The mechanism of cell locomotion of cultured osteoclasts in three-dimensions ; the observation by time-cinematography

分离ddy系小鼠破骨细胞和巨噬细胞，加或不加牙本质切片培养72小时。孵育后，用偶氮染色法检测其抗酒石酸酸性磷酸酶(TRACPase)和酸性磷酸酶(ACPase)活性。ACPasc阳性细胞吞噬印度墨水，但它们既不显示TRACPase活性，也不显示解吸活性。TRACPase阳性细胞在牙本质切片上形成陷窝，边缘皱缩，条带清晰。因此，吞噬细胞的ACPase阳性细胞可能是巨噬细胞，TRACPase阳性的细胞是破骨细胞。结果表明，在ddy小鼠培养72小时内，TRACPase活性仅针对破骨细胞，而不是巨噬细胞。光镜下观察到牙本质切片上有一个轮廓复杂的多核TRACPase阳性细胞，然后用半薄和超薄切片连续切片，观察其超微结构和三维结构。在透射电子显微镜下，该细胞具有与破骨细胞相似的结构，具有清晰的带状结构，但无褶皱边界。三维重建显示，该细胞轮廓非常平坦和复杂，细胞体延伸到牙本质切片的背面。这些结果提示该细胞可能是一种运动性破骨细胞。

Osteoclasts and macrophages of ddy strain mice were isolated, and then culturedwith or without dentine slices for 72 hours. After the incubation, tartrate-resistant acid phosphatase (TRACPase) and adic phosphateas (ACPase) activities were detected in them by the azo dye method. ACPasc-positive cells phagocytized indian inks, however, they showed neither TRACPase nor resosrptive activity. TRACPase-positive cells made lacunae on the dentine slices, and showed sthe ruffled border and clear zone. Therefore, ACPase-positive cells with phagocytosis were seemed to be macrophages, and TRACPase-positive cells were osteoclasts. These results suggested that TRACPase activity was specific for osteoclastic cells, not macrophages, in 72 hours culture of ddy mice.One multinucleated TRACPase-positive cell with comnplicated contours was observed on the dentine slice by light microscopy, and then this cell was serially sectioned by altermating semithin and ultrathin sections to observe its ultrastructure and three-dimensional sturcture. By transmission electron microscopy, this cell had the similar structure as those of osteoclasts, and possessed the clear zone-like structures, but showed no ruffled border. A three-dimensional reconstruction showed that this cell had very flat and complicated outlines, and extended its cell body to the back side of the dentine slice. These results suggested that this cell might be a locomoting osteoclast.