Effect Of Enamel Matrix Derivatives On Osteogenic Differentiation Of BMSCs Sheet On Different Titanium Surfaces

Objective:The effect of enamel matrix derivatives(EMD)on the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)sheets on different titanium surfaces was preliminarily investigated.Methods: Bone marrow mesenchymal stem cells of SD rats were cultured in vitro and identified,and their morphology was observed under an inverted microscope;alizarin red staining and oil red O staining were observed after osteogenesis and adipogenic differentiation;flow cytometry was used to analyze BMSCs surface antigen markers.The cell counting kit(CCK-8 method)was used to detect the effect of different concentrations of EMD on the proliferation of BMSCs,and the appropriate concentration of EMD was selected for subsequent experiments.BMSCs were seeded on smooth titanium surface(control group),sandblasted and acid-etched titanium surface(experimental group)and anodized titanium oxide surface(experimental group)to construct BMSCs sheet in vitro,and the morphology of cell sheet on titanium surface in each group was observed by scanning electron microscope.BMSCs sheets were cultured in 25 ug / ml EMD osteogenic medium for 7 days,14 days,and 21 days,respectively,and the absorbance of Alkaline Phosphatase(ALP)was measured.The expression of osteogenesis-related genes,such as Runt-related transcription factor-2,(RUNX2),zinc finger transcription factor Osterix(OSX),and osteocalcin(OCN).Results: The extracted cells were confirmed as bone marrow mesenchymal stem cells by morphological observation under an inverted microscope,alizarin red and oil red O staining after osteogenic and adipogenic differentiation,and identification by flow cytometry.CCK-8 test results suggest that 5-50 ug / ml EMD has a significant effect on promoting proliferation of BMSCs,and 100 ug / ml EMD has an inhibitory effect on BMSCs.Scanning electron microscopy(SEM)showed that the cell membrane of BMSCs had a single connection between cells on the smooth titanium surface,and there were breaks at the junction,and the number of cell layers was relatively small.There are many layers of cells and matrix on the surface of sandblasted acid-etched itanium,which are relatively messy and the boundaries are unclear.Uniform multi-layer distribution can be seen on the anodized surface,the matrices are connected to each other,and they are flatter and the boundaries are clearer.The ALP activity test showed that the ALP activity of the experimental group was higher than that of the control group(P < 0.05),and the ALP activity of the anodic oxidation group was higher than that of the sandblasting acid etching group at 7 and 14 days(P < 0.05),and there was no statistical difference at 21 days.The results of real-time fluorescence quantitative PCR showed that compared with the control group,the expressions of RUNX-2,OSX,and OCN in the experimental group were all up-regulated(P <0.05),the most obvious in the anodized group.The results of Western blotting showed that the expression of RUNX-2,OSX and OCN protein in the experimental group was higher than that in the control group at all time periods,and the expression was higher in the anodized group than in the control group(P<0.05).Conclusion: Our data suggest that EMD can stimulate the expression of BMSCs membrane osteogenic related proteins on the surface of sandblast and anodized titanium,and the osteogenic capacity of anodized surface is stronger.However,the role of EMD on titanium surface BMSCs diaphragm in vivo needs further study.