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Biomedical subjects

Dan L Bader

Publications and source records attributed to Dan L Bader.

At least 19 recordsLinked to original sources
PubMed2006

The relative contributions of compression and hypoxia to development of muscle tissue damage: an in vitro study.

Deep pressure ulcers develop in tissues subjected to sustained mechanical loading. Though it has been hypothesized that this damage mechanism results from local tissue ischemia, it has recently been shown with a cell model that sustained compression can cause cell deformation, leading to tissue breakdown. The present study focuses on the assessment of cell viability during compression and ischemia in an in vitro muscle model to determine their relative contributions to damage development. A model system was developed consisting of engineered skeletal muscle produced from the culture of murine muscle cells in a collagen gel. The tissue was subjected to 0, 20, or 40% compression under hypoxic or normoxic conditions. Experiments were performed on the stage of a microscope and cell viability was monitored using fluorescent markers for apoptotic and necrotic cell death. Hypoxia did not lead to significant cell death over a 22 h period. By contrast, compression led to immediate cell death that increased with time. No additional effect of hypoxia on cell death was observed. These data show that contrary to existing theories, compression can cause development of muscle damage and that hypoxia does not contribute to cell death development within 22 h in engineered muscle.

Animals↗
PubMed2006

The influence of swelling and matrix degradation on the microstructural integrity of tendon.

Tendon is multi-level fibre composite material, responsible for the transmission of forces from muscles to the skeleton. It is composed of a hierarchical arrangement of collagenous units surrounded by a proteoglycan-rich matrix, arranged to support strain transfer, and thus contribute to the mechanical behaviour of tendon. This study examines the effect of swelling and enzymatic degradation on structural integrity at different levels of the tendon hierarchy. Biochemical and microstructural analysis are used to examine the effects of incubation on the composition and swelling of the matrix, prior to a mechanical characterisation of sample integrity. Results indicated significant swelling of tendon fibrils and interfibrillar matrix after incubation in phosphate buffered saline, leading to a reduction in ultimate tensile load, with failure initiated between fibrils and sub-fibrils. In contrast, incubation with the enzyme chondroitinase ABC resulted in a total removal of glycosaminoglycan from the samples, and a subsequent reduction in the extent of swelling. These fascicles also demonstrated an increase in failure loads, with failure predominating between fibres. The findings from this work confirm the importance of the non-collagenous matrix components in controlling strain transfer within tendon structures. It also highlights the necessity to maintain samples within a suitable and controlled environment prior to testing.

Animals↗
PubMed2006

Rate of oxygen consumption by isolated articular chondrocytes is sensitive to medium glucose concentration.

Cartilage tissue engineering typically involves the culture of isolated chondrocytes within a scaffold material. The oxygen tension within the engineered tissue is known to be an essential parameter for implant success. This will be sensitive to the oxygen consumption behavior of the embedded chondrocytes, which remains to be characterized. We report that the oxygen consumption of bovine articular chondrocytes is sensitive to glucose deprivation below 2.7 mM, increasing from a basal level of 9.6x10(-16) to <18.4x10(-16) mol/cell.h in 1.3 mM glucose. Further studies examined the influence of selecting high (18.4 mM) or low (5.1 mM) glucose medium on the oxygen tension in 2 mm thick cellular agarose constructs. A relative upregulation of oxygen consumption was observed in constructs cultured in low glucose medium. This resulted in the near-anoxic oxygen concentration of 5 microM oxygen in constructs seeded with 40x10(6) cells/ml, compared to 57 microM in the corresponding high glucose culture. The upregulation of oxygen consumption generally corresponded to the inhibition of glycolysis, which is consistent with the Crabtree phenomenon. Medium osmolarity (316-600 mOsm) had minimal effects on chondrocyte oxygen consumption rate. In conclusion, glucose availability is a critical parameter that regulates the oxygen tension within tissue engineered constructs.

Animals↗
PubMed2006

Mitochondrial dynamics in chondrocytes and their connection to the mechanical properties of the cytoplasm.

BACKGROUND: The motion and redistribution of intracellular organelles is a fundamental process in cells. Organelle motion is a complex phenomenon that depends on a large number of variables including the shape of the organelle, the type of motors with which the organelles are associated, and the mechanical properties of the cytoplasm. This paper presents a study that characterizes the diffusive motion of mitochondria in chondrocytes seeded in agarose constructs and what this implies about the mechanical properties of the cytoplasm. METHOD OF APPROACH: Images showing mitochondrial motion in individual cells at 30 s intervals for 15 min were captured with a confocal microscope. Digital image correlation was used to quantify the motion of the mitochondria, and the mean square displacement (MSD) was calculated. Statistical tools for testing whether the characteristic motion of mitochondria varied throughout the cell were developed. Calculations based on statistical mechanics were used to establish connections between the measured MSDs and the mechanical nature of the cytoplasm. RESULTS: The average MSD of the mitochondria varied with time according to a power law with the power term greater than 1, indicating that mitochondrial motion can be viewed as a combination of diffusion and directional motion. Statistical analysis revealed that the motion of the mitochondria was not uniform throughout the cell, and that the diffusion coefficient may vary by over 50%, indicating intracellular heterogeneity. High correlations were found between movements of mitochondria when they were less than 2 microm apart. The correlation is probably due to viscoelastic properties of the cytoplasm. Theoretical analysis based on statistical mechanics suggests that directed diffusion can only occur in a material that behaves like a fluid on large time scales. CONCLUSIONS: The study shows that mitochondria in different regions of the cell experience different characteristic motions. This suggests that the cytoplasm is a heterogeneous viscoelastic material. The study provides new insight into the motion of mitochondria in chondrocytes and its connection with the mechanical properties of the cytoplasm.

Animals↗
PubMed2005

Cyclic tensile strain upregulates collagen synthesis in isolated tendon fascicles.

Mechanical stimulation has been implicated as an important regulatory factor in tendon homeostasis. In this study, a custom-designed tensile loading system was used to apply controlled mechanical stimulation to isolated tendon fascicles, in order to examine the effects of 5% cyclic tensile strain at 1 Hz on cell proliferation and matrix synthesis. Sample viability and gross structural composition were maintained over a 24 h loading period. Data demonstrated no statistically significant differences in cell proliferation or glycosaminoglycan production, however, collagen synthesis was upregulated with the application of cyclic tensile strain over the 24 h period. Moreover, a greater proportion of the newly synthesised matrix was retained within the sample after loading. These data provide evidence of altered anabolic activity within tendon in response to mechanical stimuli, and suggest the importance of cyclic tensile loading for the maintenance of the collagen hierarchy within tendon.

Adaptation, Physiological↗
PubMed2005

The influence of noncollagenous matrix components on the micromechanical environment of tendon fascicles.

Tendon is composed of type I collagen fibers, interspersed with proteoglycan matrix and cells. Glycosaminoglycans may play a role in maintaining the structural integrity of tendon, preventing excessive shearing between collagen components. This study tests the hypothesis that tendon extension mechanisms can be altered by modifying the composition of noncollagenous matrix. Tendon explants were treated with phosphate buffered saline (PBS) or PBS + 0.5 U ml(-1) chondroitinase ABC. Structural changes were examined using TEM and biochemical analysis, while strain response was examined using confocal microscopy and gross mechanical characterization. Chondroitinase ABC removed 90% of glycosaminoglycans from the matrix. Results demonstrated significant swelling of fibrils and surrounding matrix when incubated in either solution. In response to applied strain, PBS incubated samples demonstrated significantly less sliding between adjacent fibers than nonincubated, and a 33% reduction in maximum force. By contrast, fascicles incubated in chondroitinase ABC demonstrated a similar strain response to nonincubated. Data indicate that collagen-proteoglycan binding characteristics can be influenced by incubation and this, in turn, can influence the preferred extension mechanisms adopted by fascicles. This highlights the importance of maintaining fascicles within their natural environment to prevent structural or mechanical changes prior to subsequent analysis.

Animals↗
PubMed2005

Viscoelastic properties of single attached cells under compression.

The viscoelastic properties of single, attached C2C12 myoblasts were measured using a recently developed cell loading device. The device allows global compression of an attached cell, while simultaneously measuring the associated forces. The viscoelastic properties were examined by performing a series of dynamic experiments over two frequency decades (0.1-10 Hz) and at a range of axial strains (approximately 10-40%). Confocal laser scanning microscopy was used to visualize the cell during these experiments. To analyze the experimentally obtained force-deformation curves, a nonlinear viscoelastic model was developed. The nonlinear viscoelastic model was able to describe the complete series of dynamic experiments using only a single set of parameters, yielding an elastic modulus of 2120 +/- 900 Pa for the elastic spring, an elastic modulus of 1960 +/- 1350 for the nonlinear spring, and a relaxation time constant of 0.3 +/- 0.12 s. To our knowledge, it is the first time that the global viscoelastic properties of attached cells have been quantified over such a wide range of strains. Furthermore, the experiments were performed under optimal environmental conditions and the results are, therefore, believed to reflect the viscoelastic mechanical behavior of cells, such as would be present in vivo.

Animals↗
PubMed2005

Nutrient utilization by bovine articular chondrocytes: a combined experimental and theoretical approach.

A combined experimental-numerical approach was adopted to characterize glucose and oxygen uptake and lactate production by bovine articular chondrocytes in a model system. For a wide range of cell concentrations, cells in agarose were supplemented with either low or high glucose medium. During an initial culture phase of 48 h, oxygen was monitored noninvasively using a biosensor system. Glucose and lactate were determined by medium sampling. In order to quantify glucose and oxygen uptake, a finite element approach was adopted to describe diffusion and uptake in the experimental model. Numerical predictions of lactate, based on simple relations for cell metabolism, were found to agree well for low glucose, but not for high glucose medium. Oxygen did not play a role in either case. Given the close association between chondrocyte energy metabolism and matrix synthesis, a quantifiable prediction of utilization can present a valuable contribution in the optimization of tissue engineering conditions.

Animals↗
PubMed2004

Crosslinking density influences the morphology of chondrocytes photoencapsulated in PEG hydrogels during the application of compressive strain.

Chondrocyte deformation, which occurs during mechanical loading, is thought to play an important role in the mechanotransduction pathway. In designing a scaffold that can be gelled in situ for cartilage tissue engineering, an important consideration is the influence of mechanical loading. This study tested the hypothesis that changes in the crosslinking density of a hydrogel scaffold influence the morphology of encapsulated chondrocytes in response to an applied load. Chondrocytes were entrapped in photo-crosslinkable hydrogel scaffolds based on poly(ethylene glycol) (PEG) with two crosslinking densities, 0.119 and 0.376 mol/l, with the higher density having a 11-fold higher compressive modulus. The cell-embedded hydrogels were subjected to static compressive strains between 0% and 20% after 1 and 6 days of culture. Using confocal laser scanning microscopy, chondrocytes in the highly crosslinked gel at day 1 deformed more than gels in the more loosely crosslinked gel. By day 6, this finding was reversed. When single cells within a region were followed, heterogeneities in cell deformation were observed on both a macroscopic and microscopic scale. These heterogeneities were greater in the highly crosslinked gel. These findings demonstrate that different levels of cell deformation and heterogeneity may be obtained by varying the crosslinking density in PEG hydrogels.

Animals↗
PubMed2004

Crosslinking density influences chondrocyte metabolism in dynamically loaded photocrosslinked poly(ethylene glycol) hydrogels.

In approaches to tissue engineer articular cartilage, an important consideration for in situ forming cell carriers is the impact of mechanical loading on the cell composite structure and function. Photopolymerized hydrogel scaffolds based on poly(ethylene glycol) (PEG) may be synthesized with a range of crosslinking densities and corresponding macroscopic properties. This study tests the hypothesis that changes in the hydrogel crosslinking density influences the metabolic response of encapsulated chondrocytes to an applied load. PEG hydrogels were formulated with two crosslinking densities that resulted in gel compressive moduli ranging from 60 to 670 kPa. When chondrocytes were encapsulated in these PEG gels, an increase in crosslinking density resulted in an inhibition in cell proliferation and proteoglycan synthesis. Moreover, when the gels were dynamically loaded for 48 h in unconfined compression with compressive strains oscillating from 0 to 15% at a frequency of 1 Hz, cell proliferation and proteoglycan synthesis were affected in a crosslinking-density-dependent manner. Cell proliferation was inhibited in both crosslinked gels, but was greater in the highly crosslinked gel. In contrast, dynamic loading did not influence proteoglycan synthesis in the loosely crosslinked gel, but a marked decrease in proteoglycan production was observed in the highly crosslinked gel. In summary, changes in PEG hydrogel properties greatly affect how chondrocytes respond to an applied dynamic load.

Animals↗
PubMed2004

Confocal analysis of local and cellular strains in chondrocyte-agarose constructs subjected to mechanical shear.

Although numerous previous studies have investigated cell deformation and mechanotransduction within isolated chondrocytes compressed in agarose gel, no published studies have examined the cellular response to shear. In the present study, a novel experimental system has been used to apply precise magnitudes of simple shear strain to isolated bovine articular chondrocytes seeded in agarose. Specimens were gelled between porous endplates which enabled the specimen to be gripped within a specially designed test rig mounted on an inverted microscope. Confocal imaging of individual chondrocytes was used to determine the local and cellular shear strains at gross static shear strains up to 15%. The central region of the specimens experienced uniform local shear strain equal to the applied gross shear strain. An image analysis technique was developed to quantify the level of cell shear strain based upon the shear-induced rotation of a best-fit ellipse. Cell deformation occurred such that the magnitude of the cellular shear strain was equal to gross shear strain. This study is the first to describe the deformation of isolated articular chondrocytes subjected to shear strain. This validated experimental system will enable future studies to examine the influence of shear on chondrocyte function and the associated mechanotransduction signalling pathways.

Animals↗
PubMed2004

Anisotropic, three-dimensional deformation of single attached cells under compression.

Quantifying three-dimensional deformation of cells under mechanical load is relevant when studying cell deformation in relation to cellular functioning. Because most cells are anchorage dependent for normal functioning, it is desired to study cells in their attached configuration. This study reports new three-dimensional morphometric measurements of cell deformation during stepwise compression experiments with a recently developed cell loading device. The device allows global, unconfined compression of individual, attached cells under optimal environmental conditions. Three-dimensional images of fluorescently stained myoblasts were recorded with confocal microscopy and analyzed with image restoration and three-dimensional image reconstruction software to quantify cell deformation. In response to compression, cell width, cross-sectional area, and surface area increased significantly with applied strain, whereas cell volume remained constant. Interestingly, the cell and the nucleus deformed perpendicular to the direction of actin filaments present along the long axis of the cell. This strongly suggests that this anisotropic deformation can be attributed to the preferred orientation of actin filaments. A shape factor was introduced to quantify the global shape of attached cells. The increase of this factor during compression reflected the anisotropic deformation of the cell.

Animals↗
PubMed2004

Passage in monolayer influences the response of chondrocytes to dynamic compression.

This study tests the hypothesis that expansion by passage in monolayer influences the response of isolated articular chondrocytes to dynamic compression. Chondrocytes, isolated from bovine articular cartilage, were seeded in monolayer and passaged 4 times (P1-4). For assessment of chondrocytic and fibroblastic phenotype, freshly isolated and passaged cells were seeded on glass coverslips or in 2% alginate beads and cultured for 7 days in DMEM + 10% FCS. Samples were assayed for DNA and GAG content and stained for collagen types I and II. In separate experiments, freshly isolated or passaged chondrocytes were seeded at 10 x 10(6) cells.ml(-1) in 4% cylindrical agarose constructs and subjected to 15% dynamic compressive strain at 1 Hz for 24 hours. [(3)H]-thymidine incorporation, SO(4) incorporation and nitrite release were analysed. Immediately following isolation (P0), chondrocytes seeded in alginate expressed high levels of type II collagen, but did not stain for type I collagen. Following repeat passage the cells expressed enhanced levels of type I collagen, with an associated reduction in type II collagen staining. These data indicate a modulation to a fibroblastic phenotype during monolayer expansion which was not rapidly reversed by culture in a 3D hydrogel. Dynamic compression down-regulated SO(4) incorporation at P0, but did not affect [(3)H]-thymidine incorporation. By contrast the incorporation of both SO(4) and [(3)H]-thymidine was enhanced by dynamic compression at both P1 and to a lesser extent P2. SO(4) and [(3)H]-thymidine incorporation were inhibited at P3 and P4. Nitrite release was down-regulated by dynamic compression at all passages. These data demonstrate a clear modulation in the response of bovine articular chondrocytes to dynamic compression following passage in monolayer.

Cartilage, Articular↗
PubMed2003

Temporal regulation of chondrocyte metabolism in agarose constructs subjected to dynamic compression.

The temporal response of chondrocyte metabolism in agarose constructs subjected to different dynamic compression regimes was investigated. The current study explored the effects of continuous or intermittent compression using various duty cycles of dynamic compressive loading, over a 48 h culture period. For the continuous compression experiments, duty cycles ranged from 5400 to 172,800 and intermittent compression delivered a total of 86,400 cycles. Large numbers of duty cycles significantly stimulated proteoglycan synthesis with maximal levels obtained for constructs subjected to 12h of intermittent compression. The shortest duration of intermittent compression suggested that further cycles are inhibitory for cell proliferation. Nitrite release was independent of the length or type of compressive regime applied. The uncoupled nature of the metabolic response determined in this study suggests that mechanical conditioning regimes may be fine tuned to selectively stimulate key metabolic parameters of relevance to cartilage tissue engineering.

Animals↗
PubMed2003

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques.

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged inmonolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.

Alginates↗
PubMed2003

Influence of external uniaxial cyclic strain on oriented fibroblast-seeded collagen gels.

This study investigates the influence of cyclic tensile strain, applied to fully contracted fibroblast-seeded collagen constructs. The constructs were preloaded to either 2 or 10 mN. The preloaded constructs were subsequently subjected to a further 10% cyclic strain (0-10%) at 1 Hz, using a triangular waveform, or were cultured in the preloaded state. In all cases cellular viability was maintained during the conditioning period. Cell proliferation was enhanced by the application of cyclic strain within constructs preloaded to both 2 and 10 mN. Collagen synthesis was enhanced by cyclic strain within constructs preloaded at 2 mN only. The profile of matrix metalloproteinase (MMP) expression, determined by zymography, was broadly similar in constructs preloaded at 2 mN with or without the application of cyclic strain. By contrast, constructs preloaded at 10 mN and subjected to cyclic strain expressed enhanced levels of staining for latent MMP-1, latent MMP-9, and both latent and active MMP-2, when compared with the other conditioning regimens. The structural stiffness of constructs preloaded at 2 mN and subjected to cyclic strain was enhanced compared with control specimens, reflecting the increase in collagen synthesis. By contrast, the initial failure loads for cyclically strained constructs preloaded at 10 mN were reduced, potentially because of enhanced catabolic activity.

Collagen↗
PubMed2003

Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death.

Isolated chondrocytes stained with fluo 4-AM and visualized using standard confocal microscopy techniques exhibited Ca2- transients and oscillations. Decreasing the power of the laser light decreased the percent-age of cells exhibiting these Ca2+ signals. Treatment with the antioxidant ascorbate reduced the Ca2+ response, suggesting that it was mediated by light-induced release of reactive oxygen species (ROS). Cell viability 24 h after the 1-h confocal imaging period was approximately 90% for cells that were neither fluorescently stained nor subjected to laser excitation. By contrast, fluorescently stained cells imaged for 1 h exhibited greatly reduced viability. Treatment with ascorbate reduced the level of cell death, suggesting that the effect was mediated by release of exogenous ROS associated with the interaction of light and the fluorochrome. Ca2+ oscillations were not always associated with cell death, suggesting that separate light-sensitive pathways mediate the two processes. Light-activated Ca2+ signaling may trigger alterations in numerous cell processes and thereby represent an important and hitherto overlooked artifact in fluorescent microscopy of viable cells.

Aniline Compounds↗