Multiscale structural evolution of citrate-triggered intrafibrillar and interfibrillar mineralization in dense collagen gels.

The mineralized extracellular matrix of bone is an organic-inorganic nanocomposite consisting primarily of carbonated hydroxyapatite, fibrous type I collagen, noncollagenous proteins, proteoglycans, and diverse biomolecules such as pyrophosphate and citrate. While much is now known about the mineralization-regulating role of pyrophosphate, less is known about the function of citrate. In order to assess the effect of negatively charged citrate on collagen mineralization, citrate-functionalized, bone osteoid-mimicking dense collagen gels were exposed to simulated body fluid for up to 7 days to examine the multiscale evolution of intra- and interfibrillar collagen mineralization. Here, we show by increases in methylene blue staining that the net negative charge of collagen can be substantially augmented through citrate functionalization. Structural and compositional analyses by transmission and scanning electron microscopy (including X-ray microanalysis and electron diffraction), and atomic force microscopy, all demonstrated that citrate-functionalized collagen fibrils underwent extensive intrafibrillar mineralization within 12 h in simulated body fluid. Time-resolved, high-resolution transmission electron microscopy confirmed the temporal evolution of intrafibrillar mineralization of single collagen fibrils. Longer exposure to simulated body fluid resulted in additional interfibrillar mineralization, all through an amorphous-to-crystalline transformation towards apatite (assessed by X-ray diffraction and attenuated total reflection-Fourier-transform infrared spectroscopy). Calcium deposition assays indicated a citrate concentration-dependent temporal increase in mineralization, and micro-computed tomography confirmed that >80 vol% of the collagen in the gels was mineralized by day 7. In conclusion, citrate effectively induces mesoscale intra- and interfibrillar collagen mineralization, a finding that advances our understanding of the role of citrate in mineralized tissues.

A Dopamine Acrylamide Molecule for Promoting Collagen Biomimetic Mineralization and Regulating Crystal Growth Direction.

The reconstruction of the intra/interfibrillar mineralized collagen microstructure is extremely important in biomaterial science and regeneration medicine. However, certain problems, such as low efficiency and long period of mineralization, are apparent, and the mechanism of interfibrillar mineralization is often neglected in the present literature. Thus, we propose a novel model of biomimetic collagen mineralization that uses molecules with the dual function of cross-linking collagen and regulating collagen mineralization to construct the intrafibrillar and interfibrillar collagen mineralization of the structure of mineralized collagen hard tissues. In the present study completed in vitro, N-2-(3,4-dihydroxyphenyl) acrylamide (DAA) is used to bind and cross-link collagen molecules and further stabilize the self-assembled collagen fibers. The DAA-collagen complex provides more affinity with calcium and phosphate ions, which can reduce the calcium phosphate/collagen interfacial energy to promote hydroxyapatite (HA) nucleation and accelerate the rate of collagen fiber mineralization. Besides inducing intrafibrillar mineralization, the DAA-collagen complex mineralization template can realize interfibrillar mineralization with the c-axis of the HA crystal on the surface of collagen fibers and between fibers that are parallel to the long axis of collagen fibers. The DAA-collagen complex, as a new type of mineralization template, may provide a new collagen mineralization strategy to produce a mineralized scaffold material for tissue engineering or develop bone-like materials.