Competition of osteoblasts and fibroblasts at early stages of osseointegration

Objective:  The formation mechanisms of osseointegration for titanium (Ti) implants are still unclear. To elucidate the early stage of osseointegration, we developed a new body-simulated model to co-culture osteoblasts and fibroblasts on different materials. Method:  Two cell lines, namely fibroblasts NIH3T3 and osteoblast-like cells MC3T3-E1, in which respectively GFP and DsRed expression vectors were introduced to produce the permanent expressing clones Fib^Green and Ost^Red, were seeded together on glass disks that were beforehand vapor-deposited with a thin layer of respectively Ti, gold (Au), aluminum oxide (Al₂O₃) and zirconium oxide (ZrO₂), and cultured under static and shear-stress (shaking) conditions. In order to study which matrix molecules affect the competition for adhesion of both cells to the 4 substrates under shear-stress conditions, we also coated cell-culture plates with matrix molecules such as fibronectin, collagen Type 1, collagen Type 4, laminin, vitronectin, and osteopontin, and estimated the number of both cells types cultured. Result:  At an early stage (30 min after cell seeding), Ost^Red clearly attached more onto Ti than onto other substrates in a static culture. At 6 hrs after cell seeding, no difference in attachment was found between both cells onto Ti, while Fib^Green attached more than Ost^Red onto Au, Al₂O₃, and ZrO₂. Under shear-stress conditions, Ost^Red attached more than Fib^Green onto Ti at 6 hrs after cell seeding, but not onto the other substrates. The number of Ost^Red was significantly higher than Fib^Green in the presence of collagen Type I. Two weeks after seeding, Ost^Red was found to have differentiated to alkaline phosphatase-positive cells. After four weeks, deposition of calcium could be confirmed by Alizarin Red S staining. Conclusion:  Osteoblasts preferentially attached onto Ti under shear-stress conditions. Both the competition between fibroblasts and osteoblasts for adhesion and the shear-stress conditions were identified as factors involved in the early osseointegration process.