| Objective:Gingiva is recognized as a biological barrier to resist invasion of periodontal pathogen. Mesenchymal stem cells in gingiva play an important role in anti-inflammation and periodontal tissue regeneration. The aim of this study was to investigate the epigenetic mechamism of impaired osteo-differentiation in MSC derived from inflamed gingival and whether TSA promotes its ostegenesis.Methods:1. Isolation and identification of GMSC:Gingival tissue samples were obtained from clinic as remnant or discarded tissue following routine dental procedure. Tissues were dissociated by collagenaseI and Dispase. Cells were cultured in DMEM. Flow cytometric analysis was performed to detect the surface markers of stem cell including CD146, CD90, SSEA-4and OCT-4. Colony-forming fibroblasts unit were performed. Osteogenesis and adipogenesis were performed and evaluated by Alizarin Red, ALP and oil red O staining.2. Histone deacetylasel,3,6mRNA were detected by reverse transcription polymerase chain reaction(RT-PCR) in MSCs derived from healthy and inflamed gingival tissue.3. The effect of histone deacetylase inhibitors TSA on the osteogenic differentiatial ability of MSCs derived from healthy and inflamed the gingival tisssues.GMSC-H and GMSC-I were cultured in Osteogenic Condition Medium with100nM TSA or DMSO as control. Osteogenesis and adipogenesis were performed and evaluated by Alizarin Red, ALP and Runx2, OCN, HDAC1, HDAC3, HDAC6mRNA were detected by RT-PCR to analyze the capability of osteogenic differentiation and its causes. Differences between experimental and control groups were analyzed by a two independent t test using SPSS. Values of P<0.05were considered statistically significant.Results:1. Single cell suspensions were capable of forming adherent colonies, characteristic of other stromal stem-cell populations. Flow cytometric analysis showed hGMSC an average of49.33%positive for CD146,90.77%positive for CD90,33.69%positive for OCT-4and54.98%positive for SSEA-4. The colony forming unit was27%Alizarin red-positive nodules formed after14days of osteogenic induction. After4weeks of culture with an adipogenic medium, hGMSC were found to possess the potential to develop into Oil red O-positive lipid-laden fat cells.2. The expressions of histone deacetylase1,3,6mRNA in inflamed gingival mesenchymal stem cells were significantly higher compared to that in healthy gingival mesenchymal stem cells (P<0.05)3. Compared to the control group, cells of experimental group dyed more parts of blue by ALP staining and had higher alkaline phosphatase activity, more calcium nodules were obtained by Alizarin red staining. The expressions of OCN, Runx2mRNA expression were significantly higher in experimental group (P<0.05). The expression of HDAC1,3,6decreased in the experimental group.Conclusions:1. Acquired stem cells from human gingival by enzyme digestion and limited dilution. hGMSCs were identified as a population of highly clonogenic cells, capable of differentiating into adipocytes, osteoblasts and so forth. hGMSCs expressd surface markers of mesenchymal stem cells and embryonic stem cells, including CD146, CD90, SSEA-4and OCT-4.2. The expressions of histone deacetylase1,3,6in Inflamed gingival stem cells were elevated, suggesting that they may be involved in gene regulation of periodontal inflammatory responses.3. TSA can enhance the osteogenic potential of the gingival mesenchymal stem cells, suggesting that the TSA as a histone deacetylase inhibitor can regulate inflammatory response and induce osteoblast cell diffirentiation possibly through inhibition of HDAC1,3,6gene expressions. |
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