Combination Of Bone Mesenchymal Stem Cell Sheets And Platelet Rich Fibrin For Periodontally Accelerated Osteogenic Orthodontics In The Beagles

Objective: To Accelerated periodontal orthodontic osteogenesis(PAOO)can shorten the course of orthodontic treatment and reduce bone loss,but the effect of bone graft alone is limited.Platelet fibrin is rich in multiple growth factors(PRF),bone marrow mesenchymal stem cells(BMSCs)are excellent seed cells,and the use of stem cells patchization(BMSCs sheets)combined with PRF to repair bone defects has a broad prospect.In this study,cell experiment was conducted to explore the ability of osteogenic differentiation using PRF and BMSCs sheets in vitro,and Beagle mandibular PAOO experiment was conducted to explore the possibility of improving mandibular bone mass using BMSCs sheets/PRF together.Methods: In vitro,iliac bone marrow was extracted from beagles,and BMSCs were isolated and cultured by density gradient centrifugation.Cell surface labeled antibodies were identified by flow cytometry,and the differentiation ability of cells was identified by osteogenic,adipogenic and chondrogenic induction fluid.Extract the beagle peripheral venous blood,centrifugal preparation rich platelet fibrin(PRF),using scanning electron microscope to observe the microstructure of PRF,cell scratch experiments were used to detect the different volume PRF influence on BMSCs cell migration,CCK 8 reagent were used to detect different PRF volume effect on cell proliferation of BMSCs,using ALP staining kits PRF impact on BMSCs differentiation of development;Bone marrow mesenchymal stem cell sheets(BMSCs sheets)were obtained by inducing membrane formation of BMSCs with ascorbic acid.Cell dry markers(SOX2,OCT4,Naong)and cell growth factors(TGF-β,VEGF,HGF,FGF)of induced stem cells were detected by QPCR.PRF was used to induce BMSCs sheets in vitro,ALP staining was used to detect alkaline phosphatase activity in the compound group,ARS staining was used to detect calcium salt deposition in the compound group,and Q-PCR and WB detection were used to explore the expression of bone related markers in the m RNA and protein levels of the compound composition.In vivo,6 beagles were selected and the second premolars of the mandible were extracted on both sides.Three months later,the second premolar of the mandible was removed by PAOO surgery and the third premolar of the mandible was moved forward.BMSCs sheets/PRF complex(experimental group)was implanted in the left operative area of the mandible,leaving the right blank.The patients were sacrificed 12 weeks after the operation,and the bone changes in the left and right implanted areas were analyzed by Micro CTResults: In vitro,BMSCs were successfully isolated and cultured.The cultured cells were identified to have the surface marker antibody of stem cells and the ability of multidirectional differentiation.Canine venous blood PRF was successfully prepared,and it showed a porous fiber structure by scanning electron microscopy.Cell experiments showed that 1/2 PRF could well promote the migration,proliferation and osteogenic differentiation of BMSCs.The canine bone marrow mesenchymal stem cell patch was successfully induced,and the expression of dry genes SOX-2,OCT-4 and Naong was detected by PCR.Meanwhile,the expression of growth factor and related genes TGF-β,VEGF,HGF and FGF were increased.In the experiment of BMSCs sheets differentiation induced by PRF,ALP staining showed that the combined group had higher ALP enzyme activity,and ARS staining showed that the combined group had more calcium salt deposition.RT-PCR and WB showed that the expressions of osteogenic related molecules RUNX-2,OPN and COL-1 were significantly increased in the combined experimental group.In vivo,After PAOO,compared with the control group,the number and thickness of bone trabeculae in the experimental group were increased,trabeculae space was decreased,and bone density was increased;Histological analysis showed that HE staining and Masson staining showed faster absorption of bioss and higher bone maturity in the experimental group.Conclusion: Combined use of BMSCs Sheets /PRF can effectively increase the osteogenic effect in PAOO.