Isolation, culture and identification of osteoblasts from neonatal rabbit calvarium

doi:10.3969/j.issn.2095-4344.0576

Abstract

Abstract:

BACKGROUND: A publicly accepted way to isolate osteoblasts with high purity and efficiency has not been found.
OBJECTIVE: To explore the efficacy of advanced enzymatic digestion method to isolate and culture osteoblasts from calvarial bone of neonatal New Zealand rabbit in vitro.
METHODS: Improved enzymatic digestion method was used to isolate osteoblasts from calvarium of 3-day-old New Zealand rabbits and cells were purified via differential attachment methods. The morphology and growth of osteoblasts were observed by inverted microscope daily. Cell proliferation was detected by MTT assay and the growth curve was drawn. Alkaline phosphatase activity was measured. The expression levels of collagen type I, osteocalcin and Runt related transcription factor 2 were detected by immunohistochemical staining. Alizarin red staining was carried out to testify mineralized matrix productivity.
RESULTS AND CONCLUSION: Osteoblasts were successfully isolated, cultured and purified by enzyme digestion. The isolated osteoblasts adhered on the wall possessing typical characteristics of osteoblasts with good proliferation ability. Alkaline phosphatase staining and immunohistochemical staining on collagen type I, osteocalcin and Runt related transcription factor 2 presented positive results. Alizarin red staining showed mineralized nodules with different sizes after 21 days of culture. These findings suggest that the osteoblasts cultured by improved enzymatic digestion hold the typical characteristics of osteoblasts, with high purity and survival rate.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Skull, Osteoblasts, Cell Separation, Tissue Engineering

CLC Number:

R446
R318

Ailimaierdan•Ainiwaer1, Li Peng1, Diao Zhaofeng1, Muhetaer•Huojia2. Isolation, culture and identification of osteoblasts from neonatal rabbit calvarium[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(7): 996-1000.

Figures/Tables 1

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