Enhancing effects of basic fibroblast growth factor and fibronectin on osteoblast adhesion to bone scaffolds for bone tissue engineering through extracellular matrix-integrin pathway

The present study aimed to investigate the effects of basic fibroblast growth factor (bFGF) and fibronectin (FN) on adhesion of osteoblasts seeded into bio-derived bone scaffolds. Rat calvarial osteoblasts were separated and their osteogenic phenotypes were determined by staining for alkaline phosphatase as well as alizarin red staining. The bio-derived bone scaffolds were prepared from the metaphysis of porcine femur and their physicochemical properties were assessed by scanning electron microscopy (SEM) and X-ray diffraction analysis. An MTT assay was used to detect the effects of bFGF and FN on osteoblast adhesion or proliferation on cell/scaffold constructs through blocking the extracellular matrix FN-integrin pathway by the Gly-Arg-Gly-Asp-Ser (GRGDS) peptide. Western blot analysis was used to measure the beta 1 integrin levels. Based on the adhesion of osteoblasts stimulated by various concentrations of bFGF (0.1, 1, 10 and 100 ng/ml) on bio-derived bone scaffolds modified by various concentrations of FN (0.1, 1, 10 and 100 mu g/ml), the cell/scaffold constructs were divided into four groups: i) Control, non-stimulated and nonmodified group; ii) 10 mu g/ml FN-modified group; iii) 100 ng/ml bFGF-stimulated group; iv) 10 mu g/ml FN + 100 ng/ml bFGF group. Cell proliferation curves acquired by MTT assay and micrographs obtained by SEM showed that the combination of bFGF and FN significantly improved cell adhesion, particularly in the 10 mu g/ml FN + 100 ng/ml bFGF group vs. the other groups, and the effect on cell adhesion was inhibited by 1 mmol/l GRGDS peptide through the FN-integrin pathway. Western blot results showed that the combination of bFGF and FN significantly enhanced beta 1 integrin expression levels. These results suggested that osteoblasts stimulated by 100 ng/ml bFGF and bio-derived bone materials modified by 10 mu g/ml FN should be combined to be applied in the bone tissue engineering.