Characteristics of periodontal ligament subpopulations obtained by sequential enzymatic digestion of rat molar periodontal ligament.

Periodontal ligament (PDL) consists of different cell populations in various differentiation stages. In the present study, we isolated cell populations from rat molar PDL by sequential enzymatic digestion and characterized growth potential and mineralization activity of the PDL subpopulations (PDL-SP) to throw light on the mechanism of PDL remodeling and, in its turn, periodontal tissue regeneration. PDL attached to extracted rat molars was digested 2 mg/ml collagenase and 0.25% trypsin at 37 degrees C for 30 min. Then four consecutive digestions were performed for 20 min each in a fresh digestive solution. The solutions were centrifuged to collect released cells and 5 PDL subpopulations (30M-, 50M-, 70M-, 90M-and 110M-PDL-SP) were obtained. Light microscopic observation showed that about a half of PDL in width attached on the root surface of extracted teeth and 30M-PDL-SP was considered to contain cells mainly from middle portion of PDL. Scanning electron microscopic examination indicated that 110M-PDL-SP was enriched by root lining cementoblastic cells. 30M-PDL-SP showed a high level of proliferative activity. Although the growth potential of a subpopulation decreased in PDL-SP toward the root surface, 110M-PDL-SP had a high proliferative activity equivalent to that of 30M-PDL-SP. Analyses of alkaline phosphatase (ALP) and mineralization activities showed that higher activities in PDL-SP toward the surface of roots and that 110M-PDL-SP had the highest activity of ALP and the largest number of mineralization nodules. The present study shows as supposed by previous studies on cell kinetics in PDL that subpopulations with larger growth potential were generally located in the middle portion of PDL and those with higher mineralization activities toward the surface of the roots. It is suggested, however, that a possible pathway of PDL cell turnover may exist within the PDL-SP on the root surface in addition to the generally recognized pathway from the middle area of PDL to root surface.