No abstract

Fragments of Hertwig's epithelial root sheath persist in the periodontal ligament (PDL) in small clusters known as epithelial rests of Malassez (ERM). It is generally agreed that ERM are maintained as a quiescent and exclusively dental epithelial cluster in PDL. However, we speculate that homeostasis and cellular turnover underlies cluster maintenance. We also hypothesize that the fate of ERM clusters - diminishing or remaining - might be regulated via the presence or absence of epithelial stem cells therein. Histological analysis of aging mouse molar PDL showed that ERM clusters gradually increase in size with increasing age. Immunocytochemistry and cell culture revealed that ERM clusters contained Ki67-positive cells and were able to expand when brought in culture. The TdT-mediated biotin-dUTP nick-end labeling (TUNEL) procedure also detected signs of apoptosis. Finally, we identified putative epithelial stem cells in the clusters by 5-bromo-2'-deoxyuridine (BrdU) pulse-chase experiments and immunohistochemistry, using the stem-cell marker leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5). The results suggest that ERM clusters are maintained in the PDL, via cellular turnover, throughout life.

Identification of the specific subset of periodontal ligament-derived mesenchymal stem cells (PDLSCs) will permit the development and improvement of current periodontal regeneration therapy. A popular approach for the isolation of a subset of PDLSCs would be to use cell surface markers to identify these cells, which are effectively enriched in order to isolate stem cells. The CD146 marker is most commonly used to isolate PDLSCs from human periodontal ligament tissues (hPDL). Previous studies have shown that CD146-positive (CD146+) cells possess potencies of high self-renewal and multi-lineage differentiation. However, the capability and potency of mesenchymal stem cells (MSCs) in hPDL-derived CD146-negative (CD146-) cells have not been well established.In this study, CD146+ and CD146-cells were isolated from hPDL, and their properties, including their MSCs potential, were characterized. hPDL was obtained from healthy premolars (n = 10) extracted for orthodontic reasons. Flow cytometry analysis revealed that the average proportion of CD146+ cells was about 50%. An approximately 20% fraction of cells with the highest CD146 expression was sorted as CD146+ cells and an approximately 20% fraction with the lowest CD146 expression was sorted as CD146-cells using fluorescenceactivated cell sorting. Cell cultures were assessed for their colony-forming efficiency, proliferation and differentiation into osteoblasts, adipocytes and chondrocytes. Approximately 20% of CD146+ cells had replicative potential and formed single-cell colonies. The colony-forming efficiency of CD146+ cells was approximately twofold higher than for CD146-cells. Cell proliferation measured by cell-cycle analysis and cell counting showed that the proliferative potential of CD146+ cells was higher than that of CD146-cells. Cell differentiation potential in vitro was determined by real-time PCR and cell staining with alkaline phosphatase and Alizarin Red S for osteogenic differentiation, Oil Red O for adipogenic differentiation, and Alcian Blue for chondrogenic differentiation. The levels of osteogenic and adipogenic differentiation were significantly higher in CD146+ cells than in CD146-cells under appropriate conditions. The level of glycosaminoglycan and gene expression of cartilage oligomeric matrix protein in CD146-cell pellets were higher than in CD146+ cell pellets. These results suggest that CD146+ cells possess bi-potent differentiation potential whereas CD146-cells possess unipotent differentiation potential.