The Functional Changes Of Mitochondria In HPAA Modified Collagen Membrane Promote Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

Objectives:This study aimed to verify the role of the new guided bone regeneration(GBR)membrane---high molecular weight polyacrylic acid(HPAA)modified collagen membrane to promote the osteogenic differentiation of mouse bone marrow mesenchymal stem cells(BMSCs),and to explore the performance of mitochondrial quality control in the process of HPAA modified collagen membrane to promote BMSCs osteogenic differentiation.Further reveal the mechanism of autophagy in this process,provide a reliable basis for bone regeneration surgery in oral implant repair.Methods:The rat tail collagen was extracted to prepare a collagen membrane,and the HPAA cross-linking solution was used to process the collagen membrane to prepare a HPAA modified collagen membrane.BMSCs from mice were cultured in vitro and inoculated on collagen membranes and HPAA modified collagen membranes.The cells of the two material groups were treated for osteogenic induction for 7 days and 14 days,respectively.Immunofluorescence technology was used to detect the effect of HPAA modified collagen membrane on BMSCs apoptosis/necrosis,cell morphology and mitochondrial distribution.SEM,RT-qPCR,and Alizarin Red staining were used to detect the osteogenic differentiation ability of BMSCs.TEM and mitochondrial specific fluorescent probe(MitoTracker Green)were used to analyze mitochondrial morphological changes,RT-qPCR was used to detect mitochondrial biogenesis gene levels,and JC-1 staining was used to detect mitochondrial membrane potential.RT-qPCR,Western Blot and immunofluorescence techniques were used to analyze mitochondrial dynamic activity.TEM was used to observe autophagosomes/autophagolysosomes,Western Blot and immunofluorescence were used to detect LC3B protein expression,RT-qPCR and mitochondrial-lysosomal fluorescent probe co-localization were used to detect mitochondrial autophagy gene levels.After inhibiting/activating autophagy,MitoTracker Red was used to detect the morphological changes of mitochondria,and Alizarin Red staining was used to evaluate the osteogenic differentiation ability of BMSCs.Results:SEM results showed that the HP A A modified collagen membrane had extensive mineralization of collagen fibers with the participation of BMSCs.RT-qPCR and Alizarin Red staining results proved the expressions of osteogenic differentiation genes COL1 and OPN of BMSCs on the HPAA modified collagen membranes,and the osteogenic differentiation ability of BMSCs was also enhanced.The immunofluorescence results showed that the mitochondria on the collagen membranes were distributed near the nucleus,while the mitochondria on the HPAA modified collagen membranes were more evenly distributed in the cell.After the collagen membrane was cross-linked by HPAA,the mitochondrial morphology of BMSCs was significantly changed.The results of TEM and Mito Tracker Green showed that the mitochondria were oval on the collagen membrane and the cristae development was not complete.Compared with the collagen membrane,the mitochondria appear elongated on the HPAA modified collagen membrane,the number of mitochondria was increased,the mitochondrial membrane potential was increased,and the expressions of PGC-1?,Nrf1,Nrf2,MtTFA,and MtSSB were increased,indicating that the HPAA modified collagen membrane increased the level of mitochondrial biogenesis.RT-qPCR results showed that the expressions of mitochondrial split genes Drpl,Fis1,Mff and fusion genes Mfn1,Mfn2,Opal on the HPAA modified collagen membrane were increased after 7 days of osteogenic induction.14 days after osteogenic induction,the expressions of the mitochondrial fusion gene on the HPAA modified collagen membrane were increased,and the expression of the split gene Drp1 was decreased.Western Blot results showed that the protein expressions of Drpl and Opal were increased in BMSCs on HPAA modified collagen membrane after 7 days of osteogenic induction,and Drp1 expression decreased while Opal expression increased after 14 days of osteogenic induction.Immunofluorescence results showed that the expression of Drp1 and Mfn2 of BMSCs on HPAA modified collagen membrane increased after 7 days of osteogenic induction,and the expression of Drp1 decreased and Mfn2 expression increased after 14 days of osteogenic induction.Western Blot and immunofluorescence results showed that the expression of LC3B protein on the HPAA modified collagen membrane was increased;RT-qPCR results showed that the mitochondrial Pinkl and Parkin gene levels on the HPAA modified collagen membrane were increased;the fluorescence colocalization result of MitoTraker and LysoTracker showed The autophagy behavior of cell mitochondria on HPAA modified collagen membrane increased.Intervene the autophagy of the cells on the HPAA modified collagen membrane for 14 days of osteogenic induction.Alizarin red staining results show that inhibiting autophagy will reduce the level of osteogenic differentiation of BMSCs.Conclusions:1.The mineralization of HPAA modified collagen membrane in vitro promotes the osteogenic differentiation of BMSCs.2.The process of HPAA modified collagen membrane to promote BMSCs osteogenic differentiation in vitro is related to the increase of the level of mitochondrial biogenesis.3.The HPAA modified collagen membrane plays a role in promoting the osteogenic differentiation of BMSCs in vitro and participates in the regulation of mitochondrial dynamics balance.4.HPAA modified collagen membrane promotes the osteogenic differentiation of BMSCs by inducing autophagy.