Isolation of alkaline phosphatase-positive gingival fibroblasts from patients with chronic inflammatory periodontal disease

We have reported recently that increased expression of membrane alkaline phosphatase (ALP) activity is a phenotypical characteristic of gingival fibroblasts located in chronic inflammatory periodontal lesions. To understand the cellular properties of these cells, we isolated ALP-positive gingival fibroblasts from patients with adult periodontitis and evaluated their proliferative potential. Using an enzymatic digestion procedure, we prepared gingival cell suspensions containing ALP-positive fibroblasts without affecting their ALP activities. These cell suspensions were then subjected to 1 g sedimentation, followed by allowing cells to adhere to substrata, Using this procedure, 71.9% of isolated cells were ALP-positive. Dissociation of ALP-positive fibroblasts and contamination by non-fibroblastic cells were examined by cytochemical and immunocytochemical analyses, The proliferative capacity of ALP-positive fibroblasts in culture was assessed by monitoring the proportion of ALP-positive cells after repeated subculture passages and by labelling DNA-synthesizing cells with bromodeoxyuridine (BrdU). The proportion of ALP-positive fibroblasts decreased during cell culture passages without an apparent change in the ALP-positive phenotype. The percentage of BrdU-positive cells was significantly lower among ALP-positive than among ALP-negative fibroblasts. These results indicate that ALP-positive fibroblasts in chronic inflammatory periodontal lesions have low growth potential. We suggest that their reduced capacity to grow in vitro reflects a more differentiated state induced under inflammatory conditions in vivo.