Integrin-mediated binding of osteoblasts to titanium implant materials

Previous studies have shown that osteoblasts attach to orthopaedic implant materials in an integrin-dependent manner. The purpose of this study was to investigate how the integrin-mediated adhesion of osteoblasts to the orthopaedic implant material Ti6A14V (TIV) differs from integrin-mediated adhesion to extracellular matrix proteins. Intracellular signaling and gene expression leading to an increase in osteoblast proliferation and differentiation were investigated. Primary osteoblasts from fetal rat calvaria were plated onto TIV, fibronectin (FN, positive control) and poly-L-lysine (PLL, negative control) for various time points (20 minutes to 24 hours). The protein levels of the intracellular signaling molecules focal adhesion kinase (FAK) and mitogen activated protein kinase (MAPK) and the protein and mRNA levels of AP-1 transcription factors, c-fos and c-jun, were compared on the three substrates at various time points by Western and Northern blot analysis, respectively. Cells on all substrates showed continuous FAK phosphorylation for 24 h with a small decrease in activated FAK at 24 h in cells on PLL and TIV but not FN. Therefore FAK was activated by all substrates. However the subsequent signal transduction pathway differed on PLL compared to TIV and FN. MAPK was activated in a similar manner by osteoblast attachment to FN and TIV, although the response to TIV was delayed. Cells on PLL showed no MAPK activation. On FN and TIV c-fos and c-jun message levels were maximal at 30 min and declined at 120 min but remained elevated on PLL. C-fos protein was increased at 60 min on FN and at 30 min on TIV and remained elevated while c-fos protein in cells on PLL was negligible and undetectable at 120 min. C-jun protein levels were increased at 30 min on all three substrates, and only decreased on TIV at 120 min. Cjun protein expression on PLL was less than on the other substrates and slightly increased. The actin-based cytoskeleton of the cells was observed at various time points by phalloidin fluorescence microscopy. The cytoskeletal changes were delayed at 1 h and 4 h on TIV compared to FN but by 24 h a well spread cytoskeleton with focal adhesion sites was apparent on TIV and FN. Cells on PLL were rounded and cell spreading was minimal even at 24 h. Cell proliferation was studied by monitoring cell number on days 1, 2, 4 and 6. Cell number on FN and TIV increased significantly by 2 days and cells continued to proliferate through day 6 although initial attachment on TIV was about 50% less than on FN. Cells on PLL attached but did not proliferate. We conclude that osteoblast adhesion to TIV implants is similar to osteoblast adhesion to FN and leades to osteoblast proliferation. These data provide evidence for the biocompatibility of TIV at an intracellular level.