May L G, Gay C V
Department of Biochemistry, Pennsylvania State University, University Park 16802, USA.
In Vitro Cell Dev Biol Anim. 1996 May;32(5):269-78. doi: 10.1007/BF02723059.
Techniques for the isolation of a highly pure population of viable osteoclasts are limited. For this reason, we developed an isolation procedure that results in a high yield of osteoclast-like cells, up to 92% pure, from 3-wk-old chicken tibias. The unique feature of the method is the migration of cells from marrow-free endosteal surfaces to vitronectin-coated plates. The cells retain the osteoclast phenotype and remain viable in culture for a minimum of 1 wk. The cells were characterized and compared to two populations of authentic avian osteoclasts, which were isolated on the basis of association with fibronectin-coated plates. The cells contained substantial amounts of tartrate-resistant acid phosphatase. Alkaline phosphatase levels were negligible, suggesting little contamination by osteoblasts. Response to parathyroid hormone, dibutyryl cyclic adenosine monophosphate, calcitonin, acetazolamide, 17 beta-estradiol, and prostaglandin E2 was evident, as detected by measuring acid production. The vitronectin-associating cells contained numerous mitochondria, had the ability to resorb bone in an in vitro bone slice assay, and specifically bound biotinylated vitronectin. At 5 d of culture, the cells demonstrated marginal multinuclearity, having two to three nuclei. A large number (approximately 1 x 10(6) cells/tibia) of viable cells that exhibit characteristics of authentic osteoclasts can be obtained by the method described. Potentially, this method could be applied to other species.
分离高纯度活破骨细胞群体的技术有限。因此，我们开发了一种分离程序，可从3周龄鸡的胫骨中获得高产率的破骨细胞样细胞，纯度高达92%。该方法的独特之处在于细胞从无骨髓的骨内膜表面迁移到玻连蛋白包被的培养板上。这些细胞保留破骨细胞表型，并且在培养中至少存活1周。对这些细胞进行了表征，并与基于与纤连蛋白包被的培养板结合而分离的两个真正的禽类破骨细胞群体进行了比较。这些细胞含有大量抗酒石酸酸性磷酸酶。碱性磷酸酶水平可忽略不计，表明几乎没有成骨细胞污染。通过测量酸产生量检测到，这些细胞对甲状旁腺激素、二丁酰环磷酸腺苷、降钙素、乙酰唑胺、17β-雌二醇和前列腺素E2有明显反应。与玻连蛋白结合的细胞含有大量线粒体，在体外骨切片试验中有吸收骨的能力，并能特异性结合生物素化的玻连蛋白。在培养5天时，这些细胞表现出边缘多核性，有两到三个细胞核。通过所述方法可以获得大量（约1×10⁶个细胞/胫骨）具有真正破骨细胞特征的活细胞。该方法有可能应用于其他物种。
