Department of Mining and Materials Engineering, Faculty of Engineering, McGill University, Montreal, Quebec, Canada; Faculty of Dentistry McGill University, Montreal, Quebec, Canada; Department of Chemistry, Tianjin Key Laboratory of Molecular Optoelectronic Sciences, and Tianjin Collaborative Innovation Center of Chemical Science & Engineering, Tianjin University, Tianjin, PR China.
Department of Mining and Materials Engineering, Faculty of Engineering, McGill University, Montreal, Quebec, Canada.
J Struct Biol. 2020 Oct 1;212(1):107592. doi: 10.1016/j.jsb.2020.107592. Epub 2020 Jul 28.
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.
骨的矿化细胞外基质是一种有机-无机纳米复合材料，主要由碳酸羟基磷灰石、纤维 I 型胶原、非胶原蛋白、糖胺聚糖和各种生物分子如焦磷酸和柠檬酸盐组成。虽然现在已经了解了焦磷酸在矿化调节中的作用，但对柠檬酸盐的功能知之甚少。为了评估带负电荷的柠檬酸盐对胶原蛋白矿化的影响，我们将带有柠檬酸功能的、模仿骨类骨质的致密胶原凝胶暴露在模拟体液中长达 7 天，以检查胶原纤维内和纤维间矿化的多尺度演变。在这里，我们通过亚甲蓝染色的增加表明，通过柠檬酸功能化可以显著增加胶原蛋白的净负电荷。通过透射电子显微镜和扫描电子显微镜（包括 X 射线微分析和电子衍射）以及原子力显微镜进行的结构和组成分析，都表明在模拟体液中，柠檬酸功能化的胶原原纤维在 12 小时内经历了广泛的纤维内矿化。时间分辨、高分辨率透射电子显微镜证实了单根胶原原纤维内矿化的时间演变。更长时间的模拟体液暴露导致额外的纤维间矿化，所有这些都是通过向磷灰石的非晶到结晶转变（通过 X 射线衍射和衰减全反射傅里叶变换红外光谱评估）实现的。钙沉积测定表明，矿化随柠檬酸浓度的时间增加而增加，微计算机断层扫描证实凝胶中超过 80%的胶原蛋白在第 7 天被矿化。总之，柠檬酸有效地诱导了中尺度的纤维内和纤维间胶原蛋白矿化，这一发现加深了我们对柠檬酸在矿化组织中作用的理解。
