Extrafibrillar Mineral Research Articles

Surface-Directed Mineralization of Fibrous Collagen Scaffolds in Simulated Body Fluid for Tissue Engineering Applications.

The use of polymer additives that stabilize fluidic amorphous calcium phosphate is key to obtaining intrafibrillar mineralization of collagen in vitro. On the other hand, this biomimetic approach inhibits the nucleation of mineral crystals in unconfined extrafibrillar spaces, that is, extrafibrillar mineralization. The extrafibrillar mineral content is a significant feature to replicate from hard connective tissues such as bone and dentin as it contributes to the final microarchitecture and mechanical stiffness of the biomineral composite. Herein, we report a straightforward route to produce densely mineralized collagenous composites via a surface-directed process devoid of the aid of polymer additives. Simulated body fluid (1×) is employed as a biomimetic crystallizing medium, following a preloading procedure on the collagen surface to quickly generate the amorphous precursor species required to initiate matrix mineralization. This approach consistently leads to the formation of extrafibrillar bioactive minerals in bulk collagen scaffolds, which may offer an advantage in the production of osteoconductive collagen-apatite materials for tissue engineering and repair purposes.

Biomineralization of Collagen-Based Materials for Hard Tissue Repair.

Hydroxyapatite (HA) reinforced collagen fibrils serve as the basic building blocks of natural bone and dentin. Mineralization of collagen fibrils play an essential role in ensuring the structural and mechanical functionalities of hard tissues such as bone and dentin. Biomineralization of collagen can be divided into intrafibrillar and extrafibrillar mineralization in terms of HA distribution relative to collagen fibrils. Intrafibrillar mineralization is termed when HA minerals are incorporated within the gap zone of collagen fibrils, while extrafibrillar mineralization refers to the minerals that are formed on the surface of collagen fibrils. However, the mechanisms resulting in these two types of mineralization still remain debatable. In this review, the evolution of both classical and non-classical biomineralization theories is summarized. Different intrafibrillar mineralization mechanisms, including polymer induced liquid precursor (PILP), capillary action, electrostatic attraction, size exclusion, Gibbs-Donnan equilibrium, and interfacial energy guided theories, are discussed. Exemplary strategies to induce biomimetic intrafibrillar mineralization using non-collagenous proteins (NCPs), polymer analogs, small molecules, and fluidic shear stress are discussed, and recent applications of mineralized collagen fibers for bone regeneration and dentin repair are included. Finally, conclusions are drawn on these proposed mechanisms, and the future trend of collagen-based materials for bone regeneration and tooth repair is speculated.

Effects of bioactive glass with high phosphorus content on mineralization of type I collagen fibrils

To study effects of bioactive glass with high phosphorus content (10.8% P2O5, 54.2% SiO2, 35% CaO, mol%, named PSC) on mineralization of type I collagen fibrils. (1) PSC, and PSC combining 0.1 mg/mL, 0.5 mg/mL, or 1.0 mg/mL polyacrylic acid (PAA), were used to induce the mineralization of self-assembled type I collagen fibrils. After 3 and 7 days of mineralization, collagen fibrils were observed by transmission electron microscopy (TEM) and selected area electron diffraction (SAED). (2) PSC suspension was dialyzed in simulated body fluid (SBF), or in SBF containing 0.1 mg/mL, 0.5 mg/mL, or 1.0 mg/mL PAA, to form amorphous calcium phosphate (ACP), then observed by TEM. (1) PSC and PSC combining 0.1 mg/mL or 0.5 mg/mL PAA induced mainly extrafibrillar mineralization. PSC combining 1.0 mg/mL PAA induced both extrafibrillar and intrafibrillar mineralization. (2) The ACP induced by PSC or PSC combining 0.1 mg/mL PAA partly formed lattice structure after 24 h. The particle size of the ACP induced by PSC combining 0.5 mg/mL PAA was 100-150 nm, and that induced by PSC combining 1.0 mg/mL PAA was 30-50 nm. PSC induced mainly extrafibrillar mineralization, and PSC combining an appropriate concentration (1.0 mg/mL) of PAA induced both extrafibrillar and intrafibrillar mineralization.

Involvement of prenucleation clusters in calcium phosphate mineralization of collagen

Involvement of thermodynamically-stable prenucleation clusters (PNCs) in the biomineralization of collagen has been speculated since their existence was reported in mineralization systems. It has been hypothesized that intrafibrillar mineralization proceeds via nucleation of inhibitor-stabilized intermediates produced by liquid-liquid separation (aka. polymer-induced liquid precursors; PILPs). Here, the contribution of PNCs and PILPs to calcium phosphate intrafibrillar mineralization of collagen was examined in a model with a semipermeable membrane that excludes nucleation inhibitor-stabilized PILPs from reaching the collagen fibrils, using cryogenic electron microscopy of reconstituted fibrils and conventional transmission electron microscopy of collagen sponges. Molecular dynamics simulation with the Interface force field (IFF) was used to confirm the existence of PILPs with amorphous calcium phosphate and elucidate details of the dynamics. Furthermore, intrafibrillar mineralization of single collagen fibrils was experimentally observed with unstabilized PNCs when anionic/cationic polyelectrolytes were used to establish Donnan equilibrium across the semipermeable membrane. Molecular dynamics simulation verified PNC formation within the collagen intrafibrillar gap zones at the atomic scale and explained the role of external PILPs. The PILPs decrease the interfibrillar water content and increase the interfibrillar ionic concentration. Nevertheless, intrafibrillar mineralization of collagen sponges with PNCs alone was inefficacious, being constrained by competition from extrafibrillar mineral precipitation. Statement of SignificanceCompared with conventional PILP-based intrafibrillar mineralization, mineralization of collagen fibrils using unstabilized PNCs is constrained by competition from extrafibrillar mineral deposition. The narrow window of opportunity for PNCs to produce intrafibrillar mineralization provides a plausible explanation for the feasibility of nucleation inhibitor-free intrafibrillar apatite assembly during reconstitution of type I collagen.

Toward the Understanding of Small Protein-Mediated Collagen Intrafibrillar Mineralization.

The design of improved materials for orthopedic implants and bone tissue engineering scaffolds relies on materials mimicking the properties of bone. Calcium phosphate (Ca-PO4)-mineralized collagen fibrils arranged in a characteristic hierarchical structure constitute the building blocks of mineralized vertebrate tissues and control their biomechanical and biochemical properties. Large, flexible, acidic noncollagenous proteins (ANCPs) have been shown to influence collagen mineralization but little is known about mineralization mechanisms with the aid of small proteins. Osteocalcin (OCN) is a small, highly structured biomolecule known as a multifunctional hormone in its undercarboxylated form. Here, we examined the potential mechanism of collagen intrafibrillar mineralization in vitro mediated by OCN as a model protein. Rapid and random extrafibrillar mineralization of flakey Ca-PO4 particles was observed by transmission electron microscopy mainly on the outer surfaces of collagen fibrils of a preformed collagen scaffold in the absence of the protein. In contrast, the protein stabilized hydrated, spherical nanoclusters of Ca-PO4 on the outer surface of the fibrils, thereby retarding extrafibrillar mineralization. The nanoclusters then infiltrated the fibrils resulting in intrafibrillar mineralization with HAP crystals aligned with the fibrils. This mechanism is similar to that observed for unstructured ANCPs. Results of fibrillogenesis and immunogold labeling studies showed that OCN was associated primarily with the fibrils, consistent with ex vivo studies on mineralizing turkey tendon. The present findings contribute to expanding our understanding of collagen intrafibrillar mineralization and provide insight into design synthetic macromolecular matrices for orthopedic implants and bone regeneration.

Biomimetic mineralized collagen scaffolds and their effect on osteogenic differentiation

Background & Aim During bone healing, MSCs differentiate into bone-forming osteoblasts. This process of osteogenic differentiation is partly driven by the main noncellular constituents of bone: collagen and hydroxyapatite (HA). In terms of ultrastructure, aligned HA crystals are inside collagen fibrils (intrafibrillar) and surround fibrils (extrafibrillar). Mimicking this ultrastructure could enhance autograft alternatives in bone healing. This project aims to test ultrastructurally biomimetic mineralized collagen substrates with two extremes of extrafibrillar mineralization (Fig. 1, conditions A and B) for their effect on gene and protein expression during osteogenic differentiation in comparison to a conventional, nonbiomimetic control (Fig. 1, condition C). Methods, Results & Conclusion Methods EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)-fixed collagen suspensions were mineralized with a supersaturated Ca and P solution stabilized with poly-aspartic acid (pAsp), which permits intrafibrillar mineralization. Non-biomimetic substrates were mineralized without pAsp. Scanning electron microscopy (SEM) assessed surface mineral morphology, whereas transmission electron microscopy (TEM) assessed intrafibrillar mineral morphology and location. 2D substrates were generated by drying mineralized suspensions on tissue culture surfaces. Future work will involve testing osteogenic differentiation in 3D using similar mineralization conditions in scaffolds. For testing osteogenic differentiation of human bone marrow MSCs in different mineralization conditions, RT-PCR and immunofluorescent staining are used to quantify gene and protein expression of early (ie. runx2 and osteopontin) vs. later osteoblastic markers (ie. osteocalcin).

Mechanical behavior of biomimetically mineralized collagen matrix using the polymer – induced liquid precursor process

Biomimetic mineralization is a promising technique in biomedical applications. To understand the mechanical behavior of biomimetically mineralized collagen material (BMC), we examined the composition and structure of the mineral deposition in BMCs mineralized by the polymer-induced liquid precursor (PILP) process and applied wide angle x-ray scattering (WAXS) with in situ tensile testing to investigate the mineral-to-tissue co-deformation in the material. We found that the PILP process is able to achieve good biomimetic mineralization in bulk collagen matrix. Compositionally, the mineral deposition showed high crystallinity with no carbonation. However, the morphology of extrafibrillar mineral deposition and the preferential crystal orientation were different from natural bone. Further, the Young’s modulus and mineral-to-tissue co-deformation ratio of the BMC were significantly lower than both natural bone and partially demineralized bone with similar mineral volume fraction. It was concluded that while biomimetic mineralization can achieve good mineral deposition volume in the BMC, the mechanical behavior of the material was different from natural bone.

Modulation of biomimetic mineralization of collagen by soluble ectodomain of discoidin domain receptor 2.

Collagen fibrils serve as the major template for mineral deposits in both biologically derived and engineered tissues. In recent years certain non-collagenous proteins have been elucidated as important players in differentially modulating intra vs. extra-fibrillar mineralization of collagen. We and others have previously shown that the expression of the collagen receptor, discoidin domain receptor 2 (DDR2) positively correlates with matrix mineralization. The objective of this study was to examine if the ectodomain (ECD) of DDR2 modulates intra versus extra-fibrillar mineralization of collagen independent of cell-signaling. For this purpose, a decellularized collagenous substrate, namely glutaraldehyde fixed porcine pericardium (GFPP) was subjected to biomimetic mineralization protocols. GFPP was incubated in modified simulated body fluid (mSBF) or polymer-induced liquid precursor (PILP) solutions in the presence of recombinant DDR2 ECD (DDR2-Fc) to mediate extra or intra-fibrillar mineralization of collagen. Thermogravimetric analysis revealed that DDR2-Fc increased mineral content in GFPP calcified in mSBF while no significant differences were observed in PILP mediated mineralization. Electron microscopy approaches were used to evaluate the quality and quantity mineral deposits. An increase in the matrix to mineral ratio, frequency of particles and size of mineral deposits was observed in the presence of DDR2-Fc in mSBF. Von Kossa staining and immunohistochemistry analysis of adjacent sections indicated that DDR2-Fc bound to both the matrix and mineral phase of GFPP. Further, DDR2-Fc was found to bind to hydroxyapatite (HAP) particles and enhance the nucleation of mineral deposits in mSBF solutions independent of collagen. Taken together, our results elucidate DDR2 ECD as a novel player in the modulation of extra-fibrillar mineralization of collagen.

Generating biomimetic mineralized collagen scaffolds for bone regeneration

Background & Aim In bone healing, osteoblasts derived from periosteal mesenchymal stromal cells (MSCs) form new bone. Osteoblastic differentiation from MSCs (osteoinduction), is partly driven by the components and mechanics of the extracellular matrix (ECM). Bone ECM comprises fibrillar collagen with apatitic mineral in gap zones between collagen molecules (intrafibrillar) and surrounding fibrils (extrafibrillar). This project aims to optimize mineralization conditions for fabricating bone biomimetic hydroxyapatite (HA)-collagen scaffolds. These biomimetic scaffolds will later be tested for their osteoinductive potential in mesenchymal stromal cells (MSCs). Methods, Results & Conclusion Methods EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)-fixed fibrillar collagen scaffolds were mineralized with a metastable, supersaturated Ca and P solution for 2, 4, 6, and 8 days. This process permits biomimetic, intrafibrillar mineralization seen in native bone. Non-biomimetic scaffolds were mineralized in an unstable, supersaturated Ca and P solution. Scanning electron microscopy (SEM) assessed mineral morphology and location, and backscattered electrons detected heavier elements such as Ca and P. Transmission electron microscopy (TEM) identified HA and its alignment with selected area electron diffraction (SAED). To compare osteoinduction by biomimetic scaffolds vs. non-biomimetic scaffolds, qRT-PCR and immunofluorescent staining will be used to quantify gene and protein expression of early (ie. type I collagen and fibronectin) vs. late osteoblastic markers (ie. alkaline phosphatase and osteocalcin). Biomimetic scaffolds are expected to induce greater expression of later osteoblast markers in MSCs compared to non-biomimetic scaffolds. Results Biomimetic scaffolds mineralized for 6 and 8 days exhibited unaligned, extrafibrillar mineral compared to those mineralized for 4 days (Fig.1). SAED confirmed aligned, intrafibrillar mineral. Scaffolds mineralized for 2 days exhibited no detectable mineralization with SEM and backscatter SEM. Non-biomimetic scaffolds demonstrated extrafibrillar amorphous and crystalline mineral phases. Conclusion Mineralization for 4 days with pAsp is the optimal time point for biomimetic mineralized collagen scaffolds for future studies regarding osteogenic differentiation in MSCs. Ultimately, this biomimetic approach is expected to enhance the osteoinductivity of scaffolds as alternatives to autografts in the treatment of non-union fractures.

Polymer conjugation optimizes EDTA as a calcium-chelating agent that exclusively removes extrafibrillar minerals from mineralized collagen

During development of mineralized collagenous tissues, intrafibrillar mineralization is achieved by preventing mineralization precursor inhibitors that are larger than 40 kDa from entering the collagen fibrils. Such a property is incorporated in the design of a calcium chelator for dentin bonding in the etch-and-rinse technique that selectively demineralizes extrafibrillar apatite while leaving the intrafibrillar minerals intact. This strategy prevents complete demineralization of collagen fibrils, avoids collapse of collagen that blocks resin infiltration after air-drying, and protects the completely demineralized fibrils from bacteria colonization and degradation by endogenous proteases after resin bonding. In the present study, a water-soluble glycol chitosan-EDTA (GCE) conditioner was synthesized by conjugation of EDTA, an effective calcium chelator, to high molecular weight glycol chitosan, which exhibits weak chelation property. The GCE conjugate was purified, characterized by FTIR, 1H NMR, isothermal titration calorimetry and ICP-AES, and subjected to size exclusion dialysis to recover molecules that are >40 kDa. The optimal concentration and application time for etching dentin were determined by bond strength testing to ensure that the dentin bonding results were comparable to phosphoric acid etching, and maintained equivalent bond strength after air-drying of the conditioned collagen matrix. Extrafibrillar demineralization was validated with transmission electron microscopy. Inhibition of endogenous dentin proteases was confirmed using in-situ zymography. The water-soluble GCE dentin conditioner was non-cytotoxic and possessed antibacterial activities against planktonic and single-species biofilms, supporting its ongoing development as a dentin conditioner with air-drying, anti-proteolytic and antibacterial properties to enhance the durability of bonds created using the etch-and-rinse bonding technique. Statement of SignificanceThe current state-of-the-art techniques for filling decayed teeth with plastic tooth-colored materials require conditioning the mineralized, biofilm-covered, decayed dentin with acids or acid resin monomers to create a surface layer of completely- or partially-demineralized collagen matrix for the infiltration of adhesive resin monomers. Nevertheless, fillings prepared using these strategies are not as durable as consumers have anticipated. Conjugation of polymeric glycol chitosan with EDTA produces a new conditioner for dentin bonding that demineralizes only extrafibrillar dentin, reduces endogenous protease activities and kills biofilm bacteria. The high molecular weight glycol chitosan-EDTA is non-cytotoxic to the key regenerative players within the dentin-pulp complex. This advance permits dry bonding and the use of hydrophobic resins.

Generating biomimetic mineralized collagen scaffolds for bone regeneration

Purpose: To optimize mineralization conditions for fabricating bone biomimetic HA-collagen scaffolds. These biomimetic scaffolds will be tested in later experiments for their osteoinductive potential in differentiating MSCs into more mature osteoblasts compared to non-biomimetic HA-collagen scaffolds. Methods: To create the scaffolds, fibrillar collagen self-assembly was induced by slowly raising the pH of dissolved collagen. The scaffolds were fixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). After fixation, scaffolds were biomimetically mineralized by exposure to a supersaturated, metastable solution of calcium and phosphorus stabilized with L poly-aspartic acid (pAsp). As controls, scaffolds mineralized in the same calcium and phosphorus solution without pAsp served as non-biomimetic HA-collagen scaffolds. Scaffolds were mineralized for 2, 4, 6, and 8 days for later experiments that will assess the effect that the extent of mineralization has on osteoblast differentiation. Scanning electron microscopy (SEM) assessed mineral morphology and location. In particular, backscattered electrons from SEM were used to detect heavier elements from mineral such as Ca and P in the scaffolds. Transmission electron microscopy (TEM) was used to determine the presence of HA and alignment of mineral with selected area electron diffraction (SAED). Results: Biomimetic scaffolds mineralized for longer amounts of time (6 and 8 days) exhibit excess, unaligned extrafibrillar mineral compared to those mineralized for 4 days (Fig. 1). Scaffolds mineralized 2 days exhibited no detectable mineralization with SEM and backscatter SEM (Fig. 1 insets).Fig. 2TEM demonstrated the presence of aligned, intrafibrillar mineral, as shown by the preferentially aligned 002 arcs from the c-axis reflection of HA in the direction of the collagen fibrils (Fig. 3; diffraction pattern inset).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Yellow circle denotes positioning of selected area aperture used to generate the diffraction pattern (inset).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Conclusions: Mineralization for 4 days with pAsp is the optimal time point for “biomimetic”, intrafibrillarly mineralized collagen scaffolds for future studies regarding osteogenic differentiation. Ultimately, this bone tissue engineering project is expected to contribute to engineering bone-cartilage tissue interfaces in osteoarthritis.

MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.

Effects of Molecular Weight and Concentration of Poly(Acrylic Acid) on Biomimetic Mineralization of Collagen

Inspired by nature, poly(acrylic acid) (PAA) and other polyelectrolytes have been used as noncollagenous proteins (NCPs) surrogates for biomimetic intrafibrillar mineralization of collagen fibrils and thus, to model the ultrastructure of bone, to study the mechanism of bone mineralization and, more scarcely to fabricate scaffolds for hard tissue engineering. The objective of this study was to systematically investigate the effect of the molecular weight (MW) and the concentration of PAA on the rate and pattern of biomineralization of collagen matrices. Densified type I collagen films were mineralized in supersaturated PAA-stabilized amorphous calcium-phosphate (PAA-ACP) solutions containing increasing MW (2 kDa, 50 kDA, 450 kDa) and concentrations (10, 25, 50 mg/L) of PAA up to 7 days. The stability and physical properties of collagen-free PAA-ACP solutions were also investigated. In our system, lowering PAA MW and increasing PAA concentration resulted in solutions with increasing stability. Over stable PAA-ACP solutions that fully inhibited mineralization of the collagen matrices were achieved using PAA 2k-50. Conversely, unstable solutions were obtained using high PAA MW at low concentrations. Nucleation and growth of significant amount of extrafibrillar minerals on the collagen fibrils was obtained using these solutions. In a wide range of combined MW and concentration of PAA we obtained intrafibrillar mineralization of collagen with hydroxyapatite crystals aligned parallel to the collagen fibril as in natural tissues. Intrafibrillar mineralization was correlated with PAA-ACP stability and growth of the PAA-ACP particles in solution. Our results support using PAA to surrogate NCPs function as selective inhibitors or promoters of biological mineralization and provide parameters to manufacture new biomimetic scaffolds and constructs for bone and dentin tissue engineering.

The role of confined collagen geometry in decreasing nucleation energy barriers to intrafibrillar mineralization

Mineralization of collagen is critical for the mechanical functions of bones and teeth. Calcium phosphate nucleation in collagenous structures follows distinctly different patterns in highly confined gap regions (nanoscale confinement) than in less confined extrafibrillar spaces (microscale confinement). Although the mechanism(s) driving these differences are still largely unknown, differences in the free energy for nucleation may explain these two mineralization behaviors. Here, we report on experimentally obtained nucleation energy barriers to intra- and extrafibrillar mineralization, using in situ X-ray scattering observations and classical nucleation theory. Polyaspartic acid, an extrafibrillar nucleation inhibitor, increases interfacial energies between nuclei and mineralization fluids. In contrast, the confined gap spaces inside collagen fibrils lower the energy barrier by reducing the reactive surface area of nuclei, decreasing the surface energy penalty. The confined gap geometry, therefore, guides the two-dimensional morphology and structure of bioapatite and changes the nucleation pathway by reducing the total energy barrier.

* Engineering Osteoinductive Biomaterials by Bioinspired Synthesis of Apatite Coatings on Collagen Hydrogels with Varied Pore Microarchitectures.

Biomaterial controlled osteoinduction is influenced by the porous microenvironment and the composition of incorporated calcium orthophosphate (CaPi) polymorphs, however, for the design of materials that rival the efficacies of natural grafts a systematic approach to assessing the physicochemical properties that affects cellular differentiation is needed. In this research, we introduce a bioinspired synthetic approach to the mineralization of preformed porous collagen hydrogel scaffolds with tunable apatite coatings. Initially, collagen scaffolds are mineralized with dicalcium phosphate dihydrate (DCPD) by alternate immersion in Ca2+ and HPO42- salt solutions. Utilizing classic DCPD conversion chemistry, the surface coatings are selectively transformed to apatite by immersion of the DCPD-collagen substrate in Tris buffer at pH 7.4, 37°C, for 5 days. The composition and morphology of the deposited mineral coatings are characterized by XRD, SEM, and AFM. Variations in the porous microarchitecture of the collagen hydrogel substrate, pore size (9.5 ± 5 μm, 165 ± 50 μm) and pore alignment altered the morphology of the deposited apatite particles. Intrafibrillar and extrafibrillar mineralization of the collagen templates were observed for both investigated pore sizes. However, templates with aligned pores of both sizes were observed to restrict intrafibrillar mineralization resulting in the exclusive deposition of surface coatings. The osteoinductive activity of the apatite-collagen materials with varied pore microarchitectures was evaluated by in vitro culture of human mesenchymal stem cells for 28 days based on cellular proliferation, alkaline phosphatase activity, and the expression of RUNX2. The combined effects of apatite coatings, reduced pore size, and pore alignment contributed to reductions in cellular proliferation. However, the apatite mineral coating was determined to induce high levels of RUNX2 expression in the absence of additional osteoinductive agents, indicative of biomaterial-induced osteogenesis. This work establishes a versatile synthetic platform for the preparation of bone-like apatite collagen materials with osteoinductive activity.

On-site SEM and nanomechanical properties of human OI bone

Modulus mapping and on-site field emission-scanning electron microscopy (FE-SEM) nanoindentation are both applied for the first time on ‘undisturbed’ healthy human and osteogenesis imperfecta (OI) human cortical bones in this work. Modulus mapping reveals the elastic moduli of hydroxyapatite and collagen to be 189·9 and 2·4 GPa for the normal bone and 294·7 GPa and 2·9 GPa for the OI bone respectively. Modulus mapping also indicates a spatial variation in the elastic moduli consistent with the distribution of mineralized fibril and extrafibrillar mineral. The diameter of collagen fibers and the size of mineral crystals in OI are observed to be smaller, and the mineral crystal in OI is also observed to be less anisotropic than those from the normal bone. The median elastic moduli of the OI bone sample are greater than those of the normal bone. On-site FE-SEM nanoindentation reveals the existence of a highly mineralized inner ring with higher elastic moduli adjacent to the Haversian canal and that the elastic nanomechanical properties of interstitial lamellae are greater than those of osteonal lamellae. A periodic modulation in modulus corresponding to the width of a lamella is also observed in on-site FE-SEM nanoindentation.

In Situ Evaluation of Calcium Phosphate Nucleation Kinetics and Pathways during Intra- and Extrafibrillar Mineralization of Collagen Matrices.

We revealed that nucleation sites within collagen fibrils determined pathways for calcium phosphate (CaP) nucleation and its transformation, from amorphous species to crystalline plates, during the biomineralization process. Using in situ small-angle X-ray scattering (SAXS), we examined the nucleation and growth of CaP within collagen matrices and elucidated how a nucleation inhibitor, polyaspartic acid (pAsp), governs mineralization kinetics and pathways at multiple length scales. Mineralization without pAsp led initially to spherical aggregates of CaP in the entire extrafibrillar spaces. With time, the spherical aggregates transformed into plates at the outermost surface of the collagen matrix, preventing intrafibrillar mineralization inside. However, mineralization with pAsp led directly to the formation of intrafibrillar CaP plates with a spatial distribution gradient through the depth of the matrix. The results illuminate mineral nucleation kinetics and real-time nanoparticle distributions within organic matrices in solutions containing body fluid components. Because the macroscale mechanical properties of collagen matrices depend on their mineral content, phase, and arrangement at the nanoscale, this study contributes to better design and fabrication of biomaterials for regenerative medicine.

Selective demineralisation of dentine extrafibrillar minerals—A potential method to eliminate water-wet bonding in the etch-and-rinse technique

ObjectiveThe present study tested the central hypothesis that selective demineralisation of dentine extrafibrillar minerals by lowering the phosphoric acid concentration improves the quality of the resin-dentine interface. MethodsDentine surfaces were etched with different concentrations of phosphoric acid (1, 5, 10, 20, 30 or 40wt%). Scanning electron microscopy was used to observe the micromorphology of the etched dentine surfaces. Energy dispersive X-ray analysis was performed to determine the residual Ca-content of the demineralised dentine matrix. Atomic force microscopy-based nanoindentation was used to analyse the nanomechanical properties of the treated dentine surfaces. The influence of H3PO4 concentration on resin-dentine bond strength was evaluated by microtensile bond strength testing. One-way ANOVA was used to compare the residual Ca-content ratio, reduced elastic modulus (Er) of the treated dentine surfaces and microtensile bond strength among groups. ResultsCollagen fibrils appeared to be wider in diameter after etching with 5% and 10% H3PO4. The partially-demineralized collagen scaffold retained part of its rigidity to maintain an uncollapsed three-dimensional structure. Etching with 1% H3PO4 resulted in the highest residual Ca-content ratio and Er of demineralised dentine matrix, followed by 5% H3PO4. Those values were all significantly higher than values derived from the other groups. Etching with 30% H3PO4 resulted in the lowest Ca-content ratio and Er. Using 5% H3PO4 as etchant resulted in the highest resin-dentine bond strength. ConclusionsSelective demineralisation of the dentine matrix may be achieved by lowering the H3PO4 concentration to 5wt%, to achieve better bonding performance. Clinical relevanceBy retaining intrafibrillar minerals, more through air-drying of the partially demineralised collagen matrix may be accomplished without the need to worry about collapsing a mineral-free collagen matrix during air-drying. This may result in the elimination of water-wet bonding during the application of etch-and-rinse adhesives.

Rapid biomimetic mineralization of collagen fibrils and combining with human umbilical cord mesenchymal stem cells for bone defects healing

Collagen biomineralization is regulated by complicated interactions between the collagen matrix and non-collagenous extracellular proteins. Here, the use of sodium tripolyphosphate to simulate the templating functional motif of the C-terminal fragment of non-collagenous proteins is reported, and a low molecular weight polyacrylic acid served as a sequestration agent to stabilize amorphous calcium phosphate into nanoprecursors. Self-assembled collagen fibrils served as a fixed template for achieving rapid biomimetic mineralization in vitro. Results demonstrated that, during the mineralization process, intrafibrillar and extrafibrillar hydroxyapatite mineral with collagen fibrils formed and did so via bottom-up nanoparticle assembly based on the non-classical crystallization approach in the presence of these dual biomimetic functional analogues. In vitro human umbilical cord mesenchymal stem cell (hUCMSC) culture found that the mineralized scaffolds have a better cytocompatibility in terms of cell viability, adhesion, proliferation, and differentiation into osteoblasts. A rabbit femoral condyle defect model was established to confirm the ability of the n-HA/collagen scaffolds to facilitate bone regeneration and repair. The images of gross anatomy, MRI, CT and histomorphology taken 6 and 12weeks after surgery showed that the biomimetic mineralized collagen scaffolds with hUCMSCs can promote the healing speed of bone defects in vivo, and both of the scaffolds groups performing better than the bone defect control group. As new bone tissue formed, the scaffolds degraded and were gradually absorbed. All these results demonstrated that both of the scaffolds and cells have better histocompatibility.

Fabrication of intrafibrillar and extrafibrillar mineralized collagen/apatite scaffolds with a hierarchical structure.

A biomimetic collagen-apatite (Col-Ap) scaffold resembling the composition and structure of natural bone from the nanoscale to the macroscale has been successfully prepared for bone tissue engineering. We have developed a bottom-up approach to fabricate hierarchically biomimetic Col-Ap scaffolds with both intrafibrillar and extrafibrillar mineralization. To achieve intrafibrillar mineralization, polyacrylic acid (PAA) was used as a sequestrating analog of noncollagenous proteins (NCPs) to form a fluidic amorphous calcium phosphate (ACP) nanoprecursor through attraction of calcium and phosphate ions. Sodium tripolyphosphate was used as a templating analog to regulate orderly deposition of apatite within collagen fibrils. Both X-ray diffraction and Fourier transform infrared spectroscopy suggest that the mineral phase was apatite. Field emission scanning electron microscopy, transmission electron microscopy, and selected area electron diffraction confirmed that hierarchical collagen-Ap scaffolds were produced with both intrafibrillar and extrafibrillar mineralization. Biomimetic Col-Ap scaffolds with both intrafibrillar and extrafibrillar mineralization (IE-Col-Ap) were compared with Col-Ap scaffolds with extrafibrillar mineralization only (E-Col-Ap) as well as pure collagen scaffolds in vitro for cellular proliferation using MC3T3-E1 cells. Pure collagen scaffolds had the highest rate of proliferation, while there was no statistically significant difference between IE-Col-Ap and E-Col-Ap scaffolds. Thus, the bottom-up biomimetic fabrication approach has rendered a group of promising Col-Ap scaffolds, which bear high resemblance to natural bone in terms of composition and structure.