Fluid shear stress induces less calcium response in a single primary osteocyte than in a single osteoblast: Implication of different focal adhesion formation
Fluid shear stress induces less calcium response in a single primary osteocyte than in a single osteoblast: Implication of different focal adhesion formation
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DOI：
10.1359/jbmr.060408
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发表时间：
2006-07-01
影响因子：
6.2
通讯作者：
Takano-Yamamoto, Teruko
中科院分区：
文献类型：
--
作者：
Kamioka, Hiroshi;Sugawara, Yasuyo;Takano-Yamamoto, Teruko
引言：为了探索骨细胞群对机械应力的即时反应，我们研究了流动诱导的钙瞬变。此外，参与局灶性粘附相关的钙瞬变响应于流体流量在cells.Materials和方法：骨细胞分离从16日龄的鸡胚颅盖骨通过系列处理与EDTA和胶原酶。将没有细胞间连接的玻璃上的单细胞置于流体流动中，并使用fluo-3成像测量细胞内钙浓度。用鸡骨细胞特异性抗体OB7.3和碱性磷酸酶底物ELF-97对同一视野中的细胞群进行鉴定，随后用于成骨细胞鉴定。结果：在1.2 Pa的流体剪切力作用下，骨细胞、成骨细胞和OB7.3/ELF-97阴性细胞对流体剪切力的反应率分别为5.5%、32.4%和45.6%。此外，成骨细胞和OB7.3/ELF-97阴性细胞对2.4 Pa的反应比对1.2 Pa的反应更大，而骨细胞的反应则较低。钙瞬变相对于基线的升高在人群中没有显示出任何显着差异。为了阐明单个骨细胞对高达2.4 Pa的流体剪切应力比成骨细胞更不敏感的机制，我们研究了粘着斑相关的钙瞬变。首先，我们比较了骨细胞和成骨细胞之间的粘着斑形成，发现成骨细胞中的粘着斑数量多于骨细胞。接下来，当细胞用GRGDS（0.5 mM）预处理后再进行流动处理时，观察到成骨细胞中的钙瞬变显著减少（18%），而骨细胞中的钙瞬变没有被GRGDS改变。对照肽GRGES不降低任一细胞类型中的钙瞬变。此外，我们证实，成骨细胞在颅骨显示了一个显着的形成黏着斑在周边的细胞。然而，在颅骨骨细胞表现出微弱的黏着斑斑块仅在基地的processes.Conclusions：在玻璃上，单个骨细胞是不太敏感的流体剪切应力高达2.4 Pa的成骨细胞。钙瞬变的差异可能与粘着斑形成的差异有关。较高的剪切应力或直接变形可能是骨中骨细胞力学响应的原因。
Introduction: To explore the immediate response to mechanical stress in a bone cell population, we examined flow-induced calcium transients. In addition, the involvement of focal adhesion-related calcium transients in response to fluid flow in the cells was studied.Materials and Methods: Bone cells were isolated from 16-day-old embryonic chicken calvaria by serial treatment with EDTA and collagenase. Single cells on glass without intercellular connections were subjected to fluid flow, and intracellular calcium concentration was measured using imaging with fluo-3. The identification of cell populations in the same field was performed with a chick osteocyte-specific antibody, OB7.3, and an alkaline phosphatase substrate, ELF-97, for osteoblast identification afterward. Immunofluorescence staining of vinculin was performed to visualize focal adhesions.Results: The percentage of cells responding to fluid shear stress at 1.2 Pa was 5.5% in osteocytes, 32.4% in osteoblasts, and 45.6% in OB7.3/ELF-97-negative cells. Furthermore, osteoblasts and OB7.3/ELF-97-negative cells were more responsive to 2.4 Pa than 1.2 Pa, whereas osteocytes were less responsive. The elevation of calcium transients over baseline did not show any significant differences in the populations. To elucidate the mechanism accounting for the fact that single osteocytes are less sensitive to fluid shear stress of up to 2.4 Pa than osteoblasts, we studied focal adhesion-related calcium transients. First, we compared focal adhesion formation between osteocytes and osteoblasts and found a larger number of focal adhesions in osteoblasts than in osteocytes. Next, when the cells were pretreated with GRGDS (0.5 mM) before flow treatment, a significant reduction of calcium transients in osteoblasts (18%) was observed, whereas calcium transients in osteocytes were not changed by GRGDS. Control peptide GRGES did not reduce the calcium transients in either cell type. Furthermore, we confirmed that osteoblasts in calvaria showed a marked formation of vinculin plaques in the periphery of the cells. However, osteocytes in calvaria showed faint vinculin plaques only at the base of the processes.Conclusions: On glass, single osteocytes are less sensitive to fluid shear stress up to 2.4 Pa than osteoblasts. The difference in calcium transients might be related to differences in focal adhesion formation. Shear stress of a higher magnitude or direct deformation may be responsible for the mechanical response of osteocytes in bone.