BACKGROUND: Tissue engineering requires a lot of seed cells. Osteoblasts have become important seed cells in bone tissue engineering. However, it is difficult to culture the osteoblasts, and cell number, purity, proliferation and differentiation activity are different obtained by different culture methods.
OBJECTIVE: To identify and compare three common primary osteoblat culture methods, and to explore a method for the primary culture of osteoblasts which is easy to operate, economical and effective, in order to provide basis for the further experimental research.
METHODS: Calvarias were dissected from newborn Sprague Dawley rats in 72 hours, and osteoblasts were isolated with collagenase digestion method, sequential digestion method and bone tissue method respectively. The morphological observation and cytochemical staining were performed, the growth curve of the cells was drawn with Cell Counting Kit-8 method, and the rate of living osteobalsts was counted with trypan blue staining.
RESULTS AND CONCLUSION: The proliferation of the insolated and cultured osteoblasts was well with typical characteristics of osteoblasts, cytochemical staining results were positive. Compared with the sequential collagenase digestion method, the collagenase digestion method presented higher production of osteoblasts and higher cell survival rate (P < 0.05), and the collagenase digestion method was easier than the sequential collagenase digestion method and cost less than sequential collagenase digestion method. Bone tissue method was the easiest method with less damage to cells, but bone tissue method presented lower production of osteoblasts and cost much more time, which cannot be used in large-scale osteoblast culture. The collagenase digestion method is a simple, efficient and ideal method for isolation and culture of primary osteoblasts.