Synergetic Effect Of Topological And Chemical Signals Of Gingival Regeneration Scaffold On The Behavior Of Human Gingival Fibroblasts

BackgroundThe restoration and regeneration of gingival soft tissues play important roles in the esthetic construction of oral prosthodontics and the recovery of periodontal diseases.At present,connective tissue graft(CTG)is the gold standard method for the treatment of insufficient gingival soft tissues.However,in the case of multiple gingival recession or defects,the available amount of connective tissue for transplantation is limited,and postoperative complications increase,since there are few substitute products for gingival soft tissue transplantation.Therefore,it is of great significance to develop easily accessible substitute materials with good bioactivity for gingival soft tissue transplantation,especially tissue engineered gingiva which can simulate natural gingival tissue structure.The three major elements of tissue engineering are seed cells,scaffolds and growth factors.Seed cells are the basis of tissue engineering.The cell behaviors are influenced by many kinds of induced biomaterial signals,such as the structure of scaffolds,the release of chemical molecules and the change of mechanics factors.Therefore,exploring the effects of chemical and topographic signals on the behaviors of human gingival fibroblasts(HGFs)can provide an important reference for the research and development of gingival regeneration alternative scaffolds.In this study,random and aligned fiber scaffolds were prepared by electrospinning.Two kinds of fiber structures were used as topological signals.Polydopamine(PDA)and basic fibroblast growth factor(bFGF)were used as two kinds of chemical signals.The cell behaviors of HGFs were studied under the action of single or multiple signals.ObjectiveThe purpose of this study was to investigate the proliferation rates of HGFs and the expression levels of related genes and proteins under the action of single or multiple signals.Materials and methodsThe research was divided into the following two parts:1?Preparation and characterization of electrospinning fiber scaffolds(1)Pure PCL scaffolds with random and aligned fibers were prepared by electrospinning machine.PCL/PDA-R and PCL/PDA-A fiber scaffolds(R: Random;A: Aligned)were prepared by surface modification of polydopamine.(2)Scanning electron microscopy(SEM),X-ray photoelectron spectroscopy(XPS)and Universal capability testing machine were used to characterize the fiber scaffolds,analyzed and compared their chemical composition and mechanical properties.2? Regulation of cell behaviors by topological and chemical signals(1)The random and aligned structures of electrospun scaffolds were used as the topological signals;the PDA coated on the scaffolds and basic fibroblast growth factor(bFGF)added to the culture medium were used as the chemical signals.Eight groups were set up: PCL-R+C,PCL-R+F,PCL-A+C,PCL-A+F,PCL/PDA-R+C,PCL/PDA-R+F,PCL/PDA-A+C and PCL/PDA-A+F(C: Common culture medium;F:bFGF culture medium).(2)The growth status,morphology and skeleton of HGF were observed by SEM and laser confocal microscopy after 5 days under the action of single or multiple signals.(3)Under the action of single signal or multiple signals,the proliferation rates of HGFs after 1,3,5and 7 days were studied by CCK-8 assay.(4)The gene expression levels of collagen I(COL I),focal adhesion kinase(FAK)and fibronectin(FN)in HGFs were investigated by RT-PCR after cultured for 5 days.(5)Immunofluorescence staining and semi-quantitative analysis were used to observe and analyse the secretion and distribution of COL I,FAK and FN was carried out on the fifth day of culture.Results(1)The SEM images showed that the fibers of aligned PCL scaffolds were highly ordered,while those of random PCL scaffolds were randomly arranged.Small aggregates could be seen on PCL scaffold fibers coated with PDA.There was no significant difference in diameter between PCL group and PCL/PDA group(P >0.05).And there was significant difference in orientation angle between Aligned group and Random group(P < 0.05).(2)Mechanical tests showed that Young's modulus values,tensile strength values increased and fracture coefficient decreased with the increase of fiber ordering(P <0.05).After coated with PDA,the Young's modulus,tensile strength and fracture coefficient values were increased.(3)The results of CCK-8 assay showed that the OD value of each group increased with time,but their speeds were different.Under common medium culture conditions,the cell proliferation rate in the late stage of the Aligned group was higher than that in the random group.The OD value of PCL/PDA group was significantly higher than that of PCL group on the 3th,5th and 7th day under the same conditions of topographic signal and culture medium(P < 0.05).The difference between Common medium group(C group)and bFGF medium group(F group)was found after the third day(P < 0.05).(4)SEM and cytoskeleton staining images showed that the cell density of F and PCL/PDA groups were significantly higher than that of C and PCL groups respectively.HGFs grew highly ordered along the fiber in Aligned group,but adhered to disordered fibers in Random group.The staining results of dead and living cells showed that there were almost no dead cells in all groups.The density of cells were basically the same as those of SEM and cytoskeleton staining.(5)RT-PCR results showed that the gene expression levels of COL I,FAK and FN in Aligned group were higher than those in Random group,and their expression levels in PCL/PDA group was significantly higher than PCL group.In addition to COL I,the expression levels of FAK and FN genes in F group were higher than those in C group.(6)Immunofluorescence staining and semi-quantitative analysis showed that these three target proteins were distributed orderly on the scaffolds of Aligned group and randomly on the scaffolds of Random group.With the addition of chemicalsignals,three target proteins secretion of PCL/PDA groups were significantly higher than that of PCL group(P < 0.05).Except COL I,the secretion of FAK and FN in F group was higher than that in C group.ConclusionThe aligned scaffolds are beneficial to the proliferation and differentiation of HGFs compared with the random scaffolds.PDA and bFGF can further promote the proliferation and differentiation of cells.The induction promotion effect of PDA are the best among the three signals.Compared with the action of topological or chemical factors alone,the combination of these two factors can regulate the behaviors of HGFs in a synergetic manner.