Osteogenic Properties Of Two New Collagen Membranes For Guided Bone Tissue Regeneration In Vitro And In Vivo

Objective: To study the effect of two self-made new guided bone tissue regeneration collagen membranes(fish collagen,porcine collagen)on osteogenic differentiation of SD rat bone marrow mesenchymal stem cells(r BMSCs)and their repair effect on the cranial parietal defects of SD rats were studied.Method: Scanning electron microscopy was used to observe the surface morphology of the two membranes.The water contact angle of the two membranes was measured by contact angle measuring instrument.r BMSCs were cultured on two kinds of membranes and the blank well plates as the blank control group.The proliferation of r BMSCs was measured by CCK-8 kit at 1,3,5 and 7 days,cell adhesion and extension was observed by laser confocal microscopy,early osteogenic differentiation of r BMSCs was measured by alkaline phosphatase(ALP)staining and quantification after 5 days of osteogenesis induction.And mineralization of extracellular matrix of r BMSCs at 14 days of osteogenesis induction was assessed by alizarin red staining and semi-quantitative determination of calcium nodules.Reverse transcriptase-polymerase chain reaction(RT-PCR)was used to detect the relative expression of Bmp2,Col1 and Runx2 after 7 days of osteogenic differentiation.In vivo experiment,bone defects of 5mm in diameter were prepared on both sides of the cranial parietal bone of SD rats.In the left bone defect,fish collagen membrane or porcine collagen membrane were implanted.And no material was implanted in the right bone defect area as the blank control group.3 months later,micro-CT was used to detect the regeneration of cranial bone.Results: Scanning electron microscopy showed that the surface of fish collagen membrane was dense,while the porcine collagen membrane was a loose porous surface interlaced by disordered fibers.The contact angle measurement showed that the hydrophilicity of porcine collagen membrane was better than fish collagen membrane(P<0.05).CCK-8 results showed that cells on two membranes and blank well plates proliferated continuously within 7 days.Laser confocal microscopy showed that r BMSCs spread well on both membranes and blank well plates 24 hours later.At 5 days after osteogenic induction,ALP staining and quantitative detection showed that the ALP activity of r BMSCs on porcine collagen membrane was higher than that of fish collagen membrane and blank control group.The results of alizarin red staining and semi quantitative determination of calcium nodules showed that the mineralization level of r BMSCs extracellular matrix on porcine collagen membrane was higher than that in fish collagen membrane group and blank control group after 14 days.RT-PCR showed that the relative expression of osteogenic genes Bmp2,Col1 and Runx2 in r BMSCs cultured on porcine collagen membranes was higher than that fish collagen membranes and blank well plates after 7 days of osteogenesis induction(P<0.05).In vivo osteogenesis experiment(3 months)showed that compared with the fish collagen membrane and blank control group,porcine collagen membrane could significantly promote the regeneration of cranial parietal bone in rats(P<0.05),but there was no significant difference in the regeneration of cranial parietal bone between the fish collagen membrane group and the blank control group.Conclusion: The effect of porcine collagen membrane in promoting bone regeneration in vivo and in vitro was better than the fish collagen membrane.