| ObjectiveA new orientation is to use akermanite in skin tissue engineering.By now,only a few reports focus on the role of akermanite in enabling anti-inflammatory properties and facilitating endothelial cell proliferation in the field of wound repair.However,wound healing is a complex and precise proeess,especially in the re-epithelialization and remodeling stage,the role of akermanite in facilitating wound healing remains unclean This experiment intends to observe the effects of akermanite on cell biological behaviors such as proliferation and migration of epidermal stem cells and fibroblasts,gain an insight to the signaling pathways involved in the activation of various cations rendered by bioactive ceramics-akermanite,and indicate a new direction and experimental evidence for the use of akermanite in skin tissue engineering.Materials and methods1.Human epidermal stem cell line was used for in vitro experiments.Epidermal stem cells were cultured in different concentrations of akermanite extract.The optimum concentration of akermanite extract for culturing epidermal stem cells was screened by CCK-8 assay.The distribution of three cell subsets(G0/G1,S and G2/M)was quantitatively detected by immunofluorescence and flow cytometry to determine cell proliferation.Then the gene expressions of Itg?1,lgr4,lgr5 and lgr6 were detected by RT-q-PCR and Western blot.The possible mechanism of the stermness and proliferation of epidermal stem cells stimulated by akermanite extract was further explored.2.Human fibroblasts were extracted and cultured in vitro.The optimum concentration of akermanite extract for culturing epidermal stem cells was screened by CCK-8 assay,and the effect of akermanite extract on the proliferation of fibroblasts was quantitatively detected by flow cytometry.Then,the gene and protein expression of collagen ? and collagen ? synthesized by fibroblasts treated with akermanite extract were detected by RT-q-PCR and Western blot.In addition,the effect of extract of akermanite on the migration ability of fibroblasts was observed by cell scratch assay.The gene and protein expressions of integrin subunits related to migration ability were investigated by RT-q-PCR and western blot.3.The scald model of BALB/c mice was established and treated with and without akermanite powder respectively.The state of the wound healing was observed.After 3,7,14 and 21 days postinjury,the wound tissues were imaged and calculated to analyze the re-epithelialization,epithelium thickness,and collagen deposition by HE,immunofluorescence and Masson staining.Results1.Akermanite extracts can promote and induce the proliferation of epidermal stem cells in a time and concentration-dependent manner.Result shows that the cell viability and proliferation can be improved at the most significant level when the concentration of extracts is 1/16.The expression levels of mRNA in respect of Itg?1,lgr4,lgr,and lgr6 of the epidermal stem cells cultured with 1/16 extracts were detected through RT-q-PCR and western blot quantitative assays to up-regulate and that of Itgpl also up-regulated,suggesting that the extracts lead to better sternness and stronger proliferation of the epidermal stem cells.The mRNA and protein expression levels in respect of the representative ?-catenin,c-myc and ccndl in Wnt/?-catenin signaling pathway were detected through RT-q-PCR and western blot quantitative assay to up-regulate,suggesting that this signaling pathway was activated by the active cations in the extracts.In addition,the scratch assay showed that in contrast with negative control,1/16 extracts led to significantly stronger migration of the epidermal stem cells,the results of RT-q-PCR and western blot suggested that the extracts facilitated the up-regulation of the intracellular expressions of integrin ?5?1,?3?1 and ?6?4.2.Akermanite extracts can also promote and induce the proliferation of fibroblasts in a time and concentration-dependent manner.At this point,the cell viability and proliferation can be improved at the most significant level when the concentration of extracts is 1/64.The mRNA and protein expression levels of collagen ? and collagen ? of the fibroblasts cultured with 1/64 extracts were detected through RT-q-PCR and western blot assays quantitativly to up-regulate.In addition,the scratch assay showed that in contrast with negative control,1/64 extracts led to stronger migration of the fibroblasts,the results of RT-q-PCR and western blot suggested that the extracts facilitated the up-regulation of the intracellular expressions of integrin ?5?1 and ?3?1.3.Mice scald model experiment affirmed that akermanite can accelerate the healing of skin wounds.HE staining result showed that the re-epithelialization process in akermanite powder group was accelerated,the epithelium thickness in the mid-term healing was significantly greater than that in control group,suggesting that akermanite powder can facilitate the proliferation and differentiation of the epidermal stem cells.Immunofluorescence staining result showed that the expression levels of the integrin ?1 of the skin tissue sections in akermanite powder group were higher than that in control group,suggesting that this bioactive ceramics can enhance the sternness of epidermal cells.Moreover,the Masson-stained of the skin tissue sections showed that the wound treated with akermanite had a larger collagen deposition area and more well-arranged layout,suggesting that akermanite can facilitate the synthesis of collagen by fibroblasts,leading to better repair effect.ConclusionsIn this study,an initial insight to the role of bioactive ceramics-akermanite in facilitating the skin wound heating was gained,calcium,magnesium and silicon ions as contained in the akermanite extracts were found to activate the signaling pathway of Wnt/?-catenin,enhance the stemness of epidermal stem cells,and facilitate the proliferation and migration of epidermal cells and fibroblasts,thereby accelerating the process of epidermal re-epithelialization and collagen deposition,allowing for shorter wound healing duration,extending the use of bioactive ceramics in skin tissue engineering,which in turn indicates a new idea,direction and theoretical evidence for the clinical trials of wound healing. |
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