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测成骨因子对破骨细胞骨吸收的顺序过程的影响; 1）破骨细胞祖细胞的增殖，2）破骨细胞前体分化成多核破骨细胞，和3）功能活性破骨细胞的窝形成。破骨细胞祖细胞增殖的评价我们建立了一个两步培养系统，以确定成骨因子对破骨细胞祖细胞增殖的影响。骨髓细胞首先在各种CSF存在下在半固体甲基纤维素中培养。然后分离骨髓细胞，并在1 α，25（OH）_2D_3存在下与成骨细胞进一步共培养。共培养7天后，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和破骨细胞数，我们可以估计骨髓细胞中存在的破骨细胞祖细胞数 ...更多信息 分数利用这种方法，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.破骨细胞分化的评价我们已经报道了破骨细胞在小鼠骨髓培养中以及在小鼠成骨细胞和脾细胞的共培养中形成。利用这些培养物，我们已经建立了一个筛选系统，检查成骨因子对破骨细胞分化的影响。骨吸收因子如1 α，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.破骨细胞骨吸收活性的评价在塑料培养皿上形成的破骨细胞几乎不从培养皿表面释放。相反，当在胶原凝胶包被的培养皿上进行共培养，然后用胶原酶处理时，大多数细胞容易从培养皿中释放。破骨细胞群通过密度梯度离心富集。使用破骨细胞富集的人口和牙本质切片，我们已经开发了一个简单的骨吸收测定系统。将分离的破骨细胞与牙本质片复合培养，24小时内形成吸收陷窝，用图像分析仪定量测量吸收陷窝的面积。利用该系统，我们发现降钙素和巴非罗霉素A_1（一种空泡H^+-ATP酶抑制剂）强烈抑制破骨细胞形成小窝，这一系统可用于研究骨细胞骨吸收过程中的成骨因子的作用机制。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