Biomedical subjects

M M Knight

Publications and source records attributed to M M Knight.

At least 19 recordsLinked to original sources

PubMed2006

Chondrocyte deformation induces mitochondrial distortion and heterogeneous intracellular strain fields.

Chondrocyte mechanotransduction is poorly understood but may involve cell deformation and associated distortion of intracellular structures and organelles. This study quantifies the intracellular displacement and strain fields associated with chondrocyte deformation and in particular the distortion of the mitochondria network, which may have a role in mechanotransduction. Isolated articular chondrocytes were compressed in agarose constructs and simultaneously visualised using confocal microscopy. An optimised digital image correlation technique was developed to calculate the local intracellular displacement and strain fields using confocal images of fluorescently labelled mitochondria. The mitochondria formed a dynamic fibrous network or reticulum, which co-localised with microtubules and vimentin intermediate filaments. Cell deformation induced distortion of the mitochondria, which collapsed in the axis of compression with a resulting loss of volume. Compression generated heterogeneous intracellular strain fields indicating mechanical heterogeneity within the cytoplasm. The study provides evidence supporting the potential involvement of mitochondrial deformation in chondrocyte mechanotransduction, possibly involving strain-mediated release of reactive oxygen species. Furthermore the heterogeneous strain fields, which appear to be influenced by intracellular structure and organisation, may generate significant heterogeneity in mechanotransduction behaviour for cells subjected to identical levels of deformation.

Animals↗

PubMed2006

Cyclic compression of chondrocytes modulates a purinergic calcium signalling pathway in a strain rate- and frequency-dependent manner.

Mechanical loading modulates cartilage homeostasis through the control of matrix synthesis and catabolism. However, the mechanotransduction pathways through which chondrocytes detect different loading conditions remain unclear. The present study investigated the influence of cyclic compression on intracellular Ca2+ signalling using the well-characterised chondrocyte-agarose model. Cells labelled with Fluo4 were visualised using confocal microscopy following a period of 10 cycles of compression between 0% and 10% strain. In unstrained agarose constructs, not subjected to cyclic compression, a subpopulation of approximately 45% of chondrocytes exhibited spontaneous global Ca2+ transients with mean transient rise and fall times of 19.4 and 29.4 sec, respectively. Cyclic compression modulated global Ca2+ signalling by increasing the percentage of cells exhibiting Ca2+ transients (population modulation) and/or reducing the rise and fall times of these transients (transient shape modulation). The frequency and strain rate of compression differentially modulated these Ca2+ signalling characteristics providing a potential mechanism through which chondrocytes may distinguish between different loading conditions. Treatment with apyrase, gadolinium and the P2 receptor blockers, suramin and basilen blue, significantly reduced the percentage of cells exhibiting Ca2+ transients following cyclic compression, such that the mechanically induced upregulation of Ca2+ signalling was completely abolished. Thus cyclic compression appears to activate a purinergic pathway involving the release of ATP followed by the activation of P2 receptors causing a combination of extracellular Ca2+ influx and intracellular Ca2+ release. Knowledge of this fundamental cartilage mechanotransduction pathway may lead to improved therapeutic strategies for the treatment of cartilage damage and disease.

Animals↗

PubMed2006

Purinergic pathway suppresses the release of .NO and stimulates proteoglycan synthesis in chondrocyte/agarose constructs subjected to dynamic compression.

Mechanical loading plays a fundamental role in the physiological and pathological processes of articular cartilage. The application of dynamic compression to chondrocytes cultured in agarose, downregulates the release of nitric oxide (NO) and enhances cell proliferation and proteoglycan synthesis. We hypothesize that the observed metabolic changes in response to dynamic compression involve a purinergic signaling pathway. Chondrocyte/agarose constructs were subjected to dynamic compression (15%, 1 Hz, 48 h) in the presence of antagonists for the purinergic pathway. Gadolinium was used as a putative inhibitor of stretch-activated calcium ion channels including adenosine 5'-triphosphate (ATP) release channels; suramin was employed as a P2 receptor antagonist and apyrase was used to catalyze the hydrolysis of extracellular ATP. The data presented demonstrate that in the absence of the inhibitor, dynamic compression suppressed .NO release. Treatment with gadolinium and suramin caused a compression-induced upregulation of .NO release, a response abolished with apyrase. Compression-induced stimulation of cell proliferation was reversed with gadolinium, suramin, or apyrase. By contrast, compression-induced stimulation of proteoglycan synthesis was abolished under all treatment conditions. Thus, the purinergic pathway is important in suppressing the release of .NO and stimulation of proteoglycan synthesis. Indeed, high levels of .NO could trigger a downstream catabolic response and mediate the compression-induced inhibition of cell proliferation. The current study demonstrates for the first time the importance of a purinergic pathway in mediating the metabolic response to dynamic compression and suppressing an inflammatory effect.

Adenosine Triphosphate↗

PubMed2005

Activation of chondrocytes calcium signalling by dynamic compression is independent of number of cycles.

Mechanical loading is necessary for the development and maintenance of healthy articular cartilage through the control of extracellular matrix synthesis and catabolism. However, the underlying process of chondrocyte mechanotransduction remains unclear. This study examined the influence of cyclic compression on intracellular calcium (Ca(2+)) signalling within isolated articular chondrocytes cultured in agarose constructs. A validated experimental system was developed for applying controlled cyclic cell deformation. Cell-agarose constructs were subjected to 1Hz cyclic compression between 0 and 10% gross strain for 1, 10, 100 or 300 cycles. The cells were subsequently visualised for 300s in the unstrained state using confocal microscopy and the Ca(2+) indicator, Fluo-4 AM. Within unloaded control constructs, a sub-population of approximately 50% of chondrocytes exhibited characteristic spontaneous Ca(2+) transients each lasting approximately 40-60s. Cyclic compression, for only 1 cycle, significantly up-regulated the percentage of cells exhibiting Ca(2+) transients in the subsequent 5min period (p<0.05). Increasing the number of cycles to 10 or 100 had no additional effect. The up-regulated Ca(2+) signalling was maintained for up to 5min before returning to basal levels. By contrast, 300 cycles were followed by Ca(2+) signalling that was not significantly different from that in unloaded controls. However, this response was shown to be due to the increased time following the start of compression. In conclusion, this study indicates that chondrocyte Ca(2+) signalling is stimulated by dynamic compression, probably mediated by cyclic cell deformation. The overall response appears to be independent of the number of cycles or duration of cyclic compression. The sustained up-regulation of Ca(2+) signalling after 1, 10 or 100 cycles suggests the involvement of an autocrine-paracrine signalling mechanism. Furthermore, the reduced response following 300 cycles indicates a possible receptor desensitisation mechanism. Therefore, Ca(2+) signalling may be part of a mechanotransduction pathway through which chondrocyte populations can modulate their metabolic activity in response to changing mechanical stimuli.

Animals↗

PubMed2005

Mechanical compression and hydrostatic pressure induce reversible changes in actin cytoskeletal organisation in chondrocytes in agarose.

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.

Actin Cytoskeleton↗

PubMed2004

Increased presence of cells with multiple elongated processes in osteoarthritic femoral head cartilage.

OBJECTIVE: This study examined the morphology of chondrocytes in articular cartilage from osteoarthritic (OA) and non-OA human femoral heads and in particular the appearance of a sub-population of cells with multiple elongated processes radiating up to 30 microm into the extracellular matrix. METHODS: Cartilage explants were removed from 8 anatomical sites over the surface of OA (n=6) and non-OA (n=5) femoral heads. Cells were labeled for vimentin intermediate filaments and visualized using epi-fluorescence and confocal microscopy. The percentage of cells with elongated processes was correlated with macroscopic and histological indicators of osteoarthritis. RESULTS: Cells with processes accounted for less than 10% of the total cell population in non-OA cartilage. By contrast, in the peripheral regions of the OA femoral head these cells accounted for 20-45% of the total cell population, the differences being statistically significant. These peripheral areas are habitually non-load bearing and were also the most likely to show gross fibrillation and pannus formation. A statistically significant correlation was demonstrated between the percentage of cells with processes and the histological extent of the OA degradation, quantified in terms of the Mankin score. CONCLUSIONS: The extension of cell processes, which may be associated with localized breakdown of the pericellular matrix, will undoubtedly alter numerous aspects of cell function including phenotypic expression and mechanotransduction. Hence these significant changes in chondrocyte morphology are likely to have important implications for the aetiology of osteoarthritis and the development of potential treatment strategies.

Cartilage, Articular↗

PubMed2002

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 Young's modulus value of approx. 3 kPa.

Alginates↗

PubMed2002

Deformation properties of articular chondrocytes: a critique of three separate techniques.

This paper presents a series of techniques, which examine the deformation characteristics of bovine articular chondrocytes. The direct contact approach employs well established methodology, involving AFM and micropipette aspiration, to yield structural properties of local regions of isolated chondrocytes. The former technique yields a non-linear response with increased structural stiffness in a central location on a projected image of the chondrocyte. A simple viscoelastic model can be used with data from the micropipette aspiration technique to yield a mean value of Young's modulus, which is similar to that recently reported (Jones et al., 1999). An indirect approach is also described, involving the response of chondrocytes seeded within compressed agarose constructs. For 1% agarose constructs, the resulting cell strain, yields a gross cell modulus of 2.7 kPa. The study highlights the difficulties in establishing unique mechanical parameters, which reflect the deformation behaviour of articular chondrocytes.

Animals↗

PubMed2001

Protocol for the determination of selected neutral and acidic semi-volatile organic contaminants in fish tissue.

Progress toward the development of a protocol for the determination of a broad spectrum of organic compounds in fish tissue is reported. Finely ground and homogenized fish tissue samples were Soxhlet extracted. Phenolic compounds in the extracts were acetylated and the derivatized extract containing the acetates and neutral semi-volatiles was cleaned up with silica gel and size-exclusion column chromatography. These semi-volatile organic compounds were determined by gas chromatography-mass spectrometry. The method is evaluated for recovery and precision of selected analytes during the analysis of over 300 fish tissue samples of varying species in support of contaminant determination in fish tissue from the Columbia/Snake River watershed.

Animals↗

PubMed2001

Chondrocyte deformation within mechanically and enzymatically extracted chondrons compressed in agarose.

Within articular cartilage, the chondron microenvironment will influence chondrocyte behaviour and response to loading. Chondrons were extracted from intact cartilage using either mechanical homogenisation (MC) or enzymatic digestion (EC) and cell and matrix morphology in unstrained and compressed agarose constructs was examined. Isolated chondrocytes (IC) were used for comparison. Immunolocalisation of type VI collagen and keratan sulphate revealed differences in the structure of the pericellular microenvironment such that MC most closely resembled chondrons in situ. The unstrained cell diameters of IC and EC were larger than MC at day 1 and increased significantly over a 7 day culture period. In contrast, cell diameters for MC remained constant. Compression of constructs at day 1 resulted in cell deformation for IC and EC but not MC. The two chondron extraction methods yielded chondrons of differing matrix morphology and associated differences in cell size and cellular response to load. The results indicate that the pericellular microenvironment of MC initially possessed a greater mechanical integrity than that of EC. Although these differences may be reduced with time in culture, characterisation of mechanically isolated chondrons suggests that the stiffness of the chondrons in situ may be greater than previous estimates.

Animals↗

PubMed2001

Temporal changes in cytoskeletal organisation within isolated chondrocytes quantified using a novel image analysis technique.

This paper examines temporal changes in the organisation of the cytoskeleton within isolated articular chondrocytes cultured for up to 7 days in agarose constructs. Fluorescent labelling and confocal microscopy were employed to visualise microtubules (MT), vimentin intermediate filaments (VIF) and actin microfilaments (AMF). To quantify the degree of cytoskeletal organisation within populations of cells, a novel image analysis technique has been developed and fully characterised. Organisation was quantified in terms of an Edge Index, which reflects the density of 'edges' present within the confocal images as defined by a Sobel digital filter. This parameter was shown to be independent of image intensity and, for all three cytoskeletal components, was validated statistically against a visual assessment of organisation. Both MT and VIF exhibited fibrous networks extending throughout the cytoplasm, while AMF appeared as punctate units associated with the cell membrane. The use of the Edge Index parameter revealed statistical significant temporal variation, in particular associated with VIF and AMF. These findings indicate the possibility of cytoskeletal mediated temporal variation in many aspects of cell behaviour following isolation from the intact tissue. Furthermore, the image analysis techniques are likely to be useful for future studies aiming to quantify changes in cytoskeletal organisation.

Animals↗

PubMed2001

Compressive deformation and damage of muscle cell subpopulations in a model system.

To 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.

Animals↗

PubMed2001

Mechanical compression influences intracellular Ca2+ signaling in chondrocytes seeded in agarose constructs.

Ca2+ signaling forms part of a possible mechanotransduction pathway by which chondrocytes may alter their metabolism in response to mechanical loading. In this study, a well-characterized model system utilizing bovine articular chondrocytes embedded in 4% agarose constructs was used to investigate the effect of physiological mechanical compressive strain applied after 1 and 3 days in culture. The intracellular Ca2+ concentration was measured by use of the ratiometric Ca2+ indicator indo 1-AM and confocal microscopy. A positive Ca2+ response was defined as a percent increase in Ca2+ ratio above a preset threshold. A significantly greater percentage of cells exhibited a positive Ca2+ response in strained constructs compared with unstrained controls at both time points. In strained constructs, treatment with either Ga3+ or EGTA significantly reduced the number of positive Ca2+ responders compared with untreated controls. These results represent an important step in understanding the physiological role of intracellular Ca2+ in chondrocytes under mechanical compression.

Algorithms↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Animals↗

PubMed2000

Confocal analysis of cytoskeletal organisation within isolated chondrocyte sub-populations cultured in agarose.

This study reports the cytoskeletal organisation within chondrocytes, isolated from the superficial and deep zones of articular cartilage and seeded into agarose constructs. At day 0, marked organisation of actin microfilaments was not observed in cells from both zones. Partial or clearly organised microtubules and vimentin intermediate filaments cytoskeletal components were present, however, in a proportion of cells. Staining for microtubules and vimentin intermediate filaments was less marked after 1 day in culture however than on initial seeding. For all three cytoskeletal components there was a dramatic increase in organisation between days 3 and 14 and, in general, organisation was greater within deep zone cells. Clear organisation for actin microfilaments was characterised by a cortical network and punctate staining around the periphery of the cell, while microtubules and vimentin intermediate filaments formed an extensive fibrous network. Cytoskeletal organisation within chondrocytes in agarose appears, therefore, to be broadly similar to that described in situ. Variations in the organisation of actin microfilaments between chondrocytes cultured in agarose and in monolayer are consistent with a role in phenotypic modulation. Vimentin intermediate filaments and microtubules form a link between the plasma membrane and the nucleus and may play a role in the mechanotransduction process.

Actin Cytoskeleton↗

PubMed2000

Cognitive ability and functional status.

Significant confusion exists in the literature about functional status. Despite its importance, little attention has focused on developing and substantiating frameworks that detail the underpinnings of functional status, which has resulted in lack of agreement about its definition and dimensions. The purpose of this literature review was to examine the development of functional status and to describe the inclusion of its cognitive dimension. Cognition is one key dimension of functional status. One must 'know how' to perform to be successful in an activity. While cognitive capacity is generally considered in relation to functional status, the nature of the cognitive dimension is poorly described and poorly understood. Three databases were selected for review: Citations in Nursing and Allied Health (CINAHL), Psychology Literature (PsychLit), and the Medical data base known as MedLine. Key word searches identified thousands of sources. This analysis includes an extensive sampling of these sources from the 1960s through to 1998. The sources sorted into four primary categories and demonstrate a growing recognition of the cognitive dimension of functional status in the literature. Despite this recognition, the lack of conceptual clarity of both the term functional status and its cognitive dimension limits communication among disciplines and limits comparisons of functional status outcomes across studies. Functional status models are needed that include cognition as a core dimension. Population specific descriptions of the cognitive dimension should be guided by knowledge in the neurosciences.

Activities of Daily Living↗

PubMed1999

Application of polydivinylbenzene liquid chromatography columns to remove lipid material from fish tissue extracts for the analysis of semivolatile organics.

Liquid chromatography columns of 100% polydivinylbenzene (DVB) (packing) were used to remove lipid material from fish extracts before analysis of several semivolatile organic pollutants by gas chromatography-mass spectrometry (GC-MS). This packing material was found to be durable as the columns could be operated to about 900 p.s.i. resulting in high efficiency separation. Recoveries and relative standard deviations for 18 polynuclear aromatic hydrocarbons fortified into a fish extract and cleaned up by multiple DVB columns in series were in the range of 86 to 123% and 4 to 11%, respectively.

Animals↗