[A new method of isolating and culturing rabbit osteoblasts in vitro]

To establish an experimental model of osteoblasts to easily cause calcification of bone matrix in vitro, we took cranium of a newborn rabbit out under an aseptic condition, removed the connective tissue of the bony suture, and cut the cranium freely into the fragments of not more than 1 mm2. The we isolated and cultured the osteoblasts using tissue explant method. We observed growth status of primary osteoblasts and subcultured osteoblasts using inverted microscope. Then we conducted enzyme staining and alizarin red staining for the third generation of osteoblasts to detect the alkaline phosphatase (ALP) expression and calcified nodules. The result showed that there were calcified nodules or calcification formed after the primary osteoblasts climbing out from the bone for 1 week, and each generation of osteoblasts had the similar calcification with the primary osteoblasts, and there was an increase in calcified nodules after the continuous culture. There was a strong expression of ALP in the plasma membrane of osteoblasts. The calcified nodules were red with alizarin red staining. It is well concluded that osteoblasts isolated with this method easily cause calcification, and can be used as a new experimental model.