Osteocytes form a three-dimensional (3D) cellular network within the mineralized bone matrix. The cellular network has important roles in mechanosensation and mechanotransduction related to bone homeostasis. We visualized the embedded osteocyte network in chick calvariae and observed the flow-induced Ca signaling in osteocytes using 3D time-lapse imaging. In response to the flow, intracellular Ca ([Ca]) significantly increased in developmentally mature osteocytes in comparison with young osteocytes in the bone matrix. To investigate the differences in response between young and developmentally mature osteocytes in detail, we evaluated the expression of osteocyte-related genes using the osteocyte-like cell line MLO-Y4, which was 3D-cultured within type I collagen gels. We found that the c-Fos, Cx43, Panx3, Col1a1, and OCN mRNA levels significantly increased on day 15 in comparison with day 7. These findings indicate that developmentally mature osteocytes are more responsive to mechanical stress than young osteocytes and have important functions in bone formation and remodeling.
Osteocytes differentiated from osteoblasts play significant roles as mechanosensors in modulating the bone remodeling process. While the well-aligned osteocyte network along the trabeculae with slender cell processes perpendicular to the trabeculae surface is known to facilitate the sensing of mechanical stimuli by cells and the intracellular communication in the bone matrix, the mechanisms underlying osteocyte network formation remains unclear. Here, we developed a novel in vitro collagen matrix system exerting a uniaxially-fixed mechanical boundary condition on which mouse osteoblast-like MC3T3-E1 cells were subcultured, evoking cellular alignment along the uniaxial boundary condition. Using a myosin II inhibitor, blebbistatin, we showed that the intracellular tension via contraction of actin fibers contributed to the cellular alignment under the influence of isometric matrix condition along the uniaxially-fixed mechanical boundary condition. Furthermore, the cells actively migrated inside the collagen matrix and promoted the expression of osteoblast and osteocyte genes with their orientations aligned along the uniaxially-fixed boundary condition. Collectively, our results suggest that the intracellular tension of osteoblasts under a uniaxially-fixed mechanical boundary condition is one of the factors that determines the osteocyte alignment inside the bone matrix.
Actin polymerization-driven protrusion of the lamellipodia is a requisite initial step during actin-based cell migration, and is closely associated with attachment to the substrate. Although tremendous progress has been made in recent years toward elucidating the molecular details of focal adhesions, our understanding of the basic coordination of protrusion and adhesion, and how the two fundamental processes relate to actomyosin contractility is still inadequate. Therefore, to highlight the effect of cell-substrate interactions on the protrusive dynamics of the lamellipodia and to correlate protrusion with actomyosin activities, this study investigated the migration of fish epidermal keratocytes on fibronectin micropatterns intercalated with adhesionsuppressed gaps of varying widths. We show that insufficient adhesion associated with the gaps could limit lamellipodial protrusion such that the percentage of migrating cells decreases with an increase in gap width, and protrusion across the gaps is accompanied by ruffling. Moreover, our results suggest that up-regulating actomyosin contractility enhances the mechanical integrity of the actin cytoskeleton, leading to an increase in the width of the lamellipodia, and consequently, an increase in the percentage of cells migrating across the gaps. Thus, we demonstrate that the protrusion dynamics at the leading edge of migrating cells are functionally involved in the global mechanical regulation of actin cytoskeletal components that enable cell migration.
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