Divalent Cations Elevate Cytosolic Calcium of Chick Osteocytes

Journal of Bone and Mineral Research

Honjo

et al. 2008

Section: Methodsmentioning

confidence: 99%

Hormonal, pH, and Calcium Regulation of Connexin 43–Mediated Dye Transfer in Osteocytes in Chick Calvaria

Ishihara

Journal of Bone and Mineral Research

Honjo

et al. 2008

“…Bone cells were isolated from 16‐day‐old embryonic chicken calvaria using a modification of the technique described earlier (22) . After the periosteum had been stripped off, the calvaria were trimmed to remove noncalcified and marrow‐rich areas, as previously determined by histological analysis.…”

Section: Methodsmentioning

confidence: 99%

“…The successful isolation and maintenance of bone cells including osteocytes (22) facilitates the real‐time analysis of osteocytes and osteoblasts in isolated culture. In this study, we examined flow‐induced calcium transients in primary chick osteocytes and osteoblasts.…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, to examine the involvement of focal adhesion formation in osteocytes and osteoblasts in response to fluid shear stress, we used Arg-Gly-Aspcontaining peptides (GRGDS), which inhibit cell attachment through focal adhesions, and Arg-Gly-Glu-containing peptide, which is a control peptide for GRGDS. (21) The successful isolation and maintenance of bone cells including osteocytes (22) facilitates the real-time analysis of osteocytes and osteoblasts in isolated culture. In this study, we examined flow-induced calcium transients in primary chick osteocytes and osteoblasts.…”

Section: Introductionmentioning

confidence: 99%

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Fluid Shear Stress Induces Less Calcium Response in a Single Primary Osteocyte Than in a Single Osteoblast: Implication of Different Focal Adhesion Formation

Journal of Bone and Mineral Research

Sugawara

Murshid

et al. 2006

The immediate calcium response to fluid shear stress was compared between osteocytes and osteoblasts on glass using real-time calcium imaging. The osteoblasts were responsive to fluid shear stress of up to 2.4 Pa, whereas the osteocytes were not. The difference in flow-induced calcium may be related to differences in focal adhesion formation.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.

“…However, an intriguing method for the isolation of osteocytes with the chick osteocyte-specific antibody OB7.3, developed by Nijweide and Mulder [15], enabled us to clarify the nature of osteocytes as an active leading cell in bone tissue. We also modified the method and achieved the high-level purification of osteocyte populations through serial digestion with collagenase and EDTA treatment [31]. The successful isolation and maintenance of bone cells as well as identification of osteocytes facilitated the real-time analysis of osteocytes and other bone cells.…”

Section: Identification Of Osteocytes In Vitromentioning

confidence: 99%

Application of Bioimaging to Osteocyte Biology

Clinic Rev Bone Miner Metab

Adachi

2010

In a variety of scientific fields, it is helpful to visualize natural phenomena. Newly developed methods of visualization often lead to breakthroughs in scientific fields. Especially, in the bioscience field, it is significant to reveal temporal-spatial responses in cells while visualizing molecular phenomena. Such visualization may provide information to help understand cellular behavior in response to an extracellular stimulus. Although osteocytes are the most abundant cells in bone, it has been difficult to study their biological nature because they are embedded in hard bone tissue. So, the real 3D structure of osteocytes was not clarified until recently. On the other hand, a newly developed technique of visualization was recently introduced into bone cell biology. In this review, we introduce and review our application of a bioimaging technique to reveal the morphology of osteocytes as well as part of their biological nature.