Effects of enamel matrix derivative and transforming growth factor-β1 on human osteoblastic cells

Abstract Background Extracellular matrix proteins are key factors that influence the regenerative capacity of tissues. The objective of the present study was to evaluate the effects of enamel matrix derivative (EMD), TGF-β1, and the combination of both factors (EMD+TGF-β1) on human osteoblastic cell cultures. Methods Cells were obtained from alveolar bone of three adult patients using enzymatic digestion. Effects of EMD, TGF-β1, or a combination of both were analyzed on cell proliferation, bone sialoprotein (BSP), osteopontin (OPN) and alkaline phosphatase (ALP) immunodetection, total protein synthesis, ALP activity and bone-like nodule formation. Results All treatments significantly increased cell proliferation compared to the control group at 24 h and 4 days. At day 7, EMD group showed higher cell proliferation compared to TGF-β1, EMD + TGF-β1 and the control group. OPN was detected in the majority of the cells for all groups, whereas fluorescence intensities for ALP labeling were greater in the control than in treated groups; BSP was not detected in all groups. All treatments decreased ALP levels at 7 and 14 days and bone-like nodule formation at 21 days compared to the control group. Conclusions The exposure of human osteoblastic cells to EMD, TGF-β1 and the combination of factors in vitro supports the development of a less differentiated phenotype, with enhanced proliferative activity and total cell number, and reduced ALP activity levels and matrix mineralization.

The authors thank Mr. Roger R. Fernandes and Ms. Junia Ramos, from Cell Culture Laboratory, School of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil, for their helpful technical assistance, and Luciana Prado Maia, from the Department of Oral and Maxillofacial Surgery and Periodontology, School of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil, for the contribution in the manuscript preparation. The mouse monoclonal anti-human bone ALP antibody (B4-78), developed by Jerry A. Katzmann, and anti-rat osteopontin (MPIIIB10-1) and bone sialoprotein (WVID1-9C5) antibodies, developed by Michael Solursh and Ahnders Franzen, were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the Department of Biological Sciences of the University of Iowa (Iowa City, IA 52242).