Abstract:

BACKGROUND: Periosteal osteoblasts possess strong reproductive activity, as well as osteoblastic differentiation potential, which is an ideal seed cell if can shorten the culture time.
OBJECTIVE: Modified enzymatic digestion was used to culture rabbits’ osteoblasts, and to study the adherence and proliferation of osteoblasts on the surface of sandblasting titanium.
METHODS: Periostea were harvested from the theanteromedial surface of the proximal tibia of male, Japanese white rabbits, and cultured as follow: ①Routine method: Digested with 0.25% trypsinase at 37 ℃ for 30 minutes, followed by digestion with 0.1% type I collagenase at 37 ℃ for 30 minutes, vibration, removed trypsinase and dried. After 2 hours, DMEM containing 15% fetal bovine serums were added. ②Modified method: 30 minutes culture of type I collagenase was prolonged to 1 hour. The osteoblasts were identified by alkaline phosphatase staining and calcium node staining. The adherence and proliferation of osteoblasts cultured on sandblasting surface were measured by scanning electron microscopy and MTT.
RESULTS AND CONCLUSION: Five days after culture, the periosteal osteoblasts crawled out from tissues, gathered as monolayer with triangle or polygon at after 25 days of modified culture. After 1 month of culture, superposition growth of calcium nodus appeared. The cultured cells possessed the morphological characteristic and biological behavior of osteoblasts, which were positive to alkaline phosphorase and calcium node staining. The time of cells cultured with routine method covered flask delayed 12 days than modified method. The osteoblasts were inseted into sandblasting titatium with pseudopodium. However, the adherence and proliferation of osteoblasts cultured on sandblasting surface had no obviously difference between two culture methods. The results suggested that modified enzymatic digestion can shorten the culture time without effect on adherence and proliferation of osteoblasts.