Effect Of Biomimetic HAp With Micro-nano-structured Surface On Healing Of Bone Defects

Bone defect caused by injury、tumor and pathological fracture are queit ofen in clinic,while bone tissue engineering provide a strategy for healing of the bone defect Cells with osteogenisis potential,induced、proliferated and seeded in scaffolds with biodegradation,form tissue engeneering artificial bone,planted in bone defect,the organic of the scaffold absorbed or metabolism out of the body after degradation,while the inorganic of the scaffold become bioactivity bone with calcium in body after dissolution.In recent,many experts of surgery、biology materials and tissue engeneering are devoted to the research of bone repair materails’biomimetic and the molecular mechanism of biological adaptation.HAp is widely used in bone tissue engineering because of its good biocompatibility,osteoconduction and osteoinduction,besides biodegradability.In recent,the research about the roughness and micro-nano stucture of the scaffolds are less,template method to regulate the morphology of scaffold provides a way for the study.Human adipose-derived stem cells(hASCs)have good proliferation and multi-directional differentiation,it is one of most prospective stem cells for its rich source,simply isolated,easily cultured and their usage does not result in ethical conflict,etc.In this study,we Fabricated biomimetic HAp bioceramic scaffolds with micro-nano-structured surface,planted it into cranial defect of SD rat,observed the effect of healing bone defect in vivo,and the change expression of osteogenic related genes、integrin genes and protein、focal adhension protein.This research is divided into the following three parts1.Primary culture of hASCs,first,flow cytometry detect the surface marker of hASCs and its purity,second,osteogenic induce of hASCs to investgate the ability of osteogenic differentiation.Results,flow cytometry showed the positive surface marker:CD90(100.00%)、CD51(99.90%)、CD29(93.70%)and CD105(14.10%,ALP staining,alizarin red staining and the expression of osteogenic related protein are positive.Conclusions,primary cultured hASCs have a good proliferation and a high purity,a goog ability of osteogenic differentiation,futher more,can be used for the research of tissue engeneering.2.Synthesis of micro-HAp powders with the hydrothermal synthesis method,sheeting and high-temperature calcination to fabricate the scaffolds wih different micro-nano-surface,characterization,analysis of biological characteristics and effect of healing rat calvarial defects.Results,we fabricated successfully three types of scaffolds with different micro-nano surface topography:HAp600,HAp800,HAp1000.FTIR showed it has the same chemical component,live/dead staining and CCK-8 test indicate that the scaffolds have good biocompatibility,Biomechanical testing showed that the max load and stiffness of the tissue of calvarial defect have no significant difference with the autogenous bone graft,micro-CT showed that the BMD and BV(volume of new bone)of the calvarial defect were higher than the blank,HE and Masson staining showed that there are new bone growed even new vasculars、haversian canal and bone marrow cavity.Conclusion,the scaffolds used in our experiment have the same chemical component and different suface topography,furthermore,the scaffolds have goog biocompatibility and the ability of ptomoting osteogenesis and angiogenesis3.In this part,we want to discuss the change experession of osteogenic related genes、integrin genes and integrin protein when hASCs cultured on biomimetic HAp bioceramic scaffolds with micro-nano-structured surface.Methods,To detected the osteogenic related genes expression and integrin genesaV,α5,β1,β3by q RT-PCR,the integrin protein(α5β1,β1)were investigated with the fluorescence staining technique.Also,the focal adhesion protein(vinculin,paxillin)were detected with the fluorenscence staining technique.Results,the expression of osteogenic related genes(ALP、RUNX2、OPG)increased as the sintering temperature increased,and the expression of integrin genes(α5、αV、β1)increased as the sintering temperature increased while the interin gene β3 decreased.Conclusion,the scaffolds used in our experiment have the same chemical component and different suface topography,furthermore,the scaffolds have goog biocompatibility and the ability of ptomoting osteogenesis and angiogenesis.