Department of Biomedical Engineering, The City College of New York, USA.
Bone. 2010 Mar;46(3):577-83. doi: 10.1016/j.bone.2009.11.006. Epub 2009 Nov 17.
Osteocyte apoptosis has been linked to bone resorption resulting from estrogen depletion and other resorptive stimuli; however, precise spatial and temporal relationships between the two events have not been clearly established. The purpose of this study was to characterize the patterns of osteocyte apoptosis in relation to bone resorption following ovariectomy to test whether osteocyte apoptosis occurs preferentially in areas known to activate resorption. Moreover, we report that osteocyte apoptosis is necessary to initiate endocortical remodeling in response to estrogen withdrawal.
Adult female C57BL/6J mice (17 weeks old) underwent either bilateral ovariectomy (OVX), or sham surgery (SHAM) and were euthanized on days 3, 7, 14, or 21 days after OVX. Diaphyseal cross-sections were stained by immunohistochemistry for activated caspase-3 as a marker of apoptosis. The percentages of caspase-positive stained osteocytes (Casp+Ot.) were measured along major and minor anatomical axes around the femoral diaphysis to evaluate the distribution of osteocyte apoptosis after estrogen loss; resorption surface was measured at the adjacent endocortical regions. In a second study to test whether osteocyte apoptosis plays a regulatory role in the initiation of bone resorption, a group of OVX mice received the pan-caspase inhibitor, QVDOPh, to inhibit osteocyte apoptosis. Remaining experimental and sham groups received either QVD or Vehicle.
OVX increased osteocyte apoptosis in a non-uniform distribution throughout the femoral diaphyses. Increases in Casp+osteocytes were predominantly located in the posterior diaphyseal cortex. Here, the number of apoptotic osteocytes 4- to 7-fold higher than sham controls (p<0.005) by day 3 post-OVX and remained elevated. Increases in resorption post-OVX also occurred along the posterior endocortical surface overlying the region of osteocyte apoptosis, but these increases occurred only at 14 and 21 days post-OVX (p<0.002) well after the increases in osteocyte apoptosis. Treatment with QVD in OVX animals suppressed osteocyte apoptosis, with levels in QVD-treated samples equivalent to baseline. Moreover, the increases in osteoclastic resorption normally observed after estrogen loss did not occur in OVX mice treated with QVD.
The results of this study demonstrate that osteocyte apoptosis following estrogen loss occur regionally, rather than uniformly throughout the cortex. We also showed that estrogen loss increased osteocyte apoptosis. Apoptotic osteocytes were overwhelmingly localized within the posterior cortical region, the location where endocortical resorption was subsequently activated in ovariectomized mice. Finally, the increases in osteoclastic resorption normally observed after estrogen withdrawal did not occur in the absence of osteocyte apoptosis indicating that this apoptosis is necessary to activate endocortical remodeling following estrogen loss.
骨细胞凋亡与雌激素耗竭和其他吸收性刺激引起的骨吸收有关；然而，这两个事件之间的确切时空关系尚未明确确定。本研究的目的是描述去卵巢后骨细胞凋亡与骨吸收之间的关系模式，以测试骨细胞凋亡是否优先发生在已知激活吸收的区域。此外，我们报告骨细胞凋亡是对雌激素撤退作出反应启动内皮层重塑所必需的。
成年雌性 C57BL/6J 小鼠（17 周龄）接受双侧卵巢切除术（OVX）或假手术（SHAM），并在 OVX 后 3、7、14 或 21 天处死。通过免疫组织化学用活化的半胱天冬酶-3（Caspase-3）作为凋亡的标志物对骨干的横切面进行染色。沿股骨骨干的主要和次要解剖轴测量 Caspase 阳性染色的骨细胞（Casp+Ot.）的百分比，以评估雌激素丧失后骨细胞凋亡的分布；在相邻的内皮层区域测量吸收表面。在第二项研究中，为了测试骨细胞凋亡是否在骨吸收的起始中发挥调节作用，一组 OVX 小鼠接受了泛半胱天冬酶抑制剂 QVDOPh 以抑制骨细胞凋亡。其余的实验和假手术组接受了 QVD 或载体。
OVX 增加了股骨骨干中不均匀分布的骨细胞凋亡。Casp+骨细胞的增加主要位于骨干后侧皮质。在这里，凋亡骨细胞的数量比假手术对照组高 4-7 倍（p<0.005），并且在 OVX 后 3 天仍然升高。OVX 后吸收的增加也发生在覆盖骨细胞凋亡区域的后侧内皮层表面，但这些增加仅在 OVX 后 14 和 21 天（p<0.002）发生，远早于骨细胞凋亡的增加。在 OVX 动物中用 QVD 治疗可抑制骨细胞凋亡，QVD 处理样本中的水平与基线相当。此外，在接受 QVD 治疗的 OVX 小鼠中，通常在雌激素丧失后观察到的破骨细胞吸收的增加并未发生。
本研究结果表明，雌激素丧失后骨细胞凋亡发生在局部，而不是皮质整体。我们还表明，雌激素缺失会增加骨细胞凋亡。凋亡的骨细胞绝大多数局限于后侧皮质区域，这是在去卵巢小鼠中随后激活内皮层吸收的位置。最后，在雌激素撤退后通常观察到的破骨细胞吸收的增加在没有骨细胞凋亡的情况下并未发生，表明这种凋亡是雌激素撤退后启动内皮层重塑所必需的。
