| Background:Insufficient periodontal soft tissues can cause root caries,tooth sensitivity,and implant failure.Existing treatment methods still have certain limitations in clinical application.How to promote the regeneration and reconstruction of periodontal soft tissue efficiently and minimally invasively is the key to treatment.Tissue engineering provides a new way to solve this problem.Taking cell spheres as the main body of tissue engineering structure has the advantages of supplying seed cells and can be used as a scaffold structure itself,and is increasingly used in the field of regenerative medicine.The key to the success of tissue engineering structure transplantation is the reconstruction of the vascular system.Pre-vascularization in vitro can help the formation of blood vessels after the transplantation of cell spheroids.In order to make the constructed tissue engineering structure play a better role,a new generation of platelet concentrate-liquid phase concentrated growth factor(LPCGF)is introduced.LPCGF is rich in multiple growth factor and can promote angiogenesis and tissue regeneration.Its liquid form can be well combined with the cell spheroids structure and is convenient for application.Whether LPCGF combined with co-cultured cells has the effect of promoting periodontal soft tissue regeneration has not been reported.Aim:In vitro experiments were conducted to study the effect of LPCGF on the proliferation and migration of gingival fibroblasts(HGFs)and HGFs + human umbilical vein endothelial cells(HUVECs)under two-dimensional culture conditions.The cell spheroids structure was construct by combine LPCGF with HGFs or HGFs +HUVECs in vivo to explore whether the structure can promote the increase of periodontal soft tissue and the formation of blood vessels.Methods:1.HGFs were cultured by tissue block method and the cell morphology was observed under the microscope.Cell proliferation ability was assessed by CCK-8 assay;the cell source was identified by immunofluorescence chemical staining.Cultivate HUVECs.2.LPCGF were prepared and the universal morphology and structural characteristics of LPCGF were observed through gross observation,Giemsa staining and scanning electron microscopy.LPCGF were co-culture with HGFs and HUVECs.The effects of different concentrations of LPCGF on the proliferation of HGFs solely or co-culture with HUVECs were evaluated.The horizontal and vertical migration of different combinations of HGFs,or co-cultured with HUVECs cells and LPCGF were evaluated.3.The cell spheres was construct with HGFs,HUVECs and LPCGF by low-adhesion culture method.The morphological characteristic was observed by light microscopy,and survival state of the internal cells were analyzed by live and dead cell stain.The cell spheroids-LPCGF complex was implanted subcutaneously in nude mice,and the materials were collected,fixed,dehydrated and embedded for sectioning at 3,7and 28 days after surgery.HE staining was used to observe the morphology of the cell spheroids and surrounding tissues.Masson staining was performed to observe the formation of collagen structure and immunohistochemical staining was used to detect the expression of CD31 and neovascularization.Results:1.The primary HGFs crawl out well,and the cells are long spindle-shaped or fibroblast-like.The growth curve of HGFs cells is "S" shape,which is in accordance with the normal growth rule of cells.Immunofluorescence staining results showed that cells expressed vimtin without keratin.The HUVECs are irregular polygons,and the cells grow adherent to the wall.2.The LPCGF after the centrifugation is a palely yellow liquid with fluidity.LPCGF will gradually solidify into a gel at room temperature for a period of time.Giemsa staining and scanning electron microscopy results showed that LPCGF is intertwined by dense fibrin,in which blood cells are dispersed.The CCK-B proliferation experiment showed that the number of cells in each group increased with time.At 48 h and 72 h,the cell proliferation level of the 5% LPCGF group was significantly higher than the control group and other experimental groups(P<0.01).When the concentration of LPCGF is 5%,the cell proliferation level HGFs is higher than HGFs + HUVECs.The effects of LPCGF on the horizontal and vertical migration ability of HGFs and HGFs + HUVECs showed that the horizontal and vertical migration capacity of cells of 5% LPCGF group is higher than blank group;Under the same concentration of LPCGF,the horizontal and vertical migration capacity of cells of HGFs + HUVECs is higher than HGFs.3.The boundary of HGFs + 5% LPCGF cell spheroids were clearly observed and the HGFs + HUVECs + 5% LPCGF group had transparent gel-like structures around the cell spheroids under the microscope.Live/dead cell stain and optical density result showed that the cell spheres of two groups had both live and dead cells inside the spheroids on the 7th and 14 th days and the cells in both states were evenly distributed in the cell spheroids.The HGFs + HUVECs + 5% LPCGF group contained more viable cells inside the spheroids than the HGFs + 5% LPCGF group(P <0.01).The HGFs + HUVECs + 5% LPCGF group contained more viable cells inside the spheroids than the HGFs + HUVECs group(P <0.05).CD31 staining HE stain,Masson stain and immunohistochemistry result showed that the cell spheres aggregate with each other to form a larger cell sphere aggregates.HGFs + LPCGF group and HGFs + HUVECs + LPCGF group had no significant difference in collagen formation.The expression level of CD31 positive cells in HGFs + LPCGF group is higher than HGFs group(P> 0.05).Conclusions:In vitro study showed that HGFs,HGFs +HUVECs,HGFs + HUVECs +LPCGF can promote the cell proliferation and migration.However,in vivo study showed no significant differences was observed in collagen formation and angiogenesis between the HGFs + LPCGF group and the HGFs + HUVECs + LPCGF group after the implantation of cell spheroids. |
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