Development of titanium coated with apatite incorporating BMPs

Objective: To shorten the healing period after implantation, the titanium was coated with hydroxyapatite (HA). Bone morphogenic proteins (BMPs) can initiate the differentiation of mesenchymal cells into osteoblasts. In this study, we developed a new method for coating a titanium surface with HA incorporating bone morphogenic protein (BMP)-2. Methods: A pure titanium disc was treated in 5 N NaOH solution, and then heated at 600°C for 24 hours. The treated titanium was soaked in simulated body fluid (SBF) with or without recombinant human BMP-2 (rhBMP-2) for 3 days at 37°C. The surfaces of each specimen were examined using scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis. The existence of rhBMP-2 on the surface of the HA coating the titanium was examined using the immunogold electron microscopy method. MC3T3-E1 osteoblast-like cells were cultured on the surface of each specimen. The number and morphology of the adherent cells were observed 24 hours after cell seeding. Alkaline phosphatase activity was examined using p-nitrophenyl phosphate at 8 days after cell seeding. The expression levels of osteocalcin mRNA were examined at 2 weeks after cell seeding semi-quantitatively using RT-PCR. Results: HA was formed on the surface of alkaline-heat treated titanium after soaking in SBF for 4 days. The presence of rhBMP-2 was confirmed by the distribution of BMP-positive immunogold. The osteoblastic cells shrank on the HA surface with the incorporated rhBMP-2 compared to untreated titanium. The activity of alkaline phosphatase and the expression of osteocalcin mRNA were increased significantly in the cells on the rhBMP-2-incorporated HA titanium. Conclusions: These results demonstrate that rhBMP-2 was incorporated into HA on titanium without losing its activity using a method that combined alkaline-heat treatment with soaking in SBF with rhBMP-2.