Culture of osteogenic cells from human alveolar bone: A useful source of alkaline phosphatase

• Published in: Cell biology international 2007-11, Vol.31 (11), p.1405-1413
• Main Authors: Simão, Ana Maria S., Beloti, Marcio M., Rosa, Adalberto L., de Oliveira, Paulo T., Granjeiro, José Mauro, Pizauro, João M., Ciancaglini, Pietro
• Format: Article
• Language: English
• Subjects: Alkaline phosphatase; Alkaline Phosphatase - isolation & purification; Alkaline Phosphatase - metabolism; Bone and Bones - cytology; Bone and Bones - enzymology; Cell culture; Collagen - metabolism; Human alveolar bone; Humans; Kinetic data; Membrane solubilization; Osteoblasts - cytology; Osteoblasts - enzymology; Osteoblasts - metabolism; Osteogenic cells
• ISSN: 1065-6995; 1095-8355
• DOI: 10.1016/j.cellbi.2007.06.002

The aim of this study was to obtain membrane-bound alkaline phosphatase from osteoblastic-like cells of human alveolar bone. Cells were obtained by enzymatic digestion and maintained in primary culture in osteogenic medium until subconfluence. First passage cells were cultured in the same medium and at 7, 14, and 21 days, total protein content, collagen content, and alkaline phosphatase activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Cells in primary culture at day 14 were washed with Tris–HCl buffer, and used to extract the membrane-bound alkaline phosphatase. Cells expressed osteoblastic phenotype. The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10.0. This enzyme also hydrolyzes ATP, ADP, fructose-1-phosphate, fructose-6-phosphate, pyrophosphate and β-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of ∼120 kDa that could be solubilized by phospholipase C or Polidocanol.