Isolation and characterization of fibroblasts derived from regenerating human periodontal defects

Abstract 

In an attempt to understand better the cells responsible for periodontal regeneration, cells from human gingiva, periodontal ligament and regenerating periodontal defects treated with expanded polytetrafluorethylene membranes were isolated, cultured and characterized. Guided tissue regeneration procedures were carried out on three human volunteers around molar teeth destined for extraction. After a 6-week ‘healing phase’, fibroblast cell cultures were established from explants of the regenerating soft connective tissue (RTF), as well as from the associated periodontal ligament (PLF) and gingiva (GF). Following stimulation with platelet-derived growth factor-β (PDGF) and insulin-like growth factor-1 (IGF-1), [³H]thymidine-uptake and dye-binding assays were used to assess the rate of DNA synthesis and cell proliferation, respectively. Northern blotting was used to measure the expression of mRNA for the extracellular matrix proteoglycans decorin, biglycan and versican. The results show that the GF and RTF proliferated more quickly than the PLF. PDGF and IGF-1 were mitogenic for all three cell types. Decorin mRNA expression was stronger in the GF than the RTF and PLF, whereas versican mRNA expression was stronger in the GF and PLF than the RTF. Biglycan mRNA expression was strong in the PLF, moderate in the GF and weak in the RTF. The growth factors did not affect the mRNA expression for biglycan, but they upregulated versican and downregulated decorin mRNA. It can be concluded that RTF exhibits properties characteristic of a reparative phenotype. More specifically, it proliferates faster than PLF, from which it is derived, while exhibiting a unique pattern of proteoglycan mRNA expression. Therefore, this study demonstrates that fibroblasts obtained from the regenerating periodontal defects exhibit characteristics consistent with their ability to facilitate periodontal regeneration.

Keywords:  Decorin, Biglycan, Versican, Periodontal regeneration, Fibroblast heterogeneity

Abbreviations:  DMEM, Dulbecco modified Eagle medium, ELISA, enzyme-linked immunosorbent assay, ePTFE, expanded polytetrafluorethylene, FCS, fetal calf serum, IGF, insulin-like growth factor, PDGF, platelet-derived growth factor