Ho M L, Chang J K, Chuang L Y, Hsu H K, Wang G J
Department of Physiology, Kaohsiung Medical College, Taiwan, Republic of China.
Kaohsiung J Med Sci. 1999 May;15(5):248-55.
Primary osteoblast cultures, which reflect more phenotypic properties of normal osteoblasts than osteoblastic cell lines, can be used as an experimental tool for investigating the osteoblastic functions in vitro. Primary osteoblast cultures were obtained from the parietal bones of calvaria of fetal rats in this study. Differential characteristics of osteoblasts in our culture system were examined and fibroblast cultures were also tested for comparison. We tested the alkaline phosphatase (ALP) and von Kossa stains on osteoblast and fibroblast cultures to examine the expression of ALP and the subsequent matrix mineralization occurred at 2 and 3 weeks after cell confluence respectively. The results showed that osteoblast cultures revealed obvious positive stains of ALP and von Kossa, while fibroblast cultures revealed negative stains, suggesting the osteoblast culture system used in this study reflects the typical phenotypes of primary osteoblasts but not fibroblasts. We tested the ALP activities following various doses of PGE2 or ketorolac treatments in primary osteoblast and fibroblast cultures. The results showed that PGE2 and ketorolac stimulated intracellular ALP activities of osteoblasts in dose dependent fashions, while very low ALP activities were detected in either the control or agents treated cultures of fibroblast. These results suggest that PGE2 may be involved in osteoblastic differentiation and the stimulatory effect of ketorolac on osteoblastic ALP activity may not be PGE2 mediated. The responses of osteoblasts to both agents can be as the characteristics of primary osteoblast derived from rat calvaria.
原代成骨细胞培养物比成骨细胞系更能反映正常成骨细胞的表型特性，可作为体外研究成骨细胞功能的实验工具。在本研究中，原代成骨细胞培养物取自胎鼠颅骨的顶骨。我们检查了培养系统中骨细胞的差异特性，并对成纤维细胞培养物进行了测试以作比较。我们分别在细胞汇合后2周和3周，对成骨细胞和成纤维细胞培养物进行碱性磷酸酶（ALP）和冯·科萨染色，以检测ALP的表达及随后的基质矿化情况。结果显示，成骨细胞培养物呈现明显的ALP和冯·科萨阳性染色，而成纤维细胞培养物呈现阴性染色，这表明本研究中使用的成骨细胞培养系统反映的是原代成骨细胞而非成纤维细胞的典型表型。我们检测了不同剂量的前列腺素E2（PGE2）或酮咯酸处理后原代成骨细胞和成纤维细胞培养物中的ALP活性。结果显示，PGE2和酮咯酸以剂量依赖方式刺激成骨细胞的细胞内ALP活性，而在成纤维细胞的对照或药物处理培养物中检测到的ALP活性非常低。这些结果表明，PGE2可能参与成骨细胞分化，且酮咯酸对成骨细胞ALP活性的刺激作用可能不是由PGE2介导的。成骨细胞对这两种药物的反应可作为源自大鼠颅骨的原代成骨细胞的特征。
