Study The Influence Of Bio-inspired Bone Materials With Micro-pattern On BMSCs' Behavior And Its Preliminary Application Research

Fate control of stem cells through biophysical cues is a hotspot of stem cell research.Biophysical cues,which include topological structure and mechanical property,play a pivotal role on the upstream activity of cytoskeleton and downstream expression of gene via the interaction between cells and substrate,and finally regulate cell activities such as adhesion,proliferation,migration and differentiation.As a specific approach that can systematically influence the micro-environment of cells,patterning is a critical way which is independent on gene modification and external stimulation.For the past few years,techniques of cell-patterning,such as photolithography,soft-lithography and micro-contact,had been developed and became a powerful tool to investigate the stem cell fate.Hydroxyapatite as the main inorganic constituent of nature bone presents the desirable bio-compatibility,osteoconductivity and osteoinductivity..Since biomimetic mineralized hydroxyapatite is more analogous to natural bone on exquisite structure after the addition of the protein template,it can affect the behavior of stem cell such as adhesion and proliferation.Consequently,biomimetic mineralized hydroxyapatite is a widely used as a bone repair material.In this project,we developed a protocol t to form micro-pattern using biomimetic mineralized hydroxyapatite that was able to effectively regulate the differentiation of mesenchymal stem cells(BMSCs).Particularly,the biomimetic mineralized hydroxyapatite with the micro-pattern could control the shape of BMSCs,and accelerate osteogenic differentiation.First,PDMS template was molded through photolithography to generatean agarose stamp.Then,biomimetic mineralized hydroxyapatite with the micro-pattern was transferred from the agarose stamp to PSMD substrate through micro-contact,on which study toward mesenchymal stem cells.The results of fluorescence staining showed that biomimetic mineralized hydroxyapatite with the micro-pattern could be transferred effectively onto substrate with maintaining the stability and constrain of cells.The results of RT-PCR,flow cytometry and immunofluorescence further demonstrated that this kind of micro-pattern enabled to accelerate osteoblastic differentiation and vascularization of BMSCs.To realize the application value of micro-pattern based on biomimetic mineralized hydroxyapatite,in this research,we transferred the micro-pattern onto gelatin and took the process of cross-linking and solidification,and fabricated a bionic periosteum with certain strength.Fluorescence staining result and SEM image regarding to the artificial periosteum demonstrated that the micro-pattern was efficiently transferred to the gelatin membrane.Also,we applied this artificial periosteum into in vivo experiment to evaluate its biocompatibility and eligibility for promoting bone repair.Subcutaneous experiment suggested the bone patch exhibited an excellent biocompatibility,and proper degradability without the obvious inflammation.Besides,we also discussed the impact of micro-pattern with different shapes on inducing osteogenic differentiation of BMSCs.From the results of a series of qualitative and quantitative experiment,we concluded that line pattern showed a better performance on inducing the differentiation of BMSCs into the osteoblast than that in grid pattern,and it also exhibited a better capability in facilitating new bone generation in vivo.