The Osteogenic Capability Of Human Gingival Fibroblasts In Vitro And In Vivo

OBJECTIVE:1.Isolation and culture of human gingival fibroblasts(HGFs)in vitro,to compare the adhesion,proliferation and osteogenic differentiation of osteoinductive human gingival fibroblastswhich were stimulated by morphogenetic protein-2(BMP-2)and dexamethasone(DEX)on the periodontal diseased and healthy root slices,respectively.2.To observe the repair effect of skull defect of nude mice and the effect of BMP-2 and DEX after applying osteoinductive HGFs and Bio-Oss.3.To observe the repair effect of skull defect and the effect of BMP-2 and DEX after implanting cell sheet formed by osteoinductive HGFs,and to provide experimental evidence for the application of HGFs and cytokines in the regeneration of periodontal tissue.METHODS:1.The 4 mm×4 mm×1mm healthy and diseased root slices were prepared.HGFs were cultured by tissue piece method,and the third passage of HGFs were applied for the following experiments.The experiments were divided into 3 groups including Group A(osteogenic induction A),Group B(osteogenic induction B),and Group N(no osteogenic induction,control group).Group A: osteoinductive medium(low glucose Dulbecco's minimum essential medium(L-DMEM),3% fetal bovine serum(FBS),2×10-3 mol/L L-Glutamine,10-3 mol/L ?-sodium glycerophosphate and 5 mg/L L-ascorbic acid),10-8 mol/L DEX and 50 ng/mL BMP-2;Group B: osteoinductive medium;Group N: L-DMEM and 3% FBS.Then HGFs were inoculated onto healthy and diseased root slices respectively and the following procedures were performed at the designed time point.The number of cells attached to the slices was counted on blood cell counting plate.The viability of cells in 3 groups was detected by 3-4,5-dimethylthiazol-2-yl-5-3-carboxymethoxyphenyl-2-4-sulfophenyl-2H-tetrazolium,inner salt(MTS)assay.We used scanning electron microscopy to observe cell attachment on root slices.The activity of alkaline phosphatase(ALP)was detected by quantitative method of p-nitrophenol phosphate(PNPP)and ALP staining kit.The formation of calcium nodule was detected by alizarin red staining.2.After construction of skull defect in nude mice models,cells in three groups were mixed with Bio-Oss respectively and then implanted into the defect.After 6 weeks,the bone formation in three groups was observed.3.The cell membranes in three groups were constructed in vitro and then injected into nude mice skull defect.After 6 weeks,osteogenic regions were analyzed by HE staining and bone quantitation to evaluate the osteogenesis.RESULTS:1.The number of HGFs induced by BMP-2 combined with DEX was significantly higher than that of osteoinductive group B and the control group at 1,3,5 and 7 days(P < 0.05).2.The viability of HGFs induced by BMP-2 combined with DEX was significantly higher than that of osteoinductive group B and the control group at 3,5 and 7 days(P < 0.05).3.Scanning electron microscopy illustrated that the number of cells attached to the root in Group A was significantly higher than that of Group B and Group N,and the number of cells attached to healthy root slices was significantly higher than that attached to diseased slices at 3 and 7 days(P < 0.05).4.PNPP results showed that the osteogenic activity of HGFs induced by BMP-2 combined with DEX was significantly higher than that of osteoinductive group B and the control group at 1,3,5 and 7 days(P < 0.05).After 7 days and 21 days,ALP staining showed that the cells in group A had the strongest blue purple precipitation,and alizarin red staining showed that the cells in group A had the strongest red sediment among three groups(P < 0.05).5.The results of HE staining showed that in the A group and the B group,a large number of cord and reticular matrix were found in the skull defect.While in C group,only a small amount of matrix forming defect.The results showed that the matrix area rate of the osteogenic induction group was significantly higher than that of the non osteogenic induction group,and the matrix area rate of A group was higher than that of B group(P < 0.05).6.It took 21 days for cells in three groups to form cell sheets.The results of HE staining showed that a large number of cord and reticular matrix were formed at the edge of the osteoblast induced cell membrane,the non osteogenic induction group only a small amount of matrix formation.The results showed that the matrix area rate of the osteogenic induction group was significantly higher than that of the non osteogenic induction group,and the matrix area rate of A group was higher than that of B group(P < 0.05).CONCLUSIONS:1.The periodontal diseased root effects attachment,proliferation and osteogenic differentiation of HGFs.2.Osteoinductive HGFs were able to attach to root slices and osteogenetic differentiation.3.BMP-2 and DEX significantly promoted adhesion,proliferation and osteogenic differentiation of HGFs no matter on healthy root slices or diseased ones.4.HGFs induced by osteogenic,especially BMP-2 combined with DEX,can promote the formation of the matrix of the skull defect area,provide the necessary condition for further bone formation.