Time evolution of moduli of a polymer-induced liquid precursor (PILP) of calcium carbonate.

In situ AFM observations show that when PILP droplets contact a surface, their initial properties are either a liquid with a high interfacial tension (350 mJ m-2) or a soft gel-like material with a low modulus (less than 0.2 MPa). These findings suggest that PILP may initially be liquid-like to infiltrate collagen fibrils, enabling the production of interpenetrating composites, and/or become viscoelastic, to provide a means for moulding minerals.

Influence of Crosslinking Methods on Biomimetically Mineralized Collagen Matrices for Bone-like Biomaterials.

To assist in bone defect repair, ideal bone regeneration scaffolds should exhibit good osteoconductivity and osteoinductivity, but for load-bearing applications, they should also have mechanical properties that emulate those of native bone. The use of biomimetic processing methods for the mineralization of collagen fibrils has resulted in interpenetrating composites that mimic the nanostructure of native bone; however, closely matching the mechanical properties of bone on a larger scale is something that is still yet to be achieved. In this study, four different collagen crosslinking methods (EDC-NHS, quercetin, methacrylated collagen, and riboflavin) are compared and combined with biomimetic mineralization via the polymer-induced liquid-precursor (PILP) process, to obtain bone-like collagen-hydroxyapatite composites. Densified fibrillar collagen scaffolds were fabricated, crosslinked, and biomimetically mineralized using the PILP process, and the effect of each crosslinking method on the degree of mineralization, tensile strength, and modulus of the mineralized scaffolds were analyzed and compared. Improved modulus and tensile strength values were obtained using EDC-NHS and riboflavin crosslinking methods, while quercetin and methacrylated collagen resulted in little to no increase in mechanical properties. Decreased mineral contents appear to be necessary for retaining tensile strength, suggesting that mineral content should be kept below a percolation threshold to optimize properties of these interpenetrating nanocomposites. This work supports the premise that a combination of collagen crosslinking and biomimetic mineralization methods may provide solutions for fabricating robust bone-like composites on a larger scale.

Assessment of Optimal Conditions for Marine Invertebrate Cell-Mediated Mineralization of Organic Matrices.

Cellular strategies and regulation of their crystallization mechanisms are essential to the formation of biominerals, and harnessing these strategies will be important for the future creation of novel non-native biominerals that recapitulate the impressive properties biominerals possess. Harnessing these biosynthetic strategies requires an understanding of the interplay between insoluble organic matrices, mineral precursors, and soluble organic and inorganic additives. Our long-range goal is to use a sea anemone model system (Nematostella vectensis) to examine the role of intrinsically disordered proteins (IDPs) found in native biomineral systems. Here, we study how ambient temperatures (25-37 °C) and seawater solution compositions (varying NaCl and Mg ratios) will affect the infiltration of organic matrices with calcium carbonate mineral precursors generated through a polymer-induced liquid-precursor (PILP) process. Fibrillar collagen matrices were used to assess whether solution conditions were suitable for intrafibrillar mineralization, and SEM with EDS was used to analyze mineral infiltration. Conditions of temperatures 30 °C and above and with low Mg:Ca ratios were determined to be suitable conditions for calcium carbonate infiltration. The information obtained from these observations may be useful for the manipulation and study of cellular secreted IDPs in our quest to create novel biosynthetic materials.

Comparison of Synthetic vs. Biogenic Polymeric Process-Directing Agents for Intrafibrillar Mineralization of Collagen.

With the aging population, there is a growing need for mineralized tissue restoration and synthetic bone substitutes. Previous studies have shown that a polymer-induced liquid-precursor (PILP) process can successfully mineralize collagen substrates to achieve compositions found in native bone and dentin. This process also leads to intrafibrillar apatitic crystals with their [001] axes aligned roughly parallel to the long axis of the collagen fibril, emulating the nanostructural organization found in native bone and dentin. When demineralized bovine bone was remineralized via the PILP process using osteopontin (OPN), the samples were able to activate mouse marrow-derived osteoclasts to similar levels to those of native bone, suggesting a means for fabricating bioactive bone substitutes that could trigger remodeling through the native bone multicellular unit (BMU). In order to determine if OPN derived from bovine milk could be a cost-effective process-directing agent, the mineralization of type I collagen scaffolds using this protein was compared to the benchmark polypeptide of polyaspartic acid (sodium salt; pAsp). In this set of experiments, we found that OPN led to much faster and more uniform mineralization when compared with pAsp, making it a cheaper and commercially attractive alternative for mineralized tissue restorations.

Biotechnology Approach to Mineral Separation via Phage Flotation Collectors.

A long-standing challenge in the mining industry is the separation of mineral particles that have similar surface characteristics for which surfactant-based flotation collectors cannot discriminate. In Florida phosphate mining, this problem occurs in the separation of dolomite [CaMg(CO3)2] contaminants from the desired francolite mineral {a fluorapatite [Ca5(PO4)3(F,OH)]}. In this study, phage display techniques were used to select phage clones with specific binding affinity to francolite, which were then tested in a benchtop bubbler flotation apparatus for their ability to selectively float francolite particles from mixtures containing dolomite. Contact angles measured with the captive bubble technique were used to examine changes in the surface character of the mineral particles upon adsorption of the phage, which showed that the most selective phage led to an increase in the contact angle from 16 to 50°. Although this is below the level considered hydrophobic, the correlation between contact angles and increased flotation recovery suggests that the phage coat proteins are behaving as efficient bioamphiphiles for the attachment of the particles to air bubbles, demonstrating a new and environmentally friendly type of biocollector system. The chemical and physical characteristics of the phage "tail" peptides were evaluated to offer an explanation for the specificity of phage binding. We conclude with a discussion of the potential benefits of this biotechnology approach, even for commodity industries such as mining or other particle separation systems, when costs and sustainability are considered.

Colloid assembly and transformation (CAT): The relationship of PILP to biomineralization.

The field of biomineralization has undergone a revolution in the past 25 years, which paralleled the discovery by Gower of a polymer-induced liquid-precursor (PILP) mineralization process. She proposed this in vitro model system might be useful for studying the role biopolymers play in biomineralization; however, the ramifications of this pivotal discovery were slow to be recognized. This was presumably because it utilized simple polypeptide additives, and at that time it was not recognized that the charged proteins intimately associated with biominerals are often intrinsically disordered proteins (IDPs). Over the years, many enigmatic biomineral features have been emulated with this model system, too many to be mere coincidence. Yet the PILP system continues to be underacknowledged, probably because of its namesake, which indicates a "liquid precursor", while we now know the phase appears to have viscoelastic character. Another factor is the confusing semantics that arose from the discovery of multiple "non-classical crystallization" pathways. This review suggests a more relevant terminology for the polymer-modulated reactions is "colloid assembly and transformation (CAT)", which we believe more accurately captures the key stages involved in both biomineralization and the PILP process. The PILP model system has helped to decipher the key role that biopolymers, namely the IDPs, play in modulating biomineralization processes, which was not readily accomplished in living biological systems. Some remaining challenges in understanding the organic-inorganic interactions involved in biomineralization are discussed, which further highlight how the PILP model system may prove invaluable for studying the simple, yet complex, CAT crystallization pathway.

Enhanced silver diamine fluoride therapy using the PILP method -A nanoindentation study.

The aim of this study was to evaluate the feasibility of applying the polymer-induced liquid-precursor (PILP) method to enhance silver diamine fluoride (SDF) therapy. One hundred forty micrometer deep artificial caries lesions were treated with (A) 38% SDF solution and (B) 38% SDF containing poly-L-aspartic acid (pASP). Changes in the nanomechanical profile across the lesion were evaluated. Hydrated artificial lesions had a low reduced elastic modulus (0.3 GPa) and nanohardness (0.02 GPa) region extending about 100 µm into the lesion, with a gradual linear increase to about 168 µm where the values plateaued to around 18 GPa/1.0 GPa. Topical application of SDF resulted in significantly recovered properties (p<0.001). SDF containing pASP resulted in greater nanomechanical properties compared to SDF alone, showing similar sloped regions up to 96 µm, then SDF alone dropped while SDF containing pASP continued at a modest slope until reaching normal at 144 µm. This nanoindentation study shows enhanced SDF therapy using the PILP method.

In situ flow cell platform for examining calcium oxalate and calcium phosphate crystallization on films of basement membrane extract in the presence of urinary 'inhibitors'.

A significant portion of the population suffers from idipoathic calcium oxalate (CaOx) kidney stones, and current clinical treatments of stones have limited lasting success with a high rate of patients suffering from reoccurring stones. Understanding the role of physiologically relevant urinary species on the formation, aggregation, and growth of CaOx crystals can allow for better understanding of this complex biomineralization process and lead to more effective clinical treatments. Our prior work has focused on developing a two-stage model system, where the first stage emulates the formation of Randall's plaque, and the second stage examines the influence of the plaque on overgrowth of CaOx into a stone. Herein, we report on the development of an easy-to-use flow-cell platform that utilizes basement membrane extract (BME) as a biologically relevant crystallization substrate to study the influence of urinary 'inhibitors' on the in situ formation and growth of CaOx on BME under flow conditions. Magnesium, citrate, and osteopontin were studied because of their known ability to inhibit CaOx formation, but their influence also led to interesting modifications to the terminal crystal habit. Magnesium had little to no effect on the CaOx crystallization, but both citrate and osteopontin resulted in significant changes to the crystallization kinetics and the terminal crystal habits. Triply inhibited artificial urine solutions resulted in CaOx monohydrate formations that resembled physiological stones, and the in situ platform allowed for morphogenesis to be dynamically monitored. The BME was also used in a two-stage model system to first grow CaP that mimicked Randall's plaques, whereby the impact of the CaP crystallizing surface on CaOx formation could be studied. It was found that the CaP surface did not result in any significant changes in CaOx crystal formation or growth indicating that the urinary inhibitors and the basement membrane substrate were the dominant factors in modulating CaOx crystallization. It was also found that the basement membrane surface promoted the attachment and/or nucleation and growth of both CaOx and CaP crystals compared to bare glass surfaces, thereby enabling easy study of the urinary inhibitors. The work presented here has elucidated the terminal growth habit of different COM structures and has provided an easy to use platform that can be widely adopted by the kidney stone and other crystallization communities.

Rapid fabrication of vascularized and innervated cell-laden bone models with biomimetic intrafibrillar collagen mineralization.

Bone tissue, by definition, is an organic-inorganic nanocomposite, where metabolically active cells are embedded within a matrix that is heavily calcified on the nanoscale. Currently, there are no strategies that replicate these definitive characteristics of bone tissue. Here we describe a biomimetic approach where a supersaturated calcium and phosphate medium is used in combination with a non-collagenous protein analog to direct the deposition of nanoscale apatite, both in the intra- and extrafibrillar spaces of collagen embedded with osteoprogenitor, vascular, and neural cells. This process enables engineering of bone models replicating the key hallmarks of the bone cellular and extracellular microenvironment, including its protein-guided biomineralization, nanostructure, vasculature, innervation, inherent osteoinductive properties (without exogenous supplements), and cell-homing effects on bone-targeting diseases, such as prostate cancer. Ultimately, this approach enables fabrication of bone-like tissue models with high levels of biomimicry that may have broad implications for disease modeling, drug discovery, and regenerative engineering.

Remineralization of demineralized dentin using a dual analog system.

OBJECTIVE: Improved methods are needed to remineralize dentin caries in order to promote conservation of dentin tissue and minimize the surgical interventions that are currently required for clinical treatment. Here, we test the hypothesis that bulk substrates can be effectively mineralized via a dual analog system proposed by others, using a tripolyphosphate (TPP) "templating analog" and a poly(acrylic acid) (PAA) or poly(aspartic acid) (pAsp) "sequestration analog," the latter of which generates the polymer-induced liquid-precursor (PILP) mineralization process studied in our laboratory. MATERIAL & METHODS: Demineralized human dentin slices were remineralized with and without pre-treatment with TPP, using either PAA or pAsp as the PILP process-directing agent. A control experiment with no polymer present was used for comparison. RESULTS: No mineralization was observed in any of the PAA groups. In both the pAsp and no polymer groups, TPP inhibited mineralization on the surfaces of the specimens but promoted mineralization within the interiors. Pre-treatment with TPP enhanced overall mineralization of the pAsp group. However, when analysed via TEM, regions with little mineral were still present. CONCLUSION: Poly(acrylic acid) was unable to remineralize demineralized dentin slices under the conditions employed, even when pre-treated with TPP. However, pre-treatment with TPP enhanced overall mineralization of specimens that were PILP-remineralized using pAsp.

Development of a two-stage in vitro model system to investigate the mineralization mechanisms involved in idiopathic stone formation: stage 1-biomimetic Randall's plaque using decellularized porcine kidneys.

Idiopathic calcium oxalate (CaOx) stone formers form stones that are commonly attached to calcium phosphate (CaP) deposits in the renal tissue, known as Randall's plaques (RP). Plaques are suggested to originate in the renal tubular basement membrane, where they exhibit a morphology of concentrically laminated apatitic spherules, while in the interstitial regions, the collagen fibrils and vesicles become mineralized. We hypothesize that these minerals might form by non-classical crystallization mechanisms, such as via amorphous precursors, some of which might originate from a polymer-induced liquid-precursor (PILP) process. Thus, our goal is to identify mineralogical 'signatures' of various stone formation mechanisms. To do this for idiopathic CaOx stones, we are developing a two-stage model system of CaP-CaOx composite stones, consisting of stage (1) CaP mineralized plaque, followed by stage (2) CaOx overgrowth into a stone. For the studies presented here, decellularized porcine kidneys were mineralized with CaP using polyaspartic acid or the protein osteopontin (OPN) to induce the PILP process and create biomimetic RP. Analysis of the PILP-mineralized tissues shows features that resemble the native plaques, including mineral spherules and collagen with intrafibrillar mineral. In contrast, the classical crystallization produced large apatitic spherulites, which is a very different morphology, but one which is also found in some stones. An alternative hypothesis regarding Randall's plaque, and if or when it becomes pathological, is discussed.

Development of a two-stage model system to investigate the mineralization mechanisms involved in idiopathic stone formation: stage 2 in vivo studies of stone growth on biomimetic Randall's plaque.

Idiopathic stone formers often form calcium oxalate (CaOx) stones that are attached to calcium phosphate (CaP) deposits in the renal tissue, known as Randall's plaques (RP). Plaques are suggested to originate in the renal tubular basement membrane and spread into the interstitial regions where collagen fibrils and vesicles become mineralized; if the epithelium is breached, the RP becomes overgrown with CaOx upon exposure to urine. We have developed a two-stage model system of CaP-CaOx composite stones, consisting of Stage (1) CaP mineralized plaque, followed by Stage (2) CaOx overgrowth into a stone. In our first paper in this series (Stage 1), osteopontin (and polyaspartate) were found to induce a non-classical mineralization of porcine kidney tissues, producing features that resemble RP. For the Stage 2 studies presented here, biomimetic RPs from Stage 1 were implanted into the bladders of rats. Hyperoxaluria was induced with ethylene glycol for comparison to controls (water). After 4 weeks, rats were sacrificed and the implants were analyzed using electron microscopy and X-ray microanalyses. Differences in crystal phase and morphologies based upon the macromolecules present in the biomimetic plaques suggest that the plaques have the capacity to modulate the crystallization reactions. As expected, mineral overgrowths on the implants switched from CaP (water) to CaOx (hyperoxaluric). The CaOx crystals were aggregated and mixed with organic material from the biomimetic RP, along with some amorphous and spherulitic CaOx near the "stone" surfaces, which seemed to have become compact and organized towards the periphery. This system was successful at inducing "stones" more similar to human idiopathic kidney stones than other published models.

The influence of osteopontin-guided collagen intrafibrillar mineralization on pericyte differentiation and vascularization of engineered bone scaffolds.

Biomimetically mineralized collagen scaffolds are promising for bone regeneration, but vascularization of these materials remains to be addressed. Here, we engineered mineralized scaffolds using an osteopontin-guided polymer-induced liquid-precursor mineralization method to recapitulate bone's mineralized nanostructure. SEM images of mineralized samples confirmed the presence of collagen with intrafibrillar mineral, also EDS spectra and FTIR showed high peaks of calcium and phosphate, with a similar mineral/matrix ratio to native bone. Mineralization increased collagen compressive modulus up to 15-fold. To evaluate vasculature formation and pericyte-like differentiation, HUVECs and hMSCs were seeded in a 4:1 ratio in the scaffolds for 7 days. Moreover, we used RT-PCR to investigate the gene expression of pericyte markers ACTA2, desmin, CD13, NG2, and PDGFRß. Confocal images showed that both nonmineralized and mineralized scaffolds enabled endothelial capillary network formation. However, vessels in the nonmineralized samples had longer vessel length, a larger number of junctions, and a higher presence of αSMA+ mural cells. RT-PCR analysis confirmed the downregulation of pericytic markers in mineralized samples. In conclusion, although both scaffolds enabled endothelial capillary network formation, mineralized scaffolds presented less pericyte-supported vessels. These observations suggest that specific scaffold characteristics may be required for efficient scaffold vascularization in future bone tissue engineering strategies. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1522-1532, 2019.

Integrating the PILP-mineralization process into a restorative dental treatment.

The addition of charged polymers, like poly-aspartic acid (pAsp), to mineralizing solutions allows for transport of calcium and phosphate ions into the lumen of collagen fibrils and subsequent crystallization of oriented apatite crystals by the so-called Polymer-Induced Liquid Precursor (PILP) mineralization process, leading to the functional recovery of artificial dentin lesions by intrafibrillar mineralization of collagen. OBJECTIVE: To evaluate the feasibility of applying the PILP method as part of a restorative treatment and test for effectiveness to functionally remineralize artificial lesions in dentin. MATERIALS AND METHODS: Two methods of providing pAsp to standardized artificial lesions during a restorative procedure were applied: (A) pAsp was mixed into commercial RMGI (resin modified glass ionomer) cement formulations and (B) pAsp was added at high concentration (25mg/ml) in solution to rehydrate lesions before restoring with a RMGI cement. All specimens were immersed in simulated body fluid for two weeks to allow for remineralization and then analyzed for dehydration shrinkage, integrity of cement-dentin interface, degree of mineralization, and changes in the nanomechanical profile (E-modulus) across the lesion. RESULTS: After the remineralization treatment, lesion shrinkage was significantly reduced for all treatment groups compared to demineralized samples. Pores developed in RMGI when pAsp was added. A thin layer at the dentin-cement interface, rich in polymer formed possibly from a reaction between pAsp and the RMGI. When analyzed by SEM under vacuum, most lesions delaminated from the cement interface. EDS-analysis showed some but not full recovery of calcium and phosphorous levels for treatment groups that involved pAsp. Nanoindentations placed across the interface indicated improvement for RMGI containing 40% pAsp, and were significantly elevated when lesions were rehydrated with pAsp before being restored with RMGI. In particular the most demineralized outer zone recovered substantially in the elastic modulus, suggesting that functional remineralization has been initiated by pAsp delivery upon rehydration of air-dried demineralized dentin. In contrast, the effectiveness of the RMGI on functional remineralization of dentin was minimal when pAsp was absent. SIGNIFICANCE: Incorporation of pAsp into restorative treatments using RMGIs promises to be a feasible way to induce the PILP-mineralization process in a clinical setting and to repair the structure and properties of dentin damaged by the caries process.

Hierarchical Characterization and Nanomechanical Assessment of Biomimetic Scaffolds Mimicking Lamellar Bone via Atomic Force Microscopy Cantilever-Based Nanoindentation.

The hierarchical structure of bone and intrinsic material properties of its two primary constituents, carbonated apatite and fibrillar collagen, when being synergistically organized into an interpenetrating hard-soft composite, contribute to its excellent mechanical properties. Lamellar bone is the predominant structural motif in mammalian hard tissues; therefore, we believe the fabrication of a collagen/apatite composite with a hierarchical structure that emulates bone, consisting of a dense lamellar microstructure and a mineralized collagen fibril nanostructure, is an important first step toward the goal of regenerative bone tissue engineering. In this work, we exploit the liquid crystalline properties of collagen to fabricate dense matrices that assemble with cholesteric organization. The matrices were crosslinked via carbodiimide chemistry to improve mechanical properties, and are subsequently mineralized via the polymer-induced liquid-precursor (PILP) process to promote intrafibrillar mineralization. Neither the crosslinking procedure nor the mineralization affected the cholesteric collagen microstructures; notably, there was a positive trend toward higher stiffness with increasing crosslink density when measured by cantilever-based atomic force microscopy (AFM) nanoindentation. In the dry state, the average moduli of moderately (X51; 4.8 ± 4.3 GPa) and highly (X76; 7.8 ± 6.7 GPa) crosslinked PILP-mineralized liquid crystalline collagen (LCC) scaffolds were higher than the average modulus of bovine bone (5.5 ± 5.6 GPa).

Influence of fluoride on the mineralization of collagen via the polymer-induced liquid-precursor (PILP) process.

OBJECTIVE: The polymer-induced liquid-precursor (PILP) mineralization process has been shown to remineralize artificial dentin lesions to levels consistent with those of native dentin. However, nanoindentation revealed that the moduli of those remineralized lesions were only ∼50% that of native dentin. We hypothesize that this may be due to the PILP process having been previously optimized to obtain high amounts (∼70wt%) of intrafibrillar crystals, but without sufficient interfibrillar mineral, another significant component of dentin. METHODS: Fluoride was added to the PILP-mineralization of collagen from rat tail tendon at varying concentrations to determine if a better balance of intra- versus inter-fibrillar mineralization could be obtained, as determined by electron microscopy. Nanoindentation was used to determine if fluoridated apatite could improve the mechanical properties of the composites. RESULTS: Fluoride was successfully incorporated into the PILP-mineralization of rat tail tendon and resulted in collagen-mineral composite systems with the mineral phase of hydroxyapatite containing various levels of fluoridation. As the fluoride concentration increased, the crystals became larger and more rod-like, with an increasing tendency to form on the fibril surfaces rather than the interior. Nanomechanical testing of the mineralized tendons revealed that fluoride addition did not increase modulus over PILP mineralization alone. This likely resulted from the separated nature of collagen fibrils that comprise tendon, which does not provide lateral reinforcement and therefore may not be suited for the compressive loads of nanoindentation. SIGNIFICANCE: This work contributes to the development of minimally invasive approaches to caries treatment by determining if collagen can be functionally mineralized.

Biomimetic organization of collagen matrices to template bone-like microstructures.

The mineralized extracellular matrix (ECM) of bone is essential in vertebrates to provide structure, locomotion, and protect vital organs, while also acting as a calcium and phosphate reservoir to maintain homeostasis. Bone's structure comprises mainly structural collagen fibrils, hydroxyapatite nanocrystals and water, and it is the organization of the densely-packed collagen matrix that directs the organization of the mineral crystallites. Biogenic mineralization occurs when osteoblasts release "mineral bearing globules" which fuse into the preformed collagen matrix, and upon crystallization of this amorphous precursor, the fibrils become embedded with [001] oriented nanocrystals of hydroxyapatite. Our prior work has shown that this nanostructured organization of bone can be reproduced in vitro using the polymer-induced liquid-precursor (PILP) process. In this report, our focus is on using biomimetic processing to recreate both the nano- and micro-structure of lamellar bone. We first applied molecular crowding techniques to acidic, type-I collagen solutions to form dense, liquid crystalline collagen (LCC) scaffolds with cholesteric order. We subsequently mineralized these LCCs via the PILP process to achieve a high degree of intrafibrillar mineral, with compositions and organization similar to that of native bone and with a "lamellar" microstructure generated by the twisting LCC template. In depth characterization of the nano- and micro-structure was performed, including optical and electron microscopy, X-ray and electron diffraction, and thermogravimetric analyses. The results of this work lead us closer to our goal of developing hierarchically structured, collagen-hydroxyapatite composites which can serve as fully synthetic, bioresorbable, load-bearing bone substitutes that are remodeled by the native BRU.

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.

Multifunctional role of osteopontin in directing intrafibrillar mineralization of collagen and activation of osteoclasts.

Mineralized collagen composites are of interest because they have the potential to provide a bone-like scaffold that stimulates the natural processes of resorption and remodeling. Working towards this goal, our group has previously shown that the nanostructure of bone can be reproduced using a polymer-induced liquid-precursor (PILP) process, which enables intrafibrillar mineralization of collagen with hydroxyapatite to be achieved. This prior work used polyaspartic acid (pASP), a simple mimic for acidic non-collagenous proteins, to generate nanodroplets/nanoparticles of an amorphous mineral precursor which can infiltrate the interstices of type-I collagen fibrils. In this study we show that osteopontin (OPN) can similarly serve as a process-directing agent for the intrafibrillar mineralization of collagen, even though OPN is generally considered a mineralization inhibitor. We also found that inclusion of OPN in the mineralization process promotes the interaction of mouse marrow-derived osteoclasts with PILP-remineralized bone that was previously demineralized, as measured by actin ring formation. While osteoclast activation occurred when pASP was used as the process-directing agent, using OPN resulted in a dramatic effect on osteoclast activation, presumably because of the inherent arginine-glycine-aspartate acid ligands of OPN. By capitalizing on the multifunctionality of OPN, these studies may lead the way to producing biomimetic bone substitutes with the capability of tailorable bioresorption rates.

Biomimetic collagen-hydroxyapatite composite fabricated via a novel perfusion-flow mineralization technique.

Prevalent three-dimensional scaffolds for bone tissue engineering are mineralized collagen-hydroxyapatite (Col/HA) composites. Conventional mineralization techniques are either to coat collagen scaffold surfaces with minerals or to simply mix collagen and mineral nanoparticles together. These conventional in vitro collagen mineralization methods are different from the in vivo bone formation process and often result in scaffolds that are not suitable for bone tissue engineering. In this study, a unique perfusion-flow (i.e., dynamic) in conjunction with a previously described polymer-induced liquid-precursor (PILP) method was used to fabricate a porous Col/HA composite. The dynamic flow emulated the physiological extracellular fluid flow containing the mineralization ions, while the PILP method facilitated the deposition of the HA crystals within the collagen fibrils (i.e., intrafibrillar mineralization). By utilizing a dynamic PILP technique to mimic the in vivo bone formation process, the resultant Col/HA composite has a similar structure and compositions like human trabecular bone. A comparison of the dynamic and static mineralization methods revealed that the novel dynamic technique facilitates more efficient and homogenous mineral deposition throughout the Col/HA composite. The dynamic intrafibrillar mineralization method generated stiff Col/HA composites with excellent surface property for cell attachment and growth. The human mesenchymal stem cells cultured on the Col/HA composites quickly remodeled the scaffolds and resulted in constructs with an extensive cell-derived extracellular matrix network.