Establish of neonatal rat osteoblasts and research of osteoblasts biological activies

Objective To culture a large number of pure osteoblasts with high survival rate,which would be a foundation of bone tissue engineering. The effects of osteoblast`s biology activity were observed with using incorporation of BMP- 2 and BMP- 7. Methods 5 days old SD rats received an aseptic surgery to get its calvaria bone. An improved second enzymatic digestion method were used to separate osteoblasts. To observe the osteoblast activity distribution,the inverted phase contrast microscope observation,alkaline phosphatase staining,mineralized nodule identification and micro- titration( MTT)method were used after a culture cycle. Cells were cultured with BMP- 2 and BMP- 7 respectively and bothly. After 48 h,MTT was used to assay cell proliferation,and the ALP activity and OCN content were determined from cell lysate. Results MTT method and morphological observation showed that 10 d was the strongest activity time after osteoblasts were cultured.The neonatal rat osteoblast proliferation was stimulated with using BMP- 2 and BMP- 7,so was the incorporation of BMP-2 and BMP- 7. osteoblast ALP activity and OCN contents were significantly increased after using BMP- 2 and BMP- 7 respectively and bothly. However,using BMP- 2 and BMP- 7 respectively are not significant. Treatment with BMP- 2 and BMP- 7HA resulted in signifcant improvement when compared to BMP- 2 and BMP- 7 respectively treatmented group( P 0.05). Conclusion These results suggest that the modified secondary enzymatic digestion can be able to access a large number of osteoblasts in a short time. The biological activity of osteoblasts was significantly enhanced with incorporation of BMP- 2 and BMP- 7.