Methods: The PDL tissue was scraped from the roots of freshly extracted teeth to enzymatically digest using collagenase. The cells were sub-cultured. Flow-cytometric analysis for the MSC surface-markers CD105, CD73, CD166, CD90, CD34, CD45 and HLA-DR was performed. To confirm the phenotype, total RNA was extracted to synthesize cDNA and which was then subjected to RT-PCR. The gene-expression for Oct4A, Sox2, NANOG and GAPDH was determined by gel-electrophoresis. To assess their multilineage potential, cells were cultured with osteogenic, chondrogenic and adipogenic medium and then stained by Alizarin-red, Alcian-blue and Oil-Red-O respectively. MSCs from the bone-marrow were processed similarly to serve as controls.
Results: The cells isolated from extracted teeth expanded successfully. On flow-cytometric analysis, the cells were positive for CD73, CD90, CD105, CD166 and negative for CD34, CD45 and HLA-DR. The PDLSCs expressed Oct4A, Sox2, and NANOG mRNA with GAPDH expression. Cells cultured in the osteogenic, chondrogenic and adipogenic media stained positive for Alizarin-red, Alcian-blue and Oil- Red-O respectively. The surface marker expression and the trilineage differentiation characteristics were comparable to those of the BMMSCs.
Conclusions: The periodontal ligament tissue of extracted teeth is a potential source of therapeutically useful MSCs. Harvesting them is not invasive and are a promising source of MSC as the PDLSCs showed characteristics similar to those of the highly regarded MSC's derived from bone-marrow.
MATERIALS AND METHODS: PDLSCs were treated with 0, 5, 10 and 20 µg/mL of Escherichia coli LPS. At 48 and 96 h, total cell numbers of control and LPS treated PDLSCs were counted by haemocytometer under a microscope. The VEGF concentration in the conditioned media of the PDLSCs was measured by ELISA.
RESULTS: Rate of cell proliferation of PDLSCs decreased significantly in all LPS treated groups at both 48 h and 96 h except for the group treated with 5 µg/mL of LPS at 48 h. At both 48 and 96 h, VEGF secretion from PDLSCs was reduced significantly at all three LPS concentrations. There was no statistically significant difference in cell proliferation and the amount of VEGF secretion of PDLSCs among the groups treated with different LPS concentrations. No statistically significant change was found in cell proliferation of LPS treated PDLSCs over time, whereas VEGF secretion of PDLSCs was found to have increased significantly with time despite the LPS treatment.
CONCLUSIONS: LPS reduced cell proliferation and VEGF secretion of PDLSCs, suggesting that periodontal pathogens might reduce the capability of PDLSCs in periodontal regeneration. Yet, LPS treated PDLSCs remained viable and VEGF secretion increased significantly over time. Further research is needed to study the potential use of PDLSCs in periodontal regeneration and the relationship of biofilm LPS accumulations.