Regulation of gingival fibroblast phenotype by periodontal ligament cells in vitro

Objectives Stem cell transplantation has shown modest effects on periodontal tissue regeneration, and it is still unclear how regenerative effects utilizing this modality are mediated. A greater understanding of the basic interactions between implanted and host cells is needed to improve future strategies. The aims of this study were to investigate the effects of periodontal ligament (PDL) cells on expression of periodontal markers and alkaline phosphatase (ALP) activity of gingival fibroblasts (GF). Materials and Methods Primary human PDL cells were co-cultured with primary GF cultures either by direct co-culture with subsequent FACS sorting or indirect co-culture using transwell cultures and PDL cell conditioned medium. Expression of periodontal markers, asporin, nestin, and periostin, was assessed by qPCR and immunofluorescence staining. Alkaline phosphatase (ALP) expression was assessed by qPCR, histochemical staining, and activity assessed by para-nitrophenol enzymatic assay. Single cultures of PDL cells and GF were used as controls. The role of Wnt signaling on ALP activity was assessed via Dkk1-mediated inhibition. Results PDL cells significantly upregulated expression of PDL markers in GF with both direct and indirect co-culture methods when compared to controls (6.05 vs. 0.73 and 59.48 vs. 17.55 fold change of asporin expression). PDL/GF cell co-cultures significantly increased ALP activity in GF when compared with single GF cultures. Similar results were obtained when using conditioned medium isolated from PDL cell cultures. Dkk1 caused dose-dependent reduction in ALP activity of GF cultured in PDL cell conditioned medium. Conclusions PDL cells stimulate expression of periodontal markers and osteogenic capacity of gingival fibroblasts via paracrine signaling which can be partially inhibited with addition of the Wnt antagonist, Dkk1.Further studies are required to identify specific secreted factors responsible for this activity.