Morphology of in vitro cultured osteoblasts derived from growing-rat long bone

Objective To establish a culture system for osteoblasts from long bone of growing rats and investigate its growth and ossification process in vitro . Methods The tibiae and femora from 10-week-old Wistar rats were isolated and subjected to sequential digestions for cell isolation. Harvested cells were cultured and passaged in a DME∶F-12 1∶1 medium supplemented with 10% fetal calf serum or in medium with 50 μg/ml ascorbic acid plus 10 mmol/L beta-glycerophosphate. Cell morphology, proliferation and calcification process was observed. Results ①Cells isolated from the rat long bone showed the general morphology of osteoblasts and had population doubling time of 78 h. ② The cells synthesized spherical-like and non-shaped matrices. ③ When the cells were cultured in calcification medium and allow to grow over confluence for 3 weeks, they formed calcified nodules. ④ Most of the cells demonstrated alkaline phosphatase positive, which closely related to matrix calcification. Conclusion Osteoblasts derived from growing rat long bone by enzyme digestion exhibit some biological behaviors of osteoblasts. This simple isolation method provides a valuable model for further study of children's bone growth and regulation.