The Clinical Effect And Mechanism Of CGF In Promoting Gingival Tissue Regeneration

Objective:Gingival thickness was of importance to maintain the health of periodontal tissues.Clinically,various methods have been used to thicken gingival tissue.Previous research by our team suggested that concentrated growth factor(CGF)could increase the thickness of gingival tissue,but its underlying mechanism was unclear.The purpose of this study was to explore the clinical application and internal mechanism of CGF in promoting gingival tissue regeneration.Materials and Methods:Clinical study: Twenty patients who met the inclusion criteria were randomly divided into Bio-gide membrane group and CGF membrane group.Periodontally accelerated osteogenic orthodontic(PAOO)was performed,and cone beam computed temography(CBCT)was applied before and 6 months after surgery.The gingival thickness of the enamel cementum was measured,and the changes of the gingival thickness of the two groups were compared and studied.Experimental research: Immunofluorescence,alkaline phosphatase staining,alizarin red staining,and oil red O staining were used to detect the self-renewal and multi-directional differentiation ability of gingival mesenchymal stem cells(GMSCs).After treatment with different concentrations of CGF,the survival and proliferation ability of GMSCs was detected by dead/ living cell staining and CCK8;the migration ability of GMSCs was detected by Transwell;the fibroblast and angiogenic genes of GMSCs were observed by RT-PCR,Western Blot and cell immunofluorescence in vitro.The effect of AKT,Wnt and YAP signal pathways was detected by Western Blot;the cytoplasmic nuclear transport of YAP was observed by immunofluorescence;AKT pathway inhibitor LY294002 and YAP inhibitor CA3 were used to study the changes of AKT,Wnt and the YAP signaling pathway and the effect of CGF on GMSCs migration,fibroblasts,and angiogenesis under the action of inhibitors;the proliferation,migration,and expression of fibroblasts and angiogenic genes and proteins of YAP in GMSCs was detected through overexpression and knockdown of YAP;Construct a local injection model of rat gingiva and inject CGF and PBS on both sides of rat gingiva to observe the changes in rat gingival thickness by HE staining and immunohistochemical staining.Further experiments was designed to study the expression of proliferation and vascularization-related proteins and the changes in YAP expression.Results:Compared with Bio-gide membrane,CGF could increase gingival thickness in patients with PAOO.GMSCs showed great expansion ability and osteogenic,adipogenic and chondrogenic differentiation ability.CGF has no toxic effects on GMSCs.And the cell ability of proliferation and migration increased with the increase of CGF concentration.Stimulated by CGF,the gene and protein expression of collagen-1(Col-1),fibronectin(FN),vascular endothelial growth factor(VEGF),and angiopoietin-1(Ang-1)was up-regulated.Under the action of CGF,the phosphorylation expression of AKT and GSK3 β increased,and YAP was transferred from the cytoplasm to the nucleus.LY294002 could inhibit the phosphorylation of AKT and GSK3 β and the dephosphorylation of YAP,while CA3 could only dephosphorylate YAP,and has no significant effect on the phosphorylation level of AKT and GSK3 β.Further experiments showed that the expression of Col-1,FN,VEGF and Ang-1 genes and proteins expression in GMSCs was decreased under the action of LY294002 or CA3.Similarly,the migration ability of GMSCs after LY294002 or CA3 treatment was also significantly decreased.Transfection of YAP overexpression plasmid and si RNA revealed that GMSCs proliferation,migration,and expression of Col-1,FN,VEGF,and Ang-1 genes and proteins were changed with the changes in YAP.The model of rats was successfully established with local injection of CGF.Compared with the PBS group,CGF can increase the gingival thickness and the expression of Ki67 and CD31. The intranuclear expression of YAP also increased in GMSCs.Conclusions:1.To some extent,CGF could increase the gingival thicknes with PAOO surgery;2.CGF could promote the proliferation,migration,angiogenesis and fibroblast regeneration of GMSCs;3.CGF could promote gingival regeneration through AKT,Wnt / β-catenin,and YAP signaling pathways.AKT signaling pathway might be the potential upstream signaling pathway of Wnt / β-catenin and YAP signaling pathways;4.YAP dephosphorylation and nuclear transport might be key factors for CGF to promote gingival regeneration.