Dept. of Orthopaedics and Rehabilitation, Yale University School of Medicine, 333 Cedar St., TMP 510, New Haven, 06510, USA (e-mail,
Cytotechnology. 2002 Sep;39(3):147-54. doi: 10.1023/A:1023936503105.
Substrate attachment is crucial for normal growth and differentiation of many cell types. To better understand the role of gravity in osteoblast attachment and growth in vitro, 17-day-old embryonic chick calvarial osteoblasts were subjected to directional variations with respect to gravity. Osteoblasts, grown in MEM or DME supplemented with 10% FBS and attached to type I collagen-coated coverslips, were loaded into cylindrical containers completely filled with medium and oriented so that cells were either atop or beneath, or coverslips continuously rotated ( approximately 2 rpm) in a clinostat, thereby continuously changing their orientation with respect to gravity. Cells in these three conditions were collected daily for up to 6 days, and cell viability, two osteoblast functions, and proliferation were assessed. Data suggest the number and function of attached osteoblasts is unaltered by inversion or clino-rotation in initially confluent cultures. In sparsely plated cultures, however, osteoblast viability was significantly decreased ( approximately 50%) in inverted and rotated cultures during the first 3 days of sampling, but from days 4-6 no significant difference was found in viable cell number for the three conditions. Decreases in viable cell number within the first days of the experiments could result from death followed by detachment, detachment followed by death, differences in proliferation rate, or lag-phase duration. To help distinguish among these, BrdU labeling for 2 or 24 hr was used to assess cell proliferation rate. Log-phase growth rates were calculated and were unchanged among the three conditions tested. These results point to an increase in lag-phase duration in inverted and rotated cultures. In summary, changing the cell-substrate attachment direction with respect to gravity causes an immediate response in the form of diminished viable osteoblast number in sparse, early cultures, but the effect disappears after 3-4 days and does not occur in mature, confluent cultures.
基质附着对于许多细胞类型的正常生长和分化至关重要。为了更好地理解重力在体外成骨细胞附着和生长中的作用，将 17 日龄鸡胚颅骨成骨细胞置于相对于重力的方向变化下。将生长在 MEM 或 DME 中补充有 10％FBS 并附着在 I 型胶原涂层盖玻片上的成骨细胞装入完全充满培养基的圆柱形容器中，并定向，使细胞位于顶部或底部，或在旋转器中连续旋转（约 2rpm），从而使其相对于重力的方向不断变化。在 6 天内每天收集这三种条件下的细胞，并评估细胞活力、两种成骨细胞功能和增殖。数据表明，在最初汇合的培养物中，反转或倾斜旋转不会改变附着的成骨细胞的数量和功能。然而，在稀疏接种的培养物中，在第 1 天至第 3 天的采样过程中，倒置和旋转培养物中成骨细胞的活力显着降低（约 50％），但在第 4 天至第 6 天，三种条件下的活细胞数没有发现显着差异。实验的最初几天内活细胞数的减少可能是由于死亡后脱落、脱落后死亡、增殖率差异或迟滞期持续时间不同所致。为了帮助区分这些，使用 BrdU 标记进行 2 或 24 小时以评估细胞增殖率。计算对数期生长速率，并且在三种测试条件之间不变。这些结果表明，在倒置和旋转培养物中，迟滞期持续时间增加。总之，相对于重力改变细胞-基质附着方向会导致稀疏、早期培养物中成骨细胞活力数量的立即响应，但在 3-4 天后这种影响消失，并且在成熟、汇合的培养物中不会发生。
