Culture of osteogenic cells from human alveolar bone: A useful source of alkaline phosphatase Ana Maria S. Sim~ ao a , Marcio M. Beloti b , Adalberto L. Rosa b , Paulo T. de Oliveira b , Jose ´ Mauro Granjeiro c , Jo~ ao M. Pizauro d , Pietro Ciancaglini a, * a Depto Quı ´mica, FFCLRP, Universidade de S~ ao Paulo, Av. Bandeirantes, 3900, 14040-901, Ribeir~ ao Preto, SP, Brazil b Laborato ´rio de Cultura de Ce ´lulas, FORP, Universidade de S~ ao Paulo, Av. Cafe ´ s/n, 14040-904, Ribeir~ ao Preto, SP, Brazil c Depto Biologia Celular e Molecular, Universidade Federal Fluminense, UFF, Outeiro de S~ ao Jo~ ao Baptista, s/n 24.020-150 Campus do Valonguinho, Centro/Nitero ´i, RJ, Brazil d Depto Tecnologia, FCAV, Universidade Estadual Paulista, Via Prof. Paulo Donato Castellabe, s/n 14884-900 Jaboticabal, SP, Brazil Received 2 February 2007; revised 16 March 2007; accepted 12 June 2007 Abstract 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 TriseHCl 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 b-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of w120 kDa that could be solubilized by phospholipase C or Polidocanol. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Alkaline phosphatase; Human alveolar bone; Cell culture; Osteogenic cells; Membrane solubilization; Kinetic data 1. Introduction Biological calcification is a tightly regulated process in which different types of tissues, cells, organelles and biomole- cules participate in the coordination and regulation of the metabolic events involved in accumulating large amounts of calcium phosphate (Anderson, 1995; Hsu and Anderson, 1995; Leone et al., 1997; Boyan et al., 2000). Understanding the role of each membrane component, such as membrane pro- teins, lipids, and carbohydrates will contribute to resolving the details of the calcification process (Anderson, 1995; Hsu and Anderson, 1995, 1996; Hsu et al., 1999, 2000; Kirsch and Claassen, 2000; Kirsch et al., 2000; Milla ´n, 2006; Ciancaglini et al., 2006; Sim~ ao et al., 2007). Bone marrow cells can be isolated, cultivated and induced to differentiate into cells involved in the calcification process, such as chondrocytes or osteoblasts (Phinney, 2002; Prockop et al., 2003; Osyczka and Leboy, 2005). Several studies have shown that the stages of differentiation to achieve the osteo- blastic phenotype require the coordinated expression of many molecules (Cheng et al., 1996; Osyczka and Leboy, 2005). Additionally to the expression of collagen type I, osteopontin, bone sialoprotein and osteocalcin, high levels of tissue non- specific ecto-alkaline phosphatase, are also induced during the osteoblast differentiation for the mineralization process (Cheng et al., 1996; Osyczka and Leboy, 2005). Histological and biochemical studies have shown that membrane of mineralizing cells or matrix vesicles (MV) are * Corresponding author. Tel.: þ55 16 3602 3753; fax: þ55 16 3602 4838. E-mail address: pietro@ffclrp.usp.br (P. Ciancaglini). 1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.06.002 Cell Biology International 31 (2007) 1405e1413 www.elsevier.com/locate/cellbi