Polymer-induced Liquid-precursor Process Research Articles

Engineered phage films as scaffolds for CaCO3 biomineralization.

M13 bacteriophages (phage) were exploited as CaCO3 mineralization scaffolds for hard tissue engineering applications. M13 phage was first self-assembled into biomimetic fibrous scaffolds, followed by CaCO3 biomineralization via the polymer-induced liquid precursor process. The phage scaffolds successfully incorporated calcium carbonate, facilitating nucleation and growth of spherulitically textured calcite. The Young's modulus of the scaffolds increased by an order of magnitude after mineralization while also supporting the growth of mouse fibroblasts. These findings demonstrate that phage-based biomaterials are a feasible platform for creating biomineralized hard tissue constructs, in support of future studies in hard tissue engineering and biomedical applications.

Biomimetic Randall’s plaque as an in vitro model system for studying the role of acidic biopolymers in idiopathic stone formation

Randall's plaque (RP) deposits seem to be consistent among the most common type of kidney stone formers, idiopathic calcium oxalate stone formers. This group forms calcium oxalate renal stones without any systemic symptoms, which contributes to the difficulty of understanding and treating this painful and recurring disease. Thus, the development of an in vitro model system to study idiopathic nephrolithiasis, beginning with RP pathogenesis, can help in identifying how plaques and subsequently stones form. One main theory of RP formation is that calcium phosphate deposits initially form in the basement membrane of the thin loops of Henle, which then fuse and spread into the interstitial tissue, and ultimately make their way across the urothelium, where upon exposure to the urine, the mineralized tissue serves as a nidus for overgrowth with calcium oxalate into a stone. Our group has found that many of the unusual morphologies found in RP and stones, such as concentrically laminated spherulites and mineralized collagenous tissue, can be reproduced in vitro using a polymer-induced liquid precursor (PILP) process, in which acidic polypeptides induce a liquid phase amorphous precursor to the mineral, yielding non-equilibrium crystal morphologies. Given that there are many acidic proteins and polysaccharides present in the renal tissue and urine, we have put forth the hypothesis that the PILP system may be involved in urolithiasis. Therefore, our goal is to develop an in vitro model system of these two stages of composite stone formation to study the role that various acidic macromolecules may play. In our initial experiments presented here, the development of "biomimetic" RP was investigated, which will then serve as a nidus for calcium oxalate overgrowth studies. To mimic the tissue environment, MatriStem(®) (ACell, Inc.), a decellularized porcine urinary bladder matrix was used, because it has both an intact epithelial basement membrane surface and a tunica propria layer, thus providing the two types of matrix constituents found associated with mineral in the early stages of RP formation. We found that when using the PILP process to mineralize this tissue matrix, the two sides led to dramatically different mineral textures, and they bore a striking resemblance to native RP, which was not seen in the tissue mineralized via the classical crystal nucleation and growth process. The interstitium side predominantly consisted of collagen-associated mineral, while the luminal side had much less mineral, which appeared to be tiny spherules embedded within the basement membrane. Although these studies are only preliminary, they support our hypothesis that kidney stones may involve non-classical crystallization pathways induced by the large variety of macromolecular species in the urinary environment. We believe that mineralization of native tissue scaffolds is useful for developing a model system of stone formation, with the ultimate goal of developing strategies to avoid RP and its detrimental consequences in stone formation, or developing therapeutic treatments to prevent or cure the disease. Supported by NIDDK grant RO1DK092311.

Strong stabilization of liquid amorphous calcium carbonate by polymers and proteins

We have studied the effect of bio-inspired polymers and proteins like ovalbumin, lysozyme and silicatein, which are present in the first stage of egg shell formation or in the formation of siliceous spicules of sponges, on the homogeneous formation of the liquid-amorphous calcium carbonate (LACC) precursor, by a combination of complementary methods like in situ WAXS, light scattering, TEM and cryo-TEM. Lysozyme destabilizes the LACC emulsion, whereas ovalbumin extends the lifetime of the emulsified state. We demonstrate that ovalbumin acts as a stabilizer for a polymer-induced liquid precursor (PILP) process. We propose that the liquid amorphous calcium carbonate is affected by polymers by depletion stabilization and de-emulsification rather than induced by acidic proteins and polymers during a polymer-induced liquid precursor process. Thus, the original PILP coating effect appears to be a result of a de-emulsification process of a stabilized LACC phase. Silicatein-α is responsible for the biomineralization of silica in sponges guides the self-assembly of calcite "spicules" similar to the spicules of the calcareous sponge Sycon. The self-assembled spicules, 10-300 µm in length and 5-10 µm in diameter, are composed of aligned calcite nanocrystals. The spicules are initially amorphous but transform into calcite within months, exhibiting unusual growth along [100]. While natural spicules evidence brittle failure, the synthetic spicules show an elastic response which greatly enhances bending strength. Later stages of nucleation have been studied by "trapping" nuclei from solution by shock-freezing of droplets in liquid ethane (cryo-TEM). This yields snapshots of the structure formation process at given point. In a first step the full determination of the structure of vaterite, one of the common CaCO3 polymorphs, was solved on nanometer-sized crystallites by electron crystallography. These results demonstrate that crystals that are too small for single-crystal X-ray diffraction and too difficult to solve by powder diffraction may nevertheless be amenable to accurate structure determination by electron crystallography.

Correction: Functional Remineralization of Dentin Lesions Using Polymer-Induced Liquid-Precursor Process

There was an error in the same of the fourth author. The correct name is: Stefan Habelitz The correct citation is: Burwell AK, Thula-Mata T, Gower LB, Habelitz S, Kurylo M, et al. (2012) Functional Remineralization of Dentin Lesions Using Polymer-Induced Liquid-Precursor Process. PLoS ONE 7(6): e38852. doi:10.1371/journal.pone.0038852

Functional Remineralization of Dentin Lesions Using Polymer-Induced Liquid-Precursor Process

It was hypothesized that applying the polymer-induced liquid-precursor (PILP) system to artificial lesions would result in time-dependent functional remineralization of carious dentin lesions that restores the mechanical properties of demineralized dentin matrix. 140 µm deep artificial caries lesions were remineralized via the PILP process for 7–28 days at 37°C to determine temporal remineralization characteristics. Poly-L-aspartic acid (27 KDa) was used as the polymeric process-directing agent and was added to the remineralization solution at a calcium-to-phosphate ratio of 2.14 (mol/mol). Nanomechanical properties of hydrated artificial lesions had a low reduced elastic modulus (ER = 0.2 GPa) region extending about 70 μm into the lesion, with a sloped region to about 140 μm where values reached normal dentin (18–20 GPa). After 7 days specimens recovered mechanical properties in the sloped region by 51% compared to the artificial lesion. Between 7–14 days, recovery of the outer portion of the lesion continued to a level of about 10 GPa with 74% improvement. 28 days of PILP mineralization resulted in 91% improvement of ER compared to the artificial lesion. These differences were statistically significant as determined from change-point diagrams. Mineral profiles determined by micro x-ray computed tomography were shallower than those determined by nanoindentation, and showed similar changes over time, but full mineral recovery occurred after 14 days in both the outer and sloped portions of the lesion. Scanning electron microscopy and energy dispersive x-ray analysis showed similar morphologies that were distinct from normal dentin with a clear line of demarcation between the outer and sloped portions of the lesion. Transmission electron microscopy and selected area electron diffraction showed that the starting lesions contained some residual mineral in the outer portions, which exhibited poor crystallinity. During remineralization, intrafibrillar mineral increased and crystallinity improved with intrafibrillar mineral exhibiting the orientation found in normal dentin or bone.

A metastable liquid precursor phase of calcium carbonate and its interactions with polyaspartate

Invertebrate organisms that use calcium carbonate extensively in the formation of their hard tissues have the ability to deposit biominerals with control over crystal size, shape, orientation, phase, texture, and location. It has been proposed by our group that charged polyelectrolytes, like acidic proteins, may be employed by organisms to direct crystal growth through an intermediate liquid phase in a process called the polymer-induced liquid-precursor (PILP) process. Recently, it has been proposed that calcium carbonate crystallization, even in the absence of any additives, follows a non-classical, multi-step crystallization process by first associating into a liquid precursor phase before transition into solid amorphous calcium carbonate (ACC) and eventually crystalline calcium carbonates. In order to determine if the PILP process involves the promotion, or stabilization, of a naturally occurring liquid precursor to ACC, we have analyzed the formation of saturated and supersaturated calcium carbonate–bicarbonate solutions using Ca2+ ion selective electrodes, pH electrodes, isothermal titration calorimetry, nanoparticle tracking analysis, 13C T2 relaxation measurements, and 13C PFG-STE diffusion NMR measurements. These studies provide evidence that, in the absences of additives, and at near neutral pH (emulating the conditions of biomineralization and biomimetic model systems), a condensed phase of liquid-like droplets of calcium carbonate forms at a critical concentration, where it is stabilized intrinsically by bicarbonate ions. In experiments with polymer additive, the data suggests that the polymer is kinetically stabilizing this liquid condensed phase in a distinct and pronounced fashion during the so called PILP process. Verification of this precursor phase and the stabilization that polymer additives provide during the PILP process sheds new light on the mechanism through which biological organisms can exercise such control over deposited CaCO3 biominerals, and on the potential means to generate in vitro mineral products with features that resemble biominerals seen in nature.

Strong Stabilization of Amorphous Calcium Carbonate Emulsion by Ovalbumin: Gaining Insight into the Mechanism of ‘Polymer-Induced Liquid Precursor’ Processes

The impact of the ovo proteins ovalbumin and lysozyme--present in the first stage of egg shell formation--on the homogeneous formation of the liquid amorphous calcium carbonate (LACC) precursor, was studied by a combination of complementing methods: in situ WAXS, SANS, XANES, TEM, and immunogold labeling. Lysozyme (pI = 9.3) destabilizes the LACC emulsion whereas the glycoprotein ovalbumin (pI = 4.7) extends the lifespan of the emulsified state remarkably. In the light of the presented data: (a) Ovalbumin is shown to behave commensurable to the 'polymer-induced liquid precursor' (PILP) process proposed by Gower et al. Ovalbumin can be assumed to take a key role during eggshell formation where it serves as an effective stabilization agent for transient precursors and prevents undirected mineralization of the eggshell. (b) It is further shown that the emulsified LACC carries a negative surface charge and is electrostatically stabilized. (c) We propose that the liquid amorphous calcium carbonate is affected by polymers by depletion stabilization and de-emulsification rather than 'induced' by acidic proteins and polymers during a so-called polymer-induced liquid-precursor process. The original PILP coating effect, first reported by Gower et al., appears to be a result of a de-emulsification process of a stabilized LACC phase. The behavior of the liquid amorphous carbonate phase and the polymer-induced liquid-precursor phase itself can be well described by colloid chemical terms: electrostatic and depletion stabilization and de-emulsification by depletion destabilization.

In vitro mineralization of dense collagen substrates: A biomimetic approach toward the development of bone-graft materials

Bone is an organic–inorganic composite which has hierarchical structuring that leads to high strength and toughness. The nanostructure of bone consists of nanocrystals of hydroxyapatite embedded and aligned within the interstices of collagen fibrils. This unique nanostructure leads to exceptional properties, both mechanical and biological, making it difficult to emulate bone properties without having a bone-like nanostructured material. A primary goal of our group’s work is to use biomimetic processing techniques that lead to bone-like structures. In our prior studies, we demonstrated that intrafibrillar mineralization of porous collagen sponges, leading to a bone-like nanostructure, can be achieved using a polymer-induced liquid precursor (PILP) mineralization process. The objective of this study was to investigate the use of this polymer-directed crystallization process to mineralize dense collagen substrates. To examine collagen scaffolds that truly represent the dense-packed matrix of bone, manatee bone was demineralized to isolate its collagen matrix, consisting of a dense, lamellar osteonal microstructure. This biogenic collagen scaffold was then remineralized using polyaspartate to direct the mineralization process through an amorphous precursor pathway. The various conditions investigated included polymer molecular weight, substrate dimension and mineralization time. Mineral penetration depths of up to 100μms were achieved using this PILP process, compared to no penetration with only surface precipitates observed for the conventional crystallization process. Electron microscopy, wide-angle X-ray diffraction and thermal analysis were used to characterize the resulting hydroxyapatite/collagen composites. These studies demonstrate that the original interpenetrating bone nanostructure and osteonal microstructure could be recovered in a biogenic matrix using the PILP process.

Oriented hydroxyapatite in turkey tendon mineralized via the polymer-induced liquid-precursor (PILP) process

Bone is a hierarchically structured composite which imparts it with unique mechanical properties and bioresorptive potential. These properties are primarily influenced by the underlying nanostructure of bone, which consists of nanocrystals of hydroxyapatite embedded and uniaxially aligned within collagen fibrils. There is also a small fraction of non-collagenous proteins in bone, and these are thought to play an important role in bone's formation. In our in vitro model system of bone formation, polyanionic peptides are used to mimic the role of the non-collagenous proteins. In our prior studies, we have shown that intrafibrillar mineralization can be achieved in synthetic reconstituted collagen sponges using a polymer-induced liquid-precursor (PILP) mineralization process. This led to a nanostructured arrangement of hydroxyapatite crystals within the individual fibrils which closely mimics that of bone. This report demonstrates that biogenic collagen scaffolds obtained from turkey tendon, which consist of densely packed and oriented collagen fibrils, can also be mineralized by the PILP process. Synchrotron X-ray diffraction studies show that the mineralization process leads to a high degree of crystallographic orientation at the macroscale, thus emulating that found in the biological system of naturally mineralizing turkey tendon.

Remineralization of Artificial Dentin Lesions via the Polymer-Induced Liquid-Precursor (PILP) Process.

Acid-etched dentin samples with a zone of demineralized dentin were remineralized via the polymer-induced liquid-precursor (PILP) process. Poly-L-aspartic acid was used as the polymeric process-directing agent. Samples were incubated in the mineralization solution for 1-4 weeks. Dentin samples remineralized by the PILP process presented a surface morphology very similar to the intact mineralized dentin's architecture, in contrast to samples mineralized via the conventional nucleation and growth method (without polymer additive), which led to a superficial crust of randomly organized mineral crystals. Energy dispersive x-ray spectroscopy analysis of the PILP-mineralized samples showed the presence of calcium and phosphate ions at high levels. Since no hydroxyapatite (HA) clusters were observed on the surface of the PILP-mineralized samples, we could conclude the signal was produced from the mineral embedded within the dentin matrix. TEM and diffraction analyses suggest that both intrafibrillar and interfibrillar remineralization occurred in the demineralized dentin matrix.

Mimicking the Nanostructure of Bone: Comparison of Polymeric Process-Directing Agents

The nanostructure of bone has been replicated using a polymer-induced liquid-precursor (PILP) mineralization process. This polymer-mediated crystallization process yields intrafibrillar mineralization of collagen with uniaxially-oriented hydroxyapatite crystals. The process-directing agent, an anionic polymer which we propose mimics the acidic non-collagenous proteins associated with bone formation, sequesters calcium and phosphate ions to form amorphous precursor droplets that can infiltrate the interstices of collagen fibrils. In search of a polymeric agent that produces the highest mineral content in the shortest time, we have studied the influence of various acidic polymers on the in vitro mineralization of collagen scaffolds via the PILP process. Among the polymers investigated were poly-L aspartic acid (PASP), poly-L-glutamic acid (PGLU), polyvinylphosphonic acid (PVPA), and polyacrylic acid (PAA). Our data indicate that PASP and the combination of PGLU/PASP formed stable mineralization solutions, and yielded nano-structured composites with the highest mineral content. Such studies contribute to our goal of preparing biomimetic bone graft substitutes with composition and structure that mimic bone.

Mineralogical signatures of stone formation mechanisms

The mechanisms involved in biomineralization are modulated through interactions with organic matrix. In the case of stone formation, the role of the organic macromolecules in the complex urinary environment is not clear, but the presence of mineralogical 'signatures' suggests that some aspects of stone formation may result from a non-classical crystallization process that is induced by acidic proteins. An amorphous precursor has been detected in many biologically controlled mineralization reactions, which is thought to be regulated by non-specific interactions between soluble acidic proteins and mineral ions. Using in vitro model systems, we find that a liquid-phase amorphous mineral precursor induced by acidic polypeptides can lead to crystal textures that resemble those found in Randall's plaque and kidney stones. This polymer-induced liquid-precursor process leads to agglomerates of coalesced mineral spherules, dense-packed spherulites with concentric laminations, mineral coatings and 'cements', and collagen-associated mineralization. Through the use of in vitro model systems, the mechanisms involved in the formation of these crystallographic features may be resolved, enhancing our understanding of the potential role(s) that proteins play in stone formation.

Development of bone-like composites via the polymer-induced liquid-precursor (PILP) process. Part 1: Influence of polymer molecular weight

Bone is an organic-inorganic composite consisting primarily of collagen fibrils and hydroxyapatite crystals intricately interlocked to provide skeletal and metabolic functions. Non-collagenous proteins (NCPs) are also present, and although only a minor component, the NCPs are thought to play an important role in modulating the mineralization process. During secondary bone formation, an interpenetrating structure is created by intrafibrillar mineralization of the collagen matrix. Many researchers have tried to develop bone-like collagen-hydroxyapatite (HA) composites via the conventional crystallization process of nucleation and growth. While those methods have been successful in inducing heterogeneous nucleation of HA on the surface of collagen scaffolds, they have failed to produce a composite with the interpenetrating nanostructured architecture of bone. Our group has shown that intrafibrillar mineralization of type I collagen can be achieved using a polymer-induced liquid-precursor (PILP) process. In this process, acidic polypeptides are included in the mineralization solution to mimic the function of the acidic NCPs, and in vitro studies have found that acidic peptides such as polyaspartate induce a liquid-phase amorphous mineral precursor. Using this PILP process, we have been able to prepare collagen-HA composites with the fundamental nanostructure of bone, wherein HA nanocrystals are embedded within the collagen fibrils. This study shows that through further optimization a very high degree of mineralization can be achieved, with compositions matching that of bone. Synthetic collagen sponges were mineralized with calcium phosphate while analyzing various parameters of the reaction, with the focus of this report on the molecular weight of the polymeric process-directing agent. In order to determine whether intrafibrillar mineralization was achieved, an in-depth characterization of the mineralized composites was performed, including wide-angle X-ray diffraction, electron microscopy and thermogravimetric analyses. The results of this work lead us closer to the development of bone-like collagen-HA composites that could become the next generation of synthetic bone grafts.

Polymer-Induced Liquid-Precursor (PILP) Process in the Non-Calcium Based Systems of Barium and Strontium Carbonate

Barium and strontium carbonate with various nonequilibrium morphologies were grown in the presence of poly(acrylic acid) sodium salt to induce the polymer-induced liquid-precursor (PILP) process. Previously, the PILP process had only been demonstrated for calcium carbonate but with indirect evidence suggesting it can be induced in calcium phosphate and barium carbonate. In this report, further evidence to suggest a similar liquid-phase amorphous precursor can be generated for both barium and strontium carbonate is presented, and these phases were used to synthesize various unique morphologies in the transformed crystals, including films, fibers, and cones. On the basis of the nanogranular texture of the fibers, a new mechanism for fiber formation is proposed, which combines a permutation of our former solution−precursor−solid (SPS) mechanism with mesocrystal assembly. This hypothesis includes an autocatalytic assembly of nanodroplets/nanoparticles at high energy surface protrusions as a means for generati...

Biomimetic mineralization of collagen via an enzyme-aided PILP process

The development of bone-like collagen-hydroxyapatite composites is highly desirable because bone has outstanding mechanical properties and resorptive potential, and a combination of these characteristics could ultimately lead to a load-bearing and bioresorbable bone substitute. Our prior work has shown that intrafibrillar mineralization of collagen can be achieved using a polymer-induced liquid-precursor (PILP) mineralization process. In our in vitro model system, polyaspartate is used to mimic the acidic non-collagenous proteins involved in bone formation. We have previously shown that the anionic polypeptide sequesters ions to induce an amorphous calcium phosphate precursor, and we have put forth the hypothesis that the early-stage precursor is highly hydrated, enabling fluidic droplets to be drawn into the gaps and grooves of collagen fibrils by capillary action. Here, we further our biomimetic approach by using alkaline phosphatase to provide a slow release of inorganic phosphate ions from a phosphate ester, mimicking the biochemical processes of ion regulation found in natural bone formation. The collagen-hydroxyapatite composites were characterized using transmission electron microscopy (TEM) and selected area electron diffraction (SAED), which show that nanocrystals of hydroxyapatite are intrafibrillar and [0 0 1] oriented along the collagen fibril axis. With repeated mineralization steps, the fibrils become cemented together with a non-descript extrafibrillar mineral coating. Although the degree of intrafibrillar mineralization was not as high as our usual method, we believe that with further optimization this enzyme-aided PILP process could provide a closer mimic to the biochemical processes involved in bone formation, and serve as a useful in vitro model system for studying the mechanisms involved in bone formation.

Transition Bars during Transformation of an Amorphous Calcium Carbonate Precursor

A variety of calcium carbonate (CaCO3) biominerals are now thought to be formed via an amorphous precursor pathway. Our group has been using in vitro model systems to examine potential crystallochemical reactions that might occur during biomineralization and, in particular, has proposed that a polymer-induced liquid-precursor (PILP) mineralization process may play a fundamental role in biomineralization. The PILP process is induced with polyanionic polypeptides (containing aspartic acid and phosphoserine) to produce colloids of an amorphous mineral that are so highly hydrated that they coalesce into smooth mineral films. In situ observations, however, show that these films are not smooth throughout the reaction because transition bars form ridges in the tablets/films as the amorphous phase crystallizes. Using fluorescently labeled polymer, it was determined that the transition bars are due to exclusion of the polymeric impurity into diffusion limited zones as crystallization proceeds across the amorphous ...

Growth of nanofibrous barium carbonate on calcium carbonate seeds

Fibrous barium carbonate (BaCO 3/witherite) crystals 50–100 nm in diameter and several microns in length were grown on calcium carbonate (CaCO 3) seeds at temperatures as low as 4 °C. The BaCO 3 fibers were deposited onto calcite rhombs or CaCO 3 films using the polymer-induced liquid-precursor (PILP) process, which was induced with the sodium salt of polyacrylic acid (PAA). The structure and morphology of the resultant fibers were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), selected-area electron diffraction (SAED), and polarized light microscopy (PLM). Fibers were successfully grown on calcite seeds of various morphologies, with a range of barium concentrations, and PAA molecular weight and concentration. Two categories of fibers were grown: straight and twisted. Both types of fibers displayed single-crystalline SAED diffraction patterns, but after examining high-resolution TEM lattice images, it was revealed that the fibers were in fact made up of nanocrystalline domains. We postulate that these nanocrystalline domains are well aligned due to a singular nucleation event (i.e., each fiber propagates from a single nucleation event on the seed crystal) with the nanocrystalline domains resulting from stresses caused by dehydration during crystallization of the highly hydrated precursor phase. These BaCO 3 fibers grown on calcite substrates further illustrate the robustness and non-specificity of the PILP process.

Bone structure and formation: A new perspective

Abstract Bone is a hierarchically structured composite material which, in addition to its obvious biological value, has been well studied by the materials engineering community because of its unique structure and mechanical properties. This article will review the existing bone literature, with emphasis on the prevailing theories regarding bone formation and structure, which lay the groundwork for proposing a new model to explain how intrafibrillar mineralization of collagen can be achieved during bone formation. Intrafibrillar refers to the fact that growth of the mineral phase is somehow directed by the collagen matrix, which leads to a nanostructured architecture consisting of uniaxially oriented nanocrystals of hydroxyapatite embedded within and roughly [0 0 1] aligned parallel to the long collagen fibril axes. Secondary (osteonal) bone, the focus of this review, is a laminated organic–inorganic composite composed primarily of collagen, hydroxyapatite, and water; but minor constituents, such as non-collagenous proteins (NCPs), are also present and are thought to play an important role in bone formation. To date, there has been no clear understanding of the role of these NCPs, although it has been generally assumed that the NCPs regulate solution crystal growth via some type of ‘epitaxial’ relationship between specific crystallographic faces and specific protein conformers. Indeed, ‘epitaxial’ relationships have been calculated; but in practice, it has not been demonstrated that intrafibrillar mineralization can be accomplished via this route. Because of the difficulty in examining biomineralization processes in vivo , the authors of this article have turned to using in vitro model systems to investigate the possible physicochemical mechanisms that may be involved in biomineralization. In the case of bone biomineral, we have now been able to duplicate the most fundamental level of bone structure, the interpenetrating nanostructured architecture, using relatively simple anionic polypeptides that mimic the polyanionic character of the NCPs. We propose that the charged polymer acts as a process-directing agent, by which the conventional solution crystallization is converted into a precursor process. This polymer-induced liquid-precursor (PILP) process generates an amorphous liquid-phase mineral precursor to hydroxyapatite which facilitates intrafibrillar mineralization of type-I collagen because the fluidic character of the amorphous precursor phase enables it to be drawn into the nanoscopic gaps and grooves of collagen fibrils by capillary action. The precursor then solidifies and crystallizes upon loss of hydration waters into the more thermodynamically stable phase, leaving the collagen fibrils embedded with nanoscopic hydroxyapatite (HA) crystals. Electron diffraction patterns of the highly mineralized collagen fibrils are nearly identical to those of natural bone, indicating that the HA crystallites are preferentially aligned with [0 0 1] orientation along the collagen fibril axes. In addition, studies of etched samples of natural bone and our mineralized collagen suggest that the long accepted “deck of cards” model of bone's nanostructured architecture is not entirely accurate. Most importantly, this in vitro model demonstrates that a highly specific, epitaxial-type interaction with NCPs is not needed to stimulate crystal nucleation and regulate crystal orientation, as has long been assumed. Instead, we propose that collagen is the primary template for crystal organization, but with the important caveat that this templating occurs only for crystals formed from an infiltrated amorphous precursor. These results suggest that the 25-year-old debate regarding bone formation via an amorphous precursor phase needs to be revisited. From a biomedical perspective, in addition to providing possible insight into the role of NCPs in bone formation, this in vitro system may pave the way toward the ultimate goal of fabricating a synthetic bone substitute that not only has a composition similar to bone, but has comparable mechanical properties and bioresorptive potential as natural bone. From a materials chemistry perspective, the non-specificity of the PILP process and capillary infiltration mechanism suggests that non-biological materials could also be fabricated into nanostructured composites using this “biomimetic” strategy.

Biomimetic synthesis of calcite films by a polymer-induced liquid-precursor (PILP) process: 1. Influence and incorporation of magnesium

Magnesium-bearing calcium carbonate films have been synthesized via a polymer-induced liquid-precursor (PILP) mineralization process. A variety of morphological features of biominerals can be mimicked with this PILP process; therefore, our group has proposed that this crystallization system can be used as an effective in vitro model system for examining mechanistic issues related to biomineralization. Here, the effect of the Mg 2+/Ca 2+ ratio on the rate of transformation of the amorphous precursor films was investigated using polarized optical microscopy (POM), and the final crystalline structure and composition were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), inductively coupled plasma spectroscopy (ICP) and energy dispersive spectroscopy (EDS). The entrapment of high levels of magnesium in the deposited precursor films had a pronounced inhibitory effect on the amorphous to crystalline transformation, and furthermore, influenced the polycrystalline nature of the film. The magnesium content incorporated within the calcite lattice (8–26%) resembles the range found in biologically formed high magnesium-bearing calcite, while much lower levels were formed via the conventional solution crystallization process. The formation of non-equilibrium morphologies and similar compositions of magnesium-bearing calcite via the PILP process further supports our hypothesis that the PILP process may play a fundamental role in the formation of calcitic biominerals in nature. In the realm of biomimetic engineering, the PILP process may also establish itself as a new method to produce thin ceramic films with variable compositions under ambient conditions.

Patterning Inorganic (CaCO3) Thin Films via a Polymer-Induced Liquid-Precursor Process

The biomimetic synthesis of patterned mineral thin films, based on a combination of the microcontact printing technique and a novel crystallization process called the polymer-induced liquid-precursor (PILP) process, is demonstrated. The PILP process enables the deposition of smooth and continuous calcitic mineral films (up to 1500 nm in thickness) under low-temperature and aqueous-based processing conditions. The films are formed by deposition of colloidal droplets composed of a liquid-phase mineral precursor that is induced by a polymeric process-directing agent (polyaspartate or polyacrylate salts). The droplets can be preferentially deposited onto patterned substrates templated with self-assembled monolayers (SAMs) of alkanethiolate on gold. The droplets coalesce to form an amorphous mineral film, which then transforms (solidifies and crystallizes) while retaining the shape of the patterned template, providing a means for patterning the location and morphology of two-dimensional calcite crystals. A vertical substrate experiment supports the premise that the calcite films are created by adsorption of colloidal droplets from solution, rather than heterogeneous nucleation and growth of an amorphous phase on the SAMs. Large single-crystalline domains, on the order of 50-100 microm, can be "molded" into nonequilibrium morphologies by constraining the mineral precursor to a chemically defined "compartment". Biominerals are well recognized for their elaborate nonequilibrium molded crystal morphologies, and increasing evidence suggests that many biominerals are formed from an amorphous precursor that is stabilized by polyanionic proteins. The biomimetic system examined here, which consists of a polyanionic process-directing agent in combination with a functionalized organic template, offers a practical tool for generating complex inorganic structures such as those found in biominerals.