Martin M. Knight

Chondrocyte deformation within compressed agarose constructs at the cellular and sub-cellular levels

Mechanotransduction events in articular cartilage may be resolved into extracellular components followed by intracellular signalling events, which finally lead to altered cell response. Cell deformation is one of the former components, which has been examined using a model involving bovine chondrocytes seeded in agarose constructs. Viable fluorescent labels and confocal laser scanning microscopy were used to examine cellular and sub-cellular morphology. It was observed that cell size increased up to day 6 in culture, associated with an increase in the contents of proteoglycan and collagen. In addition, the organisation of the cytoskeleton components, described using a simple scoring scale, revealed temporal changes for actin fibres, microtubules and vimentin intermediate filaments. The constructs on day 1 were also subjected to unconfined compressive strains. A series of confocal scans through the centre of individual cells revealed a change from a spherical to an elliptical morphology. This was demonstrated by a change in diameter ratio, from a mean value of 1.00 at 0% strain to 0.60 at 25% strain. Using simple equations, the volume and surface areas were also estimated from the scans. Although the former revealed little change with increasing construct strain, surface area appeared to increase significantly. However further examination, using transmission electron microscopy to reveal fine ultrastructural detail at the cell periphery, suggest that this increase may be due to an unravelling of folds at the cell membrane. Cell deformation was associated with a decrease in the nuclear diameter, in the direction of the applied strain. The resulting nuclear strain in one direction increased in constructs compressed at later time points, although its values at all three assessment times were less than the corresponding values for cell strain. It is suggested that the nuclear behaviour may be a direct result of temporal changes observed in the organisation of the cytoskeleton. The study demonstrated that the chondrocyte-agarose model provides a useful system for the examination of compression events at both cellular and sub-cellular levels.

The influence of elaborated pericellular matrix on the deformation of isolated articular chondrocytes cultured in agarose.

This study investigates the mechanical influence of pericellular matrix on the deformation of isolated articular chondrocytes compressed within 3% agarose specimens. After 1 day in culture, the cells were associated with minimal amounts of sulphated glycosaminoglycan (GAG) and hydroxyproline and exhibited substantial deformation from a spherical to an oblate ellipsoid morphology when subjected to 20% gross compressive strain. However, over the 6 day culture period, there was a reduction in cell deformation associated with an increase in matrix content. Treatment with testicular hyaluronidase at days 3 and 6 reduced sulphated GAG content to levels observed in untreated specimens at day 1. At day 3, the resulting cell deformation during 20% compression was equivalent to that in specimens compressed at day 1. However, at day 6 cell deformation was only partially restored, suggesting the presence of additional structural matrix components, other than sulphated GAG, which were not present at day 3. Dual scanning confocal microscopy indicated that the elaborated matrix formed a pericellular shell which did not deform during compression and was therefore stiffer than the 3% agarose substrate. Therefore, the elaboration of a mechanically functional pericellular matrix within 6 days, effectively limits the potential involvement of cell deformation in mechanotransduction within cell seeded systems such as those employed for cartilage repair.

Beta transition and stress-induced phase separation in the spinning of spider dragline silk.

Spider dragline silk is formed as the result of a remarkable transformation in which an aqueous dope solution is rapidly converted into an insoluble protein filament with outstanding mechanical properties. Microscopy on the spinning duct in Nephila edulis spiders suggests that this transformation involves a stress-induced formation of anti-parallel beta-sheets induced by extensional flow. Measurements of draw stress at different draw rates during silking confirm that a stress-induced phase transition occurs.

Cell and nucleus deformation in compressed chondrocyte–alginate constructs: temporal changes and calculation of cell modulus

Mechanical loading is essential for the homeostasis of articular cartilage and may be necessary for achieving functional tissue engineered cartilage repair using isolated cells seeded in scaffolds such as alginate. Chondrocyte mechanotransduction is poorly understood, but may involve cell deformation and associated distortion of intracellular organelles. The present study used confocal microscopy to examine cell and nucleus morphology in isolated chondrocytes compressed in alginate constructs. Compression of 2% alginate resulted in cell deformation from a spherical to an oblate ellipsoid morphology with conservation of cell volume. Cell deformation was associated with deformation, to a lesser degree, of the nucleus. Despite constant cell deformation over a 25 min period of static compression, the nucleus deformation reduced significantly, particularly in the axis perpendicular to the applied compression. Constructs made of a lower alginate concentration exhibited a reduced compressive modulus with an altered cellular response to compression. In 1.2% alginate, compression resulted in cell deformation which was initially of a similar magnitude to that in 2% alginate but subsequently reduced over a 60 min period reflecting the viscoelastic behaviour of the gel. This phenomenon enabled the calculation of a stress-strain relationship for the cell with an estimated Youngs modulus value of approx. 3 kPa.

Primary cilia mediate mechanotransduction through control of ATP-induced Ca2+ signaling in compressed chondrocytes

We investigated the role of the chondrocyte primary cilium in mechanotransduction events related to cartilage extracellular matrix synthesis. We generated conditionally immortalized wild‐type (WT) and IFT88orpk (ORPK) mutant chondrocytes that lack primary cilia and assessed intracellular Ca2+ signaling, extracellular matrix synthesis, and ATP release in response to physiologically relevant compressive strains in a 3‐dimensional chondrocyte culture system. All conditions were compared to unloaded controls. We found that cilia were required for compression‐induced Ca2+ signaling mediated by ATP release, and an associated up‐regulation of aggrecan mRNA and sulfated glycosaminosglycan secretion. However, chondrocyte cilia were not the initial mechanoreceptors, since both WT and ORPK cells showed mechanically induced ATP release. Rather, we found that primary cilia were required for downstream ATP reception, since ORPK cells did not elicit a Ca2+ response to exogenous ATP even though WT and ORPK cells express similar levels of purine receptors. We suggest that purinergic Ca2+ signaling may be regulated by polycystin‐1, since ORPK cells only expressed the C‐terminal tail. This is the first study to demonstrate that primary cilia are essential organelles for cartilage mechanotransduction, as well as identifying a novel role for primary cilia not previously reported in any other cell type, namely cilia‐mediated control of ATP reception.—Wann, A. K. T., Zuo, N., Haycraft, C. J., Jensen, C. G., Poole, C. A., McGlashan, S. R., Knight, M. M. Primary cilia mediate mechanotransduction through control of ATP‐induced Ca2+ signaling in compressed chondrocytes. FASEB J. 26, 1663‐1671 (2012). www.fasebj.org

Compressive Deformation and Damage of Muscle Cell Subpopulations in a Model System

AbstractTo study the effects of compressive straining on muscle cell deformation and damage an in vitro model system was developed. Myoblasts were seeded in agarose constructs and cultured in growth medium for 4 days. Subsequently, the cells were allowed to fuse into multinucleated myotubes for 8 days in differentiation medium, resulting in a population of spherical myoblasts (50%), spherical myotubes (35%), and elongated myotubes (15%) with an overall viability of 90%. To evaluate cell deformation upon construct compression half-core shaped constructs were compressed up to 40% strain and the resulting cell shape was assessed from confocal scans through the central plane of spherical cells. The ratio of cell diameters measured parallel and perpendicular to the axis of compression was used as an index of deformation (DI). The average DI of myoblasts decreased with strain level (0.99±0.03, 0.70±0.04, and 0.56±0.10 at 0%, 20%, and 40% strain), whereas for myotubes DI decreased up to 20% strain and then remained fairly constant (0.99±0.06, 0.55±0.06, 0.50±0.11). The discrepancy in DI between spherical myoblasts and myotubes at 20% strain was explained by the relative sensitivity of the cell membrane to buckling, which is more pronounced in the myotubes. Sustained compression up to 24 h at 20% strain resulted in a significant increase in cell damage with time as compared to unstrained controls. Despite differences in membrane buckling no difference in damage between myoblasts and spherical myotubes was observed over time, whereas the elongated myotubes were more susceptible to damage.

Articular chondrocytes express connexin 43 hemichannels and P2 receptors – a putative mechanoreceptor complex involving the primary cilium?

Mechanical loading is essential for the health and homeostasis of articular cartilage, although the fundamental mechanotransduction pathways are unclear. Previous studies have demonstrated that cyclic compression up‐regulates proteoglycan synthesis via an intracellular Ca2+ signalling pathway, mediated by the release of ATP. However, the mechanism(s) of ATP release has not been elucidated. The present study examines expression of the putative mechanosensitive ATP‐release channel, connexin 43 and whether it is expressed on the chondrocyte primary cilium, which acts as a mechanosensor in a variety of other cell types. In addition the study characterized the expression of a range of purine receptors through which ATP may activate downstream signalling events controlling cell function. Bovine articular chondrocytes were isolated by sequential enzyme digestion and seeded in agarose constructs. To verify the presence of functional hemichannels, Lucifer yellow (LY) uptake into viable cells was quantified following treatment with a hemichannel agonist (EGTA) and antagonist (flufenamic acid). LY uptake was observed in 45% of chondrocytes, increasing to 83% following EGTA treatment (P < 0.001). Treatment with the hemichannel blocker, flufenamic acid, significantly decreased LY uptake to less than 5% with and without EGTA. Immunofluorescence and confocal microscopy confirmed the presence of primary cilia and the expression of connexin 43. Approximately 50% of bovine chondrocyte primary cilia were decorated with connexin 43. Human chondrocytes in situ within cartilage explants also expressed connexin 43 hemichannels. However, expression was confined to the upper 200 µm of the tissue closest to the articular surface. Immunofluorescence revealed the expression of a range of P2X and P2Y receptor subtypes within human articular cartilage. In conclusion, the expression of functional hemichannels by articular chondrocytes may represent the mechanism through which mechanical loading activates ATP release as part of a purinergic mechanotransduction pathway. Furthermore, the expression of connexin 43 on the chondrocyte primary cilium suggests the possible involvement of the cilium in this pathway.

Stem cell mechanobiology

Stem cells are undifferentiated cells that are capable of proliferation, self‐maintenance and differentiation towards specific cell phenotypes. These processes are controlled by a variety of cues including physicochemical factors associated with the specific mechanical environment in which the cells reside. The control of stem cell biology through mechanical factors remains poorly understood and is the focus of the developing field of mechanobiology. This review provides an insight into the current knowledge of the role of mechanical forces in the induction of differentiation of stem cells. While the details associated with individual studies are complex and typically associated with the stem cell type studied and model system adopted, certain key themes emerge. First, the differentiation process affects the mechanical properties of the cells and of specific subcellular components. Secondly, that stem cells are able to detect and respond to alterations in the stiffness of their surrounding microenvironment via induction of lineage‐specific differentiation. Finally, the application of external mechanical forces to stem cells, transduced through a variety of mechanisms, can initiate and drive differentiation processes. The coalescence of these three key concepts permit the introduction of a new theory for the maintenance of stem cells and alternatively their differentiation via the concept of a stem cell ‘mechano‐niche’, defined as a specific combination of cell mechanical properties, extracellular matrix stiffness and external mechanical cues conducive to the maintenance of the stem cell population. J. Cell. Biochem. 112: 1–9, 2011.

Mechanical loading modulates chondrocyte primary cilia incidence and length

The pathways by which chondrocytes of articular cartilage sense their mechanical environment are unclear. Compelling structural evidence suggests that chondrocyte primary cilia are mechanosensory organelles. This study used a 3D agarose culture model to examine the effect of compressive strain on chondrocyte cilia. Chondrocyte/agarose constructs were subjected to cyclic compression (0–15%; 1 Hz) for 0.5–48 h. Additional constructs were compressed for 48 h and allowed to recover for 72 h in uncompressed free‐swelling conditions. Incidence and length of cilia labelled with anti‐acetylated α‐tubulin were examined using confocal microscopy. In free‐swelling chondrocytes, these parameters increased progressively, but showed a significant decrease following 24 or 48 h compression. A 72 h recovery partially reversed this effect. The reduced cilia incidence and length were not due to increased cell division. We therefore propose that control of primary cilia length is an adaptive signalling mechanism in response to varying levels and duration of mechanical loads during joint activity.

Measurement of the deformation of isolated chondrocytes in agarose subjected to cyclic compression.

Mechanically induced cell deformation is one of a number of possible mechanotransduction pathways by which chondrocytes sense and respond to changes in their mechanical environment. The present study describes a system for measuring the deformation of isolated chondrocytes in agarose during both static and cyclic compression. A test rig mounted on the stage of an inverted microscope was used to apply precise levels of compressive strain to individual cell-agarose constructs bathed in culture medium. Images of the cells were recorded using a CCD video camera attached to the microscope. Cell deformation was quantified in terms of a deformation index (X/Y) representing the ratio of cell diameters measured parallel (X) and perpendicular (Y) to the axis of compression. Cyclic compression between 0 and 15% strain, at 0.3 Hz, resulted in cyclic deformation of the cells at the same frequency. However, during the unstrained phase the cells did not fully recover to their initially spherical morphology (X/Y = 1.0). During the strained phase, the level of deformation (X/Y = 0.59) was initially similar to that observed during static 15% strain. However, this level of cell deformation reduced over a 20 min period of cyclic compression (X/Y = 0.72), although during static compression the cell deformation remained constant. This system may be used to examine cellular events under a range of dynamic mechanical stimuli.