Study Of Bilayer Pifithrin-? Loaded Pocrine Small Intestinal Submucosa Extracellular Matrix/PLGA Composite Membrane For Promoting Bone Formation

Part ? Mechanism of osteogenic differentiation of MC3T3-E1 cells induced by PFTaObjective To explore the mechanism of osteogenic differentiation induced by PFT aMethod In this study,CCK-8 was used to detect the toxicity of PFTa to MC3T3-E1 cells,and the optimal concentration of PFTato induce osteogenic differentiation of MC3T3-E1 cells was screened.After the cells were treated with PFT?,the levels of NOX4 in the cells were detected by real-time PCR,western blot and immunofluorescence,and the levels of ROS were detected by flow cytometry.SiNOX4i was prepared and screened,and siNOX4 was transfected into cells.The effect of PFT? on osteogenesis induction of MC3T3-E1 cells was observed after interference with NOX4.Bone formation was observed by ALP and alizarin red staining;Nox4 was detected by qPCR,WB and IF;ROS expression was detected by flow cytometry.Results PFTa can significantly increase osteogenic differentiation of MC3T3-E1 cells.In the range of 10-80?mol,cell viability decreased with increasing PFTa concentration.At the same time,with the increase of PFTa concentration,the levels of NOX4 and ROS also gradually increased,and the osteogenesis ability increased.After transfection of siNOX4 cells,the effect of PFTa on increasing NOX4-ROS and promoting osteogenesis was significantly inhibited.Conclusion:Our data revealed that PFT-? at relatively high doses induced osteogenic activity of MC3T3-E1 cells,and this was through by induction of NOX4/ROSPart ? chitosan/nano hydroxyapatite/zoledronic acid scaffoldObjective Small intestinal submucosa extracellular matrix(SIS-ECM)composite membrane are catching eyes in tissue engineering but have been rarely studied in bone repair.In this study,we developed the unique bilayer composite membrane by assembling decellularized SIS-ECM and poly(lactic-co-glycolic acid)(PLGA)nanofibers through the electrospinning technique.Method To strengthen the bioactivity of the composite membrane,pifithrin-?(PFT?),a p53 inhibitor that can reduce the inhibitory functions of p53 in osteogenesis,was preloaded in the PLGA electrospinning solution.Scanning electron microscope(SEM)was used to analyze the structure of SIS-ECM/PLGA.MC3T3-E1 cells were cultured and observed with the process of cell adhesion,toxicity(CCK-8,hemolysis test).Furthermore,MC3T3-E1 cells were cultured in vitro to observed the osteogenic differentiation and mineralized nodule.Results The SIS-ECM/PLGA/PFT a composite membrane are porous and connected by gaps.SIS-ECM/PLGA has good cell adhesion in vitro and can promote the proliferation and differentiation of MC3T3-E1 cells.Compared with the positive control group,SIS-ECM/PLGA/PFT a membrane did not cause more red blood cell damage and had good histocompatibility.SIS-ECM/PLGA/PFT?+MC3T3-E1 cells were cultured in vitro and cultured with osteogenic inducer.The results showed that SIS-ECM/PLGA/PFTa composite membrane could promote the osteogenic differentiation and mineralization of MC3T3-E1 cells in a PFT ?-dose dependent manner in vitro.Conclusion The double SIS ECM/PLGA composite membrane loading PFT ?promoted the regeneration of vascularized bone with good histocompatibility.Part ? Osteogenesis induced by SIS-ECM/PLGA/PFT a composite membrane in vivoObjective To explore the osteogenesis ability of SIS-ECM/PLGA/PFT ? in vivoMethod Four groups were arranged according to study.Group ? was SIS-ECM(control group),Group ? was SIS-ECM/PLGA,Group ? was SIS-ECM/PLGA/PFT? 20 group,and Group IV was SIS-ECM/PLGA/PFT? 40.The corresponding materials of each group were cultured with MC3T3-E1 cells.24 rats were randomly divided into 4 groups.Each rats' left leg was made a modle of quadriceps femoris muscle bag,and the corresponding membrane of each group was implanted into the muscle bag.The rats were sacrificed 30 days after operation.The tissues around the implants were stained with hematoxylin eosin staining and Masson's trichrome staining.Sections of heart,liver,spleen,lung and kidney from rats were stained to evaluate the toxicity of the materialsResults Compared with the control group,the composite membrane has a better property of osteogenesis.The composite membrane loaded with PFTa can increase bone mineralization and vascular growth as well.No obvious damage was found in all the organs,including heart,liver,spleen,lung and kidney.Conclusion SIS-ECM/PLGA/PFT? can increase osteogenesis and bone mineralization,furthermore,promote the growth of blood vessels.This composite membrane have good biocompatibility and will be safe for use in future.