Elsevier

Journal of Biomechanics

Volume 43, Issue 8, 28 May 2010, Pages 1560-1564
Journal of Biomechanics

Correlation of cell strain in single osteocytes with intracellular calcium, but not intracellular nitric oxide, in response to fluid flow

https://doi.org/10.1016/j.jbiomech.2010.01.030Get rights and content

Abstract

Osteocytes compose 90–95% of all bone cells and are the mechanosensors of bone. In this study, the strain experienced by individual osteocytes resulting from an applied fluid flow shear stress was quantified and correlated to two biological responses measured in real-time within the same individual osteocytes: (1) the upregulation of intracellular calcium and (2) changes in intracellular nitric oxide. Osteocyte-like MLO-Y4 cells were loaded with Fluo-4 AM and DAR-4M and exposed to uniform laminar fluid flow shear stresses of 2, 8, or 16 dyn/cm2. Intracellular calcium and nitric oxide changes were determined by measuring the difference in fluorescence intensity from the cell’s basal level prior to fluid flow and the level immediately following exposure. Individual cell strains were calculated using digital image correlation. MLO-Y4 cells showed a linear increase in cell strain, intracellular calcium concentration, and nitric oxide concentration with an increase in applied fluid flow rate. The increase in intracellular calcium was well correlated to the strain that each cell experienced. This study shows that osteocytes exposed to the same fluid flow experienced a range of individual strains and changes in intracellular calcium and nitric oxide concentrations, and the changes in intracellular calcium were correlated with cell strain. These results are among the first to establish a relationship between the strain experienced by osteocytes in response to fluid flow shear and a biological response at the single cell level. Mechanosensing and chemical signaling in osteocytes has been hypothesized to occur at the single cell level, making it imperative to understand the biological response of the individual cell.

Introduction

Bone is known to adapt to its loading conditions via modeling and remolding. Osteocytes comprise 90–95% of all bone cells (Parfitt, 1977) and function as the mechanosensors of bone (Bonewald, 2006). They are located in the mineralized bone matrix within cave-like structures called lacunae. Extending from the lacunae is a network of canaliculi, which contain the extensive number of cell processes of the osteocytes. Through the establishment of this complex network of caves and canals osteocytes become ideally situated to sense the presence or absence of bone loading and respond by sending signals to the bone-forming osteoblasts and the bone-absorbing osteoclasts, thereby orchestrating the bone remodeling process. The application of force to the skeletal system produces several potential stimuli for osteocytes. Bone loading induces fluid flow and changes in hydrostatic pressure within the interstitial lacunar–canalicular network, while fluid flow across and around cells induces shear stresses. Bone tissue strain can also be transmitted to embedded cells directly though cellular adhesions and attachments, stretching and deforming the cells. Osteocytes have been shown to respond biologically to both strain via direct mechanical stimulation through membrane/cell stretching, and shear induced by fluid flow (Kamioka et al., 1995, Kamioka et al., 2006; Klein-Nulend et al., 1995a, Klein-Nulend et al., 1995b; Turner and Pavalko, 1998; Burger and Klein-Nulend, 1999; Nakamura, 1999; Cheng et al., 2001; Bonewald, 2002, Bonewald, 2004; Mullender et al., 2004; Rubin et al., 2006; Vatsa et al., 2006, Vatsa et al., 2007; Vezeridis et al., 2006).

The strain induced upon osteocytes by shear applied via fluid flow has yet to be quantified and associated with any resulting biological responses. However, fluid flow has been shown to rapidly increase intracellular calcium and nitric oxide levels in bone cells (Smalt et al., 1997b; Ajubi et al., 1999; Donahue et al., 2001; Reilly et al., 2003; Mullender et al., 2004, Mullender et al., 2006; Bacabac et al., 2005; Kamioka et al., 2006; Vatsa et al., 2006; Vezeridis et al., 2006; Genetos et al., 2007; Tan et al., 2007). In a 2006 study by Kamioka et al. it was suggested that the calcium response of bone cells under fluid flow varied in response to the number of cell adhesions. However, this relationship could be more directly related to the actual strains experienced by the individual bone cells as a result of the integrity of these adhesions. It is possible that the more tightly bound a cell is to the substrate, the less strain and deformation the cell will experience in response to fluid shear stress. In this study, the real-time upregulation of intracellular calcium and nitric oxide levels within individual osteocytes in response to an applied fluid flow was examined. The resulting imposed strain of each of the osteocytes was also quantified and correlated to a biological response.

Section snippets

Cell culture

Osteocyte-like MLO-Y4 cells were cultured on type I rat tail collagen (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) coated on 100 mm dishes in α-minimal essential medium (α-MEM) (GIBCO, Grand Island, NY, USA) supplemented with 2.5% fetal bovine serum (FBS) (Summit Biotechnology, Fort Collins, CO, USA), 2.5% calf serum (CS) (HyClone Laboratories, Logan, UT, USA), and 1% penicillin and streptomycin (PS) (Cellgro, Manassas, VA, USA). Cells were maintained at 37 °C and 5% CO2 in a

Results

Osteocyte-like MLO-Y4 cells seeded on collagen-coated glass slides were imaged prior to and immediately following exposure to laminar fluid flow resulting in shear stresses of 2, 8, and 16 dyn/cm2. The field of view for each glass slide was randomly selected from the laminar flow region and all viable cells within the field were analyzed. The upregulation of intracellular calcium levels, nitric oxide levels, and average cellular strains were calculated for a total of 96 different individual

Discussion

The purpose of this study was to measure both the real-time changes in intracellular calcium and nitric oxide levels and the mechanical strain in individual osteocyte-like MLO-Y4 cells exposed to a laminar fluid flow field. Interestingly, cells exposed to the same fluid flow experienced a wide range of strains and changes in intracellular calcium and nitric oxide concentrations, suggesting that strain at the cell level is influenced by more than just the globally applied shear rate. This

Conflict of interest statement

The authors have no conflicts of interest.

Acknowledgments

This research was funded by NIH/NIAMS P01 AR046798.

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