Degradation Behavior And Osteogenic Properties Of Chitosan-Mineralized Collagen/Mg-Ca Alloy Composites In Vitro

ObjectiveMagnesium(Mg)and its alloys are regarded as future candidates for bone defect repair due to their excellent biocompatibility,biodegradability,elastic modulus close to bone tissue,and mechanical support.The uncontrollability of its degradation rate has always been the biggest problem restricting clinical practical applications.Meanwhile,the growth and repair of bone tissue is not only related to osteoblasts,angiogenesis also closely affects the bone repair process.Therefore,in the present study,magnesium-calcium alloy(Mg-Ca)after micro-arc oxidation(MAO)was used as the matrix material,chitosan(CS)and mineralized collagen(nano-Hydroxyapatite/collagen,n HAC))were used as the reinforcement materials,the CS-n HAC/Mg-Ca composites was prepared through freeze-drying.By studying the tissue structure,in vitro corrosion behavior,osteogenic activity and angiogenesis ability of composites with different composition ratios,explore their clinical application potential as bone defect repair materials,and provide experimental basis and thinking strategy for the development and design of degradable Mg-based composites.Methods1.CS and n HAC were mixed according to the mass ratio of 2:1,1:1 and 1:2respectively,and 0.5% acetic acid solution was added to prepare 0.3g/ml colloidal solution.The colloidal solution and MAO/Mg-Ca matrix were put into the mold by stages.Then,after freeze-drying,alcohol gradient deacidification,and second freeze-drying,they were prepared into 2CS-1n HAC/Mg-Ca,1CS-1n HAC/Mg-Ca,1CS-2n HAC/Mg-Ca composites.The CS/Mg-Ca composites and MAO/Mg-Ca were used as experimental control groups.2.Scanning Electron Microscope(SEM)and Energy Dispersive Spectroscopy(EDS)were used to analyze the surface,cross-section morphology and element composition of composites.X-Ray Diffractometer(XRD)and Fourier Transform Infrared Spectrometer(FTIR)detected the phase composition and composition structure of the composites surface.Simulated Body Fluid(SBF)immersion method(including p H measurement method and weight loss method)was used to analyze the corrosion behavior and degradation rate of composites.3.CCK-8 method,rhodamine labeled phalloidin staining and direct co culture SEM were used to detect the effects of the composites and their extracts on the proliferation and adhesion of mouse pre osteoblasts(MC3T3-E1)and human umbilical vein endothelial cells(HUVECs).4.Alkaline phosphatase(ALP)activity detection and alizarin red staining method were used to determine the effect of different composites on the differentiation of MC3T3-E1 cells.Cell scratch test and Enzyme-linked immunosorbent assay(ELISA)were used to evaluate the effects of each group of composites on HUVECs cell migration and Vascular Endothelial Growth Factor(VEGF)secretion levels.Determine the osteogenic activity and angiogenesis ability of the composites.Results1.The SEM results shown that A large number of uniform pores were observed on the surface of CS/Mg-Ca,2CS-1n HAC/Mg-Ca,1CS-1n HAC/Mg-Ca,but no obvious pores were found on the surface of 1CS-2n HAC/Mg-Ca,which was covered by a large number of n HAC aggregates.Meanwhile,the cross-sectional SEM results shown that there is no obvious gap between the matrix and the reinforcement materials in 2CS-1n HAC/Mg-Ca group.In the simulated body fluid immersion experiment,2CS-1n HAC/Mg-Ca exhibits the best corrosion resistance and structural stability compared with other composition ratio composites and MAO/Mg-Ca group.2.CCK-8 and phalloidin staining results shown that the all composites are non-cytotoxic,and can promote the cell proliferation of MC3T3-E1 and HUVECs,2CS-1n HAC/Mg-Ca and 1CS-1n HAC/Mg-Ca exhibit the best cell proliferation vitality.At the same time,the direct co-cultivation SEM results also showed that compared with the MAO/Mg-Ca group,MC3T3-E1 and HUVECs adhered to the surface of the composites in a larger number and the cells stretched more obviously.3.Through ALP activity detection and Alizarin Red staining experiments,it is found that MC3T3-E1 cells cultured in 2CS-1n HAC/Mg-Ca and1CS-1n HAC/Mg-Ca groups have significantly higher ALP activity and extracellular matrix mineralization levels than other composites and MAO/Mg-Ca group.In addition,the results of cell scratches and ELISA experiments showed that the 2CS-1n HAC/Mg-Ca and 1CS-1n HAC/Mg-Ca composites can significantly promote the cell migration and VEGF secretion of HUVECs.Conclusions1.CS and n HAC as the reinforcement materials can effectively reduce the body fluid to corrode Mg-Ca matrix,and among the composites with multiple composition ratios,2CS-1n HAC/Mg-Ca exhibits the better corrosion resistance and structural stability.2.Among the various groups of composites,2CS-1n HAC/Mg-Ca and1CS-1n HAC/Mg-Ca composites have the better biocompatibility,which can not only promote the proliferation and adhesion of MC3T3-E1 cells and HUVECs,but also enhance the osteogenic activity and angiogenesis of Mg-Ca matrix.