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Objective @#To investigate the effect of silencing histone deacetylase 9 (HDAC9) expression on the proliferation and osteogenic differentiation of periodontal ligament stem cells (PDLSCs).@*Methods@# PDLSCs were isolated, cultured and identified in vitro. An siRNA construct specific for HDAC9 was transfected into PDLSCs (siHDAC9 group), and a nontargeting siRNA was used as a control (siNC group). The interference effect was determined by qRT-PCR. The cell cycle progression of PDLSCs was detected using flow cytometry. The proliferation activity of PDLSCs was detected via CCK-8 assay. Western blotting was used to detect the protein expression of proliferating cell nuclear antigen (PCNA). The mRNA expression of runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP) was investigated by qRT-PCR. The protein expression of RUNX2 was detected by western blotting. In addition, the formation of mineralized nodules was assessed by alizarin red staining. @*Results@#Compared with that in the siNC group, the mRNA expression of HDAC9 in the siHDAC9 group was lower (P < 0.01). Moreover, compared with those in the siNC group, the proliferation index (P<0.01), proliferation activity (P<0.05) and protein expression of PCNA (P<0.01) in the siHDAC9 group were all increased. Compared with the siNC group, the siHDAC9 group exhibited higher mRNA expression of RUNX2 and ALP (P < 0.05), and the protein expression of RUNX2 showed the same results (P < 0.01). The results of alizarin red staining showed that compared to the siNC group, the siHDAC9 group formed more mineralized nodules.@* Conclusion@#Silencing HDAC9 expression can promote the proliferation and osteogenic differentiation of PDLSCs.
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Objective@#To investigate the osteogenic properties of maxillary sinus membrane stem cells (MSMSCs). @*Methods @#Beagle maxillary sinus mucosa was collected, immunomagnetic bead method was applied for isolation of CD146+ cells, and MSMSCs were harvested and cultured from the canine maxillary sinus floor mucosa. The levels of the cell surface antigens CD44, CD146, and CD34 were determined at passage one by flow cytometry. Cells at passage one were cultured in basal medium and osteogenic inductive medium. Real-time PCR, immunohistochemical staining, alkaline phosphatase activity, alizarin red staining and Von Kossa staining were used to investigate the osteogenic properties in vitro. @*Results@#The canine MSMSCs were cultured successfully. The results of flow cytometry were positive for CD146 and CD44 expression but negative for CD34 expression. The relative mRNA expression of runt-related transcription factor 2 (RUNX2) (t = 14.44,P < 0.001), osteopontin (OPN) (t = 7.85,P = 0.001) and alkaline phosphatase alkaline phosphatase (t = 14.27,P < 0.001) was apparently higher in the osteoinductive medium group than in the basal medium group, the differences in relative mRNA expression between the groups were significant. The protein levels of RUNX2 and OPN increased in the osteoinductive medium group. The alkaline phosphatase activity of the MSMSCs increased when the cells were cultured in osteoinductive medium; the activity increased to a level that was significantly higher than that in basal medium, particularly at days 3 (t = 8.79, P < 0.001), 7 (t = 9.75,P < 0.001), 14 (t = 12.14,P < 0.001), 21 (t = 19.62,P < 0.001) and 28 (t = 17.53,P < 0.001). Obvious mineralized nodules were observed by alizarin red staining or Von Kossa staining.@*Conclusion @#Maxillary sinus membrane stem cells exhibit osteogenic ability.
ABSTRACT
<p><b>OBJECTIVE</b>This study investigated the effects of salvianolic acid B (Sal B), a major bioactive component of the Chinese medicine salvia miltiorrhiza, on osteogenic differentiation of human periodontal ligament cells (hPDLCs).</p><p><b>METHODS</b>Third passage PDLCs were used in this experiment. Methyl thiazolyl tetrazolium (MTT) method was employed to observe the effects of different Sal B concentrations on proliferation activity of hPDLCs. Alkaline phosphatase (ALP) activity and mineralization capability were measured, and mRNA expression of osteocalcin (OCN) was detected to investigate the effects of Sal B on osteogenesis of hPDLCs.</p><p><b>RESULTS</b>Sal B did not influence the viability of hPDLCs. The ALP activity and OCN mRNA expression levels of hPDLCs were both significantly improved (P<0.05) under treatment with different Sal B concentrations (0.5, 1, and 5 μmol·L⁻¹) compared with those in OIM group. Moreover, the number of mineralized nodules formed by hPDLCs were considerably higher under treatment with different Sal B concentrations (0.5, 1, and 5 μmol·L⁻¹) than that in the OIM group.</p><p><b>CONCLUSIONS</b>Appropriate Sal B concentration can improve the osteogenic differentiation of hPDLCs.</p>