| 20-Hydroxyecdysone, an ecdysteroid hormone, can induce proliferation and osteogenic differentiation in mesenchymal stem cells. Periodontal ligament stem cells (PDLSC) been developed from human Periodontal ligament cells (PDLC) have mesenchymal-stem-cell-like qualities. However, there are no studies describing the effect of20E on PDLC. Objective:To explore an effective culture technique of PDLC by comparing the effects of tissue explanting, enzymatic digestion, the combination of tissue explant and enzymatic digestion. To study the isolation, identification and differentiation induction of PDLSC in vitro; To study the effects of20E on PDLSC in vitro, such as the proliferation, ALP activity and osteogenic differentiation. Methods:1. PDLC were cultured using three different methods. PDLSC were isolated by limited dilute method. To characterize stem cell phenotypic markers of single-colony derived PDLSC, the expressions of STRO-1, CD146and CD45were analyzed by flow cytometry. To investigate the capacity of PDLSC for multipotential differentiation, PDLSC were performed adipogenic and osteogenic induction by adding adipogenic differentiation medium or osteogenic differentiation medium to the cultures.2. PDLSC were seeded at4×103cells/well in200ul DMEM medium containing10%FBS in96-well plates. After incubating24h, the culture medium was discarded and replaced with fresh DMEM medium containing0,50,100,200or400uM of20E. The cells were then cultured for additional1-10days. The cell line proliferation on vitro was assayed by MTT on the1-10d. Finally, the optical density(OD) was assayed by an enzyme-linked immunosorbent assay detector at490nm.3. PDLSC were seeded at 2×104cells/cm2in6-well plates. After incubating24h, the culture medium was discarded and replaced with fresh DMEM medium containing0,50,100or200uM of20E.7days later, alkaline phosphatase staining was performed.4. PDLSC were seeded at4×105cells/well in lml DMEM medium containing10%FBS in6-well plates. After incubating24h, the cells were treated with and without20E (200uM).3days later, RUNX2were analyzed by real-time PCR. The statistical significance of raw data between the groups in each experiment was evaluated by SPSS13.0, using unpaired Student’s t-test or ANOVA followed by Student-Newman-Keuls multiple comparisons post-test. P value<0.05was considered as statistically significant. The results were expressed as means±EM of the indicated n values. Results:1.The successful rates of the combination of tissue explant and enzymatic digestion, the tissue explanting, enzymatic digestion were60%,53%,40%. We observed that a high percentage of PDLSC expressed STRO-1and CD146. In contrast, only few PDLSC expressed CD45. PDLSC could different to osteoblast and adipocyte lineages in vitro under specific conditions.2. There were no significant difference between experimental groups and control group of the OD values on1d (P>0.05),on2d-10d, the OD values in experimental groups and the control group were statistically different (P<0.01),and the OD values in400uM group were lower than in control groups, the OD values in lower concentrations groups (50,100,200uM) were higher than in control group. The OD values increased with the increase of the concentrations of20E.3. Alkaline phosphatase staining was positive.4. On3d, the experimental group and control group were statistically different (P<0.01) and the RUNX2expression in the experimental group was greater than in the control group. Conclusion:1.The combination of tissue explant and enzymatic digestion has a high successful rate. Limited dilute method is an effective method to isolate PDLSC. PDLSC had the capacity for multipotential differentiation.2.400uM of20E had cytotoxic effect and restrained PDLS cell proliferation, but treatment with lower concentrations significantly increased PDLS cell proliferation in a dose-dependent manner.3.20E markedly stimulated ALP activity of PDLSC.4.20E enhanced osteogenic differentiation of PDLSC. |
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