The differentiation potential of gingival mesenchymal stem cells induced by apical tooth germ cell-conditioned medium

Gingival-derived mesenchymal stem cells (GMSCs) have recently been harvested; however, the use of GMSCs in periodontal tissue engineering requires further study. The present study established an indirect co-culture system between rat apical tooth germ-conditioned medium (APTG-CM) and GMSCs, in order to determine the effects on periodontal tissue differentiation in vitro and in vivo. Using the limiting dilution technique, single-colony derived human GMSCs and periodontal ligament stem cells (PDLSCs) were isolated and expanded to obtain homogeneous populations. PDLSCs were used as a positive control group. Cell cycle distribution, alkaline phosphatase (ALP) activity, mineralization behavior, expression of genes associated with a cementoblast phenotype (osteocalcin, bone sialoprotein, ALP, type I collagen, cementum-derived protein 23), and in vivo differentiation capacities of GMSCs/PDLSCs co-cultured with APTG-CM were evaluated. Flow cytometry indicated that GMSCs and PDLSCs were positive for STRO-1 and CD105, whereas CD45 expression was negative. The cell types were capable of forming colonies, and of osteogenic and adipogenic differentiation in response to appropriate stimuli. The induced GMSCs and PDLSCs exhibited numerous characteristics associated with cementoblast lineages, as indicated by increased proliferation and ALP activity, and upregulated expression of cementum-associated genes in vitro. In vivo, cementum/periodontal ligament-like structures were shown to form along the dentin surface and ceramic bovine bone in GMSCs and PDLSCs induced by APTG-CM group. Conversely, vertical fibers could not insert in the control group, which was not co-cultured with APTG-CM. In conclusion, GMSCs are likely to have a role in periodontal tissue regeneration. In addition, APTG-CM was able to provide a cementogenic microenvironment and promote differentiation of GMSCs along the cementoblastic lineage.