Effects of conditioned medium of human periodontal ligament stem cells on proliferation and osteogenic differentiation of inflammatory tissue-derived human periodontal ligament stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND: The conditioned medium rich in bioactive substances can maintain the stability of proliferation and biological characteristics of stem cells. Whether the conditioned medium of human periodontal stem cells derived from healthy tissues can affect the proliferation and osteogenesis of human periodontal stem cells derived from inflammatory tissue is significant for periodontal tissue regeneration and reconstruction. OBJECTIVE: To investigate the effect of human periodontal stem cells-conditioned medium derived from healthy tissue on proliferation and osteogenic differentiation of human periodontal stem cells derived from inflammatory tissue. METHODS: HPDLSCs from normal periodontal ligaments of healthy adults were isolated, purified and cultured in vitro. Human periodontal stem cells-conditioned medium was obtained by collecting the supernatants from the serum free medium which was used for the third generation cells grown up to 80% of the bottom of the bottle after 24 hours cultivation. Human periodontal stem cells derived from inflammatory tissue were obtained from pericementum of periodontitis patients, and cultured by using limiting dilution assay. Human periodontal stem cells derived from inflammatory tissue were separately cultured under conditioned medium treatment group (conditioned medium containing 50% human periodontal ligament stem cells + 50% conventional medium) and control group (conventional medium). Protein expression levels of vimentin, Pan Cytokeratin, and stromal cell antigen STRO-1 were identified by immunofluorescence staining. The proliferative activity of cells was analyzed by MTT assay and flow cytometry. After osteogenesis in vitro, alkaline phosphatase activity and the expression levels of three osteogenesis related genes (Runx2, OPN, and OCN) were detected using alkaline phosphatase kit and RT-PCR, respectively in both groups. RESULTS AND CONCLUSION: (1) Both groups of cells were in accordance with the morphological characteristics of adult stem cells, showing long fusiform or polygonal shapes. There was no significant difference in cell morphology between the two groups under inverted phase contrast microscope. (2) The immunofluorescence staining showed that cells in both groups were positive for the specific antibodies of vimentin and STRO-1, but negative for the specific antibody of Pan Cytokeratin. (3) The results of MTT assay showed that after 3, 5 and 7 days, the proliferative activity of conditioned medium treatment group was higher than that of control group (P < 0.01). (4) Cell cycle analysis showed that compared with the control group, the number of cells in G2/M phase and S phase in conditioned medium treatment group increased significantly (P < 0.05). (5) At 5 and 7 days of osteogenic induction in vitro, alkaline phosphatase activity of conditioned medium treatment group was higher than that of control group (P < 0.05). (6) After 21 days of osteogenic induction, the expression levels of osteogenic related genes Runx2, OPN, and OCN in conditioned medium treatment group were significantly higher than those in control group (P < 0.01, P < 0.05). (7) In conclusion, human periodontal stem cells-conditioned medium derived from healthy tissues can enhance the proliferation and osteogenic differentiation of human periodontal stem cells derived from inflammatory tissue.