Epidermal growth factor and bone morphogenetic proteins upregulate osteoblast proliferation and osteoblastic markers and inhibit bone nodule formation

Objective: The aim of this study was to investigate the in vitro osteogenic activity of EGF in association with bone morphogenetic proteins BMP2 and BMP7. Methods: SaOS-2 (osteoblast-like cell line from human osteosarcoma) were cultured in the presence of EGF and BMPs for various culture periods to assess (a) cell proliferation by MTT assay, (b) Runx2, alkaline phosphatase (ALP) and osteocalcin (OC) mRNA expression using quantitative RT-PCR and ELISA, and (c) bone tissue mineralization using Alizarin Red staining. Results: EGF alone was able to stimulate osteoblast growth in a time-dependent manner. When mixed with BMP2, BMP7, and their combination, EGF greatly promoted osteoblast growth, compared to the BMP- and EGF-stimulated cells, suggesting a possible synergistic effect between EGF and BMPs on osteoblast growth. Stimulation with EGF, EGF/BMP2, and EGF/BMP2/BMP7 for 7 days upregulated Runx2 mRNA expression by the osteoblasts. EGF downregulated ALP mRNA expression, which was recovered when the BMP2/BMP7 combination was added to the osteoblast culture. Tested on DC mRNA expression, EGF had no effect and inhibited the enhancing effect of BMP2 and BMP7 on osteocalcin expression. The bone mineralization assay showed that EGF reduced both the number and size of the bone nodules. This reducing effect was observable even in the presence of BMP2 and BMP7. Conclusion: This study demonstrated that EGF may act in the early phase to promote osteoblast growth and specific marker expression rather than the late phase involving cell differentiation/mineralization. (C) 2010 Elsevier Ltd. All rights reserved.