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 reevaluated 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 X 10(-3) M 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. (C) 2003 Elsevier Science Ltd. All rights reserved.