Effect And Mechanism Of PRP Composite POC/CS Scaffold On Oral Keratinized Gingival Regeneration

Objectives: The repair of oral keratinized gingival defect is a difficult problem in the field of oral implantology.It has become an urgent clinical need to find a safe,efficient and economical mean to promote keratinized gingival regeneration.The purpose of this study was to promote the regeneration of keratinized gingival by using in situ tissue engineering techniques,so as to reduce the secondary injury caused by traditional methods such as free gingival transplantation(FGG)and relieve patients’ pain.In this study,platelet-rich plasma(PRP)and poly(1,8-octanediol citrate)/ chitosan scaffolds were combined to construct novel composite scaffolds to promote tissue regeneration through slow release of growth factors.In vitro and in vivo experiments were conducted to explore the role of composite scaffolds in promoting keratosis gingival regeneration,and preliminary exploration of related molecular mechanisms.It provides a new idea and method for the application of composite stent in the regeneration of oral keratinized gingival.Methods:Part Ⅰ: Synthesis and characterization of POC/CS scaffolds1.POC/CS scaffolds with different ratios were synthesized by solution casting method based on POC and CS.2.Fourier infrared spectroscopy was used to detect the characteristic peak of the scaffold.3.The water absorption expansibility and degradability of POC/CS scaffold were tested by weighing method.4.CCK-8 and live/dead staining experiments were used to verify the biocompatibility of POC/CS scaffolds for human oral keratinocytes(h OK)and human oral mucosal fibroblasts(h OMF).5.Scanning electron microscopy was used to observe the surface and cross section of the POC/CS scaffold.Part Ⅱ: The effect of PRP composite POC/CS scaffold on the proliferation and migration of h OK and h OMF and related mechanism exploration1.PRP was prepared by one-step centrifugal method and co-incubated with POC/CS scaffold to construct PRP composite POC/CS scaffold.2.Enzyme-linked immunosorbent assay(ELISA)was used to verify the ability of PRP composite POC/CS scaffold to release PDGF-BB,VEGF,TGF-β1 and other growth factors in PRP.3.CCK-8 and cell cycle experiments were conducted to explore the effect of PRP composite POC/CS scaffold on the proliferation and cell cycle of h OK and h OMF.4.Scratch test and Tanswell to explore the effect of PRP composite POC/CS scaffold on h OK and h OMF migration.5.The effect of PRP composite POC/CS scaffold on h OK expression of integrins and changes of some epithelial-mesenchymal transition(P-EMT)related proteins were detected by Western blot and immunofluorescence,and the role of PI3K/AKT signaling pathway in this process was explored.6.Inhibitors of integrin αvβ3 and PI3K/AKT pathway proteins were added,and the changes of h OK expression of integrin αvβ3,EMT-related index proteins and PI3K/AKT phosphorylation were detected by WB.CCK-8 and scratch test were used to detect the effects on h OK proliferation and migration,and the related molecular mechanisms were explored.Part Ⅲ: In vivo experiments to verify the effect of PRP composite POC/CS scaffolds on keratinized gingiva regeneration in New Zealand white rabbits1.A model of keratinized gingiva defect was constructed in the mouth of New Zealand white rabbits using a gingival loop cutter.2.Single-lens reflex(SLR)camera was used to record the wound healing of keratinized mucosa in New Zealand rabbits,and Image J was used to analyze the effect of PRP composite POC/CS scaffold on the wound healing of keratinized mucosa in New Zealand rabbits.3.The regenerated gingival tissue was taken for HE staining to analyze the effect of PRP combined POC/CS scaffold on the regeneration of keratinized gingiva in New Zealand rabbits.4.Immunohistochemical staining was used to detect the expression of integrin and p-EMT-related proteins in regenerated gingival tissue samples.Results:Part Ⅰ:1.Different proportions of POC/CS scaffolds were synthesized by solution casting method.2.The characteristic peaks of Fourier infrared spectra confirmed that the POC/CS scaffold was synthesized successfully.3.The water absorption and expansion rate of POC/CS scaffold was about 40% ～ 50%after 12 hours,and the degradation rate of POC/CS scaffold was 8% ～ 14% after 14 days.4.POC/CS scaffolds with different proportions had good biocompatibility with h OK and h OMF cells,and the cell survival rates were all above 90%,and there was no significant statistical difference between the groups(p > 0.05).5.Scanning electron microscope results showed that the surface of POC/CS scaffold was a loose and porous structure with interconnected channels inside.Part Ⅱ:1.PRP was successfully prepared by one-step centrifugal method,and PRP composite POC/CS scaffold was successfully constructed by co-incubation with POC/CS.2.Results of ELASA experiment showed that the growth factor in PRP was released quickly within 3 days,while the growth factor in PRP composite POC/CS scaffold was released slowly for 14 days.3.The results of CCK-8 showed that PRP combined with POC/CS scaffold could promote the proliferation of h OK and h OMF more than PRP at 7 and 14 days,and the difference was statistically significant(p <0.05).The cell cycle results showed that,compared with PRP group,PRP combined POC/CS scaffold group had a higher proportion of cells in S phase and G2 phase at 7 and 14 days.4.Scratch test and Tanswell’s results showed that PRP combined with POC/CS scaffolds had more significant migration effects on h OK and h OMF at 7 and 14 days compared with PRP,with statistical significance(p <0.01).5.PRP combined with POC/CS scaffold up-regulates the expression of integrin αvβ3 in h OK,and may promote P-EMT-like changes in h OK through activation of PI3K/AKT pathway.6.The addition of integrin αvβ3 inhibitor Cyclo(-RGDfk)can inhibit h OK expression of integrin protein and partially reverse P-EMT,inhibiting h OK migration.7.The inhibitor of PI3 K pathway(LY294002)can inhibit the phosphorylation of PI3 K and downstream AKT,partially reverse EMT,and inhibit h OK proliferation and migration.Part Ⅲ:1.The quantitative analysis results of Image J showed that PRP combined with POC/CS scaffolds could significantly promote the healing of oral mucosal wounds in rabbits at day 7,and the difference was statistically significant compared with the control group(p <0.05).2.H-E results showed that the inflammatory reaction in the regenerated gingival tissue of PRP composite POC/CS scaffold group was less severe,the new gingival epithelium was thicker,and the arrangement of fibrous connective tissue was more regular.3.The results of immunohistochemistry showed that PRP combined POC/CS scaffold promoted the expression of integrin alpha V and beta 3 in the regenerated gingival tissue of New Zealand white rabbits,and the expression of integrin alpha V was statistically different from that of the control group(p <0.05).4.Immunohistochemical staining results showed that the expression of N-cadherin and Vimentin in PRP composite POC/CS scaffold group was up-regulated,and the average optical density value was significantly higher than that in the control group,and the difference was statistically significant(p <0.05),while the change of E-cadherin was not significant(p > 0.05).Conclusions:1.POC/CS scaffold has good water absorption,expansibility and degradability,and has good biocompatibility with h OK and h OMF cells.The internal structure is loose and porous,which can be an important candidate for tissue engineering scaffold.2.PRP composite POC/CS scaffolds can promote the proliferation and migration of h OK and h OMF by slowly releasing growth factors.3.PRP composite POC/CS scaffold can promote keratinized gingiva regeneration in New Zealand white rabbits in vivo.4.RPP composite POC/CS scaffold promotes h OK to up-regulate the expression of integrin αvβ3,activate PI3K/AKT pathway,promote P-EMT of h OK,accelerate re-epithelialization,and promote the regeneration of keratinized gingiva.