The effect of compressive loading magnitude on in

Authors: Ryan M J Madden SangKuy Han Walter Herzog

Publish Date: 2014/05/23

Volume: 14, Issue: 1, Pages: 135-142

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Abstract

Chondrocyte metabolism is stimulated by deformation and is associated with structural changes in the cartilage extracellular matrix ECM suggesting that these cells are involved in maintaining tissue health and integrity Calcium signaling is an initial step in chondrocyte mechanotransduction that has been linked to many cellular processes Previous studies using isolated chondrocytes proposed loading magnitude as an important factor regulating this response However calcium signaling in the intact cartilage differs compared to isolated cells The purpose of this study was to investigate the effect of loading magnitude on chondrocyte calcium signaling in intact cartilage We hypothesized that the percentage of cells exhibiting at least one calcium signal increases with increasing load Fully intact rabbit femoral condyle and patellar bone/cartilage samples were incubated in calciumsensitive dyes and imaged continuously under compressive loads of 10–40  strain Calcium signaling was primarily associated with the dynamic loading phase and greatly increased beyond a threshold deformation of about 10  nominal tissue strain There was a trend toward more cells exhibiting calcium signaling as loading magnitude increased p = 0133 These results provide novel information toward identifying mechanisms underlying calciumdependent signaling pathways related to cartilage homeostasis and possibly the onset and progression of osteoarthritisOsteoarthritis OA is a degenerative joint disease characterized by a breakdown of the articular cartilage extracellular matrix ECM resulting in joint pain inflammation and stiffness Currently the mechanisms underlying the onset and progression of OA are not well understood Under normal physiological conditions joint loading causes deformation of the cartilage and its cells Abusara et al 2011 This deformation causes a complex array of biological processes to occur resulting in the synthesis and/or degradation of structural macromolecules Sah et al 1989 These processes have been associated with the adaptive/degenerative changes involved in OA Wieland et al 2005 Despite this knowledge the complex processes behind cell mechanotransduction remain to be elucidated One of the initial steps is an influx of calcium into the cell cytoplasm known as calcium signaling Guilak et al 1999 Calcium is a second messenger directly involved in many cellular processes including gene transcription contraction and proliferation Bootman et al 2001 It can enter the cytoplasm from extracellular stores through the activation of various membrane channels or from intracellular stores such as the endoplasmic reticulum which releases calcium via channels such as the inositol145triphosphate IP3R receptor Berridge 1993 Berridge et al 2003 Huser and Davies 2007For chondrocytes calcium is involved in matrix synthesis Clark et al 1994 Valhmu and Raia 2002 cytoskeletal remodeling Erickson et al 2003 MillwardSadler and Salter 2004 cell hyperpolarization Wright et al 1996 and cell death Huser and Davies 2007 Amin et al 2009 Previous work investigating chondrocyte calcium signaling has focused on cells removed from their physiological environment such as isolated cells/cell cultures Yellowley et al 1997 Guilak et al 1999 Mizuno 2005 Chao et al 2006 Kono et al 2006 and chondrocytes embedded in gel constructs Roberts et al 2001 PingguanMurphy et al 2005 2006 It was recently shown that calcium signaling of chondrocytes in situ differs compared to isolated cells embedded in gel constructs Specifically calcium signaling in situ occurred virtually instantaneously with loading as compared to the timedelayed signaling previously observed in cellgel constructs Han et al 2012 The duration of calcium signals observed in in situ chondrocytes was also shorter than signals observed in cellgel constructs and completely isolated cells and instead were closer to the durations of osmotically induced calcium signals in intact mouse femora Han et al 2012 These results suggest that the calcium signaling behavior of chondrocytes in the intact tissue differs substantially from isolated cells and cellgel constructs Therefore further investigation is needed to understand chondrocyte mechanotransduction as it occurs in intact cartilageLoading magnitude is thought to play a key role in the calcium signaling response of chondrocytes Yellowley et al 1997 Guilak et al 1999 and the magnitude of mechanical stimuli has been shown to greatly influence Ca2+ signaling in other cell types such as endothelial cells Shen et al 1992 and osteocytes Lu et al 2012 We have shown previously that local ECM strains and chondrocyte deformations induced by mechanical loading differ between joint regions Madden et al 2013 and others have observed topographical variations in aggrecan synthesis Little and Ghosh 1997 a process that involves calcium signaling Fitzgerald et al 2004 Therefore the purpose of this study was to examine the effect of compressive loading magnitude on chondrocyte calcium signaling in intact cartilage attached to its native bone for two distinct joint regions A secondary objective was to relate previously measured local ECM strains and chondrocyte deformations Madden et al 2013 to the observed calcium response We hypothesized that the percentage of cells exhibiting at least one calcium response would increase with increasing compressive tissue loadCartilage samples were obtained from the knees of six to eightmonthold New Zealand White rabbits n = 5 All experiments were approved by the Animal Ethics Committee of the University of Calgary Femoral condyles n = 10 and patellae n = 5 were extracted from the knee joints stripped of noncartilaginous connective tissues while maintaining the underlying bone and placed in high glucose Dulbecco’s Modified Eagle’s Medium with 4 mM Lglutamine and 25 mM HEPES DMEM Gibco OR USA supplemented with 1  penicillin/streptomycin and 1 mM sodium pyruvate Tissue thickness of each sample was measured by needle indentation at two locations close to the region to be loaded Cartilage samples were then incubated in medium containing the ratiometric calciumsensitive dyes Fura Red 30 upmu M and Fluo4 15 upmu M for 1 h at 37 circ C After staining the samples were rinsed in dyefree medium for 20 min prior to testing All specimens were tested within 15 h of collection and the order of testing was randomized to mitigate the effect of timea Assembled view of the system for simultaneous loading and imaging of in situ chondrocytes b Schematic of dashed white box shown in a showing the orientation of the sample in the tissue holder the indenter and the light path of the confocal microscopea Exemplar field of view x–y plane showing fluorescently labeled cells scale bar = 50 upmu m b Calcium signal of a representative cell indicating the cellspecific threshold signal characteristics and corresponding confocal images scale bars = 5 upmu m

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