Role of fibronectin during biological apatite crystal nucleation: ultrastructural characterization.

The role of adhesion molecules like osteopontin and bone sialoprotein, both containing the Arg-Gly-Asp sequence have been shown to have a role in mineral formation, whereas fibronectin (FN), another adhesive protein, was never studied during the mineralization processes. The formation and maturation of biological apatite crystals are under matrix control, and one of the roles of specific crystal proteins is to control the nucleation and growth of biological apatite during the mineralization process (promotion or inhibition). In the case of calcium phosphate ceramic used as a bone substitute, a dissolution-precipitation process occurs after implantation before the bone ingrowth and bone mineralization. The early precipitation consists of common biological apatite crystals. These crystals are the result of secondary nucleation and a heteroepitaxic growth process on synthetic residual crystals. In in vivo studies, hydroxyapatite crystals were implanted subcutaneously into mice for 1 or 2 weeks. Fibronectin immunogold labeling of the newly formed crystals on surfaces of high-resolution transmission electron microscopy sections of retrieved implants revealed the close association of these precipitated crystals with FN. In in vitro experiments using a solution of human FN incubated in the presence of calcium phosphate crystals, we obtained apatite crystal precipitation. The fibronectin network observed in high-resolution transmission electron microscopy showed numerous clusters of very small particles (1 nm in diameter and 2 nm in length), whereas the same experiment realized as control on albumin revealed no crystal precipitation. These results demonstrate for the first time the role of FN in early biological crystal nucleation. This process could have important biological significance in accounting for ectopic calcification, primary nucleation in calcified tissue, and bone ingrowth on calcium phosphate ceramics.