Primary culture and identification of neonatal rabbit osteoblasts:modified tryptase and collagenase sequential digestion
10.3969/j.issn.2095-4344.2014.38.011
- VernacularTitle:乳兔成骨细胞原代培养与鉴定：改良胶原酶与胰酶的分段消化
- Author:
Sen YANG
;
Fuming FENG
;
Yinhui WANG
- Publication Type:Journal Article
- Keywords:
osteoblasts;
alkaline phosphatase;
osteocalcin
- From:
Chinese Journal of Tissue Engineering Research
2014;(38):6129-6135
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND:There are many kinds of ways to obtain osteoblasts at present, but how to get high-purity osteoblasts in a easy and fast way has become a hot research.
OBJECTIVE:To explore a method to get massive and high purified osteoblasts effectively by comparing three common primary osteoblast culture methods, and to observe the biological characteristics of the osteoblasts from the skul of neonatal rabbit.
METHODCalvarias were dissected from newborn New Zealand white rabbits within 24 hours, and osteoblasts were isolated with bone tissue method, col agenase digestion method and modified tryptase and col agenase sequential digestion method respectively, then the cells were subcultured in vitro. Osteoblast proliferation and osteogenic activity were identified by inverted microscope for morphology observation. The rate of living osteobalsts was counted with trypan blue staining. The growth curve of the cells was drawn with MTT method. Alizarin red staining was applied to detect alkaline phosphatase and osteocalcin protein in the cellculture supernatants. Col agen I and col agen III immunohistochemical staining was also performed. RT-PCR was used to determine the expression of osteocalcin and col agen I mRNA expression.
RESULTS AND CONCLUSION:The cultured cells showed highly homogeneous appearance with active proliferation, and they had the typical features of osteoblasts. Alizarin red staining and col agen I immunohistochemical staining were both positive, while col agen III immunohistochemical staining was negative. Alkaline phosphatase and osteocalcin protein expression in the cellculture supernatants can be detected. The expression of osteocalcin and col agen I mRNA was positive in the RT-PCR test. Compared with col agenase digestion method, the modified tryptase and col agenase I sequential digestion method cost less time, presented higher production of osteoblasts and higher cellsurvival rate (P<0.05). Bone tissue method was the easiest method and did the least damage to osteoblasts, but it presented lowest production of osteoblasts and cost the maximum time among the three methods. So it cannot be used in large-scale osteoblast culture. A large quantity of high purity osteoblasts were obtained by modified trypsase and col agenase I sequential digestion method, which can be used as a reliable and efficient way to obtain the original generation osteoblasts in vitro.
