H 2O 2-derived free radicals treated fibronectin substratum reduces the bone nodule formation of rat calvarial osteoblast

Abstract

Fibronectin (FN) is involved in various cellular activities such as adhesion, proliferation and migration as a substratum. Since the metabolic turnover of FN is much slower than other cellular components, it may be affected by the oxygen free radicals produced in the aging process. However, the effect of oxygen free radicals on FN as substratum in bone formation has not been well characterized. The objective of this study was to examine the effect on the bone forming activity of osteoblasts using an oxygen free radical treated FN substratum in vitro (H 2O 2–Cu 2+system). SDS-PAGE, Western blotting and immuno-blotting analysis revealed that FN was degradated and/or modified by H 2O 2–Cu 2+ (·OH) treatment. Bone nodule formation per well was examined for total number, total area and area per nodule, which data were then compared between non-coated and FN-coated, and between FN-coated and ·OH treated FN-coated. Bone nodule formation in the FN-coated was significantly greater than in the non-coated. Furthermore, bone nodule formation in ·OH treated FN-coated was significantly less than that of FN-coated. These findings suggested that FN plays important roles in osteoblast activity and that FN substratum damaged by the oxygen free radicals produced by the aging process may cause decline of bone nodule formation through inhibition of the proliferation, differentiation and calcification processes.