Establishment of the screening methods for bone-resorbing factors using in vitro osteoclast formation system.

我们已经开发了体外系统来检查成骨因子对破骨细胞骨吸收的顺序过程的影响；1)破骨细胞祖细胞增殖，2)破骨细胞前体向多核破骨细胞分化，3)功能活跃的破骨细胞形成窝。破骨细胞祖细胞增殖的评价我们建立了两步培养系统，以确定成骨因子对破骨细胞祖细胞增殖的影响。骨髓细胞首先在半固体甲基纤维素中培养，其中存在各种csf。然后分离骨髓细胞，并在1alpha， 25(OH)_2D_3存在下与成骨细胞共培养。共培养7 d后，对形成的破骨细胞数量进行评分。根据与成骨细胞共培养的骨髓细胞数量和形成的破骨细胞数量，我们能够估计骨髓细胞中存在的破骨细胞祖细胞的数量。通过这种方法，我们发现M-CSF是诱导破骨细胞祖细胞生长的最有效的生长因子。破骨细胞前体分化的评价我们已经报道了破骨细胞在小鼠骨髓培养和小鼠成骨细胞和脾细胞共培养中形成。利用这些培养物，我们建立了一个筛选系统来检测成骨因子对破骨细胞分化的影响。骨吸收因子如1alpha， 25(OH)_2D_3， PTH， PGE_2和IL-1同样刺激破骨细胞前体分化为功能活跃的破骨细胞。破骨细胞的骨吸收活性评价在塑料盘子上形成的破骨细胞几乎没有从盘子表面释放出来。相反，当在胶原蛋白凝胶包被的培养皿上进行共培养，然后用胶原酶处理时，大多数细胞很容易从培养皿中释放出来。密度梯度离心富集破骨细胞群体。利用富含破骨细胞的群体和牙本质切片，我们开发了一种简单的骨吸收测定系统。分离的破骨细胞在牙本质切片上培养，24小时内形成吸收坑。用图像分析仪定量测量了吸收坑的面积。利用该系统，我们发现降钙素和巴菲霉素A_1（一种空泡H +- atp酶抑制剂）强烈抑制离体破骨细胞形成窝。这些系统似乎有助于研究成骨因子在破骨细胞骨吸收中的作用机制。少

We have developed in vitro systems to examine the effects of osteotropic factors on the sequential process of osteoclastic bone resorption ; 1) proliferation of osteoclast progenitors, 2) differentiation of osteoclast precursors into multinucleated osteoclasts, and 3) pit formation by functionally active osteoclasts.1. Evaluation of proliferation of osteoclast progenitorsWe have developed a two-step culture system to determine the effect of osteotropic factors on proliferation of osteoclast progenitors. Bone marrow cells were first cultured in semisolid methylcellulose in the presence of various CSFs. Marrow cells were then isolated and further co-cultured with osteoblastic cells in the presence of 1alpha, 25(OH)_2D_3. After co-culture for 7 days, the number of osteoclasts formed were scored. On the basis of the number of marrow cells co-cultured with osteoblastic cells and that of osteoclasts formed, we were able to estimate the number of osteoclast progenitors present in marrow cell … More fractions. Using this method, we found that M-CSF was the most potent growth factor in inducing the growth of osteoclast progenitors.2. Evaluation of differentiation of osteoclast precursorsWe have reported that osteoclasts are formed in mouse marrow cultures and in co-cultures of mouse osteoblastic cells and spleen cells. Using these cultures, we have established a screening system for examining the effects of osteotropic factors on osteoclast differentiation. Bone-resorbing factors such as 1alpha, 25(OH)_2D_3, PTH, PGE_2 and IL-1 similarly stimulated differentiation of osteoclast precursors into functionally active osteoclasts.3. Evaluation of bone-resorbing activity of osteoclastsOsteoclasts formed on plastic dishes were hardly released from the dish surface. In contrast, when co-cultures were performed on collagen gel-coated dishes then treated with collagenase, most of the cells were easily released from the dishes. The osteoclast population was enriched by density gradient centrifugation. Using an osteoclast-enriched population and dentine slices, we have developed a simple bone resorption assay system. When isolated osteoclasts were cultured on dentine slices, they formed resorption pits within 24 hr. The area of resorption pits was quantitatively measured with an image analyzer. Using this system, we found that calcitonin and bafiromycin A_1 (an inhibitor of vacuolar H^+-ATPase) strongly inhibited pit formation by isolated osteoclasts.These systems appear to be useful for examining the mechanism of action of osteotropic factors in osteoclastic bone resorption. Less