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Effects of a Mixture of Growth Factors and Proteins on the Development of the Osteogenic Phenotype in Human Alveolar Bone Cell Cultures

Paulo Tambasco de Oliveira, Marcos Andrade de Oliva, William Marcatti Amarú Maximiano, Karen Elaine Vasconcelos Sebastião, Grasiele Edilaine Crippa, Pietro Ciancaglini, Márcio Mateus Beloti, Antonio Nanci and Adalberto Luiz Rosa

Cell Culture Laboratory, School of Dentistry of Ribeirão Preto (PTO,MAO,WMAM,KEVS,GEC,MMB,ALR), and Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (PC), University of São Paulo, Ribeirão Preto, São Paulo, Brazil, and Laboratory for the Study of Calcified Tissues and Biomaterials, Université de Montréal, Montréal, Quebec, Canada (AN)

Correspondence to: Prof. Dr. Paulo Tambasco de Oliveira, Division of Oral Histology, School of Dentistry of Ribeirão Preto, University of São Paulo, Av. do Café, s/n, 14040-904 Ribeirão Preto SP, Brazil. E-mail: tambasco{at}usp.br

Strategies to promote bone repair have included exposure of cells to growth factor (GF) preparations from blood that generally include proteins as part of a complex mixture. This study aimed to evaluate the effects of such a mixture on different parameters of the development of the osteogenic phenotype in vitro. Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured under standard osteogenic conditions until subconfluence. They were subcultured on Thermanox coverslips up to 14 days. Treated cultures were exposed during the first 7 days to osteogenic medium supplemented with a GFs + proteins mixture containing the major components found in platelet extracts [platelet-derived growth factor-BB, transforming growth factor (TGF)-β1, TGF-β2, albumin, fibronectin, and thrombospondin] and to osteogenic medium alone thereafter. Control cultures were exposed only to the osteogenic medium. Treated cultures exhibited a significantly higher number of adherent cells from day 4 onward and of cycling cells at days 1 and 4, weak alkaline phosphatase (ALP) labeling, and significantly decreased levels of ALP activity and mRNA expression. At day 14, no Alizarin red–stained nodular areas were detected in cultures treated with GFs + proteins. Results were confirmed in the rat calvaria–derived osteogenic cell culture model. The addition of bone morphogenetic protein 7 or growth and differentiation factor 5 to treated cultures upregulated Runx2 and ALP mRNA expression, but surprisingly, ALP activity was not restored. These results showed that a mixture of GFs + proteins affects the development of the osteogenic phenotype both in human and rat cultures, leading to an increase in the number of cells, but expressed a less differentiated state. (J Histochem Cytochem 56:629–638, 2008)

Key Words: cell culture • osteoblasts • growth factors • cell proliferation • alkaline phosphatase • mineralization

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