Primary culture and identification of neonatal rabbit osteoblasts: modified tryptase and collagenase sequential digestion

doi:10.3969/j.issn.2095-4344.2014.38.011

Abstract

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 skull of neonatal rabbit.
METHODS: Calvarias were dissected from newborn New Zealand white rabbits within 24 hours, and osteoblasts were isolated with bone tissue method, collagenase digestion method and modified tryptase and collagenase 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 cell culture supernatants. Collagen I and collagen III immunohistochemical staining was also performed. RT-PCR was used to determine the expression of osteocalcin and collagen 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 collagen I immunohistochemical staining were both positive, while collagen III immunohistochemical staining was negative.
Alkaline phosphatase and osteocalcin protein expression in the cell culture supernatants can be detected. The expression of osteocalcin and collagen I mRNA was positive in the RT-PCR test. Compared with collagenase digestion method, the modified tryptase and collagenase I sequential digestion method cost less time, presented higher production of osteoblasts and higher cell survival 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 collagenase I sequential digestion method, which can be used as a reliable and efficient way to obtain the original generation osteoblasts in vitro.

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

全文链接：

Key words: osteoblasts, alkaline phosphatase, osteocalcin

CLC Number:

R318

Yang Sen, Feng Fu-ming, Wang Yin-hui. Primary culture and identification of neonatal rabbit osteoblasts: modified tryptase and collagenase sequential digestion[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(38): 6129-6135.

Figures/Tables 4

2.1 成骨细胞形态学观察结果组织块法第5天细胞从骨片边缘爬出，呈长梭形或多边形并有突起(图1A)；胶原酶消化法分离所得的原代细胞接种5 d后细胞贴壁生长，呈长梭形、三角形或鳞片状，有两三个突起，多为单核(图1B)；改良胶原酶和胰酶分段消化法第5天细胞呈长梭形或鳞片状有两三个突起，体积较大，胞浆丰富(图1C)。3种方法所得细胞均具有成骨细胞特征。 2.2 成骨细胞苏木精-伊红染色结果原代成骨细胞传代至第3代苏木精-伊红染色，见细胞轮廓清晰，细胞呈梭形或星状，有多个突起，胞浆丰富，细胞核为单个蓝色，胞浆为红色(图2A)。 2.3 成骨细胞茜素红染色结果成骨细胞原代培养14 d后，可见细胞汇合处呈现多层重叠生长，形成不透明的钙化区，茜素红染色后，显微镜下可见大块被染成橘红色的钙化结节(图2B)。 2.4 成骨细胞增殖能力MTT测定结果接种后1-3 d细胞生长缓慢为适应期，3-6 d生长较快，为对数生长期，第7-9天生长变缓进入平台期，采用SPSS 13.0统计软件绘制细胞的生长曲线(图3A)。 2.5 碱性磷酸酶测定结果原代成骨细胞连续培养14 d，随培养时间增加，碱性磷酸酶数值先增大后降低，表示成骨细胞分泌了碱性磷酸酶，随着成骨细胞矿化成熟，碱性磷酸酶分泌减少(图3B)。 2.6 骨钙素测定结果原代成骨细胞连续培养14 d，随着培养时间的增加，细胞上清液中骨钙素含量也增加(图4A)。 2.7 Ⅰ型胶原和Ⅲ型胶原免疫组织化学染色结果结果判定参考细胞培养学[6]，Ⅰ型胶原蛋白免疫组织化学染色阳性，细胞核周围呈黄褐色着色(图4B)；阳性细胞比例达90%以上，Ⅲ型胶原蛋白免疫组织化学染色阴性(图4C)，成骨细胞偶呈淡棕色，表明成骨细胞产生少量Ⅲ型胶原。 2.8 RT-PCR检测成骨细胞Ⅰ型胶原、骨钙素、GADPH mRNA表达结果骨钙素、Ⅰ型胶原PCR凝胶电泳显示有目的基因，骨钙素、Ⅰ型胶原mRNA表达，扩增产物长度骨钙素为310 bp，Ⅰ型胶原为259 bp，内参GADPH为177 bp(图5)。 2.9 原代成骨细胞3种培养方法的比较结果改良酶消化法与胶原酶消化法比较，分离成骨细胞密度高、纯度好、消化时间短、细胞存活率高；与组织块消化法相比，分离成骨细胞数量多、耗时短，适于成骨细胞大规模培养(表1)。"

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