In vitro assay of mineralized-tissue formation on titanium using fluorescent staining with calcein blue

Many studies have examined mineralized-tissue formation on titanium in vivo and in vitro; however, no precise method for measuring the mineralized tissue produced by cultured osteoblastic cells on titanium in vitro has been established. This study developed a method for measuring mineralized-tissue formation by cultured rat osteoblastic cells on titanium in vitro, and re-evaluated the effects of modifying the titanium surface. We used calcein blue, which binds to mineralized tissue, and measured the resulting fluorescence under ultraviolet light. A 1-h incubation with 3.1×10−3 calcein blue resulted in sufficient fluorescence of bone-like nodules on the titanium. Consequently, we found that osteoblastic cells produced larger bone-like nodules on titanium than in culture dishes. Fewer bone-like nodules formed on calcium-ion impregnated titanium than on pure titanium. However, when bisphosphonate was immobilized on the calcium-ion impregnated titanium, more bone-like nodules formed than on pure titanium. The results suggest that bisphosphonate immobilization on titanium is useful for stimulating mineralized-tissue formation on titanium implants.