Bioinformatics Analysis And Identification Of Cirular RNAs Promoting The Osteogenic Differentiation Of Human Bone Marrow Mesenchymal Stem Cells On Titanium Treated By Surface Mechanical Attrition

Objective:Surface mechanical attrition treatment(SMAT)technology uses high-frequency projectiles to repeatedly impact the surface of the sample,and the surface of the material is plastically deformed to achieve grain refinement,which results in a gradient distribution structure from the surface layer to the matrix with a small to large grain size.Because there is no interface connection problem between the surface layer and the substrate,the problem of poor bonding force between the surface nanostructure layer and the substrate which is easy to peel off is fundamentally solved.In addition,the nanostructured layer affects the growth environment of the cells on the surface of the material.Its ability to absorb hydrogen is significantly improved,and it shows good reversibility of hydrogen storage and promotes surface activation.With the increase of surface activation energy,the adsorption capacity of calcium and phosphorus ions is enhanced,the hydrophilicity is enhanced,and the protein adsorption amount is increased.And the nanostructured surface will reduce the adhesion of other types of cells other than osteoblasts,especially fibroblasts.Moreover,the nanostructured surface can also inhibit the adhesion of bacteria.Therefore,the surface of the material after SMAT treatment can promote cell proliferation,adhesion and osteogenic differentiation,and improve the osteointegration ability of the material.Circular RNA(circRNA)can be used as competitive endogenous RNA(ceRNA)to bind to microRNA(miRNA)through miRNA response elements(MRE),thereby reducing the effect of messenger RNA(mRNA)on shearing,degradation or inhibiting its translation.In recent years,a large number of studies have shown that circRNA can regulate osteogenic differentiation of stem cells.To explore the effect of SMAT titanium on osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMSCs)through this experiment.To investigate the mechanism,and verify whether SMAT titanium promotes osteogenic differentiation of hBMSCs by affecting circRNA.Providing theoretical and experimental basis for the application of SMAT titanium in oral clinic.Methods:1.Material preparation: SMAT technology was used to treat medical metal pure titanium plates,and gradient nanometer metal pure titanium materials with surface grain size of nanometer grade are obtained.EDM was used to cut the materials into 11 mm,20mm diameter,2.5mm high experimental group gradient nano metal pure titanium sheets.The control group was treated with SMAT and then annealed to grow the grains and obtain annealed metal pure titanium sheets with similar surface roughness.Material treatment:Sequentially washed the materials with acetone,36%-38% dilute hydrochloric acid,absolute ethanol,and distilled water ultrasonically for 20 minutes,dried them,and stored them at high temperature and autoclave for later use.Transmission electron microscope(TEM)was used to observe the surface structure of the experimental materials so as to determine whether it has gradient nanostructures.2.Under sterile operation,cultured hBMSCs on the surface of the materials.CCK-8(Cell Counting Kits-8)method was used to detect the proliferation of hBMSCs and draw the proliferation curve of hBMSCs.3.Alkaline Phosphatase(ALP)activity assay was used to analyze the effect of each group of materials on hBMSCs osteogenic differentiation.4.Alizarin red staining method was used to analyze the effect of each group of materials on hBMSCs osteogenic differentiation.5.Bioinformation prediction software miwalk3.0 was used to construct circRNA-miRNA-mRNA networks by predicting circRNAs with osteogenesis-related mRNAs and miRNAs.Analyze their interactions,and selected 3-5 candidate circRNAs located at key positions in the network for verification.Quantitative Real-time Polymerase Chain Reaction(QRT-PCR)technology was used to detect the expression of candidate circRNAs in each group of hBMSCs.6.SPSS Statistics 20.0 software and Graphpad Prism 7 were used for statistical analysis of experimental data.Results:1.The TEM observation shows that the SMAT method can make the surface of gradient nano-metal pure titanium nanocrystallization,and the grain size is on the nanometer scale.2.CCK-8 results: HBMSCs growth curve is approximately "S" shaped.The incubationperiod after subculture is 1-3 days,the logarithmic proliferation period is 3-5 days,the cell number reaches a peak at 3and 5 days,and then enters the plateau phase.On the 5th day of culture,the difference between the SMAT titanium group and the annealed titanium group was statistically significant.3.ALP activity test results: When hBMSCs were cultured osteogenically for 3,5,7,14 days,the ALP activity of each group increased with time.At each time point,the ALP activity of the SMAT titanium group was higher than that of the annealed titanium group.On days 3 and 5,the difference between the SMAT titanium group and the annealed titanium group was statistically significant.4.Alizarin red staining results: When hBMSCs were cultured osteogenically for 7,14,21 days,the area of alizarin red staining in each group increased with time.At each time point,the area of alizarin red staining in the SMAT titanium group was larger than that of the annealed titanium group.On days14,the difference between the SMAT titanium group and the annealed titanium group was statistically significant.5.Bioinformatics prediction results: According to 20 osteogenesis-related mRNAs,8osteogenesis-related miRNAs and 51 circRNAs were predicted.Among them,30 circRNAs and 8 miRNAs constitute 6 circRNA-miRNA-mRNA networks.Circ-LTBP2 was selected as a candidate circRNA.The QRT-PCR results showed that compared with the control group,the expression levels of hsa?circ?0032599,hsa?circ?0032600,and hsa?circ?0032601 in the experimental group were up-regulated,and the hsa?circ?0032600 expression was the most up-regulated and statistically significant On days14.(fold change/FC = 4.25,P <0.05).Conclusion:1.SMAT titanium has better ability to promote osteogenic differentiation of hBMSCs than annealed titanium.2.SMAT titanium can promote hBMSCs osteogenic differentiation by affecting circRNA.