In-vitro culture of porcine periodontal ligament cells: Response of fibroblast-like and epithelial-like cells to prostaglandin E 1, parathyroid hormone and calcitonin and separation of a pure population of fibroblast-like cells

Abstract

The cells were evaluated for their responses to the calcium-regulating hormones prostaglandin E 1 (PGE 1), parathyroid hormone (PTH) and calcitonin (CT) by measuring the concentration of intracellular cyclic-3′,5′-adenosine monophosphate, cAMP. The primary cultures of mixed epithelial cells and fibroblast-like cells responded to 2.5 μg/ml of PGE 1 with an increase in cAMP of 335 pmole/10 6 cells decreasing to 40 pmole/10 6 cells in the first and second subcultures. The fibroblast-like cells were separated from the epithelial cells by sequential trypsinization and the serum-free medium method (SFM method). When second subcultures of predominantly fibroblast-like cells, prepared by sequential trypsinization, were evaluated for their response to PGE 1, an increase of 210 pmole cAMP/10 6 cells was found although the equivalent subcultures of predominantly epithelial cells prepared by the same method gave an increase in cAMP of only 25 pmole/10 6 cells, suggesting that fibroblasts. and not epithelial cells, are responsive to PGE 1. The SFM-method of purification, which gave pure cultures of fibroblast-like cells, works because they, unlike epithelial cells, contract to stellate morphology when challenged with serum-free medium. The effect of 2.5 μg/ml of PGE 1 on the second subculture of pure fibroblast-like cells prepared by the SFM-method was maximal, 400 pmole cAMP/10 6 cells, after 4 min of incubation, but decreased gradually thereafter. The cAMP level was still considerably higher than that of the control after 30 min of incubation. The response of the fibroblast-like cells to PGE 1 was dose-dependent up to the maximum concentration of 5 μg/ml used. None of the cultures responded to either PTH or CT.