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Published on: May 13, 2014
[Primary culture and identification of neonatal rat's mandibular osteoblasts with modified repeating enzymatic
Ling-Ling E1, Hong-Chen Liu, Dong-Sheng Wang
1Stomatologic Institute, General Hospital of Chinese PLA, Beijing 100853, China.
Objective:
To investigate an efficient method of primary mandibular osteoblasts (MO) culture.
Methods:
The mandible harvested from 24 h SD rats was stripped off all soft tissues including the periosteum, rinsed and cut to trivial bone block under sterile condition. Then the bone block was subcultured in culture flask after digested with modified repeating enzymatic digestion-adherent explants method. The proliferation of the acquired cells were examined with assay of methyl thiazolyl tetrazolium (MTT) and identified with invert microscope, immunohistochemistry stain of osteocalcin, alkaline phosphorase (ALP) stain and stain of calcified nodules.
Results:
MTT assay showed that the cells grew slowly in 1-3 days of post-inoculation, it was cell growth adaptation period. At seventh day, the cells growth reached highest, then the proliferation of the cells was slow gradually. The cells were identified to be osteoblasts by invert microscope, immunohistochemistry stain of osteocalcin, ALP stain and alizarin bordeaux stain of calcified nodule.
Conclusion:
The modified repeating enzymatic digestion-adherent explants method is an ideal method to obtain and culture neonatal rat's MO having typical characteristics.
