The Effects Of Basic Fibroblast Growth Factor And Fibronection On Osteoblast Adhesion On Bio-derived Bones And Its Mechanisms

ObjectiveTo investigate the effects of solo-use or co-use of basic fibroblastgrowth factor (bFGF) and fibronection (FN) on osteoblast adhesion onbio-derived bone; to explore the optimal combination of these twoextracellular matrix proteins for enhancing osteoblast adhesion andinvestigate the potential mechanism of the effects generated by these twoproteins.MethodsNeonate rat calvarial osteoblasts were cultured in vitro and theirosteogenic phenotypes were determined by alkaline phosphatase (ALP)staining and alizarin red staining method. The bio-derived bones wereprepared and their physico-chemical properties were measured. The thirdgeneration of osteoblasts were stimulated by various concentrations ofbFGF (0.1, 1, 10 and 100ng/ml) and the bio-derived bones were modifiedby various concentrations of FN (0.1, 1, 10 and 100μg/ml). Subsequently,the stimulated osteoblasts were seeded on the modified bio-derived bonesin turn. Cell adhesion was measured by methyl thiazolyl tetrazolium(MTT) assay. Cell density and appearance were observed by scanning electron microscope (SEM). According to the results of MTT assay, theoptimal combination group and control groups were chosen. Followingabove-mentioned procedure, 1mmol/L GRGDS peptide was used to blockRGD-integrinα₅β₁ adhesion pathway, then MTT assay was repeated.Whether bFGF stimulation of osteoblasts could affect the expression ofβ1 integrin subunit was detected by Western blot.ResultsCharacterizations of the osteogenic phenotype of cells cultured invitro revealed that both ALP staining and alizarin red staining werepositive. The prepared bio-derived bones reserved original bony saltstructures and natural three dimensional porous systems observed bySEM and their protein level was only 0.01±0.007% reflecting theycontained almost no proteins. MTT assay showed that both simplebFGF-stimulation of osteoblasts and simple FN-modification ofbio-derived bones could enhance osteoblast adhesion on bio-derivedbones (P＜0.05). However, the osteoblast adhesion was furtherstrengthened by the combined use of bFGF and FN. BFGF and FN had astatistic significant synergistic effect on osteoblast adhesion onbio-derived bones (P＜0.01). Among the various combinations,combination of 100ng/ml bFGF-stimulation of osteoblasts and 10μg/mlFN-modification of bio-derived bones had the biggest enhancement ofcell adhesion. Oppositely simple PLL-modification of bio-derived bones couldn't enhance osteoblast adhesion obviously(P＞0.05). PLL and bFGFhad no synergistic effect either (P＞0.05). The combined use of FN andbFGF had a better effect on cell density and appearance than the simpleuse when observed by SEM. The enhancement of cell adhesion generatedby the combined use of bFGF and FN was significantly blocked byapplication of GRGDS peptide. Expression of 131 integrin subunit inosteoblasts was enhanced obviously after bFGF stimulation.ConclusionsThe combined use of bFGF and FN has a significant synergisticeffect on osteoblast adhesion on bio-derived bones. When 100ng/mlbFGF-stimulated osteoblasts were seeded on the 10μg/ml FN-modifiedbio-derived bones, the biggest enhancement of cell adhesion could begenerated. The mechanism of the synergistic effect is probably mediatedby RGD-integrinα₅β₁ pathway.