The Effect Of Macromolecule On The Process Of CaCO₃ And HAP Biomineralization

The product of biomineralization, different from that of mineralization, has special morphologies and particular mechanical properties, so the process of biomineralization has been researched extensively. Biological macromolecules influence the process of mineralization by regulating the nucleation of inorganic minerals on the inorganic/organic interfaces. The process of crystals growing plays an important role in the process of biomineralization, so, to further elucidate the regulation mechanism of crystals growing is important to study the process of biomineralization. As two kinds of the most widely mineral substance existing in the nature, calcium carbonate（CaCO₃）, with simple structure, and hydroxyapatite（HAP）, the highest amount of mineral in human’s body, are the product of biomineralization in this study. And in this study, we research the process of CaCO₃ and HAP crystals growing by invitro experiments.In this study, we use primarily two organic templates ——silk fibroin and collagen. We prepared microspheres of silk fibroin by controlling the self –assembly of silk fibroin with electric field, and then we can get the silk fibroin microspheres, on which CaCO₃ crystals would grow, with size of less than 1μm. The template of HAP biomineralization is 3D collagen gel. In addition, we used polyaspartic acid（PASP） as an additive of biomineralization to further study the process of CaCO₃ mineralization, and recorded the process of CaCO₃ growing by time.（1） In the study of silk fibroin microspheres regulating CaCO₃ crystals growing, we discussed the influence during the mineralization, concentrations of Ca²⁺ on crystallization behavior of CaCO₃. We found that as the template, silk fibroin microspheres would absorb a great number of Ca²⁺ on their surface. With CaCO₃ crystals growing, the influence of silk fibroin microspheres on CaCO₃ was weakening gradually. Silk fibroin microspheres would not have any influence on CaCO₃ crystal when it grown to the size of 5μm. In other words, it is strictly restricted that fibroin microspheres regulate CaCO₃ crystals growing.（2） In the study of HAP growing on 3D collagen gel, compared with the experiment of HAP growing on biogalss, we found that collagen fibers had a little advantage of increasing the number of HAP nucleation, while it would slow down the rate of HAP crystals growing. The HAP crystals growing on collagen fibers have a smooth surface, but its size is small, and the crystallinity is degraded.（3） Then we use PASP as an additive in the process of CaCO₃ mineralization. In the study, we observe the real-time process of CaCO₃ mineralization by Confocal Laser Scanning Microscope. We could find that the CaCO₃ mineralization would be finished in 30 mins, and in this process, there would be a large number of amorphous calcium carbonate（ACC）. We further explain the mechanism that as a kind of precursor phases of CaCO₃ mineralization, ACC not only would transform to thermodynamically stable CaCO₃, but would participate the process of CaCO₃ crystals growing as well.In addition, we also studied the distribution and size of CaCO₃ with different concentrations of PASP and Ca²⁺, and then concluded that, first, the molecules of PASP are the sites of CaCO₃ nucleation. In general, the amounts of CaCO₃ nucleation are in direct proportion to the contents of PASP. Second, PASP could hinder the grow process of CaCO₃ crystals. On the one hand, it would slow down the rate of CaCO₃ crystals growing. On another, it could prevent CaCO₃ crystals from growing to a large size. Different with mineralization in nature, in the process of biomineralization, the sizes of CaCO₃ depend on the concentrations of Ca²⁺, which is no relevant to the amount of CaCO₃ nucleation. Yet the largest size of CaCO₃ crystals is about 5μm in presence of PASP.In this study, we mainly discussed the process of biomineralization with different protein templates and additive, which would have a practical reference value in the field of crystals growing and biomaterial.