Role Of HIF-1α/Twist1 In BMSCs Osteogenic Differentiation Induced By Cycling Stretch

Objective: During orthodontic tooth movement,the mechanical force through the teeth to the periodontal tissue,and produce a series of biological reaction,cause periodontal tissue reconstruction,eventually make the teeth move.Alveolar bone remodeling is the biological basis for the movement of orthodontic teeth under mechanical forces.Mechanical force and hypoxic stimulation are two important factors affecting bone remodeling.The osteoblast activity plays a central role in regulation of the bone remodeling process.In this research,we study the role the possible mechanism of hypoxic signaling pathway HIF-1α/Twist1 in the BMSCs osteogenic differentiation subjected to cycling stretch in vitro,and seeking more scientific and reasonable theoretical basis for orthodontic treatment.Main methods: The model of rat bone mesenchymal stem cells(BMSCs)cultured in vitro-mechanical stimulation was successfully constructed.The multichannel cell stress loading system was used to impose the cyclic stress on BMSCs for 0.5h,2h,6h 8h and 12 h respectively.The loading frequency was 1Hz,tensile deformation was 5%.,and nonstressed group as the control group,6 groups of cells cultured in the same conditions.We use western blotting and q PCR to detect the expression of Twist1 protein and mRNA in stretch-induced osteogenic differentiation of BMSCs.Stable BMSCs with RNA interference of Twist1 were constructed by lentiviral virus,and were induced by the ideal stretch,and we explore the relationship among force,HIF-1α/Twist1 and BMSCs osteogenic differentiation.To make clear the role of HIF-1α/Twist1 in the BMSCs osteogenic differentiation subjected to cycling stretch in vitro,SPSS23.0 was used to analyze the experimental data.Result: 1.Identification of rat bone mesenchymal stem cells: BMSCs were purchased and cultured in vitro,and amplified in a short period with high purity and quantity.We use light microscope to observe the formation of cells,the morphology showed a uniform long fusiform.The growth rate and state of BMSCs were stable after the recovery.Flow cytometric analysis showed that 97.9% and 91.4% of cells expressed CD44 and CD90.In contrast,expression of CD45,a pan-hematopoietic marker,was observed in a distinct population 17.5%.The results indicated that the cells obtained through isolated culture were bone marrow stromal cells.2.Observe the expression of Twist1 protein and mRNA after application of cycling stretch on BMSCs: The Twist1 mRNA level in unloaded cells was very low at first and then increased,reached a maximum at 6 h,but then decreased a little.In accordance with the changes in mRNA levels,protein levels of Twist1 increased as the stretched time prolonged in BMSCs with stretch treatment,and reached a maximum at 6h.which indicated that Twist1 alpha could make a response to mechanical stress,and hypoxic signaling pathway HIF-1α/Twist1 may be involved in BMSCs osteogenic differentiation under cyclic stress.3.Construction of stable BMSCs with RNA interference of Twist1: In this experiment,four sh RNA interference targets were designed according to the sequence of Twist1 gene in rats.The correct clone was RNA interfering lentiviral vector successfully built.Then the BMSCs of the rats were infected with the lentivirus of the four groups of the sh RNA lentivirus and the negative control group,plus the normal cell group,and there were 6 experimental groups.After infected successfully,interference effects were evaluated by q PCR,and the stable strains were obtained and successfully identified.4.Observe the role of hypoxic signaling pathway HIF-1α/Twist1 in the cycling stretchinduced BMSCs osteogenic differentiation:The expression of Twist1 protein and mRNA level in uninfected cells,Twist1-sh RNA cells,and NC cells was measured by western blotting and q PCR,respectively.Cells in three groups were applied optimal force(5%,1Hz cycling stretch for 6hs).The changes of BMP-2 and Runx2 protein and mRNA level were measured to assess the osteogenic differentiation of BMSCs.It remained unchanged in NC group(p>0.05),but infected with Twist1-sh RNA,the protein and mRNA level of BMP-2 markedly increased(p<0.05),there was no statistic difference in mRNA and protein expressions of two osteogenic genes between uninfected cells and NC group(p>0.05).The protein and mRNA level of Runx2 Show the same trend.Conclusion: 1.After force application the Twist1 mRNA and protein expression increased dramatically in a time-dependent manner.which suggest that HIF-1 alpha could make a response to mechanical stress.Low oxygen signal path HIF-1α/Twist1 plays a rival role in remodeling of periodontal tissue in vitro.2.Silent expression of the Twist1 gene,namely blocking the hypoxic signaling pathway HIF-1α/Twist1,can promote the osteogenic differentiation of rat bone marrow mesenchymal stem cells induced by periodic tensile stress.