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.

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.

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.

Association of Randall plaque with collagen fibers and membrane vesicles.

PURPOSE: Idiopathic calcium oxalate kidney stones develop by calcium oxalate crystal deposition on Randall plaque. The mechanisms involved in Randall plaque formation are still unclear. We hypothesized that Randall plaque formation is similar to that of vascular calcification, involving components of extracellular matrix, including membrane bound vesicles and collagen fibers. To verify our hypothesis we critically examined renal papillary tissue from patients with stones. MATERIALS AND METHODS: We performed 4 mm cold cup biopsy of renal papillae on 15 patients with idiopathic stones undergoing percutaneous nephrolithotomy. Tissue was immediately fixed and processed for analysis by various light and electron microscopic techniques. RESULTS: Spherulitic calcium phosphate crystals, the hallmark of Randall plaque, were seen in all samples examined, including in interstitium and laminated basement membrane of tubular epithelium. Large crystalline deposits were composed of dark elongated strands mixed with spherulites. Strands showed banded patterns similar to collagen. Crystal deposits were surrounded by collagen fibers and membrane bound vesicles. Energy dispersive x-ray microanalysis and electron diffraction identified the crystals as hydroxyapatite. Few kidneys were examined and urinary data were not available on all patients. CONCLUSIONS: Results showed that crystals in Randall plaque are associated with collagen and membrane bound vesicles. Collagen fibers appeared calcified and vesicles contained crystals. Crystal deposition in renal papillae may have started with membrane vesicle induced nucleation and grown by the further addition of crystals at the periphery in a collagen framework.

Biomimetic mineralization of woven bone-like nanocomposites: role of collagen cross-links.

Ideal biomaterials for bone grafts must be biocompatible, osteoconductive, osteoinductive and have appropriate mechanical properties. For this, the development of synthetic bone substitutes mimicking natural bone is desirable, but this requires controllable mineralization of the collagen matrix. In this study, densified collagen films (up to 100 µm thick) were fabricated by a plastic compression technique and cross-linked using carbodiimide. Then, collagen-hydroxyapatite composites were prepared by using a polymer-induced liquid-precursor (PILP) mineralization process. Compared to traditional methods that produce only extrafibrillar hydroxyapatite (HA) clusters on the surface of collagen scaffolds, by using the PILP mineralization process, homogeneous intra- and extrafibrillar minerals were achieved on densified collagen films, leading to a similar nanostructure as bone, and a woven microstructure analogous to woven bone. The role of collagen cross-links on mineralization was examined and it was found that the cross-linked collagen films stimulated the mineralization reaction, which in turn enhanced the mechanical properties (hardness and modulus). The highest value of hardness and elastic modulus was 0.7 ± 0.1 and 9.1 ± 1.4 GPa in the dry state, respectively, which is comparable to that of woven bone. In the wet state, the values were much lower (177 ± 31 and 8 ± 3 MPa) due to inherent microporosity in the films, but still comparable to those of woven bone in the same conditions. Mineralization of collagen films with controllable mineral content and good mechanical properties provide a biomimetic route toward the development of bone substitutes for the next generation of biomaterials. This work also provides insight into understanding the role of collagen fibrils on mineralization.

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.

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.

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.

Mechanism of formation of concentrically laminated spherules: implication to Randall's plaque and stone formation.

We report on the formation of calcium phosphate multi-laminated spherules via a polymer-induced liquid-like precursor (PILP) process. In this non-classical crystallization route, the precipitation of liquid-like amorphous calcium phosphate (ACP) particles is promoted using anionic polypeptide additives, and these droplets coalesce to form globules that later crystallize into spherulites. During crystallization of the amorphous globules, the polymer additive, as well as the waters of hydration, is excluded ahead of the crystallization front, but some polymer becomes entrapped within diffusion-limited zones. This results in the formation of concentric laminations with layers of variable density from organic-rich inclusions. The striking resemblance of these spherules with the crystals of the Randall's plaque and other laminated stones suggests that such biological structures may form via an amorphous precursor process as well. Given the organic-rich environment present in the urinary tract, one might expect a large amount of organic materials to become entrapped within the stratified zones of a forming stone during this type of solidification and transformation process.

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.

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.

Molding mineral within microporous hydrogels by a polymer-induced liquid-precursor (PILP) process.

Natural biominerals often have exquisite morphologies, where the cells exercise a high degree of crystallographic control through secretion of biological macromolecules and regulation of ion transport. One important example is the sea urchin spine. It has recently been shown to be formed through deposition of a transient amorphous calcium carbonate (ACC) precursor phase that later transforms to single-crystalline calcite, ultimately forming an elaborate three-dimensional microporous calcium carbonate structure with interconnected pores. Macromolecules associated with the mineral phase are thought to play a key role in regulating this transformation. The work described here mimics this type of morphological control by "molding" an amorphous calcium carbonate precursor within a porous poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel that has been prepared as a negative replica from the void space of an urchin spine. Using an acidic biomimetic polymer as a process-directing agent, we show that polyaspartic acid induces amorphous calcium carbonate (ACC) nanoparticles, which have fluidic character and therefore are able to infiltrate the PHEMA hydrogel replica and coalesce into the convoluted morphology that replicates the original microporous structure of the sea urchin spine. By "molding" calcium carbonate into a complex morphology at room temperature, using a precursor process that is induced by a biomimetic acidic macromolecule, the PILP process is a useful in vitro model for examining different aspects of the amorphous-to-crystalline transformation process that is apparently used by a variety of biomineralizing organisms. For example, although we were able to replicate the overall morphology of the spine, it had polycrystalline texture; further studies with this system will focus on controlling the nucleation event, which may help to elucidate how such a convoluted structure can be prepared with single-crystalline texture via an amorphous precursor. Through a better understanding of the mechanisms used by organisms to regulate crystal properties, such biomimetic processes can lead to the synthesis of materials with superior electronic, mechanical, and optical properties.

Periplasmic cuticular calcium oxalate crystal deposition in Dracaena sanderiana.

• The chemical nature and biological basis for crystal deposition in epidermal subcuticular areas are reported here for the angiosperm Dracaena sanderiana. • Position, development and identification of crystals in adult leaves of D. sanderiana was carried out using X-ray diffraction, crystal morphology and scanning and transmission electron microscopy techniques. • Numerous small (< 1-6 µm) calcium oxalate monohydrate crystals were found between the primary epidermal cell wall and the cuticle. Their formation was highly specific and predictable with respect to location and relative timing of development during leaf ontogeny. The crystals were perisplasmic as, at formation, the nascent epidermal cell wall was external to the crystals. Cuticular crystallization of calcium oxalate monohydrate in D. sanderiana occurred in crystal chambers situated between the plasma membrane and the primary cell wall. Crystal deposition did not occur in developing guard cells. • The spatial pattern of calcium oxalate monohydrate within the epidermal cells, orientation of the crystallographic axes and the existence of crystal chambers outside the plasma membrane suggest biologically controlled crystal deposition in D. sanderiana.

Aggregation and dispersion characteristics of calcium oxalate monohydrate: effect of urinary species.

In this research, screening and central composite experimental designs are used to determine the effect of various factors on the aggregation and dispersion characteristics of previously grown calcium oxalate monohydrate (COM) crystals in artificial urinary environments of controlled variables. The variables examined are pH and calcium, oxalate, pyrophosphate, citrate, and protein concentrations in ultrapure water and artificial urine. Optical density measurements, particle size analysis, optical microscopy, AFM force measurements, and protein adsorption have been used to assess the state of aggregation and dispersion of the COM crystals and to elucidate the mechanisms involved in such a complex system. The data indicate that our model protein, mucin, acts as a dispersant. This is attributed to steric hindrance resulting from the adsorbed mucoprotein. Oxalate, however, promotes aggregation. Interesting interactions between protein and oxalate along with protein and citrate are observed. Such interactions (synergistic or antagonistic) are found to depend on the concentrations of these species. Surface responses for these interactions are presented and discussed in this paper. In summary, solution, surface, and interface chemistries interact in a complex manner in the physiological environment to either inhibit or promote aggregation, and an understanding of such interactions may help determine and control the factors affecting kidney stone formation.

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.