| Obejective:Periodontal ligament cell (PDLC) contain postnatal stem cells (PLDSCs), isolated from healthy impacted third molars(H-PDLSC), that are capable of self-renewal, and their multipotency, including differentiating into osteoblasts, odontoblasts. But the differentiation capacity of PDLSCs of chronic periodontitis patients with(P-PDLSC) and without type 2 diabetes mellitus(D-PDLSC) is not yet well understood, In this study, on the level of gene and protein, we investigated whether diabetes primarily affects periodontitis by reducing the PDLSC differentiation capacity, we quantitatively characterized H-PDLSC,P-PDLSC and D-PDLSC to investigate their stem cell properties, including stem cell marker expression, self-renewal, and their multipotency, which may explain the clinical phenomena that type 2 diabetes mellitus patients have high prevalence of periodontitis.Methods:In this study, PDL tissues were obtained from premolar or third molars of healthy volunteers, periodontitis patients and periodontitis with type 2 diabetes mellitus, periodontal ligament fibroblasts were cultured in vitro. PDLSCs were isolated by limiting dilution technique:low-density primary cells were seeded in 96-well plates, cloned cells were digested by trypsin and regular culture when cells proliferated to the 80% bottom of well. Preliminary identification of stem cell was assayed by cloning efficiency, immunofluorescence cytochemistry staining, flow cytometry(FCM) phenotype of surface markers; proliferative ability of H-PDLSC, P-PDLSC and D-PDLSC was detected by MTT and FCM; osteogenic and adipogenic diffrerentiation were carried on H-PDLSC, P-PDLSC and D-PDLSC, then Alizarin Oil red O staining to observe the calcified nodules and lipid droplets after 21 days induction; at the same time, we also continuous observations the ALP activity of stem cells during the osteogenic induction for 9 days; additional, gene expression of osteogenic(RUNX-2, ALP, OCN, BSP, Col-1) and adipogenic(LPL and PPARy)diffrerentiation assayed by real time RT-PCR and protein expression of osteogenic diffrerentiation(RUNX-2, ALP, BSP) assayed by Western Blot before and after 7 days induction.Results:(1) Isolation and preliminary identification of PDLSC:we isolated putative postnatal dental stem cells from single cells of three different groups.10 samples from healthy volunteers were successfully cultured, but only 7 samples (periodontitis) and 3 samples(T2DM with periodontitis) from inflammatory microenvironments were successfully cultured. The morphology of two PDLSCs which from difference inflammatory tissue were similar to the healthy tissues,and the isolated cells expanded easily in vitro and most of the cells exhibited their fibroblastic spindle shape, immunofluorescence cytochemistry staining show that (a)expression of vimentin of three group of stem cells was positive, and cytokeratin was negative, which meant PDLSCs we used derived from mesoderm without epithelial cell.(b) the percentage positivity for STRO-1 and CD 146 from D-PDLSC was much lower than that of those from H-PDLSC and P-PDLSC too, the rate of STRO-1+cells was 62.6±1.6% in H-PLDSC,40.6±2.5% in P-PDLSC and 27.2±1.6% in D-PDLSC(P<0.05); the rate of CD146+cells was 69.6±3.4% in H-PLDSC,48.9±1.6% in P-PDLSC and 32.1±0.9% in D-PDLSC, the difference was a significant, the result was confirmed the source of stem cells and their stem cell properties by FCM cell cycle analysis.(2) Comparison of self-renew and proliferative ability:H-PDLSC,P-PDLSC and D-PDLSC have certain self-renewal capacity, our results showed statistically significant differences:D-PDLSC showed a weaker clonal formation unit(CFU) behavior compared to P-PDLSC and H-PDLSC after ten-day culture, the rates of CFU are 17.8±2.1%(D-PDLSC), 25.0±2.3%(P-PDLSC), and 36.8±4.9%(H-PDLSC), respectively(P<0.001). MTT assayed showed that the proliferative ability of D-PDLSC was weaker than those of H-PDLSC and P-PDLSC with the same condition after 7 days assay (P<0.01). In addition, D-PDLSC presented a lower population in cell S+M phases than those of H-PDLSC and P-PDLSC by FCM for cell phenotype analysis.(3) Comparison of Osteogenic and adipogenic differentiation:After 21 days induction, analysis alizarin red and oil red O staining for calcium deposits and globules area, the amount of mineralized matrix and lipid droplet declined with increased types of diseases, D-PDLSC decreased the alizarin red-positive area and lipid droplet area per well compared to H-PDLSC, and P-PDLSC, Correlation was observed in vitro osteogenic and adipogenic activity in different cell groups. Quantitative RT-PCR was performed to investigate the expression of genes responsible for H-PDLSC, P-PDLSC, D-PDLSC osteogenitic differentiation, results showed that the mRNA expressions of Runx-2, ALP, Col-I, BSP, OCN, LPL and PPARy in the D-PDLSC were obviously lower than those of H-PDLSC and P-PDLSC before and after osteogenitic and adipogenic induction for 7 days(P< 0.05). Consistent with RT-PCR results, protein expression of Runx-2, ALP, and BSP was also confirmed by Western blot analysis, there were significant changes among three groups.(4) Comparison of inflammatory factors:The expression of IL-6 and TNF-a were successfully quantified using RT-PCR. Results demonstrated a trend that expression levels of IL-6 and TNF-a were increased from H-PDLSC to D-PDLSC, the differences were statistically significant (P<0.05).Conclusion:Our study clearly demonstrate the successful isolation and characterization of undifferentiated progenitor cells contained in human PDLSC of chronic periodontitis patients with and without type 2 diabetes mellitus that can be expanded in vitro condition. The morphology of two PDLSCs which from difference inflammatory tissue were similar to the healthy tissue, however, reduction of STRO-1, CD146, proliferative ability, osteogenic and adipogenic differentiation ability indicated that a trend of decrease when disease status changes from "no disease" to "one disease(periodontitis)" to "two diseases(periodontitis and T2DM)". Herein, we hypothesize that T2DM status may exert an effect on the periodontium through multilineage di□erentiation potential of PDLSC, increasing the severity of periodontitis, by inhibiting repair of resorbed bone, increasing bone loss, or both. Maybe it's a better explanation to the relationship between periodontitis and type 2 diabetes mellitus. |
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