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Purpose: To investigate the expression of alkaline phosphatase (ALP) activity in gingival fibroblasts from individuals with chronic periodontitis (CP) and drug-induced gingival hyperplasia (DGH) induced by diphenylhydantoin. Methods: Gingival fragments were obtained from 13 patients (8 women and 5 men, from 22 to 74 years of age), with 4 fragments from clinically normal gingiva (NG), 5 from biopsy of periodontal pockets with CP, and 4 from DGH induced by diphenylhydantoin. Using an enzymatic digestion procedure, gingival cell suspensions containing ALP-positive fibroblasts were prepared without affecting ALP activity. Cytochemistry and histochemistry analyses were performed. Results: Fibroblasts from NG presented low levels of ALP when compared to CP and DGH, which showed elevated and intermediate levels of ALP, respectively. Little cell proliferation was observed for fibroblasts from CP and DGH as compared to NG. However, the quantity of cells recovered from the subcultures was similar to the quantity recovered from the initial cell culture for the three sources. Conclusion: The expression of ALP is increased in CP and DGH, and fibroblasts in CP and DGH show low proliferation. This suggests that periodontal inflammation and diphenylhydantoin may influence ALP expression and human gingival fibroblast expansion. Other studies are necessary to better assess the importance of ALP in the development and progression of CP and DGH. [ABSTRACT FROM AUTHOR]