| The key to tissue engineering and regenerative medicine is finding stem cells with the potential for self-renewal and multidirectional differentiation.Among different populations of stem cells,human gingival mesenchymal stem cells(hGMSCs)have attracted much attention because they are easily accessible from the oral cavity,the collection procedures do not require invasive procedures.An increasing number of studies have found that GMSCs can differentiate into chondrocytes,osteoblasts,endothelial cells and smooth muscle cells upon induction by different factors.Most importantly,GMSCs are expected to be safely and effectively applicable to clinical tissue regeneration.With the development of traditional Chinese medicine,more and more traditional Chinese medicine has been developed and applied.Natural musk has the effects of tranquilizing and allaying excitement,relieving swelling,promoting circulation of blood to eliminate stasis of blood and ameliorating infantile convulsions.Muscone,which is the main effective component,is used in the treatment of cardiovascular diseases and angina,improving the vascular remodeling of the middle cerebral artery.It has been reported that muscone exerts cytotoxic effects,induces the apoptosis of cancer cells,and influences the expression of proto-oncogenes and tumor suppressor genes to achieve antitumor effects.Studies have shown that that muscone promotes BMSCs' proliferation and osteogenic differentiation and the migration of exogenous rat BMSCs in a rat model.To our knowledge,the effect of muscone on the biological behavior of hGMSCs has not been reported.Wnt pathway is a very important signal transduction pathway,which plays a crucial role in the process of embryonic development and formation,and is also closely related to the maintenance of organism tissue homeostasis.Previous studies have shown that Wnt/?-catenin signal transduction pathway participates in and affects the activity and function of mesenchymal stem cells,including the process of proliferation and differentiation.Therefore,this study explored the effect of muscone on the proliferation,migration and differentiation of hGMSCs in vitro,providing a theoretical basis for the application of hGMSCs in tissue engineering and regenerative medicine.Objectives:The purposes of this research were to study the effects of muscone on the differentiation,proliferation and migration of hGMSCs,and to explore correlations with Wnt/?-catenin signaling during the process of differentiation of hGMSCs.We hope that the results provide a theoretical basis for the application of hGMSCs and muscone in tissue engineering and regenerative medicine.Materials and methods:(1)hGMSCs Isolation and culture in vitro:health gingival tissue was removed by outpatient dental extraction and isolated and cultured primary hGMSCs by limiting dilution method.Cells which belong to 3 to 5 generations in fine conditions were used into following process.(2)Proliferation of hGMSCs:the effects of muscone on proliferation of hGMSCs was assessed by Cell-counting kit-8(CCK-8)and colony formation assay.(3)Migration of hGMSCs:the effects of muscone on migration of hGMSCs was tested by wound healing and cell migration assays.(4)Osteogenic differentiation of hGMSCs:ALP staining and activity assays were used to detect the ALP activity of hGMSCs after 7 days and 14 days of exposure to muscone at different concentrations.Alizarin Red staining assay was used to detect the formation of calcification nodules of hGMSCs after 21 days of treatment with muscone at different concentrations,and calcium quantification was conducted.mRNA expression of osteogenesis-related genes ALP and Runt related transcription factor 2(RUNX-2)in hGMSCs were determined by real-time polymerase chain reaction Analysis(qRT-PCR).(5)Adipogenic differentiation of hGMSCs:Oil Red O staining assay was used to detect the formation of lipid droplets of hGMSCs after 21 days of treatment with muscone at different concentrations,and lipid droplets quantification was conducted.mRNA expression of adipogenesis-related genes Peroxisome proliferator activated receptor y(PPARy)and Lipoprotein lipase(LPL)in hGMSCs were determined by real-time polymerase chain reaction Analysis(qRT-PCR).(6)Mechanism of the effect of muscone on the differentiation of hGMSCs:qRT-PCR and Western Blot were used to detect the effect of lithium chloride(LiCl,an agonist of the Wnt/?-catenin signaling pathway)on the effect of muscone on promoting adipogenic differentiation and inhibiting osteogenic differentiation of hGMSCs.(7)Statistical analysis:using SPSS 20 statistical analysis software analyze the experimental data.Using One-way analysis of variance for the comparison.Using t test compared the two different groups of data.Using paired t test compared the two different groups of data at different time points,and we consider P<0.05 statistically significantly.Results:Muscone can obviously promote the proliferation and migration.When the concentration of muscone was 6mg/L,the promotion effect was the most significant(P<0.05).Muscone can also promote lipid droplets formation and inhibit ALP activity and calcification nodules formation of hGMSCs.The results of lipid droplets quantification and calcium quantification were statistically significant(P<0.05).Both ALP and RUNX-2 gene expressions were down-regulated compared with the control group,while PPARy and LPL gene expressions were up-regulated(P<0.05).In addition,lithium chloride affected the effect of Wnt/?-catenin signaling pathway on muscone of hGMSCs to inhibit osteogenic differentiation and promote adipogenic differentiation.Conclusion:(1)Muscone promotes the proliferation and migration of hGMSCs to some extent.(2)Muscone promotes the adipogenic differentiation of and inhibits osteogenic differentiation hGMSCs to some extent.(3)Wnt/?-catenin signaling pathway is closely related to the effect of muscone on hGMSCs to adipogenic differentiation of and inhibit osteogenic differentiation. |
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