人類牙齦纖維母細胞之培養與分化潛力及效能之探討

Abstract

Background: Recently, the discovery of multipotent stem cell populations residing in periodontal ligament (PDL) and dental pulp provides exciting prospects of resource for periodontal or tooth regeneration. The expense of a tooth and difficulty in cultivating PDL and pulp cells, however, hinders the clinical application of PDL and pulp cells in tissue engineering for repairing dental tissues. In contrast, gingival cells could be easily harvested and expanded in vitro. Furthermore, gingiva has been reported to develop from an ectomesenchymal origin rather than a mesenchymal one, and ectomesenchymal stem cells are believed to be pluripotent during early development. Thus, the possible existence of ectomesenchymal stem cells in gingiva makes it of great importance to investigate the putative stem cell or progenitor cell populations in gingival fibroblasts. The purpose of this study is through flow cytometry and induced differentiation to prove the existence of putative stem cell populations in gingiva. Materials and Methods: Primary human gingiva-derived fibroblast-like cells (GFs) was obtained from palatal gingiva of seven healthy adults. For phenotypic characterization, cells from passages two or three were harvested to identify stem cells or mesenchymal progenitor cells-specific surface molecules, such as CD29, CD44, CD90, CD105, CD146 and STRO-1 by flow cytometry. For investigation of osteogenic and adipogenic differentiation capacity, cells were treated with osteogenic or adipogenic induction medium. After stimulation, the results were analyzed by histochemistry staining, such as alkaline phosphatase (ALP) staining and Alizarin Red-S staining for osteogenic lineage cells and Oil Red-O staining for adipogenic lineage cells. Besides, the findings were further examined by RT-PCR for expression of osteogetic markers, such as Runx2, ALP and OCN (Osteocalcin), and adipogenic markers, such as PPARγ2 (Peroxisome proliferative activated receptor gamma 2) and LPL (lipoprotein lipase). Result: GFs demonstrated not only similar morphology to mesenchymal stem cells (MSCs), upon phenotypic characterization, they also shared almost the same cell surface markers profile with MSCs. Furthermore, under the stimulation of osteogenic or adipogenic induction medium, the positive results of ALP, Alizarin Red-S, and Oil Red-O stainings revealed that GFs possessed the capability to differentiated into osteogenic and adipogenic lineages, and these findings were further confirmed by the expression of osteoblastic and adipocytic lineage genes by RT-PCR. Conclusions: We speculate that GFs comprise of not only fibroblasts but also mesenchymal progenitor cells (MPCs) of osteogenic and adipogenic lineages and possibly some MSCs, to some extent. These observations provide clues in clinical applications of gingival cells in cell-based regeneration for dental tissue repair in future.