Mechanical compression and hydrostatic pressure induce reversible changes in actin cytoskeletal organisation in chondrocytes in agarose.
ABSTRACT In numerous cell types, the cytoskeleton has been widely implicated in mechanotransduction pathways involving stretch-activated ion channels, integrins and deformation of intracellular organelles. Studies have also demonstrated that the cytoskeleton can undergo remodelling in response to mechanical stimuli such as tensile strain or fluid flow. In articular chondrocytes, the mechanotransduction pathways are complex, inter-related and as yet, poorly understood. Furthermore, little is known of how the chondrocyte cytoskeleton responds to physiological mechanical loading. This study utilises the well-characterised chondrocyte-agarose model and an established confocal image-analysis technique to demonstrate that both static and cyclic, compressive strain and hydrostatic pressure all induce remodelling of actin microfilaments. This remodelling was characterised by a change from a uniform to a more punctate distribution of cortical actin around the cell periphery. For some loading regimes, this remodelling was reversed over a subsequent 1h unloaded period. This reversible remodelling of actin cytoskeleton may therefore represent a mechanism through which the chondrocyte alters its mechanical properties and mechanosensitivity in response to physiological mechanical loading.
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ABSTRACT: Cartilage and chondrocytes experience loading that causes alterations in chondrocyte biological activity. In vivo chondrocytes are surrounded by a pericellular matrix with a stiffness of ~25-200kPa. Understanding the mechanical loading environment of the chondrocyte is of substantial interest for understanding chondrocyte mechanotransduction. The first objective of this study was to analyze the spatial variability of applied mechanical deformations in physiologically stiff agarose on cellular and sub-cellular length scales. Fluorescent microspheres were embedded in physiologically stiff agarose hydrogels. Microsphere positions were measured via confocal microscopy and used to calculate displacement and strain fields as a function of spatial position. The second objective was to assess the feasibility of encapsulating primary human chondrocytes in physiologically stiff agarose. The third objective was to determine if primary human chondrocytes could deform in high-stiffness agarose gels. Primary human chondrocyte viability was assessed using live-dead imaging following 24 and 72h in tissue culture. Chondrocyte shape was measured before and after application of 10% compression. These data indicate that (1) displacement and strain precision are ~1% and 6.5% respectively, (2) high-stiffness agarose gels can maintain primary human chondrocyte viability of >95%, and (3) compression of chondrocytes in 4.5% agarose can induce shape changes indicative of cellular compression. Overall, these results demonstrate the feasibility of using high-concentration agarose for applying in vitro compression to chondrocytes as a model for understanding how chondrocytes respond to in vivo loading.Journal of Biomechanics 11/2013; DOI:10.1016/j.jbiomech.2013.10.051 · 2.50 Impact Factor
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ABSTRACT: This paper develops a set of digital volume correlation (DVC) algorithms to address 3-D deformation measurements of soft gels with the aid of laser-scanning confocal microscopy. As an extension of the well-developed digital image correlation (DIC) method, the present DVC approach adopts a three-dimensional zero-normalized cross-correlation criterion (3-D ZNCC) to perform volume correlation calculations. Based on a 3-D sum-table scheme and the fast Fourier transform technique, a fast algorithm is first proposed to accelerate the integer-voxel correlation computations. Subsequently, two kinds of sub-voxel registration algorithms, i.e., 3-D gradient-based algorithm and 3-D Newton–Raphson algorithm, are presented to obtain the sub-voxel displacement and strain fields of volume images before and after deformation. Both a series of computer-simulated digital volume images and an actual agarose gel sample randomly embedded with fluorescent particles are employed to verify the 3-D deformation measurement capability of the proposed DVC algorithms, which indicates that they are competent to acquire 3-D displacement and strain fields of soft gels.International Journal of Applied Mechanics 06/2011; 03(02). DOI:10.1142/S1758825111001019 · 1.29 Impact Factor
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ABSTRACT: Articular chondrocytes experience a variety of mechanical stimuli during daily activity. One such stimulus, direct shear, is known to affect chondrocyte homeostasis and induce catabolic or anabolic pathways. Understanding how single chondrocytes respond biomechanically and morphologically to various levels of applied shear is an important first step toward elucidating tissue level responses and disease etiology. To this end, a novel videocapture method was developed in this study to examine the effect of direct shear on single chondrocytes, applied via the controlled lateral displacement of a shearing probe. Through this approach, precise force and deformation measurements could be obtained during the shear event, as well as clear pictures of the initial cell-to-probe contact configuration. To further study the non-uniform shear characteristics of single chondrocytes, the probe was positioned in three different placement ranges along the cell height. It was observed that the apparent shear modulus of single chondrocytes decreased as the probe transitioned from being close to the cell base (4.1 +/- 1.3 kPa), to the middle of the cell (2.6 +/- 1.1 kPa), and then near its top (1.7 +/- 0.8 kPa). In addition, cells experienced the greatest peak forward displacement (approximately 30% of their initial diameter) when the probe was placed low, near the base. Forward cell movement during shear, regardless of its magnitude, continued until it reached a plateau at ~35% shear strain for all probe positions, suggesting that focal adhesions become activated at this shear level to firmly adhere the cell to its substrate. Based on intracellular staining, the observed height-specific variation in cell shear stiffness and plateau in forward cell movement appeared to be due to a rearrangement of focal adhesions and actin at higher shear strains. Understanding the fundamental mechanisms at play during shear of single cells will help elucidate potential treatments for chondrocyte pathology and loading regimens related to cartilage health and disease.Biomechanics and Modeling in Mechanobiology 08/2009; 9(2):153-62. DOI:10.1007/s10237-009-0166-1 · 3.25 Impact Factor