Biomedical subjects

D L Bader

Publications and source records attributed to D L Bader.

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

Computational modeling to predict the temporal regulation of chondrocyte metabolism in response to various dynamic compression regimens.

Based on previously published experimental work, computational models were developed to simulate the effect of different dynamic compression regimens on the activity of chondrocytes seeded in agarose constructs. In particular, the balance between proliferation and matrix synthesis can be adjusted by applying different intervals of continuous or intermittent mechanical compression. A phenomenological compartment based-modeling approach was used as first model. A more mechanistic cell cycle model was used as the second model. The compartment-based modeling approach was found to be useful in representing a balance between proliferation and proteoglycan synthesis, when the effect of a certain stimulation protocol is known. In order to predict the response to different intervals of mechanical stimulation, however, a more mechanistic cell cycle-based approach is required. The cell cycle model supports an important role of the onset of loading. In addition, an inhibitory effect of further loading is required, which is more likely to be related to cell cycle progression velocity than to a decreased probability of commitment to the cell cycle. The mechanisms behind this inhibitory effect and the computational implementation, however, require further investigation.

Animals↗

PubMed2006

Compression-induced deep tissue injury examined with magnetic resonance imaging and histology.

The underlying mechanisms leading to deep tissue injury after sustained compressive loading are not well understood. It is hypothesized that initial damage to muscle fibers is induced mechanically by local excessive deformation. Therefore, in this study, an animal model was used to study early damage after compressive loading to elucidate on the damage mechanisms leading to deep pressure ulcers. The tibialis anterior of Brown-Norway rats was loaded for 2 h by means of an indenter. Experiments were performed in a magnetic resonance (MR)-compatible loading device. Muscle tissue was evaluated with transverse relaxation time (T2)-weighted MRI both during loading and up to 20 h after load removal. In addition, a detailed examination of the histopathology was performed at several time points (1, 4, and 20 h) after unloading. Results demonstrated that, immediately after unloading, T2-weighted MR images showed localized areas with increased signal intensity. Histological examination at 1 and 4 h after unloading showed large necrotic regions with complete disorganization of the internal structure of the muscle fibers. Hypercontraction zones were found bilateral to the necrotic zone. Twenty hours after unloading, an extensive inflammatory response was observed. The proposed relevance of large deformation was demonstrated by the location of damage indicated by T2-weighted MRI and the histological appearance of the compressed tissues. Differences in damage development distal and proximal to the indenter position suggested a contribution of perfusion status in the measured tissue changes that, however, appeared be to reversible.

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↗

PubMed2005

Anti-inflammatory effects of IL-4 and dynamic compression in IL-1beta stimulated chondrocytes.

Mechanical loading can counteract inflammatory pathways induced by IL-1beta by inhibiting *NO and PGE2, catabolic mediators known to be involved in cartilage degradation. The current study investigates the potential of dynamic compression, in combination with the anti-inflammatory cytokine, IL-4, to further abrogate the IL-1beta induced effects. The data presented demonstrate that IL-4 alone can inhibit nitrite release in the presence and absence of IL-1beta and partially reverse the IL-1beta induced PGE2 release. When provided in combination, IL-4 and dynamic compression could further abrogate the IL-1beta induced nitrite and PGE2 release. IL-1beta inhibited [3H]thymidine incorporation and this effect could be reversed by IL-4 or dynamic strain alone or both in combination. By contrast, 35SO4 incorporation was not influenced by IL-4 and/or dynamic strain in IL-1beta stimulated constructs. IL-4 and mechanical loading may therefore provide a potential protective mechanism for cartilage destruction as observed in OA.

Animals↗

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

A new MR-compatible loading device to study in vivo muscle damage development in rats due to compressive loading.

To study the aetiology of pressure ulcers an MR-compatible loading device was developed. Magnetic resonance imaging provides the possibility of non-invasive evaluation of muscle tissue after compressive loading. Pressure was applied to the tibialis anterior region of rats by means of an indenter. The developed MR-compatible loading device allowed high quality consecutive MR measurements for up to 6h. Tissue was evaluated both during and after loading. Two loading protocols were used; a large indentation of 4.5mm (mean pressure 150 kPa) was applied for 2h and a small indentation of 2.9 mm (mean pressure 50 kPa) was applied for 4h. T2-weighted MR images after the large indentation showed an immediate increase in signal intensity, associated with damage, following load removal. After 20 h the signal intensity remained higher in the affected regions. Afterwards the tissue was perfusion fixated for histological examination. Histological evaluation revealed an inflammatory response and severe muscle necrosis. No signal increase was observed after small indentation. With this new set-up, the different factors that may play a role in the onset of muscle damage can be studied, what we believe will lead to a better understanding of the contributing factors to pressure ulcer development.

Animals↗

PubMed2005

The relative contributions of different skin layers to the mechanical behavior of human skin in vivo using suction experiments.

Although the mechanical behavior of the top layer of the skin, the epidermis, is an important consideration in several clinical and cosmetic applications, there are few reported studies on this layer. The in vivo mechanical behavior of the upper skin layer (here defined as epidermis and papillar dermis) was characterized using a combined experimental and modeling approach. The work was based on the hypothesis that experiments with different length scales represent the mechanical behavior of different skin layers. Suction measurements with aperture diameters of 1, 2 and 6 mm were combined with ultrasound and optical coherence tomography to study the deformation of the skin layers. The experiments were simulated for small displacements with a two-layered finite element model representing the upper layer and the reticular dermis. An identification method compared the experimental and numerical results to identify the material parameters of the model. For one subject the whole parameter estimation procedure was completed, leading to a stiffness of C(10,ul) = 0.11 kPa for the top-layer and C(10,rd) = 0.16 MPa for the reticular dermis. This unexpected, extreme stiffness ratio of the material parameters let to convergence problems of the finite element software for most of the individuals.

Adult↗

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↗

PubMed2005

A biomechanical analysis of an instrumented spinal fixator under torsional loads.

The effect of design features of an internal spinal fixator on the loading of its individual components is paramount to the understanding of the interaction between the fixator and the instrumented spine. Using a corpectomy injury model, a strain gauge instrumented spinal fixation device was employed to investigate the role of clamp tightening torque and the inclusion of transverse bars on the distribution of bending and torsional moments acting on the fixator under torsional loading. The increase in clamp torque from 5 to 10 Nm caused a marked decrease (40%) in torsional moments acting on the vertical rods, an increase of 24% in torsional moments acting on the screws and an increase of 44% in bending moments acting on the rods along the sagittal plane of the fixator. The inclusion of transverse elements significantly increased (132%) the bending moment acting on the rods and decreased (92%) the bending moments acting on the screws along the sagittal plane. The change in both design parameters significantly reduced the response hysteresis and decreased the asymmetry of loading. A theoretical model, developed to elucidate the load path mechanisms underlying this response, successfully predicted the external response of the fixator. This model suggested both design parameters would affect the internal force and moment distribution across the fixator and the relative role of each load response mechanism in effecting this response. The changes in load patterns across the fixator will influence both its ability to augment the process of spinal fusion and the long-term performance of its components.

Biomechanical Phenomena↗

PubMed2005

Mechanical and failure properties of single attached cells under compression.

Eukaryotic cells are continuously subjected to mechanical forces under normal physiological conditions. These forces and associated cellular deformations induce a variety of biological processes. The degree of deformation depends on the mechanical properties of the cell. As most cells are anchorage dependent for normal functioning, it is important to study the mechanical properties of cells in their attached configuration. The goal of the present study was to obtain the mechanical and failure properties of attached cells. Individual, attached C2C12 mouse myoblasts were subjected to unconfined compression experiments using a recently developed loading device. The device allows global compression of the cell until cell rupture and simultaneously measures the associated forces. Cell bursting was characterized by a typical reduction in the force, referred to as the bursting force. Mean bursting forces were calculated as 8.7+/-2.5 microN at an axial strain of 72+/-4%. Visualization of the cell using confocal microscopy revealed that cell bursting was preceded by the formation of bulges at the cell membrane, which eventually led to rupturing of the cell membrane. Finite element calculations were performed to simulate the obtained force-deformation curves. A finite element mesh was built for each cell to account for its specific geometrical features. Using an axisymmetric approximation of the cell geometry, and a Neo-Hookean constitutive model, excellent agreement between predicted and measured force-deformation curves was obtained, yielding an average Young's modulus of 1.14+/-0.32 kPa.

Animals↗

PubMed2005

Compression-induced damage in a muscle cell model in vitro.

Soft tissue breakdown can be initiated at the muscle layer associated with bony prominences, leading to the development of pressure ulcers. Both the magnitude and duration of pressure are important factors in this breakdown process. The present study utilizes a physical model, incorporating C2C12 mouse myoblasts in a homogeneous agarose gel, to examine the damaging effects of prolonged applied pressure. Identical cylindrical cores cut from the agarose/cell suspension were subjected to two separate compressive strains, of 10 and 20 per cent. The strain was applied for time periods ranging from 0.5 to 12 hours, using a specially designed loading apparatus. After each compression period, sections taken from the central horizontal plane of the individual constructs were stained using either haematoxylin and eosin or with the fluorescent probes, Calcein AM and ethidium homodimer-1, and assessed for cell damage. It was found that constructs subjected to the higher strain values demonstrated significantly higher values of non-viable cells for equivalent time points compared to the unstrained constructs. Further analysis on sections using the DNA nick-translation method suggested that this increase was primarily due to apoptosis. These findings imply a relationship between the duration of applied compression and damage to muscle cells seeded in the gel, which was particularly apparent at the strain level of 20 per cent, equivalent to a clinically relevant pressure of 32 mmHg (4.3 kPa). Such an approach might be useful in establishing damage threshold levels at a cellular level.

Animals↗

PubMed2005

Autologous chondrocyte implantation. Culture in a TGF-beta-containing medium enhances the re-expression of a chondrocytic phenotype in passaged human chondrocytes in pellet culture.

An increasing number of patients are treated by autologous chondrocyte implantation (ACI). This study tests the hypothesis that culture within a defined chondrogenic medium containing TGF-beta enhances the re-expression of a chondrocytic phenotype and the subsequent production of cartilaginous extracellular matrix by human chondrocytes used in ACI. Chondrocytes surplus to clinical requirements for ACI from 24 patients were pelleted and cultured in either DMEM (Dulbecco's modified eagles medium)/ITS+Premix/TGF-beta1 or DMEM/10%FCS (fetal calf serum) and were subsequently analysed biochemically and morphologically. Pellets cultured in DMEM/ITS+/TGF-beta1 stained positively for type-II collagen, while those maintained in DMEM/10%FCS expressed type-I collagen. The pellets cultured in DMEM/ITS+/TGF-beta1 were larger and contained significantly greater amounts of DNA and glycosaminoglycans. This study suggests that the use of a defined medium containing TGF-beta is necessary to induce the re-expression of a differentiated chondrocytic phenotype and the subsequent stimulation of glycosaminoglycan and type-II collagen production by human monolayer expanded chondrocytes.

Adolescent↗

PubMed2004

Integrin-mediated mechanotransduction processes in TGFbeta-stimulated monolayer-expanded chondrocytes.

Previous studies have demonstrated that passage in monolayer detrimentally affects the response of articular chondrocytes to the application of dynamic compression. Transforming growth factor beta (TGFbeta) is known to regulate metabolic processes in articular cartilage and can enhance the re-expression of a chondrocytic phenotype following monolayer expansion. The current study tests the hypothesis that TGFbeta also modulates the response of monolayer-expanded human chondrocytes to the application of dynamic compression, via an integrin-mediated mechanotransduction process. The data presented demonstrate that TGFbeta3 enhanced 35SO4 and [3H]thymidine incorporation and inhibited nitrite release after 48 h of culture when compared to unsupplemented constructs. Dynamic compression also enhanced 35SO4 and [3H]thymidine incorporation and inhibited nitrite release in the presence of TGFbeta3. By contrast, dynamic compression did not alter these parameters in the absence of the growth factor. The addition of the peptide, GRGDSP, which acts as a competitive ligand for the alpha5beta1 integrin, reversed the compression-induced stimulation of 35SO4 incorporation, [3H]thymidine incorporation, and suppression of nitrite release. No effect was observed when the control peptide, GRADSP, was used. The current data clearly demonstrate that the dynamic compression-induced changes observed in cell metabolism for human monolayer-expanded chondrocytes were dependent on the presence of TGFbeta3 and are integrin-mediated.

Adolescent↗

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↗

PubMed2004

An investigation into the effects of the hierarchical structure of tendon fascicles on micromechanical properties.

During physiological loading, a tendon is subjected to tensile strains in the region of up to 6 per cent. These strains are reportedly transmitted to cells, potentially initiating specific mechanotransduction pathways. The present study examines the local strain fields within tendon fascicles subjected to tensile strain in order to determine the mechanisms responsible for fascicle extension. A hierarchical approach to the analysis was adopted, involving micro and macro examination. Micro examination was carried out using a custom-designed rig, to enable the analysis of local tissue strains in isolated fascicles, using the cell nuclei as strain markers. In macro examination, a video camera was used to record images of the fascicles during mechanical testing, highlighting the point of crimp straightening and macro failure. Results revealed that local tensile strains within a collagen fibre were consistently smaller than the applied strain and showed no further increase once fibres were aligned. By contrast, between-group displacements, a measure of fibre sliding, continued to increase beyond crimp straightening, reaching a mean value of 3.9 per cent of the applied displacement at 8 per cent strain. Macro analysis displayed crimp straightening at a mean load of 1 N and sample failure occurred through the slow unravelling of the collagen fibres. Fibre sliding appears to provide the major mechanism enabling tendon fascicle extension within the rat-tail tendon. This process will necessarily affect local and cellular strains and consequently mechanotransduction pathways.

Adaptation, Physiological↗

PubMed2003

Dynamic compression counteracts IL-1 beta-induced release of nitric oxide and PGE2 by superficial zone chondrocytes cultured in agarose constructs.

OBJECTIVE: To examine the effect of IL-1 beta-induced *NO and PGE(2)release by stimulated superficial and deep chondrocyte/agarose constructs subjected to mechanical compression. DESIGN: Chondrocyte sub-populations were seeded separately in agarose constructs and cultured unstrained, within a 24-well tissue culture plate, for 48 h in medium supplemented with IL-1 beta and/or L-N-(1-iminoethyl)-ornithine (L-NIO). In a separate experiment, superficial and deep cell containing constructs were subjected to 15% dynamic compressive strain at 1 Hz, for 48 h, in the presence or absence of IL-1 beta and/or L-NIO. Nitrite was measured using the Griess assay, PGE(2)release was determined using an EIA kit and [3H]-thymidine and 35SO(4)incorporation were assessed by TCA and alcian blue precipitation, respectively. RESULTS: The current data reveal that IL-1 beta significantly enhanced *NO and PGE(2)release for superficial chondrocytes, an effect reversed with L-NIO. *NO and PGE(2)levels did not significantly change by deep cells in the presence of IL-1 beta and/or L-NIO. For both cell sub-populations, IL-1 beta inhibited cell proliferation whereas proteoglycan synthesis was not affected. Dynamic compression inhibited the release of *NO and PGE(2)in the presence and absence of IL-1 beta, for cells from both sub-populations. L-NIO reduced *NO and enhanced PGE(2)release for superficial zone chondrocytes, an effect not observed for deep cells in response to dynamic compression. The magnitude of stimulation of [3H]-thymidine incorporation was similar for both cell sub-populations and was not influenced by L-NIO, indicating an z.rad;NO-independent pathway. The dynamic compression-induced stimulation of 35SO(4)incorporation was enhanced with L-NIO for IL-1 beta-stimulated deep cells, indicating an *NO-dependent pathway. CONCLUSION: The present findings suggest that dynamic compression inhibits *NO and PGE(2)release in IL-1 beta-stimulated superficial cells via distinct pathways, a significant finding that may contribute to the development of intervention strategies for the treatment of inflammatory joint disorders.

Animals↗

PubMed2003

Compression induced cell damage in engineered muscle tissue: an in vitro model to study pressure ulcer aetiology.

The aetiology of pressure ulcers is poorly understood. The complexity of the problem, involving mechanical, biochemical, and physiological factors demands the need for simpler model systems that can be used to investigate the relative contribution of these factors, while controlling others. Therefore, an in vitro model system of engineered skeletal muscle tissue constructs was developed. With this model system, the relationship between compressive tissue straining and cell damage initiation was investigated under well-defined environmental conditions. Compression of the engineered muscle tissue constructs revealed that cell death occurs within 1-2 h at clinically relevant straining percentages and that higher strains led to earlier damage initiation. In addition, the uniform distribution of dead cells throughout the constructs suggested that sustained deformation of the cells was the principle cause of cell death. Therefore, it is hypothetised that sustained cell deformation is an additional mechanism that plays a role in the development of pressure ulcers.

Animals↗