconductor fiber formation and biological mineralization. With respect to vertebrate biominerals, I have shown that using the PILP process to generate liquid-phase mineral precursors to calcium carbonate and phosphate, intrafibrillar mineralization of re-constituted type-I collagen can be achieved. This has never been demonstrated prior, and serves as a proof of concept that an amorphous liquid-phase precursor can be used to form a fully dense bioceramic/organic composite resembling bone. Using a calcium phosphate PILP phase, it was observed that this precursor process can be used to recreate the nanostructured architecture of bone, in which each individual collagen fibril was intrafibrillarly mineralized with platy hydroxyapatite crystals oriented in the [001] direction along the long axis of the fibril. To my knowledge, this is the first time such bioceramic/organic composites, which closely mimic the biomineral morphologies observed in Nature, have been synthetically prepared, and this data suggests that biomineralization may occur via a PILP mineralization process. xi


CHAPTER 1 INTRODUCTION The importance of materials science engineering throughout history is readily apparent simply by observing the fact that historians have used the state of materials technology as nomenclature for various periods in mankinds history (e.g., stone, bronze and iron ages). Similarly, the advancement in human history can be traced through the advancement in materials processing techniques, from the development of the Damascus steel used to produce unrivaled weaponry, to the refinement of silicon for computer chips in modern computer systems (1). Yet, towards the end of the 20 th century, it was discovered that while mankind had indeed advanced exponentially in the development of technology, there were still lessons that could be learned from Nature. Through the advent of electron microscopy (EM), mankind realized that Nature synthesized hierarchical, self-assembled, organic/biomineral composites, with improved mechanical properties, at ambient conditions (e.g., brick and mortar structure of nacre, intrafibrillar hydroxyapatite mineralization of bone). With this new discovery, a new branch of materials science was developed in order to study the mechanisms of biomineral formation. Through the study of biomineralization, materials scientists can decipher how Nature creates such complex nanoscale structures, and in turn, recreate them for use in the biomedical applications. The role of the organic phase in the morphological control exerted in biomineralization may be separated into a three-component system (2-4) : i) an insoluble organic matrix, which can play a role in compartmentalization of the growing mineral, 1


2 and/or templating the nucleation for controlled crystallographic orientation and/or phase; ii) soluble acidic macromolecules (e.g., sulfated and/or phosphorylated glycoproteins containing large amounts of glutamic and aspartic acid residues), which are frequently occluded within the crystals (4) and are thought to play a role in the control of crystal shape; and iii) vesicular compartments, which provide spatial and temporal control of ion and additive transport to the mineralization front. These rules were determined empirically through decades of biomineralization research, and were subsequently corroborated through synthetic reproduction in order to show possible mechanisms and feasibility. Recently, research in the biomineralization field has uncovered certain biomineral structures, observed in both calcium carbonate (CaCO 3 ) (5) and calcium phosphate (CaP) (6) biomineral systems, which display transient amorphous mineral phases The exact role of these transient phases is not yet understood, but it is believed that they are precursors to biomineral formation. The research contained within this dissertation suggests that this transient phase possesses a fluidic nature, and can be shaped into a variety of sizes and dimensions before crystallizing, thereby rendering the many molded morphologies observed in biominerals. Using a novel biomimetic synthesis called the polymer-induced liquid-precursor (PILP) process (7), we have been able to synthetically produce such transient amorphous phases which can be used to recreate structures with morphological similarities to various biominerals (8). More specifically, we have demonstrated that through the PILP process, we can produce a fluidic amorphous mineral precursor which can be shaped or molded to conform to various substrates. The importance of the PILP process in relation to biomineralization is that by recreating mineral/organic composites that have


3 morphological similarities to biominerals, specifically bones and teeth, through biomimetic synthesis at ambient conditions, we can offer a new outlook on a viable precursor mechanism in biomineral formation. This dissertation is divided into five chapters, with the first being comprised of the introduction to biomineralization and the relevance of our research to recreating biomineral composites using a novel synthetic approach. Chapter 2 contains a thorough background on the field of biomineralization, specifically in regards to invertebrate and vertebrate systems. The various lessons introduced in Chapter 1 are expanded in Chapter 2 in order to provide a better understanding of the importance of biomineralization. Additionally, the study of biomimetics is discussed in Chapter 2 in order to demonstrate how the lessons learned from biomineralization can be applied in the field of materials engineering. Chapter 3 introduces the deposition of the CaCO 3 PILP phase upon existing calcite seed crystals to produce nano-fibrous calcite crystals through a solution-precursor-solid (SPS) process. This SPS process leads to the formation of calcite fibers similar to fibrous biominerals (such as sea urchin teeth), yet appears to be a room temperature analogue to the vapor-liquid-solid (VLS)/ solution-liquid-solid (SLS) semiconductor processing mechanisms. Because this aqueous-based SPS mechanism occurs at physiological conditions (and down to temperatures as low as 4C), it is feasible that it may be used by organisms to form their fibrous biomineral structures. This discovery suggests an interesting link between two seemingly unrelated processes, high-temperature semiconductor fiber formation and biological mineralization.


4 Chapter 4 and 5 demonstrate how the PILP phase can be used to achieve intrafibrillar mineralization of type-I collagen substrates with CaCO 3 and CaP, respectively. The prevailing theory of intrafibrillar mineralization is based upon nucleation of oriented, platy, hydroxyapatite crystals within the 40 nm hole zone created by the self-assembly of type-I collagen, followed by subsequent growth of the crystals until they fuse with other deposits along the collagen (9-11). As previously mentioned, there are also reports of amorphous CaP deposits observed within these hole zones in early mineralizing bone, suggesting a precursor mechanism might be occurring. Although nucleation and growth has been suggested as the mineralization mechanism in vivo, attempts at recreating this unique morphology in vitro has not been promising, as researchers have shown that type-I collagen serves as a substrate for nucleation of large, micron-scale hydroxyapatite (6, 12). Chapter 4 details the early works on intrafibrillar mineralization using the CaCO 3 PILP phase. Although naturally mineralizing collagen does not incorporate CaCO 3 in our preliminary work, we had not yet devised a means by which to create a CaP PILP phase, and thus we used the CaCO 3 PILP phase as a proof of concept to show intrafibrillar mineralization could occur with any Ca based mineral (8, 13). Instead of observing micron-sized crystals, as in our control, we discovered that each individual collagen fibril was infiltrated with calcite of nondescript morphology when the PILP process was applied. A topological comparison to natural bone shows that both our composite and naturally mineralized collagen has a rough, nondescript mineral coating of single fibrils. After deproteinating the composite, the calcite remained, while any unprotected collagen was removed. As expected, the centers of the mineralized fibers


5 were solid, demonstrating that the liquid-precursor had penetrated throughout the entire collagen fibril, and upon subsequent crystallization, protected the organic phase from the etchant. Using transmission electron microscopy (TEM), we observed mineralized fibrils that displayed the native 64 nm banding pattern of collagen, suggesting that the fibrils were intrafibrillarly mineralized with a single crystalline CaCO 3 mineral phase. While this data did indeed provide a proof of concept, it was still not truly bone-like due to the type of mineral present. Chapter 5 centers on the use of a CaP PILP phase in creating a composite that possesses a nanostructured architecture identical to that of natural bone. Synthetic CaP has a large number of different polymorphs, including hydroxyapatite (HAp), which is energetically favorable at higher temperatures (80C). Using the PILP process, we have been able to form HAp at physiological temperature (37C) using phosphorylated polymers in addition to the negatively charged PolyAsp. When a type-I collagen substrate is introduced to the mineralizing solution, we observed similar morphological results to the CaCO 3 mineralized substrates. Each individual collagen fibril appeared to have a rough exterior with dimensions approximately the dimensions of uncoated swollen collagen fibrils. When the sample was examined under TEM, the collagen fibrils appeared rough and platy. Selected area diffraction (SAD) revealed that the collagen fibrils were infused with HAp crystals oriented in the [001] direction along the c-axis of the collagen, yielding a diffraction pattern identical to natural bone (14). Selected area dark field (SADF) of the (002) plane revealed 25-100 nm long platy crystals along the long axis of the collagen fibril, proving that we had achieved intrafibrillar mineralization of type-I collagen with hydroxyapatite.


CHAPTER 2 BACKGROUND Biomineralization is the process by which Nature creates complex, hierarchical inorganic composites in ambient conditions while expending minimal biological activity. While most take the wonders of biomineralization for granted, there are a variety of lessons that can be learned from the exquisite composites created by Nature. In respect to the materials community, the study of biomineralization has always been present, but it has been spread amongst an assortment of scientific disciplines under the approach of studying the biological activity and mechanisms used to create these biological composite structures. Often, the resulting structure, morphology and mechanical properties were overlooked at the expense of studying the biological pathways and synthesis of the organism. From a materials standpoint, biominerals are natural composites of inorganic mineral and organic polymers, synthesized in ambient conditions. The final products most often have nanoscale morphologies and properties that far surpass that of their separate constituents (e.g., nacre of abalone shell is twice as hard and 3000 times as tough as its constituent phase, aragonite)(15). For these reasons, within the past 20 years, the materials science community has engaged in the study of biomineralization in order to learn the latent secrets locked within the seemingly quiescent biomineral structures such as seashells, diatoms, bones and teeth. The study of biomineralization can be approached from a multitude of angles, and a complete study that encompasses every aspect of biomineralization is far beyond the scope of this dissertation. As material scientists are stepping into the biomineralization 6


7 arena much later than the remainder of the scientific community, there is an abundance of information already discerned that can be utilized regarding the biological and compositional fundamentals of biominerals. The goal of the materials scientist is to apply the fundamentals of materials knowledge to the products of biomineralization while factoring in the rules learned from the preceding biological studies in order to gain a better understanding of how and why these unique structures are formed. Understanding the mechanisms by which organisms produce biominerals (e.g., ion transport, nucleation, epitaxy, etc.) provides clues as to how they can achieve the superior properties displayed. Taking lessons from Nature and applying them to synthetic materials is called biomimetics. The following background specifically details the understanding of biomineralization and a specific form of biomimetics, biomimetic mineralization. Biomineralization Biomineralization is observed throughout all five kingdoms, yet not all organisms produce biominerals. There are over 60 types of biominerals (3), ranging from the widely observed calcium carbonate (CaCO 3 ), which is found mainly in invertebrate structural components, to magnetite (Fe 2+ Fe 2 3+ O 4 ), employed by bacteria as a bio-compass. Most of the bioceramics formed have a molded or processed appearance, displaying morphologies that are far breaking from the symmetry of their synthetic counterparts (Figure 2-1). Calcium based biominerals, while observed in less than half of the species that produce biominerals, make up the majority of biominerals observed on the planet (3). For this reason, much of this article will focus mainly on calcium based biomineralization, citing two specific instances, CaCO 3 observed in sea urchin teeth and spicules, and calcium phosphate (CaP) observed in human dentin, enamel and bones.


8 As previously mentioned, biomineralization can be approached from a wide variety of paths. As material scientists, we tend to ignore the biological aspect of biomineral formation, and concentrate on the mechanisms by which the biomineral is formed, such as how the ionic components are transported to the mineralization front, the composition of the organic macromolecules associated with the biomineral, and the morphology and orientation of the final crystalline product. From a materials processing standpoint, organisms have ingeniously derived a number of mechanisms to take advantage of their surroundings to develop biominerals for structural applications and defense. Seventy years of biomineralization studies have yielded three basic observations of which organisms employ in the regulation and formation of biominerals. The morphological control exerted in biomineralization may be separated into a three-component system (2-4): i) an insoluble organic matrix, which can play a role in compartmentalization of the growing mineral, and/or templating the nucleation for controlled crystallographic orientation and/or phase; ii) soluble acidic macromolecules (e.g., sulfated and/or phosphorylated glycoproteins containing large amounts of glutamic and aspartic acid residues), which are frequently occluded within the crystals (4) and are thought to play a role in the control of crystal shape; and iii) vesicular compartments, which provide spatial and temporal control of ion and additive transport to the mineralization front. A general overview of biomineralization mechanisms will be provided, as well as a general explanation as to how researchers have studied these mechanisms in vitro. Lastly, a few specific biomineral examples, pertinent to this dissertation, will be discussed in detail.


9 Biomineralization Mechanisms Insoluble organic substrate Biomineralization involves the spatial regulation of nucleation and growth of developing mineral phases, as well as arranging nanometer to micrometer units into hierarchical structures. The use of an insoluble organic matrix, which delineates space or acts as a substrate, is usually the first component secreted by the organism. The spatial orientation and type of charged groups on the organic surface are believed to direct crystal growth. In the formation of nanoscopic iron oxide by magnetotactic bacteria, spherical vesicles comprised of a phospholipid membrane are first organized before subsequent mineralization occurs (16). The oxide crystals then deposit close to the surfaces of the charged phospholipids, growing within the contained vesicle, thereby constraining the final shape of the mineral. In vertebrates, an ordered matrix of type-I collagen is secreted by osteoblasts in bone or odontoblast in dentin, which serves as substrates for nanoscopic hydroxyapatite (HAp) crystals. The tight control of nanometer spacing serves to orient the mineral phase to create an intimately associated composite structure. Yet, the insoluble matrix is not solely responsible for the transformation of inorganic material into biominerals. This is best evidenced by the fact that type-I collagen is also observed as a major component of soft connective tissues in vertebrates, and therefore, other factors are believed to be involved in the mineralization process. In order to study the effect of template directed nucleation, there have been various approaches at mimicking the insoluble organic substrate. The first involves studying the effects of surface charge, spacing and packing at organic interfaces on Langmuir monolayers at the air-water interface (17-31). Using a Langmuir trough, a surfactant monolayer can be tailored by compressing the layer to various condensed


10 states. When the charged groups are brought closer together, it affects the spatial location of Ca/ion binding groups, thereby modifying subsequent crystal nucleation. Bruijnsters et al. (18) demonstrated that an amide-containing phospholipid serves as a template to promote the growth of [10.0] oriented calcite, regardless of phospholipid concentration, citing the bidentate orientation of the phosphate group responsible for the specific orientation of the calcite. Yet, while employing the same crystallization experiment with the addition of acidic short chained polymers to mimic biomineralization, DiMasi et al. (29) showed there was no preferential orientation of CaCO 3 crystals against a fatty-acid monolayer, and suggested that formation of crystal polymorph was kinetically driven. There has also been considerable research into using Langmuir-Blodgett (LB) or self-assembled monolayers (SAMs) to study the functionality of specific surfaces or specific terminal groups on crystal nucleation and orientation (26, 31, 32). Aizenberg et al. (33) clearly document how various functionalized SAMs can affect orientation of calcite crystals. They obtained 97-100% uniformity of crystal orientation over their films, and only noted deviations when there was an increase in defect densities of the SAMs. Additionally, SAMs have been used to demonstrate that thin single-crystalline, inorganic mineral films, such as those observed in nacre, can be spatially directed by first depositing an amorphous precursor (34-36). Another manner of studying template-mediated growth, which serves to mimic the delineation and constraint of the mineral crystal, is by using a three-dimensional organic substrate (37-45). Kanakis and Dalas determined that fibrin, a bi-product of blood coagulation, stabilizes vaterite, a highly metastable polymorph of CaCO 3 suggesting that the substrate plays a vital role in selecting the crystalline polytype (41).


11 The most biologically active biopolymer, collagen, has by far been the most studied three-dimensional substrate for mineralization. Its unique three-dimensional self-assembled structure provides a unique substrate for nucleation of HAp mineral in the formation of bone and dentin. This structure will be discussed in further detail later on in the discussion of bone and biomimetic mineralization. Soluble acidic macromolecules It is widely understood within the biomineralization community that acidic proteins can act as inhibitors to crystal nucleation or growth (2, 4, 46-48). While macromolecules are probably not involved in all forms of biomineralization, the organic extracts of many biominerals yields a micromolar amount of acidic macromolecule, usually rich in aspartic and/or glutamic acid residues (3). The high charge density of these macromolecules suggests that they are actively involved in crystal formation because they are thought to bind free ions or serve as templates for epitaxial growth. In bone, they are referred to as non-collagenous proteins (NCPs) to differentiate them from the collagen substrate (49). Since the majority of type-I collagen observed within the body does not mineralize, it is assumed that the NCPs, especially bone sialoprotein (50), play an important role in intrafibrillar mineralization (6). Similarly, phosphophoryn is thought to play a similar role in dentin, as it has a high binding capacity for calcium ions as well as a strong affinity for collagen (51). In oysters, such as the Japanese pearl oyster, a highly polyanionic macromolecule, named nacrein, has been extracted and found to have high occurrences of three different repeat sequences, [Gly-Asp-Asn], [Gly-Glu-Asn] and [Gly-Asn-Asn] (16). The high occurrence of acidic residue rich proteins, which have high affinities for binding cations such as calcium, has led researchers to


12 experiment on the effect of highly acidic polymers and proteins on synthetic crystal growth. There has been a considerable amount of research done on the in vitro analysis of polymeric additives on mineralization (7, 37, 52-60). In the case of crystal growth, it has been shown that selective inhibition of growth along stereospecific crystallographic planes can lead to a change in crystal habit (61). Teng et al. (60) used aspartic acid to demonstrate how the charged polymer absorbs to growing step edges of calcite, showing that it affects the magnitude of the surface energy, thereby altering crystal growth. In a few cases, acidic proteins have been shown to promote crystal nucleation, and particularly if the macromolecule is immobilized onto a substrate (48, 62). An alternative view regarding the role of acidic proteins has been suggested by Gower and Odum (7) and Gower and Tirrell (37). They suggested that acidic polypeptides, such as aspartic acid, could inhibit classical nucleation of calcite while inducing an amorphous liquid-phase mineral-precursor that would conform to the shape of its container, transform into a solid and then subsequently crystallize into calcite. Vesicular compartments and ion transport As previously stated, the study of biomineralization concentrates more on the final product (e.g., mineral, occluded proteins and organic matrix) than the manner in which the cells organize and produce the primary constituents. The reason is, that in many biomineral systems, it is difficult to observe mineralization in vivo, and therefore the delivery of ions is usually not readily examined. It is assumed that there is a local supersaturation at the mineralization front, but there is little known about the mechanisms of ion transport in vivo. There are three main mechanisms by which organisms raise the supersaturation; ion pumping, ion complexation and enzymatic regulation (16). Cells


13 pump ions through a metabolic process involving the hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). Through hydrolysis, H + and Ca 2+ ions are pumped out of the cell and into intracellular vesicles (16). A second manner of ion pumping is through electrochemical gradients. Cations can also be delivered to the mineralization front through complexation with ligands such as citrate or pyrophosphate (3). Once the complex reaches the target area, it is decomplexed and the cation is released. Similarly, the use of alkaline phosphatase can be used to deliver HPO 4 2to the calcification front through enzymatic regulation in matrix vesicles (16). The amount of research into mimicking these systems is scarce because scientists are more concerned with the mechanisms of crystal growth modification via organic modifiers. The main attempt at mimicking ion transport is through lipid membranes as vesicular compartments (39, 63). Murphy and Messersmith (39) demonstrated that they could separately load calcium and phosphate ions into liposomes composed of 90% 1,2-bis(palmitoyl)-sn-glycero-3-phosphocholine and 10% 1,2-bis(myristoyl)-sn-glycero-3-phosphocholine, which were stable at room temperature for weeks. When the loaded liposomes were heated to 33-37C, they released the trapped ions into solution, facilitating nucleation of CaP. Two main methods have been widely utilized in model studies of biomineralization to induce crystal growth in CaCO 3 systems. Kitano (64) demonstrated that by dissolving CaCO 3 in a solution supersaturated with CO 2 he could observe how organic additives affected recrystallization of CaCO 3 upon escape of the CO 2 gas. The second method, developed by Addadi et al. (48), utilizes the slow decomposition of ammonium carbonate into ammonia and CO 2 (g) within a closed crystallization vessel. The CO 2 (g) diffuses into solutions containing Ca 2+ and is


14 converted into CO 3 2to supersaturate the solution with respect to CaCO 3 thereby prompting nucleation and growth. While these two routes do not fully represent a biological pathway, they adequately raise supersaturation of CaCO 3 within solution at a rate that mimics organic systems, and therefore are widely used in biomimetic mineralization studies. Sea Urchins In biominerals that are formed within vesicular compartments, the final crystalline product typically adopts the shape of the compartment, and in essence is molded into the designated species-specific morphology. This is seen in organisms ranging from the intricate shells of simple unicellular protists to the macroscopic spines of the sea urchin (3, 65). In the latter case, the spines and tests of the sea urchin, which are composed of magnesium-bearing calcite, have a porous and highly convoluted shape, yet reportedly are single-crystalline calcite (Figure 2-2A and B), as observed through X-ray diffraction (46, 66). On the other hand, there have been in-depth studies of the early stages of echinoderm spiculogenesis because the mineral formation can be more readily examined in the embryonic stage (5, 67-69). Wilt (67) has shown that during the first stages of endoskeletal spiculogenesis, considerable amounts of amorphous calcium carbonate are present in the spicules, which are formed and shaped within an extracellular compartment delineated by a row of primary mesenchyme cells (PMCs). This calcium-rich amorphous phase is observed (by TEM) as granules in spicule forming cells, which are then transported to the tips of the growing spicules. It should be pointed out that analysis by TEM will dehydrate the sample and show amorphous granules. Being a highly metastable phase, the amorphous CaCO 3 then rapidly crystallizes (68). Notably, the spicules become tri-radiate as they emanate from the a-crystallographic planes of a


15 rhombohedral calcite crystal which is deposited within the PMC during gastrulation (Figure 2-3) (67, 68). It is actually quite rare to see calcite crystals of rhombohedral shape in biominerals, but this shape does not last long as the spicules form into elongated curving rod-like structures. The seed crystal apparently provides control over the crystallographic orientation, which in turn contributes to the directionality of the growing spicule. Interestingly, the authors note that the plasmalemma is tightly applied to the developing spicules, except perhaps at the elongating tips (67). This seems to indicate that there is very little solution space around the forming spicule, much less than would be expected if the crystals were grown by the traditional solution crystallization process (in which a limited concentration of ions would be present in the supersaturated solution). Another example from the sea urchin is its teeth, which are comprised of CaCO 3 mineral in both the crystalline and amorphous states (5, 70). The outer edges of the teeth are covered with platy CaCO 3 whereas the inner structure is comprised of long 5 7 m diameter crystalline rods of calcite (Figure 2-2C). At the junction of the rods and the platy outer edge, the rods are found to grow between the plates and are surrounded by amorphous calcium carbonate matrix (Figure 2-2D). Both the molded morphologies of the biominerals formed within compartments, and the fibrous structure seen in the teeth are central to the hypothesis of this dissertation. Oddly enough, the literature does not refer to these highly elongated minerals as fibers, but rather they are called rods or prisms. Presumably, this is because the word fiber implies some sort of extrusion process, and obviously, minerals cannot be melted and extruded under physiological conditions. Recent observations of amorphous phases co-existing with crystalline CaCO 3


16 in the hard tissues of sea urchins suggest that the amorphous phase appears first, followed by subsequent crystallization. Amorphous calcium carbonate (ACC) is a highly unstable phase, yet it has been observed in biominerals in a variety of different invertebrate organisms (e.g., calcareous spherules in crustacea carapace (71), cystoliths of plants (72), and the antler-shaped spicules of ascidians (73)). In at least two cases, it has been shown that both amorphous and crystalline phases can coexist within the same tissue (e.g., the teeth of sea urchins (5, 70), and the dogbone-shaped ascidian spicules (73)). Aizenberg et al. (71) have shown that macromolecules extracted from the separated phases of the ascidian spicules will produce the corresponding phase when calcium carbonate is precipitated in vitro in their presence. This group has also shown that ACC serves as a transient precursor to crystalline calcium carbonate in sea-urchin larval spicules (68). They have suggested that amorphous precursors may be more prevalent than has previously been realized because of the difficulty in detecting amorphous phases by traditional techniques (e.g., TEM, XRD), and especially in the presence of crystalline material, which masks the weaker signal emanating from the amorphous fraction. Vertebrate Teeth Vertebrate teeth, like those in sea urchins, are comprised of two different morphologies of the same mineral, hydroxyapatite. Yet, unlike the teeth of the sea urchin, which continuously grows in response to the growing edge being worn away during mastication, vertebrate teeth do not remineralize. As previously demonstrated, sea urchin teeth are composed of relatively soft CaCO 3 whereas vertebrate teeth are composed of much harder HAp. The structure of sea urchin teeth is also much more fragile than human teeth, as the scraping plates and fibrous calcite bundles are not as


17 complex as vertebrate teeth, which have much more compact triple-ply structures. Vertebrate teeth all have a similar architecture, comprised of three distinguishable levels. At the core there is unmineralized tissue called pulp which is surrounded by a mineralized type-I collagen layer called dentin (sometimes spelled dentine), and finally the crown of the tooth, called enamel, is composed of ribbon-like or fibrous hydroxyapatite (74). While this general organization is similar, the distinct differences in the details of each level between vertebrates can be as great as the difference between the structure of vertebrate and invertebrate teeth. One classic example is the enamel of rats (Figure 2-1B), which is comprised of a three-ply network of HAp rods or fibers, as compared to the two ply rods of human teeth. Rodent enamel is among the most complex enamel of all vertebrates (75). This three dimensional architecture of interwoven HAp is harder than iron, platinum and copper, which explains how rats gnaw through electrical wires (76, 77), and why it is the hardest animal tissue (78). As there are many differences between tooth morphologies of various vertebrates, and the formation of each level can be quite complex, only a general overview of teeth will be provided. Similarly, as dentin is composed of mineralized type-I collagen, as in bone, its formation and mineralization will not be covered here. The structure of enamel on the other hand, is unique from bone, and will be covered in some detail. Dentin and enamel share a unique starting point, the dentinoenamel junction (DEJ) (79), sometimes referred to as the enamel-dentine junction (EDJ) (75). From this junction, enamel crystals grow towards the chewing surface, while the dentin mineralizes inwards towards the pulp. In enamel, HAp rods are secreted from the Tomes process


18 of ameloblast cells, which are in direct apposition to the forming mineral phase (Figure 2-1B) (6, 80, 81). It is accepted that each rod is connected to two neighboring ameloblasts (82), and that the orientation of the HAp crystals is due to the movement of the amelobast during mineralization (70). During enamel formation, the cells tend to recede but stay in close contact with the mineralization front, acting almost as a biomineralization reactor (3). The ameloblasts secrete two highly acidic proteins, amelogenin and enamelin, which serve to organically mediate the mineralization process (83). Amelogenins, rich in proline, glutamine, leucine, and histidine, are observed as 20-nm nanospheres in early mineralization, but are later degraded and removed as HAp crystals mature (16). It is believed that they direct growth by binding to certain crystallographic planes, allowing growth along the c-axis of the crystal. There are also highly acidic proteins observed in enamel formation called enamelins, rich in both glutamate and aspartate (78). It is thought that their function is to form a sheath around the developing HAp crystallites (16). While the chemical nature of the organic and inorganic phases in enamel has been determined, there is a still debate as to the initial alignment of early forming enamel (79, 83-90). It has long been understood that dentine and enamel share a common starting point at the DEJ, with dentine observed mineralizing first, followed by subsequent appearance of enamel crystals. Due to the mineral nature of both phases, as well as the preferred orientation of the mineral in enamel, it has been proposed that enamel crystals are epitaxially nucleated off the ends of HAp crystals in the frayed ends of dentin fibrils at the DEJ (86, 91). In contrast, other research on the DEJ shows that enamel forms at a distance from dentin (87). Takano et al. (88) showed that enamel crystals formed at a


19 distance from the dentin, subsequently coming in contact with it after growth. Using high-resolution electron-microscopy (HREM), Bodier-Houll et al. (79) illustrated that there is no general epitaxial orientation of enamel crystals on dentin, suggesting there is a well-defined border between dentine and enamel that appears poorly-crystalline or amorphous. Bone The other major mineralized tissue in vertebrates is bone, and therefore, a longer discussion of bone will be presented. Bone is a classic example of the hierarchical levels of structure found in many biological tissues (92). A recent review article by Weiner et al. (93) breaks down the structure of bone into seven levels of hierarchy (Figure 2-4), starting with nanoscopic platelets of HAp that are oriented and aligned with the self-assembled collagen fibrils (Level 2); the collagen fibrils are layered in parallel arrangement within lamellae (Level 3); the lamellae are arranged concentrically around blood vessels, forming an osteon (Level 4); and finally the osteonal bone is either packed densely into compact bone, or is composed of a trabecular network of porous bone, referred to as spongy or cancellous bone (Level 5). On the macroscopic level, bone is a smart material that organizes itself in response to external forces, compensates organizationally for holes and empty columns (areas of classical stress concentration in solid structures) and heals itself by remodeling when damaged (94). Nanoscopically, bone is even more complex, as Nature has determined a manner in which to impregnate nanoscopic HAp crystals into a highly organized type-I collagen matrix. Many researchers have contributed to the characterization of this complex tissue (4, 6, 95-100), for which there has been consider-able controversy over its structure throughout the years. Studies on the mineralization of


20 turkey tendon have provided important information at the primary level regarding the lo-cation of HAp crystallites in the collagen fibrils of bone (noteturkey tendon naturally mineralizes, and has been utilized as a model of intrafibrillar bone structure) (98, 101). Figure 2-5 shows illustrations taken from the work of Weiner and Traub of a mineralized collagen fibril from turkey tendon and a schematic of the classic deck-of-cards arrangement of the nanocrystals within the fibril (101). The crystals are both physically aligned with the long axis of the collagen fibril, and crystallographically oriented with their c-axes (the [001] direction) parallel to the fiber axis. This most fundamental level of bone, its nanostructured architecture, is a result of intrafibrillar mineralization, where the nanocrystals of hydroxyapatite are formed within the interstices of the collagen fibrils, as well as between fibrils (i.e., interfibrillar). The term intrafibrillar is used here to specify the location of the crystallites, which Weiner and Traub (101) and Weiner, Traub and Arad (102, 103) describe as a deck-of-cards arrangement of iso-oriented nanocrystals that are embedded within the gaps and grooves of the assembled collagen fibrils (Figure 2-5). These gaps are created by the staggered arrangement of tropocollagen molecules (triple helical rods), which leads to periodicity of the hole and overlap zones. An associated periodic contrast pattern is commonly observed by transmission electron microscopy (TEM) of collagen fibers (70, 99, 104, 105) after the collagen has been stained with a organometallic, such as phosphotungstic acid. Landis et al. (10, 98) have shown evidence, from tomographic imaging of naturally mineralizing turkey tendon, that the hydroxyapatite crystals first appear within the hole zones of collagen, and then spread throughout the fibrils, leading to the array of iso-oriented nanocrystals of HAp embedded within the organic matrix


21 (Figure 2-6). From a materials engineering perspective, the nanostructure of bone is intriguing and even quite difficult to define. For example, is bone a polymer-fiber-reinforced ceramic-matrix composite; or is it a ceramic-nanoparticle-reinforced polymer-matrix composite? In other words, the two phases are so intimately linked that the mechanical properties are distinctly different from ceramics or polymers, and therefore are difficult to reproduce. In particular, the nanostructured architecture is likely to play an important role in the toughness of bone. To date, scientists do not have an understanding of how bone is formed, even at this most basic level of structure. Obviously, cellular control is important in biomineralization, and in the case of bone, helps to build its hierarchical structure (i.e., lamellae and osteons), but even the physicochemical mechanism for generating this nano-architecture has not been elucidated. Because intrafibrillar mineralization does not occur simply by trying to crystallize collagen in vitro using supersaturated solutions of HAp (crystals only nucleate heterogeneously on the surface of the collagen fibers), it is generally assumed that nucleating proteins or collagenous domains must be located at the gap regions of the collagen fibrils (although they apparently are not there during in vitro mineralization studies). Formation The mechanism of bone formation differs substantially between primary and secondary osteogenesis. Epiphysial cartilage, which serves as the basis for primary bone formation, is a combination of ground substance and very loose, small (10-20 nm in diameter) fibril bundles of type-I collagen (~17 % in rat cartilage) (106). There is a high occurrence of matrix vesicles, which are believed to deliver either a high concentration of ions to the mineralization front (107), or crystals which serve as the nidus for further


22 crystallization. The earliest mineral forms in the ground substance as clusters of HAp crystals, sometimes referred to as calcospherites or calcification nodules (107). As the metaphysis is approached, the clusters grow larger until they have formed a calcified cylinder. The mineralization is relatively rapid and unorganized, forming a woven bone microstructure. Although collagen is present in this ground substance, it is not organized into lamellae, as occurs when osteoblasts secrete collagen during secondary bone formation. Cameron suggested that the collagen fibrils found in cartilage are too narrow for the mineral to deposit within them, thereby resulting in the observed interfibrillar mineralization (106). In this instance, the collagen does not appear to play an appreciable role in directing the mineralization process, and therefore this type of bone formation is not the focus of this dissertation. On the other hand, in secondary bone formation, in which the primary bone is remodeled into a more optimal structure, mineralization is intimately associated with the collagen, and the organization of the crystals is directed by the collagen scaffold upon which it forms (6). The collagen fibrils, which assemble after secretion by the osteoblasts, are larger (20-80 nm in diameter) than those in primary bone, and are somehow assembled into a close-packed lamellar structure (how this self-organization occurs is apparently unknown). Close to the mineralization front, there are also highly charged NCPs, which are thought to play an important role in the mineralization process. Therefore, many studies have focused on determining the nucleating or inhibitory activity of the NCPs on HAp (6, 108). Mineral phase The composition of the mineral substance in bone is described as consisting of non-stoichiometric hydroxyapatite (calcium deficient), although it usually contains


23 varying degrees of carbonate, magnesium, and other substitutions. Even the degree of hydroxylation in the hydroxyapatite has been difficult to determine. Early on, there was much debate as to the morphology of the HAp crystals within bone, whether it was comprised of platelet shaped, or needle-, filament-, or ribbon-like crystals (105, 109-113). It has become generally accepted that the needle-like appearance was due to the edge-on view of very thin platelets, but the actual location of the crystals remained uncertain. Using TEM stereomicrographs, Weiner et al. (95) demonstrated that the plate shaped crystals were preferentially located at the gap zones of the collagen, and were stacked much like a deck of cards. The issue of interversus intrafibrillar location was further verified by Landis et al. (10, 114) using three-dimensional high voltage tomographic reconstructions of naturally mineralizing turkey tendon. Their three-dimensional renderings nicely demonstrated that the HAp was indeed platelet shaped crystals which first nucleated within the gap zones of the collagen fibrils. The size of bone crystals reported in the literature varies, with values ranging from length: 30-50 nm; width: 15-30 nm; and thickness: 2-10 nm (105, 112, 113). This is in part due to sample preparation, but more importantly, the type of mineralized tissue (e.g., animal species, maturation, and location of the tissue examined). Such extremely small nanocrystals, of only a few unit cells in thickness, would not normally be thermodynamically stable if it were not for being embedded within the tissue. In the case of mineralizing turkey tendon, Landis et al. (10) found that crystal widths varied from 30-45 nm, but thickness was uniform at ~ 4-6 nm. Organic matrix In order to fully understand the role of the collagen matrix in human bone, it is necessary to examine the structure of the fully mineralized composite to gain an


24 appreciation for the extent of mineral loading and organization of the mineral phase. As is true of many biominerals, the organic matrix not only influences the mechanical properties, generating bioceramic composites with both unique structures and properties, but it also plays an important role in regulating the formation of the composite. While the collagen matrix is not the only factor in the formation of bone tissue (i.e., type-I collagen is found in other tissues that do not mineralize), its organization and chemical structure are clearly central to understanding how secondary bone is formed. Therefore, there is a vast amount of information in the literature regarding the structure of collagen in mineralizing tissues. Perhaps the first well accepted model for the organization of the collagen molecules was proposed by Hodge and Petruska, who described the quarter-staggered structure of collagens triple-helices (tropocollagen units) which results in hole and overlap zones (99). Such overlap zones lead to electron-dense regions, which can be observed as a periodic banding pattern in TEM (especially with staining) and more recently by AFM. A variety of modifications to this quarter-staggered model have also been proposed, such as the alignment of gaps to form grooves, which are proposed to help account for the fact that the dimensions of the HAp crystals extracted from bone are larger than the dimensions of the gap zones, where they presumably are formed. Clearly before one can gain a better understanding of how intrafibrillar mineralization occurs, there needs to be greater advances in the analysis and organization of the organic matrix, both before and after mineralization. Much of the current data stems from demineralized tissues, which is inherently problematic due to changes in the organization and dimension of the original organic matrix after the demineralization process.


25 Mineralization mechanism Two opposing themes can be found in the literature regarding collagen-associated mineralization in secondary bone formation. The prevailing theory is that the crystals nucleate within the hole zones of collagen and subsequently grow from the primary nuclei until fusing with crystals from neighboring zones. An alternative view argues that initially, amorphous inorganic substance in bands is deposited within the hole zones (6, 10, 110, 115, 116), followed by crystallization into hydroxyapatite. The first case, where an insoluble organic substrate templates the nucleation event, occurs presumably via spatial organization of ions interacting with functional groups of the matrix (117). As previously mentioned, hydroxyapatite nuclei have been observed to first occur in the hole zones of the collagen (96, 118-121). Yet, the lattice mismatch between collagen and HAp (122), as well as collagens relatively weak ability to bind Ca 2+ ions, suggest that the collagen substrate itself would not provide a good template for directing the nucleation of hydroxyapatite. Indeed, this has been shown to be the case through in vitro studies, in which hydroxyapatite nucleated on the surface of type-I collagen fibrils (12) in a rather loose and non-specific fashion, and only on the surface (not intrafibrillar). In order to account for this, the rational provided for the preferential nucleation within the hole zones has now turned towards the non-collagenous proteins (NCPs), such as phosphoproteins and osteonectin, which are observed in close association with the mineralization front (123, 124). These proteins, which have high affinities for collagen and binding Ca 2+ are believed to sit within the hole zones (123) and promote the formation of HAp nuclei. Proposed mechanisms for protein regulation of mineral formation generally fall into two categories. The first possible route is the protein may have a structure that


26 places charged groups with a periodicity roughly matching the spacing between ions for a particular crystal face of the mineral phase. This fit, or lattice matching, of solution ions to the charged groups in the protein could serve as a nucleator of mineralization; this model is commonly referred to as epitaxy (or pseudoepitaxy). Using a steady state gel system, Hunter et al. established that Bone Sialoprotein (BSP) is one of the few matrix proteins which truly promotes rather than inhibits mineralization of HAp (125). However, it is difficult to prove if epitaxy is really at play, or if there are less specific interactions stemming from interfacial energetics. In the second model, the specific arrangement of the charged groups on the protein is less critical; instead, the function of the protein is to sequester ions and increase the local concentration so that a critical nucleus of ions can be formed, leading to the formation of the mineral. One might consider this a non-specific promoter of nucleation. Molecular modeling and the recent crystal structure of osteocalcin (126), an inhibitor of HAp formation, demonstrate a possible epitaxial match with HAp lattice spacings. However, the relatively little structure observed in nuclear magnetic resonance (NMR) solution studies of BSP to date indicates that the latter mechanism may be more apropos. Mineral precursor phases While the final product of intrafibrillar mineralization in bone is oriented, platy hydroxyapatite crystals embedded within a type-I collagen matrix, the initial phase observed within newly deposited collagen is considered by some to be amorphous (6). There are even reports that suggest that bone apatite is paracrystalline, an intermediate between amorphous and crystalline calcium phosphate (127). Recent research in the biomineralization field is pointing towards the use of amorphous phases in the formation of several calcium carbonate biominerals (68, 71, 128-131), but generally little


27 connection between CaCO 3 and CaP biomineralization mechanisms are considered because their final morphologies are so distinctly different. The presence of CaP precursor phases in vertebrate biominerals has long been debated (both for octacalcium phosphateOCP, and amorphous calcium phosphateACP), both with respect to the highly organized HAp crystals formed within the collagenous matrices of bone and dentin (132), as well as the elongated prismatic morphology of HAp crystals in dental enamel (6, 133, 134). Bonnuci reports that during the first stage of secondary bone formation, a more electron dense, finely granular inorganic substance (with an amorphous appearance) is deposited in the hole zones of collagen (132). During the second stage, platy HAp crystals are formed, eliminating any evidence of an initial amorphous phase, which is not easily detected. It has also been suggested that octacalcium phosphate (OCP) might act as a precursor to HAp (135). These concepts are based on the Ostwald-Lussac Rule of Stages, which although empirical, finds that the most soluble (least stable) phase forms first during a sequential precipitation, when the solution is supersaturated with respect to multiple phases. As both amorphous calcium phosphate (ACP) and OCP are known to be precursors to HAp in in vitro studies of calcium phosphate precipitation, these data support the concept that bone mineralization could occur via a metastable precursor mechanism. Of course, once the precursor phase is formed, crystals can then subsequently nucleate (i.e., phase transformation) into crystalline phase. The research contained within this dissertation will show, synthetically, that this is a feasible mechanism. Bone Graft Substitutes The impetus for this research is two-fold, first to better understand the use of amorphous precursors in biomineralization, and secondly, to use this knowledge to


28 synthetically recreate mineralized tissues, such as bone, for bone graft substitutes. Presently, the gold standard for bone graft substitutes is autograft tissue, which is living tissue removed from a secondary location within the body and used as a graft at the damaged site. Bone is the second most common tissue transplanted behind blood (136), with over 2.2 million replacements are done throughout the world. Autograft tissue serves three major elements necessary for a proper replacement of bone: it provides a scaffold for osteoconduction (denotes site on which new bone can grow), contains growth factors needed for osteoinduction (biological components induce bone to grow), and finally, has progenitor cells for osteogenesis (growth of new bone) (137). Yet, there are two major drawbacks to autograft bone substitutes. As the donor site for autografts is most often the iliac crest of the hip (because of the bone quality and volume), two sites of surgery are required, thereby causing additional pain and extra recovery time (136). Additionally, there may be iatrogenic complications stemming from fracture at the site of the iliac crest (138). In order to avoid these problems, the most common alternative is an allograft (donor tissue), which circumvents a second point of bodily intrusion of the patient. Allografts are applied in two different forms, mineralized and demineralized (139), each with a variety of different forms. Mineralized allografts can be used fresh, frozen or freeze-dried, and while they have limited osteoinductivity, they are very osteoconductive. Fresh allografts invoke a severe immune response (140-142), and therefore they are rarely used. By freezing or freeze-drying the tissue, it can be retained for longer periods of time and cleansed of viruses and bacteria (139). The only concerns with either of these techniques are the decrease in osteoinductivity and mechanical


29 properties. In order to improve the osteoinductivity of allografts, the tissue can be demineralized, thereby allowing important bone growth factors to be exposed. These factors include, bone morphogenetic proteins (BMPs) and NCPs (osteopontin, osteocalcin, and osteonectin)(139). The efficacy of demineralized bone matrix (DBM) is highly dependent upon processing, as the introduction of chemicals used to cleanse and preserve the tissue can have adverse effects on site of implantation. The inherent complication of all allograft tissue though, are the major concerns of transferring contaminants, toxins or infections from the donor and limited availability (e.g., shelf-life at 20C for fresh frozen bone tissue is 1 year (143), and 5 years at 70C (144)). Donor screening and advance processing techniques are often employed in order to prevent transmission of diseases and improve shelf life. The application of the preventative measures puts the risk of transmitting a disease like HIV at 1 in 1.6 million (145). In order to preserve the tissue, it must be chemically treated, which can lower efficacy of the donor tissue. One solution to the problems surrounding auto and allograft tissues is to use synthetically manufactured biomaterials. The field of materials science has made great strides in the synthesis and study of biomedical materials, enough so that as of 1996, 10% of all bone graft substitutes were synthetic (146). The major advantages of synthetic material implants is that the supply is endless, and there is little concern over biological contamination, such as bacteria or viruses. Yet, with the use of synthetic materials, there are numerous complexities. The major concern is that the material must be biocompatible, that is to say that it will not cause an adverse response from the body. A second, less biological, more material concern, is the mechanical properties of the material. If a material does not have the


30 correct materials properties, the body will undergo undue stresses and cause increased damage to the site of implantation. Two examples of this are adhesion of the implant surface to the tissue, and stress shielding. If the adhesion to the tissue is not sufficient, there may be loosening and subsequent failure of the implant (147). While there are many types of implant failures (148), in over 80% of clinical revisions, aseptic loosening of cemented implants are to blame for failure (149, 150). Stress shielding is a process that occurs when the material is too stiff and the local load near the implant is redistributed to the adjacent bone tissue. Since bone remodels according to the mechanical forces placed upon it (Wolffs Law), the added stress causes undue remodeling at the point of implant insertion. These challenges have not stopped the materials science and biomaterials community from developing a wide variety of synthetic materials for bone graft substitutes. A brief discussion will ensue, highlighting a few of the various materials attempts, including ceramics, metals and polymers, at replacing damaged or missing bone. Metallic substitutes Metallic replacements are used for a variety of biomedical applications, but one of the major uses is that of joint replacements. Two of the major advances in metal prosthesis in bone replacement are the use of the Ti and Co metal alloys, TiAl 6 V 4 and CoCrMo, respectively, due to their biocompatability and corrosion resistance (151, 152). When used as a hip replacement, these metals can be either be cemented or uncemented at the stem. When the stem is first placed into the femoral cavity, the majority of the contact bone is spongy cancellous bone, which allows two different techniques to be used to implant the stem. If the surface of the metal rough, that is, it is machined to have many small pores, no cement has to be used, and the spongy bone can grow into the pores. If a


31 smooth metal stem is used, polymer cement must be used to bond the metal surface to the cancellous bone. While the mechanical strength and biocompatibility of these metals is beneficial, the fact that they often become dislodged, cause stress shielding, and most importantly, are not resorbed, demonstrates why they are not a perfect bone graft substitute. Polymeric substitutes In the arena of bone graft substitutes, polymers are used mainly as adhesives or scaffolds. A common bone paste, polymethylmethacrylate (PMMA) is often used to cement in prosthesis. While PMMA provides good mechanical bonding, the initial polymerization is often exothermic, causing local tissue damage. Additionally, water molecules within the body fluid hydrolyze the interface between the paste and metal, weakening the bond (153). Polymers can also be fabricated into scaffolds to acts as a carrier for bone growth factors, such as BMPs (154-159). Recent work by Shoichet, Davies and co-workers using poly(lactide-co-glycolide) (PLGA) porous substrates seeded with subcultured rat bone marrow cells has shown promise (159). The idea is that the seeded cells can induce growth into the porous scaffold, which bridges the defect. As the new bone appears, the porous scaffold dissolves, leaving behind new bone. This approach has also been used with the organic constituent of natural bone, type-I collagen, which brings the scaffold closer to the final product, but still lacks mechanical stability (160). While the use of polymers and scaffolds shows a great deal of promise, the byproducts of the dissolution of synthetic polymers can include acids which can change the local chemistry, thereby damaging healthy surrounding tissue.


32 Ceramic substitutes As bone derives the majority of its strength from the inorganic mineral, another common attempt at replacing damaged bone is by using ceramics to match the stiffness, while coming closer to the composition of bone. There are three distinct uses for ceramics in bone graft substitutes, as solid grafts, as coatings, and as pastes or fillers. While large ceramic pieces have the static mechanical capabilities for large grafts, such as femoral stems, they are susceptible to crack formation and failure, and therefore they are often used to coat metal grafts to improve bonding to the bone. There are a variety of different coatings used to coat metal implants, ranging from calcium phosphates to alumina, to more complex ceramic structures such as CaO-TiO 2 -ZrO 2 -P 2 O 5 (161-168). The obvious choice is to use calcium phosphate coatings in hopes that the surrounding bone will recognize it as being similar in composition (169-174). Another type of material is Bioglass, invented at the University of Florida in 1969, is a soda-calcia-phospho-silicate glass which forms a layer of hydroxyapatite on its surface and bonds to natural tissue (175). Yet, synthetic HAp is not bioresrobable, and its bond strength with the metal prosthesis is often a problem. Ceramics, such as alumina and zirconia, have also been used as ball in femoral prosthesis (176-181). While these provide a very smooth surface with low friction, they are brittle and tend to fracture, wear or chip, causing pain and discomfort for the recipient (179-181). Lastly, ceramics can be used as replacement for bone in the form of pastes, cements or free form fabricated structures. A great deal of research has gone into calcium phosphate bone pastes and cements within the past 10 years (182). The final product of most of these pastes is an apatitic calcium phosphate (183, 184). While these pastes form a final product that may have the


33 same composition as bone, they do not easily resorb and do not permit bone ingrowth due to their dense nature (182). This problem may be solved by introducing pores into the cement, but this would weaken the overall strength of the graft. In order to overcome this problem, researchers are now creating free form structures through ink-jet processes (185, 186). Using HAp inks, three-dimensional structures with regular periodicity can be created which has stiffness close to that of bone and onto which bone cells can be attached. This intriguing approach offers a structure that is comprised of the same mineral phase as observed in bone, is porous and is load bearing. The problem with these structures is that although they can bear a static load, they are composed completely of a brittle ceramic and therefore would likely fail in the event of dynamic loading or high impact. They may also not be bioresorbable due to the micron sized hydroxyapatite structures. One of the main advantages of synthetic materials over autografts or allografts is that there is an abundance of supplies from which to synthesize them. Conversely, the properties of these synthetic materials pale in comparison to that of natural tissue. A perfect solution is to combine these two ideas together by biomimetically synthesizing a material that has the same structure as natural bone. Biomimetic Mineralization Materials science is the study of understanding materials properties through various characterization techniques in order to advance materials processing techniques. The two most recent advances in materials science research are composite design and the drive towards nanoscale processing. Through biomineralization, Nature has long since achieved both of these goals through the combination of organic macromolecules and inorganic minerals into precisely controlled, complex biomineral structures under


34 ambient processing conditions. The study of biomineralization will help to unlock the latent processing techniques organisms employ to create such complex biominerals. With this knowledge, materials scientists can apply the lessons learned through biomineralization to the field of biomimetics to engineer better materials. As previously mentioned, the field of biomimetics draws from a wide range of biological examples, biomimetic mineralization being a key example for this dissertation. One of the most extensive fields of biomimetic mineralization is focused upon recreating mineralized tissues for graft substitutes, specifically bone. Therefore, this example of biomimetic mineralization will be covered in detail. It would be desirable to synthetically prepare a bone graft substitute which matches both the chemical and mechanical properties of bone. Such a material could be both load-bearing (with the appropriate modulus, strength and toughness), yet bioresorbable to allow for the bodys own tissue repair processes to regenerate natural bone. The most obvious choice of materials for such a synthetic bone substitute would be a collagen-HAp composite; indeed, many have tried to mineralization collagen in vitro (187-195); but the preparation of such a composite has been limited by the inability to achieve the high mineral loading that is attained biologically by intrafibrillar mineralization. The sheer abundance of literature regarding bone graft substitutes that involve the use of synthetic metals, plastics and ceramics suggests the difficultly of re-creating a collagen/HAp composite that has the high mineral loading and nano-structure of bone (152, 196-205). There are two reasons why materials scientists have not placed greater attention at recreating natural bone through synthetic methods. The first is that a bulk-dense collagen sample is extremely hard to create, as collagen gels at low concentrations.


35 Secondly, as demonstrated in the previous section on bone, researchers still do not fully understand the mechanisms by which collagen is intrafibrillarly mineralized. There are two main schools of thoughts regarding biomimetic mineralization of type-I collagen. The first deals with mineralizing existing collagen substrates, both modified and unmodified, and the second regards mineralizing in the presence of fibrillogenesis. As osteoblasts first lay down an organic substrate of type-I collagen, the first route is closer to nature, but it is the second process that has achieved the most success in regards to orientation of HAp. Yet, there is little evidence to suggest that either procedure has been able to fully demonstrate high mineral loading and intrafibrillar mineralization. Collagen is observed in all connective tissues in the body, as it is the major protein observed in the body. There are over 20 different types of collagen, although type-I collagen is the most prevalent, observed as the major component in both hard and soft tissues. Interestingly enough, type-I collagen is not the only type that mineralizes. This suggests that while type-I collagen is a factor in mineralization, it is not the major factor. Regardless, it is believed that the quarter-staggered structure and 40 nm gaps zones that play an important role in the promotion of mineralization. This is well evidenced through the fact that in early bone and tendon mineralization, mineral nuclei are located in these hole zones (102). A significant amount of research has gone into studying the effect of the collagen substrate, as well as other model systems, on mineralization of both CaCO 3 and CaP (191, 192, 206-219). Some of the more relevant results will be discussed here. CaCO 3 mineralization While CaCO 3 is not observed in vertebrate hard tissues, it is a major component in invertebrate structural components, and therefore has warranted some attention as a


36 biomaterial in combination with collagen. Gelatin (atellocollagen units that have not gone through fibrillogenesis) and dissolved collagen have been utilized as substrates and crystal growth modifiers in order to study the effects on CaCO 3 crystallization. Falini (215) and Falini et al. (213) observed that the spatial organization of stretched gelatin films with entrapped polypeptides (e.g., poly-aspartic acid) induced the formation of oriented vaterite, while aragonite formed on unstretched films. When glutamic acid was used instead of aspartic acid, there was no orientation of the mineral phase in stretched films. They concluded that the -sheet conformation adopted by the aspartic acid on the stretched collagen matrix promoted orientation and specific polytype of the deposited mineral. Shen et al. (218) determined that dissolved collagen does not affect the polytype of CaCO 3 formation, as calcite, the equilibrium morphology of calcium carbonate, is always expressed in the presence of collagen additives. They concluded that at increasingly higher concentrations, collagen affected the crystallographic planes expressed. Researchers have yet to study the effect of reconstituted type-I collagen on CaCO 3 mineralization, presumably because calcite normally forms large crystals, and therefore could not possibly nucleate within the hole zones of type-I collagen; unlike hydroxyapatite, which crystallizes as nanometer scale platelettes. Thus, is not surprising that there has been little research performed with CaCO 3 to achieve intrafibrillar mineralization of type-I collagen. The majority of collagen mineralization research is focused upon creating hydroxyapatite/collagen composites, both as biomaterial composites and as biomineralization models.


37 HAp mineralization There are two manners in which researchers have attempted to mineralize type-I collagen in order to recreate intrafibrillar mineralization. The first, by using a pre-existing collagen substrates to induce CaP mineralization, and the second to mineralize CaP in conjunction with fibrillogenesis. In natural bone formation, the type-I organic matrix is first deposited and then subsequently mineralized. Therefore, there have been many attempts at duplicating intrafibrillar mineralization using pre-existing collagen substrates (188, 192, 195, 211, 214, 220-228). One of the more predominant methods of mineralization is placing a collagen substrate into simulated body fluid (SBF) for a certain amount of time in order to induce calcification (224-226, 228-230). Girija et al. (224) reported the synthesis of spherical -type carbonate apatite deposits nucleating along the collagen fibrils (Figure 2-7A). On the other hand, Rhee and Tanaka (225) determined that the formation of carbonate-apatite only formed on collagen membranes soaked in SBF in the presence of citric acid, citing the strong chelating effect of citric acid on calcium ions (Figure 2-7B). In either case, the apatite mineral nucleates as spherulites on the collagen substrate, and is most often referred to as bone-like apatite because its platy appearance resembles the nanoscopic dimensions of natural bone apatite. This use of nomenclature is misleading because it suggests that the apatite is intrafibrillarly mineralized, when in actuality it only describes a platy HAp morphology. Using Fourier transform infrared spectroscopy (FTIR), Zhang et al. (230) demonstrated that hydroxyapatite nucleation occurred on the surface of collagen fibrils due to charged groups, indicating that carboxylate groups played a major role in the mineralization While these results suggest that calcium phosphate can be mineralized on collagen


38 substrates, there has yet to be data that demonstrates intrafibrillar mineralization with this mineralization process. The most interesting result from this process is that the charged groups on the collagen substrate can induce hydroxyapatite. Researchers have also been testing the idea that the spatial charge distribution of organic macromolecular substrates, both collagenous and synthetic, can be used to induce oriented mineralization (231, 232). Stupp and co-workers demonstrated that by designing fibrous peptide amphiphiles (PA) with periodic, highly negatively charged surfaces, thereby mimicking the charged surface of non-collagenous proteins (NCPs)/collagen in bone, they could induce hydroxyapatite crystals which were oriented with respect to the long axis of the PA fibril (Figure 2-8A & B) (231). Goissis et al. (232) were able to form a hydroxyapatite coating on the surface of collagen fibrils which were modified through hydrolysis of the asparagine and glutamine carboxyamide side chains to acidic forms (Figure 2-8C). They claim that amide hydrolysis occurred preferentially near the hole and overlap zones, as suggested by the decreased interband distances of the polyanionic collagen, which they suggest increased the calcium binding sites at those regions. These studies suggest that surface modification of type-I collagen can provide a means for promoting crystallization, which in the case of the natural environment, might result from either glycosylation of the collagen, or adsorption of non-collagenous proteins. There is still little evidence that a type-I collagen substrate has induced oriented intrafibrillar mineralization of hydroxyapatite, and therefore research has branched into nucleating hydroxyapatite in the presence of fibrillogenesis (191, 199, 219, 233-237). Recent research within the past decade has demonstrated that re-naturing of collagen in the presence of hydroxyapatite mineralization produces an oriented mineral


39 phase along the c-axis of the collagen fibrils. The first to suggest this process was Trentler et al. (191), and although they did not achieve oriented mineralization, they demonstrated that nanometer needle-like hydroxyapatite was in close association with the gap zones of collagen during fibrillogenesis (Figure 2-9A & B). Rhee et al. (234) were the first to use this idea to achieve aligned hydroxyapatite on collagen fibrils. They added dissolved collagen to a phosphate solution and then mixed it with a calcium hydroxide solution (Figure 2-9C). Interestingly enough, they showed arcing of the (002) plane in diffraction patterns of single mineralized fibrils, but no native banding pattern of collagen was observed. Similar results were observed by Kikuchi et al. (199) using a process close to that of Rhee and co-workers (Figure 2-9D). Kikuchi and co-workers found that their composite structure, which had a nanostructure similar to naturally mineralized collagen, was highly bioactive, being resorbed by osteoclasts and allowing subsequent osteoblastic activity to build new bone. While these results are quite promising for bone graft substitutes, they do not mimic the formation of naturally mineralizing collagen. The arcing of the (002) planes displayed in the work of Rhee et al. (234) and Kikuchi et al. (199) suggest that there is oriented hydroxyapatite along the collagen fibrils, but the absence of electron dense banding patterns in their TEM data suggests that they have not achieved intrafibrillar mineralization. These composite structures demonstrate that the scientific community is coming closer to creating bone-graft substitutes that fully mimic the nanostructure of bone, but they do not lend any information as to how bone is naturally mineralized. The work contained in this dissertation serves to demonstrate feasibility of this phenomenon, which should automatically qualify it as a suitable bone-graft substitute.


40 A) B) C) D) Figure 2-1. Scanning electron micrographs (SEM) of various highly organized biominerals observed in nature, composed of a range of inorganic material, from calcium carbonate (CaCO 3 ), to calcium phosphate (CaP), to silica (SiO 2 ). A) The keel region a sea urchin tooth (Arbacia tribuloides) contains calcite rods, 5-7 m in diameter, embedded in an amorphous CaCO 3 matrix. Bar = 20 m. B) The ultrastructure of enamel from a rat incisor is more complex because it is woven into a cross-ply architecture by the ameloblast cells. Of relevance to this report is the fibrous morphology of the crystals, which in the vertebrates are polycrystalline bundles of HAp, rather than single-crystalline calcite rods, as in the urchin tooth. Bar = 50 m. C) Fracture surface of the cortical equine bone taken transverse to the long axis. The concentric mineralized lamella of collagen can be seen around the dark Haversian canals. Bar = 200 m. D) Diatom composed of amorphous silica. The molded appearance of the diatom is a perfect example of Natures nanoscale processing capabilities. Bar = 10 m.


41 B) A) D) C) Figure 2-2. Electron micrographs illustrating various morphologies of calcite observed within the structure of a sea urchin. A) Spines of a sea urchin (Arabacia Punctulata) composed of magnesium-bearing calcite, have a porous and highly convoluted shape, yet reportedly are single-crystalline calcite. Bar = 50 m. B) The single crystalline nature of the spine can be observed through the concoidal fracture pattern resulting from fracture. Bar = 10 m. C) The tooth of the Arbacia Punctulata illustrates the dual morphology of calcite used for two distinct purposes. The fibers in the keel region reinforce the platy region which are located on the scraping edge. Bar = 50 m. D) The transition region between the fibrous reinforcing phase and the platy scraping edge is composed of fibrous calcite encased in amorphous calcium carbonate, sandwiched between calcite platelettes. Bar = 10 m.


42 B) A) C) Figure 2-3. Tri-radiate growth of sea urchin spicules from a single calcite seed crystal. Schematics A) of single synthetic calcite crystal oriented in the same direction as a sea urchin spicule B) (3). C) SEM micrograph of the early stages of spiculogenesis that shows the tri-radiate spicule formation on the central calcite rhomb. Sea urchin spicules diffract as a single calcite crystal. Note the remaining sharp peak of the calcite rhomb in the middle of the sample (16). Bar = 1 m.


43 Figure 2-4. 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 (left), 200 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)(93). Reprinted, with permission, from the Annual Review of Materials Science,Volume 28 1998 by Annual Reviews


44 A) B) Figure 2-5. Figures copied from a paper by Weiner and Traub showing the deck-of-cards arrangement of crystallites within a collagen fibril. A) Transmission electron micrograph of mineralized collagen fibril from turkey leg tendon, showing layers of platy crystals. The banding pattern is due to more mineral being present in the gap regions of the collagen than in the overlap regions. The fibril is unstained and embedded in a thin layer of vitreous ice (Bar = 200 nm). B) Schematic illustration demonstrating that the plate-shaped crystals are all similarly oriented with their c-axes along the fibril axis. The crystals are arranged in parallel coplanar arrays forming grooves through the fibrils. The grooves are separated by four layers of triple-helical collagen molecules (not drawn to scale) (101).


45 Figure 2-6. Schematic illustrating the prevailing theory on intrafibrillar mineralization of type-I collagen in hard tissues. The collagen self assembles into a quarter-staggered structure providing 40 nm gaps, which subsequently align to form grooves (rectangular boxes), between the tropocollagen units (cylinders). Hydroxyapatite nucleates within the gaps and continues to grow along the fibrils until fusing with like growing zones (11).


46 B) A) Figure 2-7. Examples of bone-like apatite nucleated on collagen fibrils when collagen substrates are introduced into simulated body fluid (SBF). A) Spherulitic -type hydroxyapatite nucleated on reconstituted type-I bovine collagen fibrils placed in SBF (224). No scale bar indicated in publication.Reprinted, with permission, from E. K. Girija, Y. Yokogawa, F. Nagata, Bone-like apatite formation on collagen fibrils by biomimetic method. Chemistry Letters, 702 (Jul 5, 2002). Copyright (2002) Chemical Society of JapanB) Hydroxyapatite spherulites nucleated on a collagen sponge (225). Bar = 2m. Reprinted with permission of The American Ceramic Society, Copyright 1998. All rights reserved. Note that in either case the bone-like apatite has formed in a spherulitic manner, nucleated on the fibrils.


47 B) A) A) C) Figure 2-8. Electron micrographs of mineral deposited on functionalized polymer and collagen surfaces. A) TEM micrograph of hydroxyapatite crystals (arrows) mineralized on synthetically tailored self-assembled peptide-amphiphiles (231). Bar = 20 nm. B) Electron diffraction pattern taken from mineralized peptide-amphiphile bundles demonstrating the presence of the (002) and (004) planes of hydroxyapatite (231). C) SEM micrograph of hydrolized collagen that has been mineralized with hydroxyapatite. Arrow points to mineralized belts formed along the mineralized fibril (232). Bar = 200 nm.


48 B) A) C) Figure 2-9. Mineralization of HAp in the presence of fibrillogenesis of type-I collagen. A) SEM micrograph showing the presence of needle-like HAp crystals in association with collagen fibrils, both transverse and parallel to the long axis (191). Bar = 1m. Reprinted with permission from (191). Copyright 1999 American Chemical Society. B) TEM micrograph illustrating the oriented mineralization of hydroxyapatite (inset) formed during fibrillogenesis of type-I collagen (234). Bar = 100 nm. Reprinted with permission of The American Ceramic Society, Copyright 1998. All rights reserved. C) TEM micrograph also illustrating the oriented mineralization of hydroxyapatite (inset) formed during fibrillogenesis of type-I collagen (199). Note the arcing in the (002) planes indicating the mineral is not completely iso-oriented. Bar = 200 nm. Reprinted from Biomaterials, Vol. 22, M. Kikuchi, S. Itoh, S. Ichinose, K. Shinomiya, J. Tanaka, Self-organization mechanism in a bone-like hydroxyapatite/collagen nanocomposite synthesized in vitro and its biological reaction in vivo, pg. 1705, (2001), with permission from Elsevier.


CHAPTER 3 SYNTHESIS OF FIBROUS MINERAL VIA A SOLUTOIN-PRECURSOR-SOLID (SPS) MECHANISM Introduction The classical solution precipitation of calcium carbonate (CaCO 3 ) produces crystals which are usually composed of one of three polymorphs, vaterite, aragonite or calcite, in which calcite is the equilibrium phase, most commonly adopting a rhombohedral morphology (Figure 3-1A and B). In biologically produced minerals, the rhombohedral morphology is rarely adopted, as CaCO 3 is molded and shaped into a wide variety of non-equilibrium morphologies, including fibrous structures. Two relevant examples are the single-crystalline calcite fibers found in the keel and stone regions of sea-urchin teeth (Figure 3-1C and D), such as in Paracentrotus lividus (5, 69), and cave deposits and soils, which are geologic deposits, but are thought to arise from bacterial origin (238-240). In either case, such naturally occurring fibrous growths are an enigma since they do not follow the crystallographic symmetry of calcite, and calcite has never before formed this morphology in vitro. However, other inorganic materials can be produced synthetically into nanofiber morphologies using molten flux techniques, such as the vapor-liquid-solid (VLS) and solution-liquid-solid (SLS) mechanisms; but clearly, these calcitic biominerals could not be formed by a similar mechanism since they are formed under physiological, low temperature conditions. Or could they? Herein, we have evidence to suggest they can. 49


50 The VLS mechanism, first proposed by Wagner (241) and Wagner and Ellis (242), is a mechanism by which liquid flux droplets are created by heating metal particles above their melting temperature and placing them in an appropriate vapor atmosphere. The crystallizing constituents in the vapor phase are incorporated into the flux droplet at the vapor-liquid interface, which slowly raises the supersaturation within the flux droplet. The crystal face with the lowest liquid-solid interfacial energy nucleates at the solid-liquid interface. As the process continues, the fiber tip retains the flux and continually deposits upon the existing crystalline face epitaxially; yet the crystallizing area is constrained by the size of the flux droplet that provides the reactants, leading to one dimensional growth as the flux droplet is forced outward from the substrate. A key characteristic feature of this type of crystal growth mechanism is the appearance of a bobble (remnant of the liquid flux) on the tips of the fibers. Numerous high aspect ratio fibers, such as Si, Ge, and GaN, have been synthesized through this VLS mechanism (243-245). A similar mechanism was recently reported by Buhro and co-workers (246, 247) using solution chemistry to synthesize nano-fibers of III-V semiconductors at lower temperatures (200C), through a mechanism identified as a solution-liquid-solid (SLS) process. The SLS is described as being analogous to the VLS in that the process begins with low melting metal, such as Indium or Gallium, contained in an organometallic solution. The flux phase remains as a droplet on the tip of the fiber, continually being fed through solution; but in this case, there is no solid substrate upon which the fibers nucleate and grow, thus creating crystalline nano-fibers suspended in the solution. This synthesis has the ability to produce nano-fibers of InP, InAs and GaAs at temperatures


51 ranging from 110-200C, considerably lower than the VLS mechanism, which requires higher temperatures (950C) (241, 242). Yet, as has been demonstrated by nature, the ability to fabricate highly-organized nanofibrous composite structures can be achieved, and at even lower temperatures. This is the fundamental basis of biomimetic engineering, to understand how nature can create these exquisite structures at ambient conditions with minimal expenditure of energy. In previous reports we have proposed that biominerals, many of which have a sculpted or molded appearance, may not be synthesized through simple nucleation events, but might be a result of a precursor mechanism induced by acidic polypeptides, which we call the polymer-induced liquid-precursor (PILP) process (7, 8). The PILP process is induced using short-chained acidic polymers, such as polyaspartic acid (Poly-Asp) or its synthetic counterpart, polyacrylic acid (PAA), which are simple mimics to the highly acidic proteins associated with biominerals. The PILP process has been described in detail in our previous reports (7, 8, 37). In brief, we have shown that acidic polymers can sequester a high concentration of ions, while inhibiting crystal nucleation, to induce liquid-liquid phase separation of droplets of a metastable precursor within the crystallizing media. The nanoscopic droplets grow to a size of about a couple of microns, at which point they can be seen on an optical microscope. Usually the droplets settle on the substrate and coalesce, and upon solidification and crystallization, form mineral films. Under certain conditions, we have also observed calcitic fibers, which have been described in our previous report. However, in that study, the fibers were larger (circa 2 m in diameter)(8) and the fiber growth mechanism seemed to be quite different. It appeared to be a consequence of extrusion of the


52 precursor phase from gelatinous globules which had become encrusted with solidified calcium carbonate and, upon rupture of the mineralized shell (presumably due to a build-up of osmotic pressure), released streams of PILP phase (8). In the work reported here, we have been able to produce calcite fibers by introducing calcite substrate crystals of traditional rhombohedral morphology into a PILP crystallizing solution. The mechanism appears to be different from our primary report, and involves a flux droplet that restricts the crystallization to one-dimensional growth. Materials and Methods Synthesis Crystal substrates for fiber growth Substrate crystals were synthesized in two different manners, which provided calcite crystals of typical rhombohedral habit, but which differed in size and defect texture. For the first set of experiments, micron sized calcite rhombs (20-40 m) were deposited on glass slides via a vapor diffusion method (48). For comparison, larger, millimeter sized calcite rhombs were grown through a metasilicate gel method. For the first type, glass substrates (22 mm diameter microscope coverslips) were placed into small petri dishes containing 3 ml of a 24 mM CaCl 2 H 2 O (Sigma) solution, and this crystallizing solution was placed in an enclosed chamber (e.g., a desiccator) containing small vials of freshly crushed ammonium carbonate. The carbonate powder decomposes into a vapor (CO 2 and NH 3 ) at room temperature, which slowly diffuses into the calcium solution, providing a means for gradually raising the supersaturation of CaCO 3 The larger 0.5 mm calcite crystals were fabricated in a gel medium through a controlled chemical reaction method. A sodium metasilicate gel was prepared using 0.5 M CaCl 2 H 2 O (Sigma) in a 0.35 M Na 2 O 3 Si9H 2 O (Sigma) solution, which was allowed to


53 set in 15 mm dia/127 mm long glass test tubes overnight until gelation. Subsequently, 0.1 M ammonium carbonate solution was added to each test tube and then covered with parafilm. The ammonium carbonate was allowed to diffuse into the gel over a period of 1 month, at which time large calcite crystals were gently removed with tweezers and rinsed several times with deionized water. The larger calcite crystals were analyzed using EDS in order to assure no Si remained on the surface of the crystals. Fiber growth The substrate crystals were introduced into the crystallizing system by placing the glass coverslips which had nucleated the 20 40 m calcite rhombs (or contained 3 to 4 of the larger gel grown rhombs) in the bottom of a small petri dish filled with3 ml of a 12 mM calcium chloride dihydrate solution, containing 0.24 mM MgCl 2 6H 2 O or SrCl 2 6H 2 O, along with 200 g/mL of polyacrylic acid (5,100 M.W., Sigma) to induce the PILP process (7). The Mg 2+ and Sr 2+ which are routinely found in naturally occurring calcite morphologies, were added because we have determined that they enhance the PILP process (inhibit solution crystal nucleation). CO 2 (g) was delivered to the sample solutions via the decomposition of crushed ammonium carbonate powder in the same manner as described above for synthesizing substrate crystals. Each of the sample solutions and ammonium carbonate vials were covered with parafilm, into which small holes were punched in order to slowly raise the supersaturation of the calcium carbonate in solution. All components of the reaction were then placed in an enclosed chamber, which allowed diffusion of the carbonate into the solution. Control experiments, containing no polymer, were performed alongside each experiment. For further characterization, the coverslips containing the small rhomb substrate crystals (or


54 individual large gel-grown calcite rhombs) were carefully removed from solution with tweezers and rinsed with ultrapure H 2 O and then with ethanol (by gently dipping coverslip in a rinse beaker to avoid dislodging the fragile fibers) in order to remove any soluble salts. Analysis Polarized optical light microscopy (POM) Samples were examined on the glass slides in transmission mode using an Olympus BX60 polarized optical microscope with first-order red wave-plate. The wave-plate allows one to observe both amorphous and crystalline phases, which is quite useful for examining crystallization reactions that proceed via precursor mechanisms. In this case, it was also useful for examining the crystallographic orientation of the fibers. Under plane polarized light (PPL), a crystal will appear dark (extinct) under crossed polars when its vibration directions are oriented parallel to one of the polars (indicated as p and a for polarizer and analyzer direction, respectively). When a gypsum plate is inserted, the crystal in the extinct position will exhibit the same retardation color as the magenta background. Maximum birefringence will occur when the crystal is rotated 45 from the analyzer and polarizer due to constructive interference of the light waves. Calcite is an optically negative material, therefore when the slow vibration direction of the wave-plate (indicated as sd double arrow) is parallel with the slow axis of the calcite (indicated by short arrow in NE corner), the combined vibrations will destructively interfere and the fiber will appear blue (a fall in interference color determined from Michel Levy chart). Conversely, the yellow color in the opposite quadrants indicates the slow ray of the crystal is parallel to the slow direction of the accessory plate. This type of analysis allows one to identify if there is a uniform


55 orientation of the crystallographic axes within irregular shaped crystals, such as the fibers in our system. Scanning electron microscopy (SEM) The samples were dried overnight, fixed to an aluminum stub using carbon paste or double-sided copper tape and then Au/Pd coated. The samples were then examined with either a JEOL 6400 SEM or a JEOL 6335F FEGSEM instrument, both equipped with energy dispersive spectrometers (EDS), at an accelerating voltage of 15kV. Transmission electron microscopy (TEM) In order to examine the fibrous calcium carbonate, the samples were first scratched with a razor blade to dislodge some of the fibers from the rhomb substrate. A small aliquot of ethanol was then dispensed onto the scratched area and immediately drawn up using a micropipette. The removed aliquot was then dropped onto a 200 mesh copper TEM grid coated with thin layers of Formvar and amorphous carbon. The grid was air-dried and subsequently sputter coated with amorphous carbon for stability. The sample was examined on a JEOL 200cx transmission electron microscope at 200 kV in brightfield (BF) and selected area diffraction (SAD) modes. Results and Discussion Fiber growth The original intent of this study was to use seed crystals to control the crystallographic orientation of calcitic films, which are typically formed by settling and coalescence of PILP droplets. Normally, when the PILP films are deposited onto non-specific substrates, such as glass coverslips, a mosaic of randomly-oriented crystalline patches is formed (7, 37). We hypothesized that by depositing the precursor phase onto pre-existing calcite seed crystals, the crystal orientation of the films could be regulated by


56 iso-epitaxy. This idea was based on biomineralization, in which there is evidence to suggest that seed crystals are used to control the nucleation event in order generate single-crystalline structures (e.g., sea urchin spicules)(34, 67, 248), or control crystallographic orientation (e.g., mollusk nacre)(249, 250). In order to test the influence of a crystalline surface on the deposition of the PILP phase, calcite seed crystals were nucleated on glass coverslips (Figure 3-1A). As expected, in the absence of polymeric additives, surface roughening occurred due to iso-epitaxial overgrowth on the substrate crystals, while the overall rhombohedral morphology was maintained (Figure 3-2A and B). When polymer was introduced into the system, due to the large size of the seed crystals, most of the PILP film deposited only on top of the rhombs (rather than on the surrounding glass coverslip), where it was difficult to determine if the seeds induced iso-epitaxial growth. Nevertheless, interesting results were found. Fiber outgrowths were observed to emanate from the surface of the calcite rhombs (Figure 3-2A and B, and 3-3). From here on out, we will refer to the calcite rhombs as substrate crystals rather than seed crystals because the rhombs are much larger than the films or fibers which are deposited on them (which is not typical of the traditional usage of the term seed), and because the unexpected results of fiber growth show that the rhombs are providing a specific modulating substrate, rather than just seeding epitaxial growth of the same structure. Fibers (100-800 nm in diameter and varying in length from 5-80 m) were observed to grow from every face of the 20-40 m calcite substrate crystals (Figure 3-2C). We also tested substrate crystals that were prepared by growing large calcite rhombs in a metasilicate gel (Figure 3-1B). Similar fiber morphologies were observed to


57 grow off the gel-grown rhombs; yet the fiber density increased (Figure 3-2D). This is most likely a direct result of the larger amount of defect sites on the gel-grown substrate crystals, as can be seen in Figure 3-1B. Fibers deposited on both types of substrate crystals were observed to grow at both room temperature (27C) and 4C, and at times as short as 24 hours (Figures 3-2 & 3-3). SPS mechanism Based on what is known about existing fiber growth mechanisms, such as VLS and SLS, we propose that these crystalline calcitic fibers are produced via an analogous solution-precursor-solid (SPS) mechanism, but with a distinct difference in that the reactant flux is a liquid-phase mineral precursor, rather than a molten metal. The first indication is a bobble head observed at the tips of various fibers (arrows in Figure 3-3A). This bobble signifies, as noted by Wagner and Ellis (242) and Buhro and co-workers (246, 247, 251), that the tip of the fiber is a liquid phase that acts as a flux in which nucleation of the fiber begins. However, whereas the flux droplet in the VLS and SLS mechanisms is a molten metal, the flux droplet in our system consists of a polymer-induced liquid-precursor (PILP) phase. The PILP droplets, which contain a high concentration of the reacting species (the ions), are delineated by phase boundaries, a necessary requirement for generating one-dimensional growth, which arises from the spatial constraints of the flux droplet providing the reactants. Ultimately, the metastable precursor phase overcomes the inhibitory action of the polymer and crystallizes into the solid mineral phase, leaving behind the signatory remnant bobble on the tip of the fibers. A second indication of this mechanism is the morphology of the fibers, which are not always straight, as one would expect from the more traditional solution crystallization


58 process (e.g., aragonite needles grown in solution). In some experiments, the fibers had a tendency to grow in a serpentine fashion, most likely caused by temperature fluctuations in our lab (Figure 3-3C). Similar serpentine growths are reported for the VLS and SLS mechanisms (242, 246). The single crystalline nature of our fibers was first observed using polarized optical microscopy (POM), which shows that each birefringent fiber has a uniform retardation color and extinction pattern typical of single crystals (Figure 3-4A). Interestingly, the single crystalline nature of the calcite remains intact, even when the fiber growth direction has changed. For example, as shown in Figure 3-4A, the interference patterns of a single fiber which is bent at an angle of 50 demonstrates the uniform orientation of the crystallographic axes throughout the fiber, regardless of the change in fiber direction, exemplifying the symmetry breaking aspects of this mechanism. Very similar structures are found for the fibers extracted from the sea urchin tooth as well (Figure 3-4B). This is unlike the silicon fibers grown via the VLS mechanism (241), where a change in fiber growth direction is usually associated with a change in crystallographic orientation. The single crystalline nature of the calcite fibers was confirmed through electron diffraction of isolated fibers (Figure 3-5A). A common growth direction amongst all the fibers could not be discerned from diffraction analysis, as various fibers displayed different diffraction planes along the long axis of the fiber. Comparing this data to the continuous birefringence observed for curved fibers using optical microscopy suggests that while there may not be one particular growth direction for all the fibers, the crystallographic direction of an initiated fiber frequently remains constant. The fibers in Figure 3-2C and 3-3D appear to exhibit an epitaxial relationship


59 with the substrate crystal, but such a relationship is less clear in other samples, and especially if the fibers are densely packed with curvature causing them to stray from any initial growth direction. Further studies must be performed to elucidate if there is an epitaxial relationship between the fibers and substrate crystal which dictates this initial crystallographic orientation, and how substrates could be designed for controlling fiber outgrowths. At higher magnification under SEM, the surface of some of our fibers appears to be covered in micro-crystalline calcite rhombohedra (Figure 3-3E). Based on the surface appearance of these fibers, one might presume that these fibers are polycrystalline; but this is not supported by the birefringence or electron diffraction evidence. These tiny crystals all appear to be iso-oriented with the long axis of the fiber, and may simply be micro-facets arising from surface re-organization of the high energy curved surfaces towards the lower energy {10.4} planes. Iso-oriented nanoscopic rhombohedral facets on the surface of the fibers can also be seen in some TEM bright field images (Figure 3-5B). One aspect of the SPS mechanism which likely differs from the VLS and SLS mechanisms is the replenishment of the flux droplets, which presumably must be continuously supplied with reacting species (ions in our case) in order to sustain fiber growth. We consider two means by which this replenishment might occur. In the first case, precursor droplets start each fiber, with replenishment of cations and anions from the surrounding crystallizing solution across the liquid-liquid phase boundary. This scenario would be quite similar to the VLS/SLS mechanisms. Alternatively, based on prior observations of coalescence of the PILP droplets, the mechanism of fiber growth may be somewhat different than the VLS or SLS in that the surrounding solution contains


60 nanoscopic PILP droplets that can preferentially stick to the liquid droplet at the fiber tip. As the schematic in Figure 3-6 illustrates, we believe that precursor droplets are physisorbed onto the surface of the rhombohedral calcite substrates. Once initial droplets are deposited, they either solidify or remain as a liquid, depending on the metastability of the precursor phase. This phase change is important because if the droplet remains a liquid, subsequent droplets, theoretically, would be able to coalesce with the primary droplet; whereas if the first droplet solidified, secondary droplets would wick to the sides of the solidified droplet due to surface tension. We observed that surfaces that facilitate fiber growth also seem to contain numerous droplets that have solidified and coalesced into films (Figure 3-3D). The numerous defect sites on the gel grown substrate crystal seem to favor fiber formation, possibly because the defect sites help to accumulate and retain the liquid state of the PILP phase. Similar to the VLS mechanism, the phase boundary between the primary liquid droplet and the solid calcite surface begins to epitaxially nucleate calcite, thus providing a physical basis for the extension of the fiber upon continual accretion of secondary droplets. As long as the liquid flux droplet is maintained (i.e., doesnt solidify or re-dissolve), it should facilitate further coalescence of secondary droplets and lengthening of the fiber. We feel that, while this SPS mechanism is similar to the VLS or SLS mechanism, it is intriguing in that it occurs under physiological conditions and offers a viable explanation for the formation of fibrous biominerals. SPS in relation to fibrous biominerals As previously mentioned, fibrous biominerals are often observed in echinoderm (sea urchin) structural components, mainly in the Aristotles Lantern region, which contains five teeth. The fibrous composite structure of the teeth is well documented (5,


61 67), and is found to be reinforced by S-shaped fibers composed of single-crystalline Mg-bearing calcite (Figure 3-1C); but little is known about their mechanism of formation. On the other hand, there have been in-depth studies of the early stages of echinoderm spiculogenesis (5, 67-69) because the organism can be more readily examined in the embryonic stage. Wilt (67) has shown that during the first stages of endoskeletal spiculogenesis, considerable amounts of amorphous calcium carbonate are present in the spicules, which are formed and shaped within an extracellular compartment delineated by a row of primary mesenchyme cells (PMCs). This calcium-rich amorphous phase is observed (by TEM) as granules in spicule forming cells, which are then transported to the tips of the growing spicules. It should be pointed out that analysis by TEM will dehydrate the sample and show amorphous granules, which originally may have been PILP droplets. In fact, a liquid-phase amorphous precursor could easily be transported and would readily coalesce with the growing spicule. In either case, being a highly metastable phase, the amorphous CaCO 3 then rapidly crystallizes (68). Notably, the spicules become tri-radiate as they emanate from the a-crystallographic planes of a rhombohedral calcite crystal which is deposited within the PMC during gastrulation (67, 68). It is actually quite rare to see calcite crystals of rhombohedral shape in biominerals, but this shape does not last long as the spicules form into elongated curving rod-like structures. The substrate crystal apparently provides control over the crystallographic orientation, which in turn contributes to the directionality of the growing spicule. Interestingly, the authors note that the plasmalemma is tightly applied to the developing spicules, except perhaps at the elongating tips (67). This seems to indicate that there is very little solution space around the forming spicule, much less than would be expected if


62 the crystals were grown by the traditional solution crystallization process (in which a limited concentration of ions would be present in the supersaturated solution), further supporting our hypothesis that the spicule is formed from an amorphous liquid-phase precursor. There has been increasing evidence of amorphous mineral precursors in marine invertebrates (5, 71, 128, 131, 252), and particularly as a transient stage in the development and shaping of the biomineral. We have proposed that this amorphous precursor may in fact be a polymer-induced liquid-precursor (7, 8), which could essentially be molded and shaped by the biological compartment within the organism. But in order to create the complex single-crystalline morphologies that are the hallmark of biomineralization, there clearly needs to be some control exerted over the nucleation/transformation of the precursor phase. For this reason, we have been examining the influence of a variety of substrates (both organic and inorganic) on the transformation of the PILP phase. The observation of a calcite rhomb during the early stages of spiculogenesis, prior to the tri-radiate extensions of distinct crystallographic orientation, seems to suggest that there may be an epitaxial relationship. As we demonstrate here, a pre-existing crystalline surface (e.g., a rhombohedral calcite substrate) causes the formation of a fibrous mineral morphology which is not attributed to the equilibrium polymorphs of calcium carbonate (such as the acicular nature of aragonite). In regards to spiculogenesis, we suggest that through evolution, nature has determined a manner in which to control fiber growth at specific locales, such as on an existing substrate crystal surface. For example, while the substrate crystal provides the desired epitaxial substrate for activating the outward growth and controlling


63 crystallographic orientation, the plasmalemma membrane which envelops the forming spicule could limit the location of crystal outgrowths. Epitaxy appears to play a key role in initiating and directing the fiber growths in our system. Both of these factors that are found within the echinoderms, i.e., the fibrous calcite crystals in the teeth and the use of an epitaxial substrate crystal during spiculogenesis, seem to correspond well with our preliminary observations of the factors involved in the SPS mechanism, and may provide insight into the evolutionary development of the phylum Echinodermata. SPS in relation to geological CaCO 3 The study of calcium carbonate biomineralization has often been confined to the realm of marine invertebrates, yet there is an abundance of highly modified calcium carbonate biominerals observed in cave and soil deposits. There is still debate as to whether these fibers found in cave deposits and soils are formed via a geologic mechanism, or through bacterial biosynthesis (240), With respect to geological biomineralization, an interesting feature of our fiber growths is the formation of double fibers, which occurred when the fibers were grown on the larger, 0.5 mm, gel grown substrate crystals (arrows in Figure 3-3B & F). These double fibers, which appear to grow simultaneously from a single site, have a strikingly similar appearance to geological fibers, in which there is a range of single, double, quad and even hex fiber growths or bundles (239, 240). While we did not observe any fiber bundles higher than multiples of two, and the fact that our double fiber diameters are larger than those found in nature, the mechanism seems to be related to the defect texture of the substrates, and might be similar for higher order bundles nucleating on other geological substrate crystals. In addition to the appearance of multiple bundle fibers in geological settings, a wide variety of calcite fibers are possible, including single crystalline fibers, such as those reported by


64 Borsato et al., which are mono-crystalline nanofibers elongated along their c-axes (238). Although these moonmilk calcite fibers are single crystalline, they were observed to have epitaxial calcite plates overgrown on the surface. Self and Phillips noted that various single-crystalline calcite fibers, found in Australian calcrete soils had conspicuous curves and anhedral lumps at the terminations. Our fibers also displayed spot patterns (Figure 3-5A) that match exactly with the diffraction results of Phillips and Self (239). All of these features are consistent with our newly proposed SPS mechanism, which occurs under similar environmental conditions (i.e., low temperature, aqueous-based solutions). As technology continues to be scaled toward the nano-scale, the ability to synthesize nano-materials using simple, low cost, low temperature mechanisms is indispensable. Our SPS mechanism appears to be related to both the VLS and SLS mechanisms, yet produces morphologies that are consistent with naturally occurring calcite deposits. The PILP process was discovered in the CaCO 3 system, but we have evidence that demonstrates the PILP process is non-specific with respect to other minerals, such as BaCO 3 SrCO 3 and CaP (unpublished results) (8). This suggest that other mineral fibers could be formed via the SPS mechanism, including oriented hydroxyapatite fibers, such as those observed in vertebrate dental enamel. Although high temperature mechanisms, such as the VLS and SLS, have been employed to create nano-fibrous materials, nature still owns the patent on creating oriented nano-fibrous materials at ambient conditions. We have developed a synthesis that incorporates minute amounts of a mimetic polymer in order to create single crystalline calcite nano-fibers at temperatures as low as 4C, but we still have a long way to go before we can direct the growth of these fibers into organized composite structures as found in biominerals.


65 However, given this potential link between these presumed different systems, perhaps some of the state-of-the-art nano processing techniques that are currently being developed could be applied to a new set of materials not previously considered.


66 A) B) D) C) Figure 3-1. SEM micrographs depicting the differences in morphology of synthetically grown calcite (A and B) versus that of biologically formed calcite (C and D). A) Calcite substrate crystal grown by the classical solution growth mechanism through a vapor diffusion method (see Methods section). Bar = 10 m. B) A much larger calcite rhombohedral crystal synthesized in sodium metasilicate gel. Note the high degree of surface defect texture of gel grown substrate crystals. Bar = 200 m. C) and D), SEM micrographs of various regions of a sea urchin tooth, Arbacia Punctulata, showing the variability in calcite morphology within the composite structure of the tooth. C) The keel region of the tooth is comprised of S-shaped, single crystalline, Mg-bearing calcite fibers with diameters ranging from 10-15 m in diameter. Bar = 500 m. D) The boundary of the chewing tip of the Arbacia Punctulata tooth contains 1.5-3.0 m diameter calcite fibers which originate from the keel region (left side of figure) and intersect with the primary calcite plates (right side of figure). Bar = 50 m.


67 A) B) C) D) Figure 3-2. SEM micrographs of calcium carbonate deposited onto rhombohedral substrate crystals in the presence and absence of micromolar amounts of acidic polymer. A) Calcite overgrowth on calcite substrates in the absence of polymeric additives. Bar = 10 m. B) Calcite overgrowth on gel-grown calcite substrates, again without polymer. Bar = 500 m. C) Calcite fibers grown on a solution-grown calcite substrate. In this case, the fibers appear to exhibit an iso-epitaxial relationship with the underlying substrate crystal. Bar = 20 m. D) Fibrous calcite grown on the surface of a gel-grown calcite substrate crystal. An epitaxial relationship cannot be discerned for these crystals (although it could exist at the base of the fiber). Bar = 20 m.


68 A) B) C) D) E) F) Figure 3-3. SEM micrographs demonstrating mechanistic aspects of the SPS mechanism. A) Bobbles on the tips of the fibrous growths (arrows), which are thought to be remnants of the PILP flux droplets, have a similar appearance to those described for the VLS growth mechanism. Bar = 20 m. B) Higher magnification of the bobbles on the tips of fibers, as well as double fiber formation. Bar = 5 m. C) Serpentine growth, as seen here, is also observed in VLS and SLS fiber growth mechanisms. Bar = 5 m. D) Only a few extended fibers are growing off this substrate, yet there are large amounts of stunted droplets that appear to have solidified before subsequent droplets could coalesce and build a fiber. Bar = 10 m. E) Stacks of iso-oriented microfacets, presumably arising from surface re-organization, can be seen on some of the calcite fibers. In this case, the similarly aligned microfacets on neighboring fibers supports the premise of an iso-epitaxial relationship with


69 the substrate. Bar = 1 m. F) Fiber multiplets (arrow) similar to those seen here are often observed in naturally occurring calcite fibers in cave and soil deposits. A midline in the fibers pictured in Figure 3-3B can also be discerned (arrows). Bar = 5 m. A) B) Figure 3-4. Single crystalline analysis of calcite fibers. A) This series of polarized optical micrographs shows a highly birefringent fiber (which had broken off the substrate rhomb), demonstrating the uniform crystallographic orientation arising from the single-crystalline nature of the calcite fiber. Upon rotation of the sample, these interference patterns demonstrate that there is a uniform orientation of the crystallographic axes throughout the fiber, regardless of the change in fiber direction. See experimental section for detailed optical analysis. Bar = 20 m. B) Polarized optical micrograph of a fiber separated from the composite structure of a sea urchin tooth, Arbacia Punctulata. As in our synthetic fibers, this biomineral fiber exhibits a uniform crystallographic orientation even though the fiber direction has changed. Bar = 20 m.


70 A) B) Figure 3-5. Electron microscope analysis of the fibers. A) A representative Selected Area Diffraction (JEOL 200cx TEM) pattern typical of the single-crystalline calcite fibers grown via the SPS mechanism. Beam Direction B = [1 -2 1 0]. B) Bright field TEM micrograph of a calcite fiber illustrating the microfacets and surface reorganization seen on some of the fibers. Bar = 660 nm.


71 A) B) C) D) Figure 3-6. Schematic depicting the proposed solution-precursor-solid (SPS) mechanism. A) The acidic polymer induces liquid-liquid phase separation in the crystallizing solution, creating droplets of mineral precursor. B) The PILP droplets physisorb onto calcite substrate crystals, perhaps preferentially accumulating at surface defects. Dark-filled spheres depict solidified droplets, and unfilled spheres depict liquid-phase droplets. C) Liquid droplets wick to the sides of solidified droplets, and only coat the solid substrate, whereas droplets that remain fluidic coalesce with subsequent droplets, forming the primary flux droplet that leads to one-dimensional growth. As the flux droplet solidifies and eventually crystallizes, it forces the fiber outwards from the substrate crystal, which continues to grow as long as the flux droplet on the tip is maintained. D) Eventually the flux droplet solidifies, leaving a bobble remnant on the tip. Bobbles are not always present, in which case the flux droplet, being a metastable phase, most likely dissolved back into the solution.


CHAPTER 4 MINERALIZATION OF TYPE-I COLLAGEN VIA A CALCIUM CARBONATE PILP PHASE Introduction Calcified tissues, such as bone and teeth, are highly complex composites comprised mainly of collagen, apatite mineral, and water (123). The intimate association between the mineral phase and collagen fibrils provides bone with remarkable strength and toughness (74). Most descriptions of the inorganic phase of bone are derived from ex situ transmission electron microscopy (TEM) examination. The actual morphology of the inorganic phase in bone was, at first, heavily debated (105, 111, 113, 116, 253); for example, whether the hydroxyapatite (HAp) crystals were in the form of needles, ribbons, or plates. This stemmed from the fact that TEM is a two-dimensional analysis, and platelets viewed edge on resemble needles. It is now generally accepted that the inorganic phase, which is often simply called hydroxyapatite [Ca 10 (PO 4 ) 6 (OH) 2 ] (but is more accurately identified as non-stoichiometric carbonated apatite), exists in its final form as nanoscopic platelets that form preferentially in the hole zones of the collagen fibrils (254-256). Weiner and Traub (101) have described the nanostructured architecture of mineralized turkey tendon (which is considered a model of bone structure) as similar to a deck of cards, with nanoscopic platelets of aligned and oriented hydroxyapatite crystals embedded within the collagen fibrils. In this chapter, we will consider this type of structure as being produced by intrafibrillar mineralization, to distinguish it from interfibrillar mineralization that is typical for collagen mineralized in vitro, in which the 72


73 crystals essentially nucleate heterogeneously on the surface of fibrils, and do not lead to a highly mineralized matrix with an intimate association between matrix and crystals (188-190, 193, 195, 257). There are very few examples of topographic bone analysis because it does not effectively show the internal relationship between collagen and apatite at the nanoscopic level. One exception is the work done by Bagambisa et al. (258), in which scanning electron microscopy (SEM) was used to examine the resting surface of a canine femur. Figure 4-1 shows a SEM micrograph reprinted from their work, which provides a somewhat different perspective on the morphology of the mineral phase. It can be seen that the collagen fibers are encased with a fairly continuous but rough mineral coating, which has very little resemblance to the nanoscopic HAp platelets represented in the multitude of TEM micrographs in the literature (96, 101, 104, 105, 193). The work reported herein will use SEM and TEM, in order to characterize a mineralized collagen composite structure generated in vitro via a polymer-induced liquid-precursor (PILP) process. The PILP process uses acidic polymers that are simple mimics to the acidic proteins associated with biominerals, to transform the classical solution crystallization of an inorganic salt into a precursor process (3, 7, 37). For example, when polyaspartic acid (PolyAsp) is added in low concentration (micromolar) to a crystallizing solution that is slowly raised in supersaturation for calcium carbonate, the polymer sequesters calcium and carbonate ions (while inhibiting nucleation), thus inducing liquid-liquid phase separation within the crystallizing media (7). The minor phase consists of nanometer to micron-sized droplets, which ultimately transforms (dehydrates/solidifies/crystallizes)


74 into crystalline calcium carbonate. As these droplets are initially liquid-like in nature, they can be deposited as films or coatings because they conform to the shape of their container and coalesce upon contact. It should be noted that this is not a gel phase, which is known to occur at high concentrations of ionic salts (and in particular for calcium phosphates). The fluidic nature of the PILP phase is significant because it can take on a variety of morphologies, and since the crystals retain the shape delineated by the phase boundaries of the precursor, a variety of non-equilibrium morphologies are generated (e.g., crystal "drops", fibers, and films). We have proposed that the PILP process may play a fundamental role in both vertebrate and invertebrate biomineralized tissues (8), and its potential relevance to bone formation will be described here. Bone consists of a hierarchy of structural features, and to produce such a hierarchical structure within the foreseeable future, without cellular control, is beyond the capabilities of the materials engineer. Even the most fundamental level of structure, the nanostructured arrangement of iso-oriented HAp crystallites embedded within collagen fibrils, remains a mystery as to how it is formed. Here, we present a model system for intrafibrillar mineralization which may help to shed a new light on understanding this most fundamental level of bone formation. We propose that the collagen fibrils can be infiltrated with a liquid-phase mineral precursor via capillary action created by the gaps and grooves of the collagen matrix. This hypothesis is based on various observations of the PILP phase, in which it is observed to seep into cracks and crevices due to capillary forces acting on the phase boundaries of the precursor phase. With respect to collagen mineralization in bone formation, once this liquid-phase mineral precursor was drawn into the fibrils, it would be expected to solidify (the waters of hydration are driven off by


75 thermodynamic driving forces), and subsequently crystallize into the more stable HAp phase. The crystals would likely retain the shape of the space within which they were deposited, as has been observed for our other studies on the PILP process (7), thus forming crystals of nanoscopic dimensions delineated by the surrounding collagenous matrix. This work is intended to demonstrate proof-of-concept for the ability to generate intrafibrillar mineralization of collagen using a liquid-phase mineral precursor. The mineral used in this study, however, is calcium carbonate (CaCO 3 ). Because CaCO 3 is not the mineral constituent of bone, it was not anticipated that this mineral system would lead to a nanostructural arrangement which mimics that of bone because calcium carbonate generally forms much larger crystals than hydroxyapatite. Selective etching techniques were applied to the composites, using either weak hydrochloric acid or dilute bleach solution, to remove the organic or inorganic phase respectively, in order to determine whether intrafibrillar mineralization was achieved. Materials and Methods Synthesis The synthesis of the polymer-induced liquid-precursor (PILP) phase of calcium carbonate has been described in detail in our previous reports (7, 37, 259). The process involves slowly raising the supersaturation of a calcium chloride solution with carbonate counterion, in the presence of micromolar quantities of a soluble, short-chained acidic polymer. The PILP process was originally discovered using the additive poly(aspartic acid), which is a simple mimic to the acidic proteins associated with biominerals; but polyacrylic acid (PAA) has been found to exhibit very similar behavior with respect to inducing the PILP process, and being less expensive, was used here. The counterion is


76 slowly added via vapor diffusion of the decomposition products of crushed ammonium carbonate (CO 2 and NH 3 ) into a solution containing calcium chloride and the polymeric additive. In the studies presented here, instead of depositing films of PILP phase onto glass coverslips, as described in our previous reports, collagen sponges were placed in the mineralizing solution as substrates. The collagen sponges (Cellagen sponge: ICN) are 1 mm thick, and composed of reconstituted type-I collagen from bovine tendon. Samples were prepared by cutting the Cellagen sponge into 3.25 x 1.6 mm 2 strips and placing them into a Falcon polystyrene petri dish (3.5 cm diameter), to which 1.5 mL of a filtered 24 mM calcium chloride solution was added. Appropriate L quantities (ranging from 0 333 g/mL) of dissolved polymer (PAA: M w = 5100, Aldrich) and distilled water were added to the petri dishes using a micropipette, to bring the final volume to 3 mL. A control dish, containing no PAA additive, was run along aside each set of experiments. All solutions were prepared with doubly distilled water and filtered using 0.2 m Gelman Acrodisc filters. Once the solutions were prepared, the dishes were covered with stretched Parafilm, into which three holes were punched with a needle to allow for slow vapor diffusion of the carbonate species into the solution. The crystallizing dishes were placed in an enclosed chamber (33 cm Nalgene desiccator), along with three small vials (5 mL) of crushed ammonium carbonate. The vials were also covered with stretched Parafilm and had one needle hole punched in the center to allow for diffusion of the decomposition products into the calcium/polymer crystallizing solutions. The desiccators were then held at 4C for three days, at which time the collagen was removed from the solution and rinsed with deionized water and ethanol, to remove any loose particulate matter and soluble salts.


77 After each 3-day interval, one sample was removed from the solution, while each of the remaining samples was placed into fresh calcifying solutions and the reaction continued. Thus, samples were sequentially mineralized up to five times, with reaction time periods of 3, 6, 9, 12, and 15 days. Analysis Acid etch and bleach treatment Since there is a large surface area of collagen fibers relative to the amount of PILP phase generated per crystallization, the samples were not homogeneously mineralized. Some regions had very thick mineral coatings, in which the fiber texture was no longer apparent, while other regions were uncoated. A weak acid etch was used to dissolve away some of the thick mineral coating. Samples were exposed to a 0.1M HCl solution for 15 minutes at 25C to preferentially etch the CaCO 3 In order to remove the organic matrix and determine the extent of mineral penetration, the samples were exposed to a dilute bleach solution (0.5 vol% NaOCl, prepared from a 1:10 dilution of 5% household bleach) for 15 minutes at 25C. After the etch treatments, the samples were thoroughly rinsed in deionized water to remove any remnant acid or bleach. Scanning electron microscopy (SEM) The samples (untreated, mineralized, and mineralized followed by subsequent acid etch or bleach treatment) were dried under vacuum at 30 40C overnight and sputter coated with Au/Pd. The samples were then examined with a JEOL 6400 SEM instrument, equipped with an energy dispersive spectrometer (EDS), at an accelerating voltage of 15kV.


78 X-ray diffraction (XRD) The PILP process produces non-equilibrium morphologies of mineral salts (7, 37, 260); thus identification of a specific crystallographic phase using SEM is impossible since the characteristic habit is not observed (i.e., rhombohedral calcite crystals). Therefore, x-ray analysis was used to determine the crystal structures of samples mineralized in the absence and presence of polymeric additives. 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. Transmission electron microscopy (TEM) Samples were prepared for transmission electron microscopy (TEM) following the protocols performed on bone and naturally mineralized tendon by Weiner and Traub. 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 staining with optional 1% phosphotungstic acid (PTA) in a PBS buffer to enhance the collagen contrast. The samples were then analyzed using a 200cx JEOL TEM at 200kV in brightfield (BF), selected area darkfield (SADF) and selected area diffraction (SAD) modes. Results and Discussion Collagen is considered the most important biopolymer in the regulation of bone structure. However, it is clearly not the sole source responsible for the regulation of bone mineralization since the majority of the body is composed of collagenous tissues that never mineralize (96, 132). Likewise, mineralization of type-I collagen in vitro by many investigators does not appear to reproduce the collagen/mineral structure of bone at the


79 nanoscopic level (191, 214). Thus, the role of the non-collagenous proteins associated with bone and dentin are deemed significant with respect to either inhibitory or promotory interactions during crystal nucleation and growth (100, 123, 261). Some of these proteins are highly acidic, and include proteins that are enriched in aspartic or glutamic acid residues, or phosphorylated serine/threonine (or sulfated glycoproteins) (3, 4, 262-265). We hypothesize that a possible function of these acidic proteins may be to serve as a process-directing agent, in which the polymer transforms the solution crystallization process into a precursor process. As we have demonstrated here, and in our other reports (7, 8, 37), the PILP process is an inherently different crystallization mechanism from standard solution processes, and it can have a profound influence on the crystal morphologies that result. Reconstituted type-I collagen has been used in a wide variety of biological applications, from the study of mineralization to skin grafts (191, 266). While CaCO 3 is not utilized in the mineralization of vertebrate structures, its use in studying biomineralization has been applied to various biological substrates, including elastin, chitin, collagen and cellulose (38, 41, 210, 213, 215, 267, 268). The Cellagen sponge (shown in Figure 4-2A and B) is reconstituted bovine collagen consisting of unoriented fibers of type-I collagen, ranging from 250 670 nm in diameter (269-271). When the sponge is placed in a mineralizing solution in the absence of polymeric additives, it serves as a substrate which provides heterogeneous nucleation sites for the calcite, as is shown in Figure 4-2C. Large rhombohedral crystals of calcite (20 40 m in diameter) are found randomly distributed along the entire surface of the collagen substrate. This is the characteristic morphology that would be expected for calcite grown via the classical


80 solution crystal growth process, in the absence of polymer at standard temperature and pressure (37). With the addition of micromolar quantities of PAA (200 g/ml), a patchy film-like coating of mineral was deposited onto the sponge substrate (Figure 4-3A). At higher magnification, it can be seen that the mineral film is actually quite thick because it had encased the individual collagen sub-fibers (Figure 4-3B). It is not immediately obvious that the mineral deposited on the fibers is calcite because the non-equilibrium morphologies produced by the PILP process lack the crystal facets that are typical of classical solution growth crystals. Therefore, energy dispersive spectroscopy (EDS) was used to confirm that the coated collagen surfaces had high calcium content, and x-ray diffraction (XRD) was used to identify the mineral phase as being calcite (see separate section on XRD analysis below). In comparison to other studies using collagen substrates (187, 188, 190, 191, 195, 215), a very different morphology is produced when the collagen substrate is mineralized using the PILP process. Instead of oriented crystallites, non-faceted crystals are deposited in the form of a mineral coating, which upon repeated application, fully infiltrates the matrix to form a highly mineralized composite structure. SEM analysis has been used to demonstrate intrafibrillar mineralization of collagen. Although the data presented in this paper is based upon the mineralization of type-I collagen with CaCO 3 whereas natural mineralization in vertebrates utilizes calcium phosphate, the emphasis is placed here on the mechanism of mineralization. The results obtained from this model system support our hypothesis that a plausible mechanism for intrafibrillar mineralization of collagen in bone and dentin


81 formation may occur via a PILP process, in which a liquid-phase precursor provides the appropriate medium for capillary action. In the initial experiments, only a patchy surface coating of mineral was achieved due to the small amount of PILP phase produced per batch relative to the total surface area of the collagen substrate. In order to more fully mineralize the substrate, repeated mineralization steps (up to 5 times) were used to transform the collagen sponge into a collagen-mineral composite (Figure 4-3). Using sequential mineralization steps, a collagenous sponge was transformed into a highly mineralized composite structure with an intimate association between the organic and inorganic phase. The samples became thicker upon sequential mineralizations (Figure 4-3C & D), as well as stiffer, as the mineral phase continued to infiltrate the matrix. The thickness increase is related to the encapsulation of the individual collagen sub-fibers, as is demonstrated by the step-edge of the mineralized patch in Figure 4-3B. The non-mineralized region of the sponge surrounding this patch became compact upon drying for SEM analysis, while the mineralized region retained the dimensions of the hydrated state of the collagen. Likewise, in the top-left region of Figure 4-3D, it can be seen that the dehydrated fibers collapsed, but were held apart by the surrounding mineralized regions. The banding of type-I collagen arises from the quarter-staggered structure of the micro-fibrils, which generates overlap and hole-zones of variable electron density, leading to the characteristic banded appearance at the nanoscopic level (64-67 nm hole zones are seen in TEM and AFM). Nucleation of crystals within hole-zones of reconstituted collagen has been observed using CaP by Glimcher and Krane (272); thus the idea of the holes zones acting as nucleating sites due to increased surface energy (as opposed to the phenomenon being


82 chemically or epitaxially driven) is plausible. In the early stages of our mineralization process, bands of calcite disks are observed, which lie perpendicular to the c-axis of the collagen fibers, with a spacing of roughly 250 500 nm. In the later stages of mineralization, the fibers become fully coated with a non-descript mineral phase, which bears some similarity to natural bone (as imaged by SEM). In order to determine the extent of mineral penetration, the samples were exposed to either a weak acid solution (0.1M HCl) or a dilute bleach solution (0.5 vol% NaOCl) for 15 minutes to preferentially etch the CaCO 3 or dissolve the collagen, respectively, and then examined by SEM (Figures 4-4). Since there is such a large surface area of collagen fibers relative to the amount of PILP phase, the samples were not homogeneously mineralized. Some regions had very thick mineral coatings, in which the fiber texture was no longer apparent, while other regions had fibers which were not coated at all. A weak acid etch was used to dissolve away some of the thick mineral coating, leaving behind only the CaCO 3 that was embedded within the collagen fibers, which apparently provided some protection to the mineral. Interestingly, the remaining intrafibrillar mineral exhibits a banded pattern, consisting of calcite disks spaced approximately 250 500 nm apart, with the disks oriented perpendicular to the c-axis of the collagen fibers (Figures 4-4A & B). Acid etching reveals that banded patterns of disk-like calcite are embedded within the encapsulated fibers. When the highly mineralized samples are exposed to a dilute bleach solution to remove the collagen, a coherent mineral structure is maintained (although not all of the collagen is necessarily removed if it is encased in the mineral). This seems to suggest that the composite consists of an interpenetrating network of


83 organic-inorganic phases. The extent of mineral penetration is also observed in cross sections of dissolved collagen fiber bundles, in which the CaCO 3 crystals appear to span completely across the pre-existing collagen bundles. In the samples exposed to the dilute bleach solution, the collagen was slowly dissolved, leaving mainly inorganic phase. Dissolution of the collagen has yielded varying results. In sections where the sample was highly mineralized (e.g., Figure 4-4C), the collagen cannot be seen due to its complete encapsulation within the CaCO 3 coating. Yet, in regions where the sample is not completely mineralized and the collagen fibers are accessible, the collagen appears to have been removed (Figure 4-4D), leaving behind disk-like crystals with a similar arrangement as the acid etched samples (Figures 4-4A & B). Figures 4-5A & B show higher magnification SEM images of the ends of two fiber bundles (10-15 m diameter) that were treated with the dilute bleach solution. These bundles are larger than most, and the end-on view shows a large amount of mineral throughout the cross-section of the fiber, which seems to indicate that the mineral had been deposited throughout the fibers, and not solely as disks in the banded regions. The structure of the composite is revealed upon etching, in which it is seen that the mineral phase spans across the diameter of the individual fibers, with a remnant pattern replicating the fibrous nature of the organic matrix that served as its template. Interestingly, after the bleach treatment, the remaining inorganic phase in some of the fibers appears to have a platy appearance (Figure 4-5B), and the platelets are oriented similarly to the platelets of bone (parallel to the fiber axis), resembling the deck of cards structure described by Weiner and Traub (101). However, these calcitic platelets


84 are an order of magnitude larger than hydroxyapatite platelets in bone, which is perhaps to be expected since calcite generally forms much larger crystals than HAp. Banding patterns were also seen in samples that were not etched with acid, and thus are not a result of a dissolution and recrystallization process. It is our contention that the liquid-phase mineral precursor is drawn into the collagen fiber by capillary action, which would be expected to preferentially occur at the hole-zones, thus leaving these regions of the collagen more fully entrenched with mineral. However, we did not expect the mineral to penetrate entirely across the sub-fibers in this transverse fashion, leading to the disk-shaped entities (it remains to be determined if these are single crystals, and if there is any preferential crystallographic orientation). The complete penetration across the sub-fibers seems to imply that large slots exist all along the collagen fibers; but this seems very unlikely unless the precursor phase caused some type of re-assembly of the collagen. An alternative explanation is that the gelatinous nature of collagen may allow for its molecular constituents to be encapsulated by the precursor phase, and ultimately become embedded within the solidified crystals. This would explain why the mineral traverses the diameter of the fibers, but it does not explain the banding pattern. For this aspect, we note that incremental growth steps, called transition bars, sometimes occur during the solidification and transformation of the PILP phase (7), which in this case could account for the periodic crystallization pattern. These transition bars are thought to arise from exclusion of impurities (i.e., the polymer), which in this case would include some of the collagen matrix, such that periodic increments of higher organic content would be generated.


85 The crystallographic phase in each sample was determined by x-ray diffraction (XRD). In all mineralized samples examined, the dominant crystallographic phase is calcite (Figure 4-6). The broad background peak (Figure 4-6-line A) corresponds to an as received sample of Cellagen sponge. Upon mineralization of the sponge without polymeric additive, large (~ 20-40 m) rhombohedral shaped crystals, which is the typical habit for solution grown calcite, formed on the surface of the sponge (Figure 4-2B). X-ray analysis showed an intense peak at 29.5, which corresponds to the (104) planes of calcite, confirming the identity of the calcite phase above the collagen background (Figure 4-6line B). Lastly, when the sponge was mineralized in the presence of PAA, which generated the CaCO 3 -collagen composite, the same calcite peaks were produced (Figure 4-6line C). However, there appears to be significant broadening of the peaks, which may indicate that the individual calcite crystals are of nanoscopic dimensions (or alternatively, that they are strained). The broadening of calcite peaks in the XRD spectra indicates that the mineral within the composite might be nanoscopic, which could be suggestive of intrafibrillar mineralization. In naturally mineralizing type-I collagen, intrafibrillar mineralization leaves mineral infused throughout the collagen matrix, indicated by electron dense regions in TEM micrographs of mineralized collagen. The native banding pattern of type-I collagen is not visible in TEM unless it is stained with an organometallic or is fully mineralized. Imaging our unstained, mineralized collagen composite, the 64 nm banding pattern of a single fibril is observed in Figure 4-7A. When a selected area aperture is placed over the center of the fibril and diffraction is performed, a nearly single crystalline pattern is obtained (Figure 4-7B). As calcium carbonate normally mineralizes in large


86 crystals, these data suggest that the PILP phase was drawn into the collagen, subsequently solidifying and then crystallizing into single crystals of CaCO 3 constrained by the collagen. While this is in sharp contrast to naturally mineralizing collagen, which displays arcs in diffraction due to a slight misorientation of platy hydroxyapatite along the collagen fibrils, it is not unexpected, as single crystals are a signature of CaCO 3 PILP phase mineralization. Based on experimental in situ observations from previous experiments (7, 37, 259), in which the liquid-phase CaCO 3 precursor was observed to form mineral coatings, it is concluded that, in this case, the PILP phase not only coated the individual collagen sub-fibers, but was also drawn into the swollen collagen matrix, presumably by capillary action. Phase boundaries exist between the PILP phase and the surrounding solution (as observed by optical microscopy (7)); therefore, capillary forces can be generated due to a liquid surface boundary of the appropriate dimensions and curvature. We have observed capillary effects in other experiments, in which the precursor phase seeps into cracks and crevices within our crystallizing apparatus. The metastable PILP phase then solidifies, resulting in space-filling crystals. Due to these observations, we have hypothesized that the space-filling properties of the PILP phase could provide a relatively simple biological means for "molding" crystals (8, 260), providing a plausible explanation for many of the elaborate morphologies of calcitic biominerals. Since the amorphous precursor phase is liquid-like in nature, it apparently coalesces after infiltrating the fibers and eventually crystallizes into a fused composite structure. This is a difficult mechanism to prove, but thus far, the evidence supports our hypothesis. Other mechanisms are conceivable, such as infiltration of the polymer additive into the collagen matrix, with subsequent


87 nucleation of crystals within the swollen fibers. However, this scenario seems unlikely in our system given that we observe that the crystallization proceeds via an amorphous precursor. Likewise, similar observations have been made regarding the existence of an amorphous precursor to HAp in secondary bone formation (96, 132). Applying three-dimensional tomographic imaging techniques to the early stages of calcification in naturally mineralizing turkey tendon (which is considered a model of secondary bone formation and structure), Landis (273) and Glimcher and co-workers (255) have demonstrated that the inorganic phase appears first at the gap regions within the collagen microfibrils. In their studies, the newly formed crystals had a platy appearance. However, Bonnuci (132) has observed bands of amorphous inorganic phase in the early stages of bone formation. Because these electron dense bands were aligned perpendicular to the c-axis of the collagen fibers (as seen by TEM), he referred to them as "inorganic substance in bands". These biological examples seem to correlate well with our in vitro model system. As a final comparison, consider the SEM analysis of dog femur by Bagambisa et al. (258). Although caution should be exercised when comparing microscopic features between two different mineral systems, we believe that the similar non-descript features of the mineral morphology of the HAp in their micrograph (Figure 4-1) to our mineralized collagen (Figure 4-3B & C) could be indicative of its formation via a precursor mechanism. It should be indicated that the micrograph in Figure 4-1 is not a faithful recreation of the appearance of natural bone in vivo, as this bone was prepared for SEM, which included removing any topographic soft organic matter (e.g., cells and connective tissue). The image is meant to illustrate the topographic data of the hard mineral phase of natural bone. Regardless, scanning electron microscopy was found


88 to be a useful tool for examining the extent of mineral infiltration in this model system, and provides a different perspective on the mineral morphology of natural bone.

PAGE 100

89 400 nm Figure 4-1. SEM micrograph of native bone at the surface of a canine femur diaphysis. Bundles of collagen fibers are described as being densely invested in mineral, sometimes causing bulging and clubbing of the fibers, and a granular surface texture (#s 1-4). The authors noted that some indications of collagen periodicity could be discerned in the loosely packed fibers (see lower left and upper right regions of the image). Note that this image of natural bone was taken after processing for SEM. The image is not meant to be faithful representation of bone in vivo, but meant to illustrate the surface topography of hard mineral (258). Bar = 400 nm. Used with permission of author.

PAGE 101

90 A) B) C) Figure 4-2. EM micrographs of Cellagen sponge samples. A) The sponge, as received, has randomly organized fibers which are relatively loosely packed (as compared to the collagen fibers in tendon or lamellae of bone), and have a diameter ranging from 250 670 nm. Bar= 20 m B) Bright field image of single stained Cellagen fibril (inset is selected area diffraction of center of fibril demonstrating amorphous diffraction pattern.) Bar = 200 nm. C) Collagen sponge mineralized without polymeric additive (control reaction) only provided a substrate for heterogeneous nucleation, in which large (40-50 m diameter), rhombohedral crystals of calcite were deposited randomly along the surface of the fibers. Bar = 100 m.

PAGE 102

91 A) B) C) D) Figure 4-3. SEM micrographs representing the sequential PILP mineralization process of Cellagen sponges in the presence of polyacrylic acid. A) After one mineralization step (a 3 day reaction), patchy calcitic films were deposited on the surface of the sponge. Bar = 100 m. B) A higher magnification view of the edge of one of the mineralized patches shows that individual sub-fibers are encased in mineral. Due to beam damage, a crack occurred in the region of the substrate that was not coated and protected by the mineral. There is likely high stress in this region due to the restraints applied by the encapsulating mineral during dehydration (vacuum drying for SEM). Bar = 5 m. C) After a second PILP mineralization (6 days), the amount of CaCO 3 deposited into the collagen sponge increased. This region shows that a fairly thick mineralized matt has formed (thickness indicated by arrow), which does not compact substantially upon dehydration. Bar = 50 m. D) After five sequential mineralizations (15 days), the composite has become a rigid brick, although a few regions remain open and porous. Bar = 10 m.

PAGE 103

92 B) A) C) D) Figure 4-4. SEM micrographs of mineralized samples subjected to etching, for determination of the extent of mineral infiltration. (A & B) These mineralized Cellagen samples were treated with 0.1M HCl for 15 minutes to remove excess surface coverage of mineral coating. A roughly periodic banding pattern is presented by the calcite disks, which are always observed to lie perpendicular to the c-axis of the collagen fibers. A) Bar = 5 m. B) Bar = 5 m. (C & D) These mineralized Cellagen samples were treated with dilute bleach solution (0.5 vol% NaOCl) for 15 minutes in order to remove the organic matrix. C) Fully mineralized samples which have a complete CaCO 3 coating are either not exposed to the bleach due to encapsulation and protection by the CaCO 3 coating, or the removal of collagen within can not be observed. Bar = 10 m. D) Surprisingly, after the bleach treatment, a coherent structure remained, which was comprised of disks of calcite that were evidently embedded within the pre-existing fibers. Bar = 5 m.

PAGE 104

93 A) B) Figure 4-5. SEM micrographs of mineralized collagen bundles that had been treated with a dilute bleach solution for 15 minutes. A) This cross-sectional view of a large 18 m diameter collagen bundle demonstrates the depth of mineral penetration into the pre-existing fibers. In this case, a banding pattern is not apparent, but instead, small crystallites appear to traverse completely across the bundle and link together the collagen sub-fibers. Bar = 10 m. B) The end of this fiber bundle shows aligned calcite crystals which exhibit a platy habit, somewhat similar to the deck of cards arrangement described by Weiner and Traub (101) regarding the spatial alignment of hydroxyapatite nanocrystals in naturally mineralizing turkey tendon. Bar = 5 m.

PAGE 105

94 Arbitrary Units (A.U.) Figure 4-6. XRD spectra of the different conditions of Cellagen mineralization. A) An XRD spectrum of the untreated Cellagen sponge, corresponding to the sample imaged in the SEM micrograph of Figure 4-2A, shows a diffuse amorphous peak as expected since the matrix material is non-crystalline and isotropically oriented. B) Spectrum-B is of the mineralized sample of Cellagen in the absence of polymeric additives, corresponding to the SEM micrograph in Figure 2b. The large rhombohedral crystals observed in Figure 4-2B are clearly identified as calcite by the XRD peaks, especially the most intense peak at 29.5 corresponding to the (104) planes of calcite. C) Spectrum-C is that of the Cellagen sample mineralized in the presence of PAA, corresponding to the SEM micrograph in Figure 4-3A. The XRD peaks are the same as those in spectrum-B (except broader), indicating that the non-descript thin films encasing the collagen are in fact calcite

PAGE 106

B) A) Figure 4-7. Transmission electron micrographs of mineralized Cellagen. A) Bright field image of type-I collagen fibril mineralized with CaCO 3 PILP phase. The differences in electron density demonstrates intrafibrillar mineralization, as more mineral is located within the hole zones. Bar = 66 nm. B) Selected area diffraction of the center of the fibril in A. The nearly single crystalline diffraction pattern of CaCO 3 H 2 O suggests that although the mineral is intrafibrillar, it is uniaxially oriented within the fibril. 95

PAGE 107

CHAPTER 5 INTRAFIBRILLAR MINERALIZATION OF TYPE-I COLLAGEN VIA A CALCIUM PHOSPHATE PILP PHASE Introduction In order to truly understand bones exceptional properties, it is necessary to examine the foundation, intrafibrillarly mineralized collagen fibrils. The intimate relationship between the uniaxially oriented, nanometer sized hydroxyapatite crystals and the self-assembled fibrillar collagen provide bone with its remarkable mechanical and bioresorbable properties. From a materials science viewpoint, the extent of mineral penetration into the collagen fibrils makes it difficult to classify bone as one specific type of composite. The dense nature of the mineral phase, encasing the fibrillar collagen, suggests that bone is a fiber-reinforced ceramic-matrix composite. In sharp contrast to this observation is the fact that ex-situ examination of deproteinated bone reveals 25-50 nm platy hydroxyapatite crystals, implying that bone may be better labeled as a nanoparticle-reinforced polymer-matrix composite. The morphology of hydroxyapatite (HAp) might be an indication as to why this mineral was evolutionarily selected for vertebrate hard tissues, as opposed to CaCO 3 which is the main mineral phase observed in invertebrates. CaCO 3 forms very large crystal structures, and although we have shown that type-I collagen can be intrafibrillarly mineralized with such a mineral phase, the structure appears to be large crystals formed within the collagen. Structurally, this is not beneficial because it allows crack propagation to occur rapidly through the solid composite. Fortunately, the platy phase of 96

PAGE 108

97 hydroxyapatite in bone is not completely uniaxially oriented. As observed through electron microscopy, the plates, while considered to be oriented in the [001] along the long axis of the collagen fibril, have a slight misorientation from the [001] direction (10, 11, 14). Because the mineral phase is platy and slightly misoriented, crack propagation would be deterred at each plate interface because of the increasingly tortuous path. The prevailing theory on the mineralization of collagen in hard tissues, first proposed by Glimcher and later supported by Weiner et al. and Landis et al., is the idea that HAp is nucleated in the hole zones of collagen, and subsequent mineral continues to grow on these initial mineral deposits such that the mineral grows along the tropocollagen, eventually fusing with other mineral deposits (10, 11, 103, 105, 118). The organic matrix of bone, type-I collagen, is thought to play a key role in the calcification due to its unique hierarchical organization of tropocollagen units in a quarter-staggered structure which allows 40 nm gaps zones between each unit where nucleation is first observed (99). Initially, hydroxyapatite deposits within the 40 nm hole zones between the ends of collagen fibrils and continues to grow along the fibrils until fusing with similarly mineralized deposits (6). The collagen structure is obviously not the main factor, as the lattice mismatch between collagen and hydroxyapatite is unfavorable for nucleation, as well as the observance of type-I collagen in connective tissues that do not undergo calcification throughout the body (122). There have been numerous studies on the early stages of mineralization, both in bone and in naturally mineralizing turkey tendon. Naturally mineralizing turkey tendon serves as a model system of intrafibrillar mineralization, in which primary deposition of mineral is observed in the hole zones (6, 96, 118, 121). Of these studies, there are a handful that indicate a deposition of a

PAGE 109

98 transient amorphous calcium phosphate or octacalcium phosphate precursor that is primarily deposited within the whole zones, both of which subsequently transform into hydroxyapatite (116, 127, 253). Yet, in vitro replication has been far from complimentary to this natural system, as achieving a fully dense, oriented, intrafibrillarly mineralized typeI collagen substrate has eluded researchers. Researchers have attempted to achieve in vitro intrafibrillar mineralization for decades. Scientists first introduced reconstituted bovine or porcine type-I collagen to simulated body fluid (SBF), only to find that while the collagen substrates served as an excellent substrate for nucleation of large mineral crystals on the surface of the fibrils, there was little evidence of intrafibrillar mineralization (12). Since that time, there have been many attempts at both modeling and recreating this phenomenon using synthetic and natural materials. The emphasis was placed on creating an environment that would facilitate preferential nucleation and subsequent orientation of calcium phosphate on a biocompatible substrate, such as synthetic amphiphiles and reconstituted collagen (191, 199, 231, 235). A great deal of success has been achieved in demonstrating that site specific nucleation can encourage hydroxyapatite to nucleate and grow along the [001] direction, as is seen in naturally mineralized collagen (199, 231, 235). However, there has been lack of evidence to suggest that a composite has been synthesized that has an oriented apatite phase that fully penetrates the organic substrate, thereby achieving high mineral loading. Using a biomimetic mineralization synthesis called the polymer-induced liquid-precursor (PILP) process, we have demonstrated the ability to create a highly loaded mineral/collagen composite in which the mineral is comprised of platy hydroxyapatite oriented in the [001] direction along the long axis of the collagen fibrils.

PAGE 110

99 Methods and Materials Synthesis We mineralized a reconstituted collagen sponge (Cellagen, ICN Biomedicals Inc.), which is comprised of pure type-I bovine collagen, with HAp mineral using the PILP process. This process has been documented in the case of forming CaCO 3 biomimetic minerals (e.g., thin films resembling nacre and fibrous calcite structures that have striking similarity to echinoid teeth), but never CaP biominerals. The process involves using a 1 mM CaCl 2 2H 2 O solution in combination with 200 g/mL Poly(ba-DL-Aspartic acid) [Polyasp] and 200 g/mL Poly(vinyl phosphonic acid) [PVPA]. The collagen sponge is cut in to a 3 x 3 cm 2 sample and placed in the mineralizing solution. This solution is then placed, uncovered, in a desiccator with three equal sized petri dishes, uncovered, and filled with crushed diammonium hydrogen phosphate. Uncovered is stressed in this explanation because in the CaCO 3 PILP process, all of the dishes are covered with parafilm into which three holes are punched to allow diffusion. The ammonium phosphate decomposes much slower than the ammonium carbonate, therefore the reaction kinetics had to be increased. Additionally, to decompose the ammonium phosphate more rapidly, and to mimic physiological conditions, the temperature was raised to 37C. The ammonium phosphate slowly decomposes and feeds the counterion into the mineralizing solution. The samples are left to mineralize between 1-4 weeks at which time they are removed from solution and rinsed in both water and ethanol to remove any soluble salts.

PAGE 111

100 Analysis Scanning electron microscopy (SEM) 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 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) Samples were prepared for transmission electron microscopy (TEM) following the protocols performed on bone and naturally mineralized tendon by Weiner and Traub (101). 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 staining with 1% phosphotungstic acid (PTA) in a PBS buffer. The samples were then analyzed using a 200cx JEOL TEM at 200kV in brightfield (BF), selected area darkfield (SADF) 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. When an objective aperture is placed over a diffraction spot (diffraction plane), the only electrons that are available for imaging are those originating from that plane. This converts a BF image into a SADF image, in which the only

PAGE 112

101 sections of the image that are illuminated are those that are from the selected diffraction spot. Results and Discussion In previous studies we have demonstrated a synthetic method by which to generate such a liquid precursor phase to Ca based biominerals, such as CaCO 3 and CaP, through the addition of micromolar amounts of highly charged acidic polymers to supersaturated crystallizing solutions (7, 37). We consider this a biomimetic method because these polymers are analogous to acidic residue rich polypeptides observed in biominerals. The charged polymer inhibits nucleation while promoting phase separation in solution. Using this polymer-induced liquid-precursor (PILP) process, we have shown an ability to mold CaCO 3 into patterned thin films and deposit calcite nanofibers on calcite rhombs, duplicating various biomineral morphologies (35, 274). Initial attempts at recreating intrafibrillar mineralization were limited to the use of CaCO 3 due to synthetic difficulties in creating a CaP PILP phase, yet the results served as proof of concept, as the data showed that each collagen fibril could be infiltrated with non-descript calcite mineral phase, resulting in a highly loaded collagen/mineral composite (8, 13). Through the addition of micromolar quantities of low molecular weight polyvinyl phosphonic acid (PVPA) and poly(,-D,L) aspartic acid, and diffusion of the phosphate counterion through evaporation of an ammonium dihydrogen phosphate solution, we have successfully generated a precursor to CaP mineral using the PILP process. With this breakthrough, we have been able to achieve intrafibrillar mineralization of a reconstituted type-I collagen sponge to synthetically recreate the nanostructured architecture of bone.

PAGE 113

102 We propose that collagen in mineralized tissues does not initially nucleate within the hole zones, but is delivered as a liquid-phase amorphous mineral precursor, which is drawn into the collagen fibrils via capillary action and subsequently mineralizes, leaving the collagen embedded with nanoscopic platelets of hydroxyapatite. The crystallites are aligned and oriented by the collagen (Figure 5-1). As the organic component in bone is type-I collagen, we chose a reconstituted type I bovine-collagen sponge to serve as a substrate (Fig. 5-2A). The reconstituted bovine collagen is type-I collagen, as seen by the native 64-67 nm banding pattern of type-I collagen, visualized using a 1% phosphotungstic acid in 0.1M PBS negative stain. While type-I collagen diffracts x-rays (275), a single fibril of collagen does not diffract electrons, as evidenced by the amorphous diffraction pattern of a single collagen fibril (inset in Figure 5-2A). Therefore, any diffraction observed in our studies may assuredly be from the mineral phase. In both bone and naturally mineralizing turkey tendon, the organic substructure is organized before mineralization occurs, therefore, instead of attempting to mineralize in the presence of fibrillogenesis, in which other groups have had partial success (199, 235), we felt that in order to fully recreate the nanoscale morphology of bone, a pre-existing collagen substrate should be used. They collagen sponge was placed in the mineralizing solution and was allowed to interact with the PILP phase. In the early stages of mineralization, 50-70 nm amorphous droplets (arrows) are observed in close association with single collagen fibrils (Figure 5-2B). The dimensions of the amorphous nanoscopic droplets are identical to those in our preliminary work with the HAp PILP phase, which eventually transform to a stable hydroxyapatite phase. The

PAGE 114

103 remnant particles in Figure 5-2B appear to be attached and intimately associated with the stained collagen fibril (arrows), and while they are now solid (due to vacuum drying in sample preparation), in solution the droplets are a liquid-like phase. As they are fluidic, they could accumulate on the collagen surface and subsequently be drawn into the 40 nm hole zones via capillary action, thereby fully infiltrating each collagen fibril with the mineral precursor. Upon solidification, in which the waters of hydration are thermodynamically driven off the metastable precursor phase, the fibrils would be embedded with nanoscopic crystals of the mineral. Once the PILP phase fully infiltrates the collagen substrate, it subsequently solidifies and crystallizes into crystalline HAp. As we have previously shown with CaP, we can create collagen/mineral composites in which each individual collagen fibril is fully encased in mineral through the PILP phase (8, 13). The same non-descript high mineral loading has can also be demonstrated using the CaP PILP process (Figure 5-2C-F). When the collagen substrate is introduced into the crystallizing solution, it swells allowing the PILP phase to be drawn in via capillary action. As the amorphous precursor solidifies and crystallizes, it entraps the collagen fibrils in the dimensions of the hydrated state. When the sample is dehydrated, the unmineralized fibrils dehydrate and shrink, while the mineralized sections retain the shape of the swollen state (Figure 5-2C). It is generally accepted that non-collagenous proteins (NCPs), observed in the regions of bone growth, play a major role in calcification due to their ability to bind calcium and high affinity for collagen (276). NCPs, such as osteonectin and phosphoproteins, contain many acidic residues. We argue that the role of the acidic proteins may be to serve as a process directing agents that transform the solution

PAGE 115

104 crystallization process to a precursor process, in which solidification provides a powerful means of controlling crystal morphology. As previously mentioned, there have been various observations of amorphous calcium phosphate deposits, described inorganic substance in bands (ISBs), detected in hole zones in early-mineralized bone (6). Synthetically, amorphous calcium phosphate, a common precursor to hydroxyapatite, has only been described as a gel phase, formed due to a high concentration of ions. A weak acid etch was used to dissolve away some of the thick mineral coating, leaving behind only the CaP that was embedded within the collagen fibers, which apparently provided some protection to the mineral. Interestingly, the remaining intrafibrillar mineral exhibits a banded pattern, consisting of calcite disks spaced approximately 250-500 nm apart, with the disks oriented perpendicular to the c-axis of the collagen fibers (Figures 5-3A & B). Acid etching reveals that banded patterns of disk-like calcite are embedded within the encapsulated fibers. When the highly mineralized samples are exposed to a dilute bleach solution to remove the collagen, a coherent mineral structure is maintained (although not all of the collagen is necessarily removed if it is encased in the mineral). This seems to suggest that the composite consists of closely associated organic-inorganic phases. The extent of mineral penetration is also observed in cross sections of dissolved collagen fiber bundles, in which the CaP crystals appear to span completely across the pre-existing collagen bundles. When the collagen is etched away using a 0.1% NaOCl solution, the mineralized fibrils display completely solid structures (Figures 5-3C & D), suggesting that the mineral has fully penetrated the collagen fibrils thereby protecting coated sections from the etchant. If the collagen fibrils were simply coated with mineral, it would be expected

PAGE 116

105 that the collagen would be etched away, thereby leaving mineral tubules. The surfaces of the mineralized collagen fibrils (Figure 5-2C) are similar to those observed in natural bone (13). Yet, we did image a mineralized fibril that appeared to have oriented platy crystals along its c-axis (Figure 5-2F). The 50-100 nm long platy crystals, while reminiscent of those observed in deproteinated bone, do not provide pure evidence of crystal structure or orientation. Using transmission electron microscopy (TEM), we observed a section of mineralized collagen that shows intrafibrillarly-mineralized hydroxyapatite aligned in the [001] direction along the long axis of the collagen fibrils, comparable to the mineral found in bone (Figure 5-3). Figure 5-3A is a single isolated collagen fibril mineralized with HAp PILP phase. Although the mineral is not easily visualized in the brightfield image, using selected area diffraction (SAD) of the middle of the fiber yields a diffraction pattern identified as HAp (Figure 5-3B). More importantly, the (002) and (004) planes are aligned with the long axis of the collagen fibril (arrow), identical to diffraction patterns observed in mineral in human femur (14). The arcing of the (002) and (004) planes suggests that the hydroxyapatite crystals are not uniaxially oriented along the c-axis of the collagen fibril, an observation which has been corroborated in naturally mineralizing collagen through three dimensional electron-diffraction tomography (10). In order to visualize the dimensions of the platy crystals, an objective aperture was placed over the (002) diffraction plane and then visualized in BF mode (described as selected area dark field (SADF)). By limiting the illumination intensity to that diffracting from the (002) plane, only the crystals satisfying the Bragg condition in that plane would illuminate in the BF image, thereby illustrating their spatial coordinates and orientation

PAGE 117

106 within the field. The resultant image, Figure 5-3C, clearly illustrates 25-100 nm long needle/platy crystals oriented along the long axis of the collagen fibril. As morphological data derived from TEM data is only a two-dimensional projection, the platy crystals observed in the SADF image could be located on the surface of the collagen fibril, as is the case in Figure 5-2F. Therefore, we prepared the TEM sample used to obtain data for Figure 5-4A-C for SEM. Upon locating the exact fibril used to show oriented intrafibrillar mineralization, the surface of the 200 nm diameter mineralized fibril appear smooth, thereby confirming that the 25-100 nm long crystals are indeed within the type-I collagen framework. This discovery of intrafibrillar mineralization of type-I collagen with oriented HAp mineral via a synthetic polymer-induced liquid-precursor (PILP) process not only illustrates the possibility of mineralization without site-specific nucleation and growth, but also sheds new light onto existing theories of calcification of mammalian collagenous structures. The orientation of the hydroxyapatite mineral was quite unexpected because we did nothing to specifically modify the collagen to induce intrafibrillar mineralization. We speculate that because the amorphous mineral phase is shaped by the collagen before crystallization occurs, the charge groups on the collagen might be responsible for orienting the transformation from amorphous to crystalline HAp. By using acidic-rich biomimetic polymers, corollaries to NCPs in natural bone, we have demonstrated that an amorphous mineral precursor phase similar to that observed in naturally mineralizing collagenous tissues, can be used to create a highly loaded mineral/organic composite with 25-100 nm long platy HAp mineral oriented in the [001] along the long axis of the collagen fibril. By synthetically recreating the nanostructure of natural bone at the

PAGE 118

107 nanoscale in a petri dish, we feel that this technology might be used to build composites that not only have the organization of bone, but also match its unique bioresorbable and mechanical properties.

PAGE 119

108 Figure 5-1. Schematic illustrating the mineralization of type I collagen via a polymer-induced liquid-precursor (PILP) process. A) Type-I collagen fibril composed of quarter-staggered atellocollagen units, comprised of a triple helix of 300 nm long collagen units. B) Type-I collagen substrate introduced into a PILP crystallizing solution swells and absorbs the 50-100 nm liquid-amorphous droplets via capillary action, beginning in the hole zones and subsequently drawn along the atellocollagen subunits. C) After each individual collagen fibril is infiltrated with the PILP phase, the amorphous precursor subsequently crystallizes, leaving nanocrystals aligned with the long axis of the collagen fibril, resulting in an intrafibrillarly-mineralized composite.

PAGE 120

109 B) A) D) C) E) F) Figure 5-2. Electron micrographs illustrating various stages of mineralization using the PILP process. A) Brightfield (BF) micrograph of unmineralized type-I collagen that has been stained with 1% phosphotunstic acid (PTA) in phosphate buffered solution (PBS) depicting the native 64 nm banding pattern perpendicular to the long axis of the collagen fibril. (inset: selected area diffraction (SAD) of collagen fibril (arrow). Note that only an amorphous halo is observed, suggesting there is no existing mineral deposited within the collagen.) Bar = 200 nm. B) BF image illustrating amorphous PILP droplets

PAGE 121

110 on the order of 40-50 nm in diameter (arrows) in close association with collagen fibrils. Faint outlines of platy contrast appear along the long axis of the collagen fibril. C) SEM micrograph illustrating a partially mineralized section of collagen. The dehydrated collagen fibrils (left) can be traced across the sponge through the mineralized portion (right) in which the each of the fibrils is fully infiltrated with mineral, thereby retaining its hydrated dimensions. Note that the mineral phase is has a rough, non-descript appearance, much like that of naturally mineralized bone (258). A few solidified PILP droplets are also present. Bar = 10 m. E) SEM micrograph of mineralized collagen fibrils. Bar = 10 m. F) Higher magnification of a single HA PILP mineralized fibril from area marked with a # in D). The appearance of platy crystals along the c-axis of the fibril (arrow) suggest the mineral is oriented parallel to the fiber, similar to bone. Bar = 200 nm. G) Higher magnification of a bungle of mineralized fibrils from area marked with a in D. Bar = 1 m.

PAGE 122

111 A) B) C) D) Figure 5-3. SEM micrographs of mineralized samples subjected to etching, for determination of the extent of mineral infiltration. (A & B) These mineralized Cellagen samples were treated with 0.1M HCl for 15 minutes to remove excess surface coverage of mineral coating. A roughly periodic banding pattern is presented by the calcite disks, which are always observed to lie perpendicular to the c-axis of the collagen fibers A) Bar = 5 m. B) Bar = 5 m. (C & D) These mineralized Cellagen samples were treated with dilute bleach solution (0.5 vol% NaOCl) for 15 minutes in order to remove the organic matrix. C) Fully mineralized samples which have a complete CaP coating are either not exposed to the bleach due to encapsulation and protection by the CaP coating, or the removal of collagen within can not be observed. Bar = 10 m. D) Surprisingly, after the bleach treatment, a coherent structure remained, which was comprised of disks of calcite that were evidently embedded within the pre-existing fibers. Bar = 10 m.

PAGE 123

112 G) F) E) D) C) B) A) Figure 5-4. Electron micrographs of mineralized collagen fibrils. A) TEM Bright Field image of a PILP mineralized collagen fibril. Note the 64 nm banding pattern of type-I collagen fibril that appears perpendicular to the long axis of the collagen fibril (highlighted by the arrow). Bar = 100 nm. B) Selected area diffraction (SAD) pattern of middle of the fiber in A, indicating that the mineral is hydroxyapatite. Note the 002 and 004 planes are oriented along the long axis of the collagen fibril (long arrow) specifying that the mineral is

PAGE 124

113 oriented in the [001] direction. The arching of these planes indicates that the HA crystals are not uniaxially aligned with the long axis, but have a slight misorientation, which is also observed in natural mineralizing turkey tendon (10). C) Selected area dark field (SADF) of fiber in A, highlighting the (002) plane. Note the illuminated needlelike crystals are 25-50 nm long and oriented along the long axis of the collagen fibril. Bar = 200 nm. D) SEM micrograph of mineralized collagen fibril in A). There appear to be no platy crystals on the surface, suggesting that the collagen fibril is intrafibrillarly mineralized. Bar = 100 nm. E) BF micrograph of 50-year-old human femur showing platy appearance of mineralized collagen fibrils. Bar = 50 nm (277). F) BF micrograph of increase magnification of section in E). Bar = 50 nm (277). G) Electron diffraction pattern taken at the edge of F) showing the arcing of the oriented hydroxyapatite mineral, suggesting there is slight misorientation along the c-axis (arrow) (277). Figures E-G were Reprinted from the Journal of Structural Biology, Vol. 126, S. Weiner, W. Traub, H. D. Wagner, Lamellar bone: Structure-function relations, pg. 241, (1999), with permission from Elsevier.

PAGE 125

CHAPTER 6 CONCLUSIONS The work contained within this dissertation will one day play a pivotal role in the understanding of amorphous precursor phases observed within biomineralization. We suggest that certain types of biomineralization could feasibly proceed via a liquid-phase mineral precursor mechanism, induced by soluble acidic proteins. Although a liquid phase precursor mechanism has yet to be observed in an in vivo biomineralization system, the ex situ evidence of amorphous deposits and granules, which eventually crystallize, points towards its plausibility. We feel confident in stating that through the addition of highly charged acidic proteins and polymers (corollaries to soluble biological proteins observed occluded within biominerals) to Ca-based mineral crystallizing solutions, we can synthetically generate such a liquid-phase mineral precursor. Using this polymer-induced liquid-precursor (PILP) process, we can synthesize composites with morphologies similar to biominerals such as teeth, soil and bones. Eventually, in vivo research of biominerals will reveal that Nature does indeed mold biominerals through a precursor phase, and as the evidence is reported in the biomineralization community, this work will be highly cited as the first synthetic analog. The impetus of this research was to demonstrate that by using the PILP process, the intricate morphologies observed in biominerals could be synthetically reproduced, thereby justifying a necessity for researchers to investigate biomineralization for such a precursor. The preceding studies performed on the PILP phase demonstrated its fluidic nature and means by which to control its deposition onto various micro-patterned, 114

PAGE 126

115 functionalized substrates (35, 260). This research advances these concepts by applying the PILP phase to various substrates observed in biomineralization processes. The first substrate, a calcite rhomb, demonstrates how the formation of calcite fibers, specifically those found in sea urchin spiculogenesis and in the formation of teeth, could occur naturally. The second and arguably more important substrate is reconstituted type-I collagen, which is the organic matrix into which hydroxyapatite intrafibrillarly mineralizes to form the basis of mammalian dentin and bones. As mentioned above, observed in ex situ observations of both of these processes are the presence of amorphous deposits and granules that have dehydrated due to the various analysis techniques performed in order to study crystallinity and composition. The feasibility of these amorphous phases has been demonstrated synthetically, but only in the form of a gel, never as a liquid. By demonstrating a liquid-phase mineral-precursor, synthesized under biological temperatures and pressures, and used to create morphologies similar, if not identical, to those observed in biominerals, we feel that this research will assist in re-defining the manner in which biomineralization is studied. Even though CaCO 3 is not the mineral utilized by Nature to form bone, applying the CaCO 3 PILP phase to a type-I collagen substrate gave us our first glimpse of intrafibrillar mineralization. We determined that a type-I collagen reconstituted bovine collagen, Cellagen, was a good substrate to use for mineralization because it was comprised of 200-400 nm diameter type-I collagen fibrils, thereby providing us with a uniform substrate. Each individual fibril in the substrate was mineralized with a non-uniform, rough coating which had an appearance much like that of natural bone. Etching studies, to either remove the organic matrix or preferentially dissolve the mineral, were

PAGE 127

116 done in order to show the extent of mineralization. When the collagen was removed by dilute hypochlorite (NaOCl), the mineralized fibrils remained solid; suggesting that the intrafibrillar mineralized collagen was protected from the etchant. Fully mineralized composites etched with dilute hydrochloric acid (HCl) demonstrated disks perpendicular to the long axis of the collagen fibril, suggesting that preferential mineralization was occurring, as the remaining mineral was deposited in the hole zones of the collagen making it less soluble to the acid. This was clearly demonstrated using transmission electron microscopy (TEM), as a single mineralized, un-stained fibril showed the 64 nm banding pattern of collagen, caused by a difference in electron density of the mineral within the hole zones. Electron diffraction of the fibril showed a single crystalline diffraction pattern, indexed as a hydrate form of CaCO 3 The single crystalline nature of the mineral suggests that although the mineral is separated by the tropocollagen units, it retains a single orientation along the fibril. At this point in the research, the ability to form CaP PILP phase had not yet been achieved, and therefore, a brief research interlude occurred. Initially the deposition of the CaCO 3 PILP phase onto calcite rhombs was performed in order to show the effect of epitaxy on the PILP phase. The preliminary work performed on the PILP phase was confined to amorphous fused silica glass (7), and therefore we wanted to illustrate how a crystalline surface would interact with the thin amorphous PILP coating. Yet, a curious phenomenon occurred, as 100-800 nm diameter single crystalline calcite fibers were observed growing off the calcite seed crystals. Interestingly, sea urchins employ the same seed crystal mechanism in growing spicules. During spiculogenesis, a single calcite seed is deposited in the plasmalamella, followed

PAGE 128

117 by deposition of amorphous granules onto discrete positions on the rhomb, resulting in tri-radiate spicule growth. Comparing our synthetic results with the phenomenon of spiculogenesis, we feel that the data are similar enough to suggest that the amorphous granules observed in spiculogenesis could in fact be PILP droplets. The proposed mechanism and morphology of these single crystalline calcite fibers has also prompted a comparison to the high temperature synthesis of semiconductor fibers. Semiconductor materials, such as GaN and Si, can be synthesized in a fibrous form through two different high temperature mechanisms, the vapor-liquid-solid (VLS) and solution-liquid-solid (SLS) processes. A single metal flux droplet (flux is defined as a single piece of metal raised above its melting temperature) is used to sequester ions, from either vapor or solution, at which point a supersaturation is achieved within the flux and the semiconductor fibrous crystal emerges from the flux. Since the flux melts at a lower temperature than the crystal, it remains as a liquid, retaining subsequent ions to increase the length of the fiber. The key characteristic feature is a remnant bobble retained on the head of the fiber. Although we are not using a metallic flux droplet, we also observed large remnant bobbles on the heads of our fibers, which we attribute to a continual addition of flux droplets to a primary deposited PILP droplet. Upon depositing onto a crystalline surface, the PILP droplets undergo surface recognition and begin to solidify at the droplet/crystalline surface, while the top of the droplet remains as a liquid. Subsequent droplets then agglomerate onto the tops of primary droplets, further extending the fiber. It is important to again note that synthetic calcite does not adopt a fibrous morphology, and it is because the amorphous precursor is shaped into a fiber and constrained within a phase boundary, that calcite crystallize only in the shape of that

PAGE 129

118 container (in this case a fiber). As this mechanism was modeled after the VLS and SLS, we denoted it as the solution-precursor-solid (SPS) process, a process that provides a tangible explanation as to spiculogenesis in sea urchins and correlates two seemingly unrelated fields, biomineralization and high temperature semiconductor material synthesis. After wrestling with this scientific anomaly, and spending a deal of time developing the SPS theory, it seemed necessary to return my efforts back to the collagen mineralization work. Fortunately, at this time we had just started to develop a CaP PILP phase. Through the addition of a phosphorylated polymer, polyvinyl-phosphonic acid, and the diffusion of phosphate ions through vapor diffusion of ammonium dihydrogen phosphate, we were able to create a CaP PILP phase that behaved similarly to the CaCO 3 PILP phase. Applying this to the Cellagen sponge, we were able to fully recreate the nanostructure of natural bone through intrafibrillar mineralization. As was reported with the calcite mineralization, each individual collagen fibril was mineralized, and the composite structure behaved similar to etching studies. Yet, unlike the CaCO 3 work, we were able to accurately demonstrate the orientation of the mineral phase in the collagen fibrils. Using selected area diffraction (SAD) and selected area dark field (SADF), we illustrated that the collagen was infiltrated with 25-100 nm long platy hydroxyapatite crystals oriented in the [001] direction along the long axis of the fibril. The orientation of the mineral phase matched so closely to natural bone that even the (002) and (004) planes arced approximately 15 from either side of normal along the c-axis, identical to that reported in mineralizing turkey tendon (a naturally mineralizing collagen that has been used to model bone mineralization) and in human bone.

PAGE 130

119 A final discussion must be made concerning the exact mechanism of the type-I collagen mineralization using a liquid-phase mineral precursor. From the initial work done on the PILP process, it was observed that precursor phase was indeed liquid-like in nature (7, 37). As this phase was observed to flow in small spaces, it was assumed that it was subject to capillary action due to its physical nature (260). Type-I collagen has a quarter-staggered structure, which in the swollen state has 40 nm hole zones between the tropocollagen units. We assume that these nanometer zones can impart capillary action on the PILP phase, thereby drawing it in along the collagen fibril, where it subsequently crystallizes. This is assumed because as our control experiments, as well as other research, has shown, the collagen substrate alone does not serve to induce intrafibrillar mineralization, but only to nucleate large crystals on the surface of the fibrils (225, 230). While we have demonstrated intrafibrillar mineralization with a liquid-phase mineral precursor, we have yet to fully demonstrate that the PILP droplets are drawn into the collagen via capillary action. The research included within this dissertation illustrates that intrafibrillar mineralization is achievable using a precursor process, and future research will be aimed at proving the exact mechanism by which this occurs. This can possibly be achieved by examining the samples through nuclear magnetic resonance (NMR) at various times during mineralization to show how the amorphous phase crystallizes within the collagen. NMR has been used to study the mineral composition of natural bone (278-284), and therefore, there is a wealth of data to which we can compare our results. Using standard NMR techniques, we can determine how the CaP PILP phase is progressing, and compare it to literature on synthetic HAp and other naturally mineralizing samples.

PAGE 131

120 Using solid-state nuclear magnetic resonance (SSNMR), we can determine the phase of the CaP PILP as it mineralizes a type-I collagen sponge, such as Cellagen. Comparing the spectrum at various times to standards of collagen and CaP spectrum, we can deduce the phase of the mineral. One drawback to this process will be the amount of sample needed for analysis and sample preparation. In order to do these NMR techniques, a large amount of sample (~150 mg) is required. Generating enough composite material is difficult because of the low density of collagen, and the scalability of the PILP phase. Additionally, the NMR tubes are rather small, and a large amount of PILP mineralized composite would need to be crushed in order to fit enough of it into the tube. At later stages of mineralization, this would not be a problem, but at early stages, where the mineral is supposedly amorphous, a severe roadblock might arise. Because the PILP phase is highly metastable, crushing of the sample might induce crystallization, thereby sabotaging the purpose of the experiment, to observe the early amorphous phase. While these experiments would still not fully answer the questions regarding capillary action, if we could get enough sample into a sample tube without altering the mineral phase, it would demonstrate that the mineral does not nucleate intrafibrillarly, but begins as an amorphous phase that infiltrates each fibril before crystallization occurs. The results of this work clearly demonstrate how a liquid-phase mineral-precursor can be synthetically generated to recreate composite morphologies similar to those observed in biominerals. There have been a variety of discussion as of late within the biominerals community as to the existence of amorphous precursor phases, and this research takes that idea one-step further by suggesting that future studies of biomineralization should be conscience that these amorphous phases might be fluidic in

PAGE 132

121 nature. Eventually, we feel that an addendum will be added to the three main tenants of biomineralization, under the role of soluble acidic proteins playing a significant role in the formation of biominerals. It will include the idea that soluble acidic proteins lead to induction (stabilization) of an amorphous liquid-phase mineral precursor, which can be molded or shaped into a variety of sizes and dimensions before subsequently crystallizing. Application of this work stretches far beyond the re-creation of biomineralization, as it has profound significance in the material science community. It demonstrates that ceramics can be synthesized through a liquid-precursor mechanism at ambient conditions. As already illustrated by this work, various composite structures with morphologies identical to those observed in Nature can be synthesized for use in the biomedical field as graft substitutes, including bones and teeth. Yet, future applications might include the production of thin film ceramic materials for the computer and defense industry.

PAGE 133

LIST OF REFERENCES 1. R. E. Hummel Understanding Materials Science : History, Properties, Applications (Springer, New York, 1998), pp. 407. 2. S. Mann, Mineralization in biological systems. Structure and Bonding 54, 125 (1983). 3. H. A. Lowenstam, S. Weiner, On Biomineralization (Oxford University Press, New York, 1989), pp. ix, 324. 4. S. Weiner, L. Addadi, Acidic macromolecules of mineralized tissues the controllers of crystal-formation. Trends in Biochemical Sciences 16, 252 (1991). 5. R. Z. Wang, L. Addadi, S. Weiner, Design strategies of sea urchin teeth: Structure, composition and micromechanical relations to function. Philosophical Transactions of the Royal Society of London Series B-Biological Sciences 352, 469 (1997). 6. E. Bonucci, Calcification in Biological Systems (C R C Press LLC, Boca Raton, 1992), pp. 43. 7. L. B. Gower, D. J. Odom, Deposition of calcium carbonate films by a polymer-induced liquid-precursor (PILP) process. Journal of Crystal Growth 210, 719 (2000). 8. M. J. Olszta, D. J. Odom, E. P. Douglas, L. B. Gower, A new paradigm for biomineral formation: Mineralization via an amorphous liquid-phase precursor. Connective Tissue Research 44, 326 (2003). 9. S. Weiner, W. Traub, Organization of hydroxyapatite crystals within collagen fibrils. Febs Letters 206, 262 (Oct 6, 1986). 10. W. J. Landis, M. J. Song, A. Leith, L. McEwen, B. F. McEwen, Mineral and organic matrix interaction in normally calcifying tendon visualized in 3 dimensions by high-voltage electronmicroscopic tomography and graphic image-reconstruction. Journal of Structural Biology 110, 39 (1993). 122

PAGE 134

123 11. W. J. Landis, K. J. Hodgens, M. J. Song, J. Arena, S. Kiyonaga, M. Marko, C. Owen, B. F. McEwen, Mineralization of collagen may occur on fibril surfaces: Evidence from conventional and high-voltage electron microscopy and three-dimensional imaging. Journal of Structural Biology 117, 24 (1996). 12. W. C. Thomas, A. Tomita, Mineralization of human and bovine tissue in vitro. American Journal of Pathology 51, 621 (1967). 13. M. J. Olszta, E. P. Douglas, L. B. Gower, Scanning electron microscopic analysis of the mineralization of type I collagen via a polymer-induced liquid-precursor (PILP) process. Calcified Tissue International 72, 583 (2003). 14. V. Ziv, I. Sabanay, T. Arad, W. Traub, S. Weiner, Transitional structures in lamellar bone. Microscopy Research and Technique 33, 203 (1996). 15. A. Sellinger, P. M. Weiss, A. Nguyen, Y. F. Lu, R. A. Assink, W. L. Gong, C. J. Brinker, Continuous self-assembly of organic-inorganic nanocomposite coatings that mimic nacre. Nature 394, 256 (Jul 16, 1998). 16. S. Mann, Biomineralization : Principles and Concepts in Bioinorganic Materials Chemistry (Oxford University Press, Inc., New York, 2001), pp. 198. 17. D. J. Ahn, A. Berman, D. Charych, Probing the dynamics of template-directed calcite crystalization with in situ FTIR. Journal of Physical Chemistry 100, 12455 (1996). 18. P. Buijnsters, J. Donners, S. J. Hill, B. R. Heywood, R. J. M. Nolte, B. Zwanenburg, N. Sommerdijk, Oriented crystallization of calcium carbonate under selforganized monolayers of amide-containing phospholipids. Langmuir 17, 3623 (2001). 19. E. Dalas, P. G. Koutsoukos, The crystallization of vaterite on cholesterol. Journal of Colloid and Interface Science 127, 273 (Jan, 1989). 20. B. R. Heywood, "Template-directed nucleation and growth of inorganic materials," in Biomimetic Materials Chemistry S. Mann, Ed. (VCH Publ., Inc., N.Y., 1996) pp. 383. 21. B. R. Heywood, S. Rajam, S. Mann, Oriented crystallization of CaCO3 under compressed monolayers .2. Morphology, structure and growth of immature crystals. Journal of the Chemical Society-Faraday Transactions 87, 735 (1991). 22. C. Jego, B. Agricole, M. H. Li, E. Dupart, H. T. Nguyen, C. Mingotaud, A proposed strategy to design molecules predisposed to in-plane orientation in Langmuir-Blodgett films. Langmuir 14, 1516 (1998). 23. A. Litvin, L. A. Samuelson, D. L. Kaplan, C. Sung, P. M. McCarthy, D. H. Charych, W. Spevak, paper presented at the Smart Structures and Materials 1995.

PAGE 135

124 24. S. Mann, B. R. Heywood, S. Rajam, J. B. A. Walker, Crystal engineering of inorganic materials at organized organic-surfaces. Acs Symposium Series 444, 28 (1991). 25. K. Sato, Y. Kumagai, T. Tanaka, Apatite formation on organic monolayers in simulated body environment. Journal of Biomedical Materials Research 50, 16 (2000). 26. K. Sato, T. Kogure, Y. Kumagai, J. Tanaka, Crystal orientation of hydroxyapatite induced by ordered carboxyl groups. Journal of Colloid and Interface Science 240, 133 (2001). 27. E. DiMasi, V. M. Patel, M. Sivakumar, M. J. Olszta, Y. P. Yang, L. B. Gower, Polymer-controlled growth rate of an amorphous mineral film nucleated at a fatty acid monolayer. Langmuir 18, 8902 (2002). 28. E. DiMasi, L. B. Gower, Synchrotron x-ray observations of a monolayer template for mineralization. Materials Research Society Symposium Proceedings 711, 301 (2002). 29. E. DiMasi, M. J. Olszta, V. M. Patel, L. B. Gower, When is template directed mineralization really template directed? Crystengcomm 5, 346 (2003). 30. E. M. Landau, S. G. Wolf, J. Sagiv, M. Deutsch, K. Kjaer, J. Alsnielsen, L. Leiserowitz, M. Lahav, Design and surface synchrotron x-ray structure-analysis of Langmuir films for crystal nucleation. Pure and Applied Chemistry 61, 673 (Apr, 1989). 31. E. M. Landau, S. G. Wolf, M. Levanon, L. Leiserowitz, M. Lahav, J. Sagiv, Stereochemical studies in crystal nucleation oriented crystal-growth of glycine at interfaces covered with Langmuir and Langmuir-Blodgett films of resolved alpha-amino-acids. Journal of the American Chemical Society 111, 1436 (Feb 15, 1989). 32. B. R. Heywood, S. Mann, Template-directed nucleation and growth of inorganic materials. Advanced Materials 6, 9 (Jan, 1994). 33. J. Aizenberg, A. J. Black, G. H. Whitesides, Oriented growth of calcite controlled by self-assembled monolayers of functionalized alkanethiols supported on gold and silver. Journal of the American Chemical Society 121, 4500 (1999). 34. J. Aizenberg, D. A. Muller, J. L. Grazul, D. R. Hamann, Direct fabrication of large micropatterned single crystals. Science 299, 1205 (2003). 35. Y.-Y. Kim, Ph.D. Dissertation, University of Florida (2003). 36. Y.-Y. Kim, L. B. Gower, paper presented at the Materials Research Society, San Francisco 2003.

PAGE 136

125 37. L. A. Gower, D. A. Tirrell, Calcium carbonate films and helices grown in solutions of poly(aspartate). Journal of Crystal Growth 191, 153 (1998). 38. F. Manoli, E. Dalas, Calcium carbonate overgrowth on elastin substrate. Journal of Crystal Growth 204, 369 (1999). 39. W. L. Murphy, P. B. Messersmith, Compartmental control of mineral formation: adaptation of a biomineralization strategy for biomedical use. Polyhedron 19, 357 (2000). 40. F. Manoli, S. Koutsopoulos, E. Dalas, Crystallization of calcite on chitin. Journal of Crystal Growth 182, 116 (1997). 41. J. Kanakis, E. Dalas, The crystallization of vaterite on fibrin. Journal of Crystal Growth 219, 277 (2000). 42. J. Kanakis, P. Malkaj, J. Petroheilos, E. Dalas, The crystallization of calcium carbonate on porcine and human cardiac valves and the antimineralization effect of sodium alginate. Journal of Crystal Growth 223, 557 (2001). 43. S. Koutsopoulos, E. Dalas, The calcification of fibrin in vitro. Journal of Crystal Growth 216, 450 (2000). 44. E. Dalas, J. K. Kallitsis, P. G. Koutsoukos, Crystallization of hydroxyapatite on polymers. Langmuir 7, 1822 (Aug, 1991). 45. E. Dalas, J. Kallitsis, P. G. Koutsoukos, The growth of sparingly soluble salts on polymeric substrates. Colloids and Surfaces 53, 197 (Feb 28, 1991). 46. L. Addadi, S. Weiner, Control and design principles in biological mineralization. Angew. Chem. Int. Ed. Engl. 31, 153 (1992). 47. S. Mann, "Crystallochemical strategies in biomineralization," in BiomineralizationChemical and Biochemical Perspectives S. Mann, J. Webb, R. J. P. Williams, Eds. (VCH Publishers, N. Y., 1989) pp. 35-62. 48. L. Addadi, J. Moradian, E. Shay, N. G. Maroudas, S. Weiner, A chemical-model for the cooperation of sulfates and carboxylates in calcite crystal nucleation relevance to biomineralization. Proceedings of the National Academy of Sciences of the United States of America 84, 2732 (1987). 49. A. Nanci, Content and distribution of noncollagenous matrix proteins in bone and cementum: Relationship to speed of formation and collagen packing density. Journal of Structural Biology 126, 256 (1999). 50. R. W. Kinne, L. W. Fisher, Keratan sulfate proteoglycan in rabbit compact-bone is bone sialoprotein-Ii. Journal of Biological Chemistry 262, 10206 (Jul 25, 1987).

PAGE 137

126 51. M. T. Dimuzio, A. Veis, Phosphophoryns major non-collagenous proteins of rat incisor dentin. Calcified Tissue Research 25, 169 (1978). 52. G. F. Xu, N. Yao, I. A. Aksay, J. T. Groves, Biomimetic synthesis of macroscopic-scale calcium carbonate thin films. Evidence for a multistep assembly process. Journal of the American Chemical Society 120, 11977 (1998). 53. S. Sarig, Crystal habit modification by water-soluble polymers. Journal of Crystal Growth 24, 338 (1974). 54. G. Falini, M. Gazzano, A. Ripamonti, Crystallization of calcium-carbonate in presence of magnesium and polyelectrolytes. Journal of Crystal Growth 137, 577 (1994). 55. L. M. Qi, H. Colfen, M. Antonietti, M. Li, J. D. Hopwood, A. J. Ashley, S. Mann, Formation of BaSO4 fibres with morphological complexity in aqueous polymer solutions. Chemistry-a European Journal 7, 3526 (2001). 56. W. J. Benton, I. R. Collins, I. M. Grimsey, G. M. Parkinson, S. A. Rodger, Nucleation, growth and inhibition of barium sulfate-controlled modification with organic and inorganic additives. Faraday Discussions, 281 (1993). 57. S. H. Yu, H. Colfen, M. Antonietti, Control of the morphogenesis of barium chromate by using double-hydrophilic block copolymers (DHBCs) as crystal growth modifiers. Chemistry-a European Journal 8, 2937 (2002). 58. A. Tsortos, G. H. Nancollas, The role of polycarboxylic acids in calcium phosphate mineralization. Journal of Colloid and Interface Science 250, 159 (2002). 59. H. H. Teng, P. M. Dove, J. J. De Yoreo, Kinetics of calcite growth: Surface processes and relationships to macroscopic rate laws. Geochimica Et Cosmochimica Acta 64, 2255 (2000). 60. H. H. Teng, P. M. Dove, C. A. Orme, J. J. De Yoreo, Thermodynamics of calcite growth: Baseline for understanding biomineral formation. Science 282, 724 (1998). 61. L. Addadi, Z. Berkovitchyellin, I. Weissbuch, J. Vanmil, L. J. W. Shimon, M. Lahav, L. Leiserowitz, Growth and dissolution of organic-crystals with tailor-made inhibitors implications in stereochemistry and materials science. Angewandte Chemie-International Edition in English 24, 466 (1985). 62. E. M. Greenfield, D. C. Wilson, M. A. Crenshaw, Ionotropic nucleation of calcium carbonate by molluscan matrix. Amer. Zool. 24, 925 (1984). 63. R. Naumann, D. Walz, S. M. Schiller, W. Knoll, Kinetics of valinomycin-mediated K+ ion transport through tethered bilayer lipid membranes. Journal of Electroanalytical Chemistry 550, 241 (Jul 17, 2003).

PAGE 138

127 64. Y. Kitano, The behavior of various inorganic ions in the separation of calcium carbonate from a bicarbonate solution. Bulletin of the Chemical Society of Japan 35:12, 1973 (1962). 65. S. Mann, J. Webb, R. J. P. Williams, Eds., Biomineralization Chemical and Biochemical Perspectives (VCH Publishers, Inc., N. Y., 1989), pp. 541. 66. K. Okazaki, R. M. Dillaman, K. M. Wilbur, Crystalline axes of the spine and test of the sea urchin Strongylocentrous purpuratus: determination by crystal etching and decoration. Biol. Bull. 161, 402 (1981). 67. F. H. Wilt, Matrix and mineral in the sea urchin larval skeleton. Journal of Structural Biology 126, pg. 216 (1999). 68. E. Beniash, J. Aizenberg, L. Addadi, S. Weiner, Amorphous calcium carbonate transforms into calcite during sea urchin larval spicule growth. Proceedings of the Royal Society of London Series B-Biological Sciences 264, 461 (1997). 69. A. Berman, J. Hanson, L. Leiserowitz, T. F. Koetzle, S. Weiner, L. Addadi, Biological-control of crystal texture a widespread strategy for adapting crystal properties to function. Science 259, 776 (1993). 70. J. G. Carter, Ed., Skeletal Biomineralization: Patterns, Processes and Evolutionary Trends vol. 1 (Van Nostrand Reinhold, N.Y., 1990). 71. J. Aizenberg, G. Lambert, L. Addadi, S. Weiner, Stabilization of amorphous calcium carbonate by specialized macromolecules in biological and synthetic precipitates. Advanced Materials 8, 222 (1996). 72. H. J. Arnott, "Calcification in higher plants," in The Mechanism of Mineralization in the Invertebrates and Plants N. Watabe, K. M. Wilbur, Eds. (Univ. South Caroline Press, Columbia, SC, 1976) pp. 55-78. 73. Y. Levi-Kalisman, S. Raz, S. Weiner, L. Addadi, I. Sagi, X-Ray absorption spectroscopy studies on the structure of a biogenic "amorphous" calcium carbonate phase. Journal of the Chemical Society-Dalton Transactions, 3977 (2000). 74. J. F. Vincent, Structural Biomaterials (Princeton University Press, Princeton, ed. Revised, 1990), pp. 256 75. A. K. Mathur, Polly, P.D., The evolution of enamel microstructure: How important is amelogenin? Journal of Mammalian Evolution 7, 23 (2000). 76. A. Jodaikin, W. Traub, D. Worms, S. Weiner, Structural aspects of the organic matrix of rat tooth enamel. Journal of Dental Research 63, 546 (1984). 77. A. Jodaikin, W. Traub, S. Weiner, Structural studies of rat tooth enamel. Journal of Dental Research 63, 590 (1984).

PAGE 139

128 78. S. Carlson, "Vertebrate dental structures," in Skeletal Biomineralization: Patterns, Processes and Evolutionary Trends J. G. Carter, Ed. (Van Nostrand Reinhold, N.Y., 1990), vol. 1, pp. 531-556. 79. P. Bodier-Houlle, P. Steuer, J. M. Meyer, L. Bigeard, F. J. G. Cuisinier, High-resolution electron-microscopic study of the relationship between human enamel and dentin crystals at the dentinoenamel junction. Cell and Tissue Research 301, 389 (Sep, 2000). 80. T. Sasaki, Cell Biology of Tooth Enamel Formation H. M. Myers, Ed., Monographs in Oral Science (Karger, N.Y., 1990). 81. A. G. Fincham, J. Moradian-Oldak, J. P. Simpson, The structural biology of developing dental enamel matrix. J. Structural Biology 126, 270 (1999). 82. A. Boyde, "Amelogenesis and the structure of enamel," in Scientific Foundations of Dentistry B. Cohen, I. R. H. Kramer, Eds. (Wilim Hieneman Medical Books, Ltd., London, 1976) pp. 335-352. 83. H. A. Lowenstam, Minerals Formed by Organisms. Science 211, 1126 (1981). 84. G. W. Bernard, Ultrastructural observations of initial calcification in dentin and enamel. Journal of Ultrastructure Research 41, 1 (1972). 85. E. J. Reith, Early stage of amelogenesis as observed in molar teeth of Yorun rats. Journal of Ultrastructure Research 17, 503 (1967). 86. A. L. Arsenault, B. W. Robinson, The dentino-enamel junction a structural and microanalytical study of early mineralization. Calcified Tissue International 45, 111 (Aug, 1989). 87. T. Diekwisch, B. J. Berman, S. Gentner, H. C. Slavkin, Initial enamel crystals are not associated with mineralized dentin. Journal of Dental Research 73, 112 (1994). 88. Y. Takano, Y. Hanaizumi, H. Ohshima, Occurrence of amorphous and crystalline mineral deposits at the epithelial-mesenchymal interface of incisors in the calcium-loaded rat: Implication of novel calcium binding domains. Anatomical Record 245, 174 (Jun, 1996). 89. D. McConnell, Apatite; its Crystal Chemistry, Mineralogy, Utilization, and Geologic and Biologic Occurrences (Springer-Verlag, New York,, 1973), pp. xvi, 111. 90. E. T. Degens, W. A. P. Luck, D. D. Perrin, Inorganic Biochemistry Topics in current chemistry ; 64 (Springer-Verlag, Berlin ; New York, 1976), pp. 225. 91. Y. Hayashi, High-resolution electron-microscopy of the initial mineral deposition on enamel surface. Journal of Electron Microscopy 42, 342 (Oct, 1993).

PAGE 140

129 92. E. Baer, J. J. Cassidy, A. Hiltner, "Hierarchical structure of collagen composite systems: Lessons from biology," in BiomimeticsDesign and Processing of Materials M. Sarikaya, I. A. Aksay, Eds. (AIP Press, Woodbury, NY, 1995) pp. 13-34. 93. S. Weiner, H. D. Wagner, The material bone: Structure mechanical function relations. Annual Review of Materials Science 28, 271 (1998). 94. R. B. Martin, D. Burr, N. Sharkey, Skeletal Tissue Mechanics (Springer-Verlag New York Incorporated, New York, 1998), pp. 406. 95. S. Weiner, W. Traub, H. D. Wagner, Lamellar bone: Structure-function relations. Journal of Structural Biology 126, 241 (1999). 96. M. J. Glimcher, S. M. Krane, "The organization and structure of bone, and the mechanism of calcification," in Treatise on Collagen: Volume 2Biology of Collagen B. S. Gould, Ed. (Academic Press, N.Y., 1968). 97. M. J. Glimcher, Recent studies of the mineral phase in bone and its possible linkage to the organic matrix by protein-ligand phosphate bonds. Philos. Trans. R. Soc. London Ser. B 304, 479 (1984). 98. W. J. Landis, J. Moradian-Oldak, S. Weiner, Topographic imaging of mineral and collagen in the calcifying turkey tendon. Connective Tissue Research 25, 181 (1991). 99. A. J. Hodge, J. A. Petruska, "Recent studies with the electron microscope on ordered aggreagates of the tropocollagen molecule," in Aspects of Protein Structure G. N. Ramanchandran, Ed. (Academic Press, London, 1963) pp. 289-300. 100. P. Bianco, "Structure and mineralization of bone," in Calcification in Biological Systems E. Bonucci, Ed. (CRC Press, Boca Raton, 1992) pp. 243-268. 101. S. Weiner, W. Traub, "Organization of crystals in bone," in Mechanisms and Phylogeny of Mineralization in Biological Systems S. Suga, H. Nakahara, Eds. (Springer-Verlag, N.Y., 1991), vol. Biomineralization '90 pp. 247-253. 102. W. Traub, T. Arad, S. Weiner, Growth of mineral crystals in turkey tendon collagen-fibers. Connective Tissue Research 28, 99 (1992). 103. S. Weiner, T. Arad, W. Traub, Crystal organization in rat bone lamellae. Febs Letters 285, 49 (1991). 104. E. P. Katz, E. Wachtel, M. Yamauchi, G. L. Mechanic, The structure of mineralized collagen fibrils. Connective Tissue Research 21, 479 (1989).

PAGE 141

130 105. W. Traub, T. Arad, S. Weiner, 3-Dimensional ordered distribution of crystals in turkey tendon collagen-fibers. Proceedings of the National Academy of Sciences of the United States of America 86, 9822 (1989). 106. D. A. Cameron, The fine structure of bone and calcified cartilage. A critical review of the contribution of electron microscopy to the understading of osteogenesis. Clin Orthop 26, 199 (1963). 107. J. Sela, Z. Schwartz, L. D. Swain, B. D. Boyan, "The role of matrix vesicles in calcification," in Calcification in Biological Systems E. Bonucci, Ed. (CRC Press, Boca Raton, 1992) pp. 73-105. 108. A. Veis, "Biochemical studies of vertebrate tooth mineralization," in BiomineralizationChemical and Biochemical Perspective S. Mann, J. Webb, R. J. P. Williams, Eds. (VCH Publ., N.Y., 1989) pp. 189-216. 109. J. C. Elliott, G. R. Davis, P. Anderson, F. S. L. Wong, S. E. P. Dowker, C. E. Mercer, Application of laboratory microtomography to the study of mineralised tissues. Anales De Quimica 93, S77 (1997). 110. J. C. Elliot, The problem of the composition and structure of the mineral components of the hard tissues. Clinical Orthopaedics and Related Research 93 (1973, 1973). 111. R. A. Robinson, An electron-microscopic study of the crystalline inorganic component of bone and its relationship to the organic matrix. Journal of Bone and Joint Surgery-American Volume 34-A, 389 (1952). 112. R. A. Robinson, M. L. Watson, Collagen-crystal relationships in bone as seen in the electron microscope. Anatomical Record 114, 383 (1952). 113. S. A. Jackson, A. G. Cartwright, D. Lewis, Morphology of bone-mineral crystals. Calcified Tissue Research 25, 217 (1978). 114. W. J. Landis, K. J. Hodgens, J. Arena, M. J. Song, B. F. McEwen, Structural relations between collagen and mineral in bone as determined by high voltage electron microscopic tomography. Microscopy Research and Technique 33, 192 (1996). 115. A. Ascenzi, E. Bonucci, Bocciare.Ds, An electron microscope study of osteon calcification. Journal of Ultrastructure Research 12, 287 (1965). 116. E. D. Eanes, "Physico-chemical principles of biomineralization," in Bone Regulatory Factors Morphology, Biochemistry, Physiology and Pharmacology NATO ASI Series A, Life Sciences A. Pecile, B. De Bernard, Eds. (Perseus Publishing, Ca mbridge, 1990), vol. Vol. 184, pp. 302.

PAGE 142

131 117. A. H. Heuer, D. J. Fink, V. J. Laraia, J. L. Arias, P. D. Calvert, K. Kendall, G. L. Messing, J. Blackwell, P. C. Rieke, D. H. Thompson, A. P. Wheeler, A. Veis, A. I. Caplan, Innovative materials processing strategies a biomimetic approach. Science 255, 1098 (1992). 118. M. J. Glimcher, Molecular Biology of Mineralized Tissues with Particular Reference to Bone. Reviews of Modern Physics 31, 359 (1959). 119. H. R. Dudley, D. Spiro, Fine structure of bone cells. Journal of Biophysical and Biochemical Cytology 11, 627 (1961). 120. R. R. Cooper, J. W. Milgram, R. A. Robinson, Morphology of osteon an electron microscopic study. Journal of Bone and Joint Surgery-American Volume A 48, 1239 (1966). 121. A. Bigi, A. Ripamonti, M. H. J. Koch, N. Roveri, Calcified turkey leg tendon as structural model for bone mineralization. International Journal of Biological Macromolecules 10, 282 (1988). 122. E. Schiffmann, G. R. Martin, E. J. Miller, Matrices that calcify. Biol. Calcif.: Cell. Mol. Aspects, 27 (1970). 123. A. Veis, Mineral matrix interactions in bone and dentin. Journal of Bone and Mineral Research 8, S493 (1993). 124. J. D. Termine, H. K. Kleinman, S. W. Whitson, K. M. Conn, M. L. McGarvey, G. R. Martin, Osteonectin, a bone-specific protein linking mineral to collagen. Cell 26, 99 (1981). 125. G. K. Hunter, B. L. Allen, M. D. Grynpas, P. T. Cheng, Inhibition of hydroxyapatite formation in collagen gels by chondroitin sulfate. Biochemical Journal 228, 463 (1985). 126. Q. Q. Hoang, F. Sicheri, A. J. Howard, D. S. C. Yang, Bone recognition mechanism of porcine osteocalcin from crystal structure. Nature 425, 977 (Oct 30, 2003). 127. E. J. Wheeler, D. Lewis, X-Ray study of paracrystalline nature of bone apatite. Calcified Tissue Research 24, 243 (1977). 128. J. Aizenberg, S. Weiner, L. Addadi, Coexistence of amorphous and crystalline calcium carbonate in skeletal tissues. Connective Tissue Research 44, 20 (2003). 129. S. Weiner, Y. Levi-Kalisman, S. Raz, L. Addadi, Biologically formed amorphous calcium carbonate. Connective Tissue Research 44, 214 (2003).

PAGE 143

132 130. E. D. Eanes, Amorphous intermediates in the formation of biological apatites. Colloques Internationaux du Centre National de la Recherche Scientifique 230, 295 (1975). 131. J. Aizenberg, G. Lambert, S. Weiner, L. Addadi, Factors involved in the formation of amorphous and crystalline calcium carbonate: A study of an ascidian skeleton. Journal of the American Chemical Society 124, 32 (2002). 132. E. Bonucci, "Role of collagen fibrils in calcification," in Calcification of Biological Systems E. Bonucci, Ed. (CRC Press, Boca Raton, 1992) pp. 19-39. 133. F. J. G. Cuisinier, J. C. Vogel, Structure of initial crystals formed during human amelogenesis. J.Crystal Growth 116, 314 (1992). 134. W. J. Landis, M. Navarro. Calcif Tissue Intl. 35, 48 (1983). 135. W. E. Brown, M. U. Nylen, Role of octacalcium phosphate in formation of hard tissues. Journal of Dental Research 43, 751 (1964). 136. R. R. Betz, Limitations of autograft and allograft: New synthetic solutions. Orthopedics 25, S561 (May, 2002). 137. A. R. Vaccaro, The role of the osteoconductive scaffold in synthetic bone grafts (vol 25, pg S571, 2002). Orthopedics 25, 1224 (Nov, 2002). 138. R. W. Hu, H. H. Bohlman, Fracture at the iliac bone-graft harvest site after fusion of the spine. Clinical Orthopaedics and Related Research, 208 (Dec, 1994). 139. A. R. Poynton, J. M. Lane, Review of the state of the art: Allograft-based systems for use as bone graft substitutes. Bone Graft Substitutes, [Proceedings of a Workshop], Orlando, FL, United States, Nov., 2000, 13 (2003). 140. D. M. Strong, G. E. Friedlaender, W. W. Tomford, D. S. Springfield, T. C. Shives, H. Burchardt, W. F. Enneking, H. J. Mankin, Immunologic responses in human recipients of osseous and osteochondral allografts. Clinical Orthopaedics and Related Research, 107 (May, 1996). 141. S. Stevenson, Q. L. Xiao, B. Martin, The fate of cancellous and cortical bone after transplantation of fresh and frozen tissue-antigen-matched and mismatched osteochondral allografts in dogs. Journal of Bone and Joint Surgery-American Volume 73A, 1143 (Sep, 1991). 142. S. Stevenson, The immune-response to osteochondral allografts in dogs. Journal of Bone and Joint Surgery-American Volume 69A, 573 (Apr, 1987). 143. H. S. Sandhu, H. S. Grewal, H. Parvataneni, Bone grafting for spinal fusion. Orthopedic Clinics of North America 30, 685 (Oct, 1999).

PAGE 144

133 144. D. M. Ehrler, A. R. Vaccaro, The use of allograft bone in lumbar spine surgery. Clinical Orthopaedics and Related Research, 38 (Feb, 2000). 145. T. Boyce, J. Edwards, N. Scarborough, Allograft bone The influence of processing on safety and performance. Orthopedic Clinics of North America 30, 571 (Oct, 1999). 146. K. U. Lewandrowski, J. D. Gresser, D. L. Wise, D. J. Trantolo, Bioresorbable bone graft substitutes of different osteoconductivities: a histologic evaluation of osteointegration of poly(propylene glycol-co-fumaric acid)-based cement implants in rats. Biomaterials 21, 757 (Apr, 2000). 147. D. C. Wirtz, B. Lelgemann, F. Jungwirth, F. U. Niethard, R. Marx, A new method to optimize the adhesion between bone cement and acetabular bone in total hip arthroplasty. Zeitschrift Fur Orthopadie Und Ihre Grenzgebiete 141, 209 (Mar-Apr, 2003). 148. D. C. Wirtz, F. U. Niethard, Causes, diagnosis and therapy of aseptic hip prostheses loosening A current concept review. Zeitschrift Fur Orthopadie Und Ihre Grenzgebiete 135, 270 (Jul-Aug, 1997). 149. H. Malchua, P. Sodermann, P. Herberts, paper presented at the 67th Annual Meeting of the AAOS, Orlando 2000. 150. H. Malchua, P. Herberts, paper presented at the 63rd Annual Meeting of the AAOS, Atlana 1996. 151. J. Black, G. W. Hastings, Handbook of Biomaterial Properties (Chapman & Hall, London ; New York, ed. 1st ed., 1998), pp. xxvi, 590. 152. J. E. Lemons, Implant retrieval and its medical interest. Anales De Quimica 93, S83 (1997). 153. H. Erli, R. Marx, O. Paar, F. Niethard, M. Weber, D. Wirtz, Surface pretreatements for medical application of adhesion. Biomedical Engineering Online 2 (2003). 154. C. E. Holy, M. S. Shoichet, J. E. Davies, Engineering three-dimensional bone tissue in vitro using biodegradable scaffolds: Investigating initial cell-seeding density and culture period. Journal of Biomedical Materials Research 51, 376 (Sep 5, 2000). 155. J. M. Karp, P. D. Dalton, M. S. Shoichet, Scaffolds for tissue engineering. Mrs Bulletin 28, 301 (Apr, 2003). 156. J. M. Karp, M. S. Shoichet, J. E. Davies, Bone formation on two-dimensional (DL-lactide-co-glycolide) (PLGA) films and three-dimensional PLGA tissue engineering scaffolds in vitro. Journal of Biomedical Materials Research Part A 64A, 388 (Feb 1, 2003).

PAGE 145

134 157. C. E. Holy, J. A. Fialkov, J. E. Davies, M. S. Shoichet, Use of a biomimetic strategy to engineer bone. Journal of Biomedical Materials Research Part A 65A, 447 (Jun 15, 2003). 158. J. A. Fialkov, C. E. Holy, M. S. Shoichet, J. E. Davies, In vivo bone engineering in a rabbit femur. Journal of Craniofacial Surgery 14, 324 (May, 2003). 159. J. M. Karp, K. Rzeszutek, M. S. Shoichet, J. E. Davies, Fabrication of precise cylindrical three-dimensional tissue engineering scaffolds for in vitro and in vivo bone engineering applications. Journal of Craniofacial Surgery 14, 317 (May, 2003). 160. D. Iejima, T. Saito, T. Uemura, A collagen-phosphophoryn sponge as a scaffold for bone tissue engineering. Journal of Biomaterials Science-Polymer Edition 14, 1097 (2003). 161. R. Torensma, P. J. ter Brugge, J. A. Jansen, C. G. Figdor, Ceramic hydroxyapatite coating on titanium implants drives selective bone marrow stromal cell adhesion. Clinical Oral Implants Research 14, 569 (Oct, 2003). 162. J. K. Bibby, P. M. Mummery, N. Bubb, D. J. Wood, "Novel bioactive coatings for biomedical applications," in Bioceramics 15 (2003), vol. 240-2, pp. 279-282. 163. E. Verne, M. Bosetti, C. V. Brovarone, C. Moisescu, F. Lupo, S. Spriano, M. Cannas, Fluoroapatite glass-ceramic coatings on alumina: structural, mechanical and biological characterisation. Biomaterials 23, 3395 (Aug, 2002). 164. R. Kato, S. Nakamura, K. Katayama, K. Yamashita, "Acceleration of bone-like crystal growth on polarized plasma sprayed HAp in SBF," in Bioceramics 14 (2002), vol. 218-2, pp. 145-148. 165. K. Schneider, R. B. Heimann, G. Berger, Plasma-sprayed coatings in the system CaO-TiO2-ZrO2-P2O5 for long-term stable endoprostheses. Materialwissenschaft Und Werkstofftechnik 32, 166 (Feb, 2001). 166. P. Torricelli, E. Verne, C. V. Brovarone, P. Appendino, F. Rustichelli, A. Krajewski, A. Ravaglioli, G. Pierini, M. Fini, G. Giavaresi, R. Giardino, Biological glass coating on ceramic materials: in vitro evaluation using primary osteoblast cultures from healthy and osteopenic rat bone. Biomaterials 22, 2535 (Sep, 2001). 167. B. Dubini, A. Krajewski, M. Mazzocchi, M. G. P. Bossi, A. Ravaglioli, G. Rizzi, F. Rustichelli, V. Stanic, R. Giardino, N. Nicoli-Aldini, E. Verne, C. V. Brovarone, "Glass-ceramics as coatings for prostheses," in Bioceramics (2000), vol. 192-1, pp. 279-282. 168. M. Hamadouche, A. Meunier, D. C. Greenspan, C. Blanchat, J. P. Zhong, G. P. La Torre, L. Sedel, "Bioactivity of bioactive sol-gel glasses coated alumina implants," in Bioceramics (2000), vol. 192-1, pp. 413-416.

PAGE 146

135 169. K. A. Gross, C. C. Berndt, H. Herman, Amorphous phase formation in plasma-sprayed hydroxyapatite coatings. Journal of Biomedical Materials Research 39, 407 (1998). 170. P. Habibovic, F. Barrere, C. A. van Blitterswijk, K. de Groot, P. Layrolle, Biomimetic hydroxyapatite coating on metal implants. Journal of the American Ceramic Society 85, 517 (2002). 171. N. Costa, P. M. Maquis, Biomimetic processing of calcium phosphate coating. Medical Engineering & Physics 20, 602 (1998). 172. S. A. Bender, J. D. Bumgardner, M. D. Roach, K. Bessho, J. L. Ong, Effect of protein on the dissolution of HA coatings. Biomaterials 21, 299 (2000). 173. C. B. Mao, H. D. Li, F. Z. Cui, Q. G. Feng, H. Wang, C. L. Ma, Oriented growth of hydroxyapatite on (0001) textured titanium with functionalized self-assembled silane monolayer as template. Journal of Materials Chemistry 8, 2795 (1998). 174. L. Jonasova, F. A. Muller, A. Helebrant, J. Strnad, P. Greil, Biomimetic apatite formation on chemically treated titanium. Biomaterials 25, 1187 (Mar-Apr, 2004). 175. L. L. Hench, H. A. Paschall, Direct chemical bond of bioactive glass-ceramic materials to bone and muscle. Journal of Biomedical Materials Research 7, 25 (1973). 176. I. C. Clarke, G. Pezzotti, S. Sakakura, N. Sugano, "Phase transformation and residual stresses in retrieved zirconia ball implant," in Bioceramics 15 (2003), vol. 240-2, pp. 777-780. 177. M. Manaka, I. C. Clarke, A. Gustafson, A. Imakiire, "Impingement in ceramic hip patient a retrieval and wear-scar analysis of femoral head," in Bioceramics 15 (2003), vol. 240-2, pp. 847-848. 178. M. M. Mak, Z. M. Jin, Analysis of contact mechanics in ceramic-on-ceramic hip joint replacements. Proceedings of the Institution of Mechanical Engineers Part H-Journal of Engineering in Medicine 216, 231 (2002). 179. M. R. Norton, R. Yarlagadda, G. H. Anderson, Catastrophic failure of the Elite Plus total hip replacement, with a Hylamer acetabulum and Zirconia ceramic femoral head. Journal of Bone and Joint Surgery-British Volume 84B, 631 (Jul, 2002). 180. C. R. McLean, H. Dabis, D. Mok, Delayed fracture of the ceramic femoral head after trauma. Journal of Arthroplasty 17, 503 (Jun, 2002). 181. M. Hasegawa, T. Ohashi, T. Tani, Poor outcome of 44 cemented total hip arthroplasties with alumina ceramic heads Clinical evaluation and retrieval analysis after 10-16 years. Acta Orthopaedica Scandinavica 72, 449 (Oct, 2001).

PAGE 147

136 182. M. Nilsson, E. Fernandez, S. Sarda, L. Lidgren, J. A. Planell, Characterization of a novel calcium phosphate/sulphate bone cement. Journal of Biomedical Materials Research 61, 600 (Sep 15, 2002). 183. B. R. Constantz, I. C. Ison, M. T. Fulmer, R. D. Poser, S. T. Smith, M. Vanwagoner, J. Ross, S. A. Goldstein, J. B. Jupiter, D. I. Rosenthal, Skeletal repair by in-situ formation of the mineral phase of bone. Science 267, 1796 (Mar 24, 1995). 184. E. Fernandez, F. J. Gil, M. P. Ginebra, F. C. M. Driessens, J. A. Planell, S. M. Best, Production and characterization of new calcium phosphate bone cements in the CaHPO4-alpha-Ca-3(PO4)(2) system: pH, workability and setting times. Journal of Materials Science-Materials in Medicine 10, 223 (Apr, 1999). 185. P. Calvert, Inkjet printing for materials and devices. Chemistry of Materials 13, 3299 (Oct, 2001). 186. J. A. Lewis, Direct-write assembly of ceramics from colloidal inks. Current Opinion in Solid State & Materials Science 6, 245 (Jun, 2002). 187. C. Du, F. Z. Cui, X. D. Zhu, K. de Groot, Three-dimensional nano-HAp/collagen matrix loading with osteogenic cells in organ culture. Journal of Biomedical Materials Research 44, 407 (1999). 188. M. Iijima, K. Iijima, Y. Moriwaki, Y. Kuboki, Oriented growth of octacalcium phosphate crystals on type-I collagen fibrils under physiological conditions. Journal of Crystal Growth 140, 91 (1994). 189. A. Bigi, M. Gandolfi, N. Roveri, G. Valdre, In vitro calcified tendon collagen: An atomic force and scanning electron microscopy investigation. Biomaterials 18, 657 (1997). 190. Y. Doi, T. Horiguchi, Y. Moriwaki, H. Kitago, T. Kajimoto, Y. Iwayama, Formation of apatite-collagen complexes. Biomedical Materials Research 31, 43 (1996). 191. J. H. Bradt, M. Mertig, A. Teresiak, W. Pompe, Biomimetic mineralization of collagen by combined fibril assembly and calcium phosphate formation. Chemistry of Materials 11, 2694 (1999). 192. J. Mythili, T. P. Sastry, M. Subramanian, Preparation and characterization of a new bioinorganic composite: collagen and hydroxyapatite. Biotechnology and Applied Biochemistry 32, 155 (2000). 193. W. Traub, T. Arad, S. Weiner, Origin of mineral crystal-growth in collagen fibrils. Matrix 12, 251 (1992).

PAGE 148

137 194. P. Calvert, P. Rieke, Biomimetic mineralization in and on polymers. Chem. Mater. 8, 1715 (1996). 195. M. Iijima, Y. Moriwaki, Y. Kuboki, In-vitro crystal-growth of octacalcium phosphate on type-I collagen fiber. Journal of Crystal Growth 137, 553 (1994). 196. S. Itoh, M. Kikuchi, K. Takakuda, Y. Koyama, H. N. Matsumoto, S. Ichinose, J. Tanaka, T. Kawauchi, K. Shinomiya, The biocompatibility and osteoconductive activity of a novel hydroxyapatite/collagen composite biomaterial, and its function as a carrier of rhBMP-2. Journal of Biomedical Materials Research 54, 445 (2001). 197. S. Kamakura, Y. Sasano, T. Shimizu, K. Hatori, O. Suzuki, M. Kagayama, K. Motegi, Implanted octacalcium phosphate is more resorbable than betatricalcium phosphate and hydroxyapatite. Journal of Biomedical Materials Research 59, 29 (2002). 198. M. S. Widmer, P. K. Gupta, L. C. Lu, R. K. Meszlenyi, G. R. D. Evans, K. Brandt, T. Savel, A. Gurlek, C. W. Patrick, A. G. Mikos, Manufacture of porous biodegradable polymer conduits by an extrusion process for guided tissue regeneration. Biomaterials 19, 1945 (1998). 199. M. Kikuchi, S. Itoh, S. Ichinose, K. Shinomiya, J. Tanaka, Self-organization mechanism in a bone-like hydroxyapatite/collagen nanocomposite synthesized in vitro and its biological reaction in vivo. Biomaterials 22, 1705 (2001). 200. A. K. Garg, "Grafting materials in repair and restoration," in Tissue EngineeringApplications in Maxillofacial Surgery and Periodontics S. E. Lynch, R. J. Genco, R. E. Marx, Eds. (Quintessence Publishing Co., Inc., Chicago, 1999) pp. 83-101. 201. R. Vago, D. Plotquin, A. Bunin, I. Sinelnikov, D. Atar, D. Itzhak, Hard tissue remodeling using biofabricated coralline biomaterials. Journal of Biochemical and Biophysical Methods 50, 253 (2002). 202. D. L. Wise, Biomaterials Engineering and Devices: Human Applications (Humana Press, Totowa, NJ, 2000), pp. v. 1-2. 203. D. Togawa, T. W. Bauer, I. H. Lieberman, G. L. Lowery, S. Takikawa, Histology of tissues within retrieved human titanium mesh cages. Spine 28, 246 (Feb 1, 2003). 204. T. J. Wu, H. H. Huang, C. W. Lan, C. H. Lin, F. Y. Hsu, Y. J. Wang, Studies on the microspheres comprised of reconstituted collagen and hydroxyapatite. Biomaterials 25, 651 (Feb, 2004). 205. M. Kikuchi, H. N. Matsumoto, T. Yamada, Y. Koyama, K. Takakuda, J. Tanaka, Glutaraldehyde cross-linked hydroxyapatite/collagen self-organized nanocomposites. Biomaterials 25, 63 (Jan, 2004).

PAGE 149

138 206. R. V. Sugars, A. A. Milan, J. O. Brown, R. J. Waddington, R. C. Hall, G. Embery, Molecular interaction of recombinant decorin and biglycan with type I collagen influences crystal growth. Connective Tissue Research 44, 189 (2003). 207. A. T. C. Wong, J. T. Czernuszka, Constant-composition study of the precipitation behavior of calcium phosphate in the presence of non-collagenous bio-chemicals. Hydroxyapatite Relat. Mater., 155 (1994). 208. D. L. Christiansen, F. H. Silver, L. Addadi, Mineralization of an axially aligned collagenous matrix a morphological-study. Cells and Materials 3, 177 (1993). 209. R. Kniep, S. Busch, Biomimetic growth and self-assembly of fluorapatite aggregates by diffusion into denatured collagen matrices. Angewandte Chemie-International Edition in English 35, 2624 (1996). 210. G. Falini, S. Fermani, M. Gazzano, A. Ripamonti, Biomimetic crystallization of calcium carbonate polymorphs by means of collagenous matrices. Chemistry-a European Journal 3, 1807 (1997). 211. M. Iijima, Y. Moriwaki, Y. Kuboki, Oriented and lengthwise growth of octacalcium phosphate on collagenous matrix in vitro. Connective Tissue Research 36, 51 (1997). 212. T. Saito, A. L. Arsenault, M. Yamauchi, Y. Kuboki, M. A. Crenshaw, Mineral induction by immobilized phosphoproteins. Bone 21, 305 (1997). 213. G. Falini, S. Fermani, M. Gazzano, A. Ripamonti, Oriented crystallization of vaterite in collagenous matrices. Chemistry-a European Journal 4, 1048 (1998). 214. C. Du, F. Z. Cui, W. Zhang, Q. L. Feng, X. D. Zhu, K. de Groot, Formation of calcium phosphate/collagen composites through mineralization of collagen matrix. Journal of Biomedical Materials Research 50, 518 (2000). 215. G. Falini, Crystallization of calcium carbonates in biologically inspired collagenous matrices. International Journal of Inorganic Materials 2, 455 (2000). 216. K. Naka, Y. Chujo, Control of crystal nucleation and growth of calcium carbonate by synthetic substrates. Chemistry of Materials 13, 3245 (2001). 217. W. Tong, S. J. Eppell, Control of surface mineralization using collagen fibrils. Journal of Biomedical Materials Research 61, 346 (2002). 218. F. H. Shen, Q. L. Feng, C. M. Wang, The modulation of collagen on crystal morphology of calcium carbonate. Journal of Crystal Growth 242, 239 (2002).

PAGE 150

139 219. N. Roveri, G. Falini, M. C. Sidoti, A. Tampieri, E. Landi, M. Sandri, B. Parma, Biologically inspired growth of hydroxyapatite nanocrystals inside self-assembled collagen fibers. Materials Science & Engineering C-Biomimetic and Supramolecular Systems 23, 441 (2003). 220. M. Iijima, Y. Moriwaki, Y. Kuboki, Oriented growth of octacalcium phosphate on and inside the collagenous matrix in vitro. Connective Tissue Research 32, 519 (1995). 221. C. Combes, C. Rey, M. Freche, In vitro crystallization of octacalcium phosphate on type I collagen: influence of serum albumin. Journal of Materials Science-Materials in Medicine 10, 153 (1999). 222. A. C. Lawson, J. T. Czernuszka, Production and characterization of a collagen-calcium phosphate composite for use as a bone substitute. Materials Research Society Symposium Proceedings 550, 273 (1999). 223. A. John, L. Hong, Y. Ikada, Y. Tabata, A trial to prepare biodegradable collagen-hydroxyapatite composites for bone repair. Journal of Biomaterials Science-Polymer Edition 12, 689 (2001). 224. E. K. Girija, Y. Yokogawa, F. Nagata, Bone-like apatite formation on collagen fibrils by biomimetic method. Chemistry Letters, 702 (Jul 5, 2002). 225. S. H. Rhee, J. Tanaka, Hydroxyapatite coating on a collagen membrane by a biomimetic method. Journal of the American Ceramic Society 81, 3029 (1998). 226. S. H. Rhee, J. D. Lee, J. Tanaka, Nucleation of hydroxyapatite crystal through chemical interaction with collagen. Journal of the American Ceramic Society 83, 2890 (2000). 227. N. J. Mathers, J. T. Czernuszka, Growth of hydroxyapatite on type 1 collagen. Journal of Materials Science Letters 10, 992 (1991). 228. D. Lickorish, J. A. M. Ramshaw, J. A. Werkmeister, V. Glattauer, C. R. Howlett, Collagen-hydroxyapatite composite prepared by biomimetic process. Journal of Biomedical Materials Research Part A 68A, 19 (Jan 1, 2004). 229. S. H. Rhee, J. Tanaka, Effect of citric acid on the nucleation of hydroxyapatite in a simulated body fluid. Biomaterials 20, 2155 (1999). 230. L. J. Zhang, X. S. Feng, H. G. Liu, D. J. Qian, L. Zhang, X. L. Yu, F. Z. Cui, Hydroxyapatite/collagen composite materials formation in simulated body fluid environment. Materials Letters 58, 719 (Feb, 2004). 231. J. D. Hartgerink, E. Beniash, S. I. Stupp, Self-assembly and mineralization of peptide-amphiphile nanofibers. Science 294, 1684 (2001).

PAGE 151

140 232. G. Goissis, S. V. D. Maginador, V. D. A. Martins, Biomimetic mineralization of charged collagen matrices: In vitro and in vivo study. Artificial Organs 27, 437 (2003). 233. M. Kikuchi, S. Itoh, H. N. Matsumoto, Y. Koyama, K. Takakuda, K. Shinomiya, J. Tanaka, Fibrillogenesis of hydroxyapatite/collagen self-organized composites. Bioceramics 15 240-2, 567 (2003). 234. S. H. Rhee, Y. Suetsugu, J. Tanaka, Biomimetic configurational arrays of hydroxyapatite nanocrystals on bio-organics. Biomaterials 22, 2843 (2001). 235. W. Zhang, S. S. Liao, F. Z. Cui, Hierarchical self-assembly of nano-fibrils in mineralized collagen. Chemistry of Materials 15, 3221 (2003). 236. A. Tampieri, G. Celotti, E. Landi, M. Sandri, N. Roveri, G. Falini, Biologically inspired synthesis of bone-like composite: Self-assembled collagen fibers/hydroxyapatite nanocrystals. Journal of Biomedical Materials Research Part A 67A, 618 (Nov 1, 2003). 237. W. Pompe, H. Worch, M. Epple, W. Friess, M. Gelinsky, P. Greil, U. Hempel, D. Scharnweber, K. Schulte, Functionally graded materials for biomedical applications. Materials Science and Engineering a-Structural Materials Properties Microstructure and Processing 362, 40 (Dec 5, 2003). 238. A. Borsato, S. Frisia, B. Jones, K. Van der Borg, Calcite moonmilk: Crystal morphology and environment of formation in caves in the Italian Alps. Journal of Sedimentary Research 70, 1171 (2000). 239. S. E. Phillips, P. G. Self, Morphology, crystallography and origin of needle-fiber calcite in quaternary pedogenic calcretes of South-Australia. Australian Journal of Soil Research 25, 429 (1987). 240. E. P. Verrecchia, K. E. Verrecchia, Needle-fiber calcite a critical-review and a proposed classification. Journal of Sedimentary Research Section a-Sedimentary Petrology and Processes 64, 650 (1994). 241. R. S. Wagner, "VLS mechanism of crystal growth," in Whisker Technology A. P. Levitt, Ed. (Wiley-Interscience, New York, (1970) pp. 47-117. 242. R. S. Wagner, W. C. Ellis, Vapor-liquid-solid mechanism of single crystal growth ( new method growth catalysis from impurity whisker epitaxial + large crystals Si E ). Applied Physics Letters 4, 89 (1964). 243. G. Gu, M. Burghard, G. T. Kim, G. S. Dusberg, P. W. Chiu, V. Krstic, S. Roth, W. Q. Han, Growth and electrical transport of germanium nanowires. Journal of Applied Physics 90, 5747 (2001).

PAGE 152

141 244. J. V. Milewski, F. D. Gac, J. J. Petrovic, S. R. Skaggs, Growth of beta-silicon carbide whiskers by the VLS process. Journal of Materials Science 20, 1160 (1985). 245. M. K. Sunkara, S. Sharma, R. Miranda, G. Lian, E. C. Dickey, Bulk synthesis of silicon nanowires using a low-temperature vapor-liquid-solid method. Applied Physics Letters 79, 1546 (2001). 246. T. J. Trentler, K. M. Hickman, S. C. Goel, A. M. Viano, P. C. Gibbons, W. E. Buhro, Solution-liquid-solid growth of crystalline Iii-V semiconductors an analogy to vapor-liquid-solid growth. Science 270, 1791 (1995). 247. T. J. Trentler, K. M. Hickman, S. C. Goel, A. M. Viano, P. C. Gibbons, W. E. Buhro, Solution-liquid-solid (SLS) growth of crystalline semiconductor fibers and whiskers a variation on VLS. Abstracts of Papers of the American Chemical Society 210, 5 (1995). 248. J. Aizenberg, J. Hanson, T. F. Koetzle, S. Weiner, L. Addadi, Control of macromolecule distribution within synthetic and biogenic single calcite crystals. Journal of the American Chemical Society 119, 881 (1997). 249. R. Giles, S. Manne, S. Mann, D. E. Morse, G. D. Stucky, P. K. Hansma, Inorganic overgrowth of aragonite on molluscan nacre examined by atomic force microscopy. Biological Bulletin (Woods Hole, MA, United States) 188, 8 (1995). 250. T. E. Schaffer, C. IonescuZanetti, R. Proksch, M. Fritz, D. A. Walters, N. Almqvist, C. M. Zaremba, A. M. Belcher, B. L. Smith, G. D. Stucky, D. E. Morse, P. K. Hansma, Does abalone nacre form by heteroepitaxial nucleation or by growth through mineral bridges? Chemistry of Materials 9, 1731 (1997). 251. T. J. Trentler, S. C. Goel, K. M. Hickman, A. M. Viano, M. Y. Chiang, A. M. Beatty, P. C. Gibbons, W. E. Buhro, Solution-liquid-solid growth of indium phosphide fibers from organometallic precursors: Elucidation of molecular and nonmolecular components of the pathway. Journal of the American Chemical Society 119, 2172 (1997). 252. S. Raz, S. Weiner, L. Addadi, Formation of high-magnesian calcites via an amorphous precursor phase: Possible biological implications. Advanced Materials 12, 38 (2000). 253. A. S. Posner, "Bone mineral and the mineralization process," in Bone and Mineral/5 W. A. Peck, Ed. (Elsevier Science Publishers, Amsterdam, (1987) pp. 65. 254. E. Johansen, H. F. Parks, Electron microscopic observations on the 3-dimensional morphology of apatite crystallites of human dentine and bone. Journal of Biophysical and Biochemical Cytology 7, 743 (1960).

PAGE 153

142 255. W. J. Landis, M. C. Paine, M. J. Glimcher, Electron-microscopic observations of bone tissue prepared anhydrously in organic-solvents. Journal of Ultrastructure Research 59, 1 (1977). 256. Bocciare.Ds, Morphology of Crystallites in Bone. Calcified Tissue Research 5, 261 (1970). 257. M. Iijima, Y. Moriwaki, Lengthwise and oriented growth of octacalcium phosphate on cation selective membrane in a model system of enamel formation. Crystal Growth 112, 571 (1991). 258. F. B. Bagambisa, U. Joos, W. Schilli, A scanning electron-microscope study of the ultrastructural organization of bone-mineral. Cells and Materials 3, 93 (1993). 259. L. A. Gower, Morphological control in biomineralization Is it simpler than we thought? Abstracts of Papers of the American Chemical Society 217, 040 (1999). 260. L. A. Gower, Ph.D. Dissertation, University of Massachusetts at Amherst (1997). 261. L. W. Fisher, J. D. Termine, Non-collagenous proteins influencing the local mechanism of calcification. Clin. Orthop. 200, 362 (1985). 262. L. Addadi, S. Weiner, Interactions between acidic proteins and crystals stereochemical requirements in biomineralization. Proceedings of the National Academy of Sciences of the United States of America 82, 4110 (1985). 263. M. E. Marsh, Biomineralization in the presence of calcium-binding phosphoprotein particles. Journal of Experimental Biology 239, 207 (1986). 264. M. A. Crenshaw, in Biological Mineralization and Demineralization G. H. Nancollas, Ed. (Springer-Verlag, N.Y., (1982) pp. 243-258. 265. A. P. Wheeler, C. S. Sikes, "Matrix-crystal interactions in CaCO 3 biomineralization," in Biomineralization Chemical and Biochemical Perspectives S. Mann, J. Webb, R. J. P. Williams, Eds. (VCH Publ., N. Y., (1989) pp. 95-131. 266. J. F. Burke, I. V. Yannas, W. C. Quinby, C. C. Bondoc, W. K. Jung, Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury. Annals of Surgery 194, 413 (1981). 267. E. Dalas, P. G. Klepetsanis, P. G. Koutsoukos, Calcium carbonate deposition on cellulose. Journal of Colloid and Interface Science 224, 56 (2000). 268. G. Falini, S. Fermani, M. Gazzano, A. Ripamonti, Polymorphism and architectural crystal assembly of calcium carbonate in biologically inspired polymeric matrices. Journal of the Chemical Society-Dalton Transactions, 3983 (2000).

PAGE 154

143 269. T. Elsdale, J. Bard, Collagen substrata for studies on cell behavior. Journal of Cell Biology 54, 626 (1972). 270. H. K. Kleinman, E. B. McGoodwin, S. I. Rennard, G. R. Martin, Preparation of collagen substrates for cell attachment effect of collagen concentration and phosphate buffer. Analytical Biochemistry 94, 308 (1979). 271. H. K. Kleinman, R. J. Klebe, G. R. Martin, Role of collagenous matrices in the adhesion and growth of cells. Journal of Cell Biology 88, 473 (1981). 272. M. J. Glimcher, Krane, S.M., Biology of Collagen B. S. Gould, Ed. (Academic Press, London, 1968). 273. W. J. Landis, The strength of a calcified tissue depends in part on the molecular-structure and organization of its constituent mineral crystals in their organic matrix. Bone 16, 533 (1995). 274. M. Olszta, S. Gajjeraman, M. Kaufman, L. Gower, Nano-fibrous calcite synthesized via a solution-precursor-solid (SPS) mechanism. Chemistry of Materials Accepted (2004). 275. P. Fratzl, K. Misof, I. Zizak, G. Rapp, H. Amenitsch, S. Bernstorff, Fibrillar structure and mechanical properties of collagen. Journal of Structural Biology 122, 119 (1998). 276. E. D. Eanes, Dynamics of calcium phosphate precipitation. Calcif. Biol. Syst., 1 (1992). 277. V. Ziv, H. D. Wagner, S. Weiner, Microstructure-microhardness relations in parallel-fibered and lamellar bone. Bone 18, 417 (1996). 278. Y. Wu, M. Glimcher, L. Graham, D. Chesler, Y. Wang, J. Ackerman, Noninvasive measurement of degree of bone mineralization with solid state proton and phosphorus MRI. Bone 32, S177 (May, 2003). 279. Y. Wu, J. L. Ackerman, E. S. Strawich, C. Rey, H. M. Kim, M. J. Glimcher, Phosphate ions in bone: Identification of a calcium-organic phosphate complex by P-31 solid-state NMR spectroscopy at early stages of mineralization. Calcified Tissue International 72, 610 (May, 2003). 280. G. Y. Cho, Y. T. Wu, J. L. Ackerman, Detection of hydroxyl ions in bone mineral by solid-state NMR spectroscopy. Science 300, 1123 (May 16, 2003). 281. Y. T. Wu, J. L. Ackerman, H. M. Kim, C. Rey, A. Barroug, M. J. Glimcher, Nuclear magnetic resonance spin-spin relaxation of the crystals of bone, dental enamel, and synthetic hydroxyapatites. Journal of Bone and Mineral Research 17, 472 (Mar, 2002).

PAGE 155

144 282. L. T. Kuhn, Y. T. Xu, C. Rey, L. C. Gerstenfeld, M. D. Grynpas, J. L. Ackerman, H. M. Kim, M. J. Glimcher, Structure, composition, and maturation of newly deposited calcium-phosphate crystals in chicken osteoblast cell cultures. Journal of Bone and Mineral Research 15, 1301 (Jul, 2000). 283. Y. T. Wu, M. J. Glimcher, C. Rey, J. L. Ackerman, A unique protonated phosphate group in bone-mineral not present in synthetic calcium phosphates identification by P-31 solid-state NMR-spectroscopy. Journal of Molecular Biology 244, 423 (Dec 9, 1994). 284. J. P. Yesinowski, H. Eckert, Hydrogen environments in calcium phosphates H-1 mas NMR at high spinning speeds. Journal of the American Chemical Society 109, 6274 (Oct 14, 1987).

PAGE 156

BIOGRAPHICAL SKETCH Matthew John Olszta was born on January 5 th 1977, to Coralie and John Olszta in famed Joliet, IL, a town made famous by the opening scene of the 1980 hit movie, The Blues Brothers. He is kin to two younger brothers, Andrew and Michael. He attended Joliet West Township High School, where he graduated as the class salutatorian in 1995. Based on his love for math and science, he applied, and was accepted to the engineering college at the University of Illinois, at Champaign/Urbana. After spending his first year deciding a major, Matthew followed on his love for polymers to the number one ranked Materials Science and Engineering Department at Illinois. In his time there, he worked as an undergraduate researcher for Dr. James Economy doing chemical modification of carbon fiber networks. During his tenure at Illinois, he performed an engineering co-op at Motorola in the Chicago suburb of Northbrook. It was here that the first seeds of graduate school were planted in his head by fellow co-workers. In the final year of his undergraduate career, he decided to apply to graduate school in sunnier pastures in the great state of Florida. After graduating with a bachelors degree in materials science and engineering in the spring of the year 2000, he moved to Gainesville to enroll in the Department of Materials Science and Engineering at the University of Florida. In his first semester at Florida, he was introduced to the glorious experience that is Florida Gator football, as well as being provided an interesting research opportunity by the professor for which he was serving as a teaching assistant, Dr. Laurie Gower. He was given the chance to do research on synthetic bone through a new biomimetic mineralization technique. Through a stroke of luck, the graduate student who was just starting this project was leaving the school to join his wife at another university. For the 145

PAGE 157

146 next 4 years Matthew spent his time working on a variety of biomimetic mineralization techniques, further expanding the original work of Dr. Gower to include the formation of nano-fibrous mineral deposits through a process, coined by Matthew and Dr. Gower, as the solution-precursor-solid (SPS) process, as well as demonstrate the ability to synthetically recreate the nanostructured architecture of natural bone. These two phenomenal discoveries served as the basis of his dissertation on the way to graduating from the Department of Materials Science and Engineering at the University of Florida in May of 2004 with his doctorate.