Dan L. Bader

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.

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

Abstract 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 mechano-transduction 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.

In vitro fatigue of human tendons

The purpose of this study was to determine the fatigue behaviour of human tendons in vitro. The testing was accomplished with the use of specially designed grips and the local measurement of tendon cross-sectional area. Ninety specimens prepared from Extensor digitorum longus (EDL) tendons of the foot were subjected to a cyclic square tension-tension stress waveform at physiological frequencies. The maximum tensile stress was normalised to values corresponding to prescribed levels between 10% and 90% of the calculated ultimate tensile strength (UTS) of 100 MPa. The minimum stress was set at 1% of the UTS. A replication of 10 specimens per stress level allowed the use of statistical models for the distribution of fatigue life. Results followed a linear model, of form S = 101.3 - 14.8 log(N), relating the normalised stress to the median number of cycles to failure, therefore suggesting the absence of an endurance limit. The Weibull distribution was found to describe adequately the probability of failure at each stress level. A model which takes into account in vivo healing was proposed. This model was able to explain the presence of intact tendons throughout the lifetime of an individual.

Can Loaded Interface Characteristics Influence Strain Distributions in Muscle Adjacent to Bony Prominences

Pressure distributions at the interface between skin and supporting tissues are used in design of supporting surfaces like beds, wheel chairs, prostheses and in sales brochures to support commercial products. The reasoning behind this is, that equal pressure distributions in the absence of high pressure gradients is assumed to minimise the risk of developing pressure sores. Notwithstanding the difficulty in performing reproducible and accurate pressure measurements, the question arises if the interface pressure distribution is representative of the internal mechanical state of the soft tissues involved. The paper describes a study of the mechanical condition of a supported buttock contact, depending on cushion properties, relative properties of tissue layers and friction. Numerical, mechanical simulations of a buttock on a supporting cushion are described. The ischial tuberosity is modelled as a rigid body, whereas the overlying muscle, fat and skin layers are modelled as a non-linear Ogden material. Material parameters and thickness of the fat layer are varied. Coulomb friction between buttock and cushion is modelled with different values of the friction coefficient. Moreover, the thickness and properties of the cushion are varied. High shear strains are found in the muscle near the bony prominence and the fat layer near the symmetry line. The performed parameter variations lead to large differences in shear strain in the fat layer but relatively small variations in the skeletal muscle. Even with a soft cushion, leading to a high reduction of the interface pressure the deformation of the skeletal muscle near the bone is high enough to form a risk, which is a clear argument that interface pressures alone are not sufficient to evaluate supporting surfaces.

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.

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.

Deep tissue injury: how deep is our understanding?

Deep pressure ulcers, necessarily involving deep tissue injury (DTI), arise in the muscle layers adjacent to bony prominences because of sustained loading. They represent a serious type of pressure ulcer because they start in underlying tissues and are often not visible until they reach an advanced stage, at which time treatment becomes problematic. Underlying mechanisms of DTI require further investigation if appropriate preventive measures are to be determined. The present commentary illustrates a hierarchic research approach selected to study these mechanisms. To differentiate between the individual roles of deformation and ischemia in the onset of skeletal muscle damage, 2 complementary approaches have been selected. In an in vivo animal model, the effects of ischemia combined with deformation and ischemia per se were studied. An in vitro muscle model was used to study the separate effects of deformation and several aspects of ischemia, including hypoxia, glucose depletion, and tissue acidification, in more detail. Based on the results of both models a sequence of events leading to cell necrosis is proposed. Deformation levels exceeding a threshold value can result in rapid tissue damage that may persist, whereas ischemia has a more gradual effect as a result of glucose depletion and tissue acidification.

Compression Induced Cell Damage in Engineered Muscle Tissue: An In Vitro Model to Study Pressure Ulcer Aetiology

AbstractThe 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.

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 in monolayer 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.