Effects Of Transforming Growth Factor-? 1 And Bone Morphogenetic Protein-2 On Proliferation And Differentiation Of Gingival Mesenchymal Stem Cells

Periapical membrane is the periodontal membrane located in the apical part of the root canal.Once the bacterial infection breaks through the apical foramen,it will cause the destruction of periapical membrane,cementum and alveolar bone,and form periapical lesions.Conventional periapical lesions can be effectively treated by root canal therapy and apical surgery.However,for a large area of periapical lesions,conventional periapical surgery can heal slowly,and periapical tissue usually heals by repair rather than regeneration.The combination of guided tissue regeneration(GTR)and apical surgery is beneficial to the healing of periapical lesions.With the development of tissue engineering technology,the application of growth factors and stem cells provides a new research idea for periapical tissue regeneration.Gingival mesenchymal stem cells(GMSCs),as a new kind of stem cells,have attracted more and more attention due to their good self-renewal,multi differentiation potential and immune regulation ability.Transforming growth factor-?1(TGF-?1)is a multifunctional cytokine,which regulates the proliferation of a variety of cells.In addition,for most types of cells,TGF-? 1 has a very significant role in promoting cell fibrosis.Bone morphogenetic protein-2(BMP-2)is one of the strongest osteoinductive factors known so far,which has the strongest osteogenic induction ability.However,exogenous BMP-2 is unstable and can lose its biological activity rapidly.In order to maintain sufficient BMP-2 in bone defect area,the clinical use of excessive BMP-2 not only greatly increases the cost burden of patients,but also leads to a series of cytotoxic reactions and clinical complications.Therefore,how to improve the efficiency of BMP-2 and ensure its role in bone differentiation stage is the key of regenerative medicine.Objective:The purpose of this study is to study the effects of transforming growth factor?1(TGF-?1)and bone morphogenetic protein 2(BMP2)on the proliferation and differentiation of gingival mesenchymal stem cells(GMSCs).By determining the optimal concentration of TGF-?1 and the optimal concentration of BMP-2 to promote bone differentiation,the effects of TGF-? 1 and BMP-2 on the osteogenesis and fibroblast differentiation of GMSCs were studied.In order to replace periodontal stem cells with GMSCs,it could provide theoretical basis for the selection of "seed cells" and the application of human gingival mesenchymal stem cells in tissue regeneration.Method:Small pieces of gingival tissue were collected from healthy periodontal patients(18 to 25 years old)with impacted mandibular third molars who needed extraction for gingival repair.Human GMSCs were isolated and purified by tissue block method combined with limited dilution method.The expression of stem cell markers on the surface of human GMSCs was detected by flow cytometry,The optimal concentration of TGF-?1 on human GMSCs was determined by Transwell assay.The optimal concentration of BMP-2 on human GMSCs was determined by CCK8 and ALP activity assay.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the effect of the optimal concentration of TGF-?1 and BMP-2 alone or in combination on the gene expression levels of osteogenic and fibroblastic markers.The experimental data were analyzed by one-way ANOVA with Graph Pad prism 8.0 software package,and the statistical results were expressed as mean,standard deviation(SD),P < 0.05,with statistical significance.Result:1.Isolation,culture and dryness identification of human GMSCs: human GMSCs were isolated and purified by tissue block method combined with limited dilution method and multiple passage adherent culture.The primary mesenchymal stem cells cultured in vitro were long fusiform and had the typical characteristics of fibroblasts.The results of flow cytometry showed that the surface markers CD90 and CD105 of human GMSCs were positive,while the surface markers CD34 and CD45 of hematopoietic stem cells were negative.GMSCs induced in vitro could differentiate into adipogenic and osteogenic cells under specific induction conditions,indicating that they have the potential of multi-directional differentiation.2.TGF-?1 can promote the proliferation and migration of human GMSCs: CCK8 results showed that 1 ng / ml and 5 ng / ml TGF-?1 could significantly promote the proliferation of human GMSCs(1 ng / ml,P < 0.01;5 ng / ml,P < 0.001);Transwell experiment showed that 1 ng / ml TGF-?1 could significantly promote the migration of human GMSCs(P < 0.01).3.BMP-2 can promote the proliferation and ALP activity of human GMSCs:CCK8 results showed that 10 ng / ml BMP-2 can promote the proliferation of human GMSCs compared with the blank control group(P < 0.05);ALP activity test results showed that 10 ng / ml BMP-2 can significantly enhance the ALP activity of human GMSCs compared with the osteogenic induction group after 7 days of osteogenic induction(P < 0.01).4.TGF-?1 promotes the fibroblast differentiation of human GMSCs: after fibroblast induction for 7,14 or 21 days,qRT-PCR results showed that 1 ng / ml TGF-?1 could significantly up regulate the gene expression levels of collagen type I(COL1),collagen type III(COL3),fibroblast specific protein-1(FSP-1),periodontal ligament associated protein-1(PLAP-1)and alpha smooth muscle actin(?-smooth muscle actin,?-SMA)(P < 0.05).5.BMP-2 promotes the osteogenic differentiation of human GMSCs: after osteogenic induction for 7,14 or 21 days,qRT-PCR results showed that 10 ng / ml BMP-2 could significantly up regulate the gene expression levels of ALP,runt related transcription factor 2(Runx2),osteopontin(OPN),COL1(P < 0.05).6.The combined application of TGF-?1 and BMP-2 could not promote the proliferation,osteogenesis,and fibroblast differentiation of human GMSCs more effectively compared with the single growth factor: CCK8 results showed that compared with the blank control group,the combination of TGF-?1 and BMP-2 could not promote the proliferation of human GMSCs,The results of qRT-PCR showed that the combination of 1 ng / ml TGF-?1 and 10 ng / ml BMP-2 had no significant effect on the proliferation of human GMSCs(P > 0.05);there was no significant difference between 1 ng / ml TGF-?1 and 10 ng / ml BMP-2 alone and 1 ng / ml TGF-?1 alone(P > 0.05);qRT-PCR results showed that there was no significant difference between the combined use of 1 ng / ml TGF-?1 and 10 ng / ml BMP-2 and 10 ng / ml BMP-2alone(P > 0.05).Conclusion:1.1 and 5 ng / ml TGF-?1 could promote the proliferation of human GMSCs;1ng / ml TGF-?1 could promote the migration of human GMSCs.2.10 ng / ml BMP-2 could promote the proliferation of human GMSCs;10 ng / ml BMP-2 could enhance ALP activity of human GMSCs.3.1 ng / ml TGF-?1 could significantly up regulate the gene expression levels of fibroblast related markers in human GMSCs after 7,14 or 21 days;10 ng / ml BMP-2could significantly up regulate the gene expression levels of osteogenic related markers in human GMSCs after 7,14 or 21 days.4.The combination of 1 ng / ml TGF-?1 and 10 ng / ml BMP-2 did not significantly promote the proliferation of human GMSCs;the combination of 1 ng / ml TGF-?1 and 10 ng / ml BMP-2 did not significantly improve the proliferation of human GMSCs compared with single factor.