Establishment And Application Of The Apatite Crystal Mineralization Model In Vitro

Biomineralization is a mineral forming process in organism, it is a physiological process that regulated precisely by numerous factors. Its research involves disciplines like biology, chemistry, crystallography, material science, mineralogy medicine and so on, is a multi-disciplines field overlapping several front disciplines. Mechanisms researches of the regaulation process are largely depend on the establishment and application of the mineralization system in vitro. Monolayer system and gel system are the most widely used models in this field, but for their own flaw, they are not the best choice for the mechanism research of dental tissue. In this paper, we use Cation-selective membrane(CMV) and dialysis membrane to construct a dual-membrane mineralization system, by using this model, the inorganic and organic factors involved in biomineralization process of dental tissue were investigated to reveal the regaluation mechanism of the mineralization process.Part 1 Establishment and test of the dual-membrane mineralization systemThe model was constructed by using the cation-selective membrane and dialysis membrane. The reliability and stability of the system was tested. Results showed that, after 3 days'reaction, OCP crystal can deposite effectively on the CMV, extended reaction time caused an increased failure rate of the in vitro system without any significent changes of crystal components. When 1ppm fluoride was added into the reaction space, large number of short, rod-like crystal deposited on the CMV, the product was a mixture of OCP and apatite; When cell culture medium supplemented with 10%FBS was added, plate-like OCP was the main component deposited on the CMV. After some improvements, the in vitro mineralization model was established.Part 2 Effects of inorganic factors on apatite mineralization in vitroUsing the dual-membrane system, effects of F-, Mg2+ and HCO3- on apatite mineralization were investigated. Results showed that, flouride can change the crystal shape and component effectively, even at very low concentration(1ppm). Compared with the worm-like crystal derived from DDW(double-distilled water) group, when flouride was added, oriented rod-like crystal was the main component of the product on CMV. When flouride concentration rised to or exceeded 5ppm, large amount of rod-like flouroapatite crystal deposited in the phosphate solution beside the CMV surface. When Mg2+ was added to the system, deposition of OCP on CMV was inhibited accompany with the decreased crystallinity. HCO3- ions can accelerate the mineralizaton process of OCP, and take part in the crystal lattice directly.These results revealed that, floride can accelerate the transform process of OCP to apatite and cause the change of the crystal shape. Mg's inhibit effects of OCP mineralization can be attributed to the competition of Mg with Ca, for their similar ion diameter. HCO3- can replace the phosphate in the OCP lattice and modify the local pH value of the mineralization environment, that promoted the mineralizaton process of OCP. The inorganic factors play important rule during the nucleation and growth process of the apatite crystal.Part 3 Effects of organic factors on apatite mineralization in vitroThe effects of single-component organic molecule(BSA, P-Amel and MEPE), multi-component (the culture medium of rat DPC after mineralization induction), were inverstigated using the dual-membrane system. When mineralization related proteins(Amel and MEPE) were applied, OCP crystals aggregated to form crystal sphere, which could not be found in BSA group. All the organic molecules tested can inhibit the growth of OCP crystal, Amel and BSA had stronger effect. The induced culture medium of DPC showed impressive effects on OCP crystal's shape when compared with control group, large amount of well oriented, plate-like crystals were deposited on CMV surface.All the evidences indicated that, organic molecules'effects on crystal mainly focus on the crystal morphology. They may concentrate Ca ions and act as the crystal nuclui directly. Also, the interaction of their functional groups and specific crystal surface may play critical rules of the oriented growth of the apatite crystal.Part 4 Co-effects of organic/inorganic factors on apatite crystal mineralization in vitroThe co-effects of BSA and fluoride, Amel and fluoride on apatite crystal mineralization were tested. when BSA was applied accompany with fluoride, morphology of the deposites changed, interestingly, at the same time, XRD results revealed that, the crystal component changed from OCP to another HAP precursor, DCPD. While in the Amel and fluoride group, the crystal component changed from OCP to HAP. That means the Amel may regulated the HAP nuclearation and growth through some specific effects that BSA doesn't have.In summary, both organic and inorganic factors play critical rules when regulating the biomineralization process. The inorganic ions mainly affect the component of the apatite through chemistry reactions, while the organic molecules affect the crystal nuclearation and morphology processes through their functional groups. The co-regulation of the organic and inorganic factors is the key element of biomineralization.