Uniaxially fixed mechanical boundary condition elicits cellular alignment in collagen matrix with induction of osteogenesis

Abstract

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.

Osteoblast-Induced Collagen Alignment in a 3Din vitroBone Model

“…In this study we developed a human MSC-based 3D cell culture using uniaxially fixed fibrin matrices, in which osteogenic differentiation, collagen matrix development, and the collagen-cell interface were studied. In accordance with previous reports (Iordachescu et al, 2018; Kim et al, 2021; Matsumoto et al, 2007; Sasaki et al, 2010; Sasaki et al, 2015), we observed that the uniaxial fixation of the culture induced alignment of the cells and the deposited collagen matrix solely through the activity of osteoblasts and osteocyte-like cells. Using a 3D multiscale imaging approach, combining 3D array tomography SEM, live fluorescence imaging, and 3D FIB/SEM (Figure 5), we were able to reveal structural details of the resulting culture that allow us to propose that the alignment of the newly deposited collagen matrix derives from the migration of the osteogenic cells as detailed below.…”

Lateral bone ridge expansion and internal tissue replacement for vertebral body growth in Pacific bluefin tuna Thunnus orientalis

“…Previous reviews have hypothesized that osteoblasts, rather than osteocytes, are involved in mechanosensing in teleost acellular bone (Davesne et al, 2019; Shahar & Dean, 2013), and it is possible that osteoblasts in the tuna vertebrae function in mechanosensing despite the presence of osteocytes in the vertebrae. In addition, the regulation of osteoblast and osteocyte alignment was investigated using a culture system with controlled mechanical loading (Inaba et al, 2017; Kim et al, 2021). Further studies using these systems may help understand the processes and functions of osteoblast embedding.…”

Osteocyte Dysfunction in Joint Homeostasis and Osteoarthritis

“…Contractions of actin filaments of the cytoskeleton are believed to introduce intracellular tension in osteocytes. This allows the cell to sense the stress exerted by the extracellular collagen matrix [ 86 ]. Collagen orientation is in turn controlled by the dominant loading orientation, either longitudinally with tensile stress or transversely with compression [ 87 , 88 , 89 ].…”