Quantitative analysis of the in vitro mineralization of preosteoblasts after exposure to ionizing radiation under standard and osteogenic conditions

Abstract

Transforming growth factor-beta 1 (TGF-β1) is an important mediator of cell growth, but conflicting results have been reported regarding its effect on osteoblastic cell differentiation. Our previous study on calvaria-derived osteogenic primary cultures had demonstrated that TGF-β1 inhibited the differentiation of osteoblastic cells; they revealed typical fibroblasts morphology with no extracellular matrix formation and therefore with few collagen fibrils. This in vitro study aimed to evaluate the effects of TGF-β1 (5 ng/μL), with different supplementation time periods (7, 10 and 14 days) on the establishment of osteogenic phenotype of MC3T3-E1 line cell. For comparison, ascorbic acid (AA; 5 μg/mL)/β-glycerophosphate (βGP; 10 mM), dexamethasone (Dex; 10−7 M) and TGF-β1/Dex were also added. We have performed quantitative PCR and Western blotting to evaluate the alkaline phosphatase (ALP), type I collagen (COL I) and osteocalcin (OC) expression after these treatments. The results have shown that ALP, COL I and OC expression was enhanced by AA/βGP treatment. In contrast, TGF-β1 alone or supplemented with Dex decreased the expression of these proteins in all studied period times. The present results demonstrated that the TGF-β1 pathway may interfere on the biomineralization and differentiation of osteoblastic cells by reducing the expression of key extracellular matrix proteins on bone development. Grants are from FAPESP 10/00352-6 and 10/