Preparation And Biological Properties Evaluation Of Calcium Phosphate Ceramics With Different Micro/Nano Topography

Calcium Phosphate Ceremics(CPC), which has the similar crystal structure and Ca/P ratio to natural bone and forms chemical bonds with live tissues, has recognized as bioactive ceramics for clinical appliations. Meanwhile, more and more researches showed that micro/nano topography could effectively regulate adhesion,stretch, proliferation and differentiation of pluripotent stem cells. Based on all of this, it was expected to prepare hydroxyapatite with different micro-nano topography and to study the osteogenesis mechanism.In this paper, RNA-seq was applied to research the molecular mechanism of osteoinduction, which was invovled in calcium ion released from CPCs, then the interaction of hydroxyapatite molecule and fibronection(FN) was researched to prove the bioactivities of CPCs in the level of molecule by the means of molecular docking. Finally different micro/nato topographical CPCs were prepared by mechanical way and hydrothermal synthesis, following by different experiments to research the phisical, chemical and biological properties. The main conclusions were drown as follows:Firstly, BMSCs were harvested to extract mRNA for the sequencing of transcriptome after cultured for 1, 4 and 7 days in DMEM with 10 mM calcium respectively and the negative control were the BMSCs which cultured in DMEM. The sequences were analyzed by Tophat, Cufflinks, and DAVID successively, and the results showed that calcium ions prompted the expression of BMP2 and inhibited the expression of Bmper ,which played the positive effect and negative effect in the BMP signaling pathway respectively. Further analysis showed that calcium ion inhibited ERRR, wnt and TGF-? signaling pathways which involved in the osteogenesis.Meanwhile, Auto DOCK was selected to analyze the interaction between hydroxyapatite molecule and partial fragments of fibronection(FN-?7-10) based on the theory of molecular docking. It was revealed that the FN-?10 was the most important fragment among FN-?7-10 in promoting fibronectin binding to hydroxyapatite molecule by minimising the interaction energy; arginine was observed to directly interact with the hydroxyl group of hydroxyapatite molecule through electrostatic forces and hydrogen bonding. Besides, it was found that the binding sites of hydroxyapatite molecule on FN-?10 were mainly located at the RGD loop, which did not interrupt the interaction between the fibronectin and cellular receptors to form focal adhesions.Then,in the hydrothermal condition, different micro/nano-structural calcium phosphate coating were sythesized on the surface of hydroxyapatite scaffolds in the solution with calcium ion, phosphate ion and inositol hexaphosphate (IP6) at 150 ? for 3 h after the scaffolds were immersed into the solutions of phosphate and calcium salts successively for 3 times. The results showed that it was the IP6 that prompt the precipitation of calcium phosphate on the surface of hydroxyapatite scaffolds and the micro/nano topography of the coating was composed of axiolitic particles. If there was not IP6 in the hydrothemal solution,the coating was composed of hexagonal prism, and the thickness was smaller.Micro/nano-topographical CPCs released calcium ions and the release rate of axiolitic coating was larger than that of hexagonal prism. It was also proved that all the micro/nano-structural coating were biocompatibility and axiolitic coating benefited the osteo differentiation by prompting the expression of BMP2 and YAP/TAZ compared with other samples.Finally, hydroxyapatite ceramics, which were fabricated by different techniques, were polished to expose the inner micropores, and scanning electron microscopy (SEM) , X-ray diffraction (XRD) were used for the analysis of topography and components, and the results showed that there were many micro/nano pits, which were different on size and distribution.It was proved that micro/nano pits tailored the ability of fibronectin adsorption , made effects on the cell topography, ALP activity, and osteogenetic genes expression.In conclusion, micro/nano topography of CPCs made great effects on the adhesion and osteo differentiation of preosteoblst by regulating protein adsorption, calcium ions release.Progress in this work would provide reference for the fabrication of micro/nano topography of biomaterials, and new ideas for the research on biomaterials, especially on calcium phosphate ceramics.