Culture and Behavior of Osteoblastic Cells Isolated from Young Growing Rat Long Bone.

Objective To establish osteoblasstsic cell isolaton and in vitro culturing method from young growing rat long bone. Methods Tibiae and femora from 10 week old Wistar rat were isolated and subjected to sequential digestions in 0.25% trypsin and 0.1%collagenase.Harvested cells were cultured in a DME:F 12 1∶1 medium supplemented with 10% fetal calf serum,in which the cell growth and proliferation were observed.In addition,the studies utilized established protocols for the characterization of these cells as osteoblasts by means of alkaline phosphatase activity,Van Gieson and von Kossa staining. Results ①Cells released from young growing rat long bone had the general morphology of osteoblasts and undergo proliferation and differentiation.The population doubling time was 78 hr.②Most of the cells were alkaline phosphatase positive.There was a little elevated positive rate after passage.③The cells retained the ability to produce and synthesize collagen.④When the cell layers were supplemented with both 50μ g/ml ascorbic acid and 10 mM beta glycerophosphate and allow to grow past confluency for 3 weeks,they formed calcified nodules. Conclusions Osteoblastic cells derived from young growing rat long bone by enzymatic digestion exhibited some biologic behaviors of osteoblasts.This simple isolation technique now facilitated the availability of young growing rat osteoblastic cells for further investigation of bone growth regulation,metabolism and biology.