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

Compressive deformation and damage of muscle cell sub-populations 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. © 2001 Biomedical Engineering Society

Predicting the optimal geometry of microneedles and their array for dermal vaccination using a computational model

Microneedle arrays have been developed to deliver a range of biomolecules including vaccines into the skin. These microneedles have been designed with a wide range of geometries and arrangements within an array. However, little is known about the effect of the geometry on the potency of the induced immune response. The aim of this study was to develop a computational model to predict the optimal design of the microneedles and their arrangement within an array. The three-dimensional finite element model described the diffusion and kinetics in the skin following antigen delivery with a microneedle array. The results revealed an optimum distance between microneedles based on the number of activated antigen presenting cells, which was assumed to be related to the induced immune response. This optimum depends on the delivered dose. In addition, the microneedle length affects the number of cells that will be involved in either the epidermis or dermis. By contrast, the radius at the base of the microneedle and release rate only minimally influenced the number of cells that were activated. The model revealed the importance of various geometric parameters to enhance the induced immune response. The model can be developed further to determine the optimal design of an array by adjusting its various parameters to a specific situation

Investigating the influence of intermittent and continuous mechanical loading on skin through non-invasive sampling of IL-1α

\u3cp\u3ePressure ulcers (PUs) are a major burden to both patients, carers and the healthcare system. It is therefore important to identify patients at risk and detect pressure ulcers at an early stage of their development. The pro-inflammatory cytokine IL-1α is a promising indicator of tissue damage. The aim of this study was to compare the temporal skin response, by means of IL-1α expression, to different loading regimens and to investigate the presence of individual variability. The sacrum of eleven healthy volunteers was subjected to two different loading protocols. After a baseline measurement, the left and right side of the sacrum were subjected to continuous and intermittent loading regimen, respectively, at a pressure of 100 mmHg. Data was collected every 20 min, allowing for a total experimental time of 140 min. Sebum, collected at ambient conditions using Sebutape, was analyzed for the pro-inflammatory cytokine IL-1α. Most robust results were obtained using a baseline normalization approach on individual data. The IL-1α level significantly changed upon load application and removal (p<0.05) for both loading regimens. Highest IL-1α ratio increase, 3.7-fold, was observed for 1 h continuous loading. During the refractory periods for both loading regimen the IL-1α levels were still found to be up-regulated compared to baseline (p<0.05). The IL-1α level increased significantly for the two initial loading periods (p<0.05), but stabilized during the final loading period for both loading regimens. Large individual variability in IL-1α ratio was observed in the responses, with median values of 1.91 (range 1.49–3.08), and 2.52 (range 1.96–4.29), for intermittent and continuous loading, respectively, although the differences were not statistically significant. Cluster analysis revealed the presence of two distinct sub-populations, with either a low or high response to the applied loading regimen. The measurement after the first loading period proved to be representative for the subsequent measurements on each site. This study revealed that trends in normalized IL-1α provided an early indicator for tissue status following periods of mechanical loading and refractory unloaded conditions. Additionally, the observed individual variability in the response potentially identifies patients at risk of developing PUs.\u3c/p\u3

Myoglobin and troponin concentrations are increased in early stage deep tissue injury

\u3cp\u3e Pressure-induced deep tissue injury is a form of pressure ulcer which is difficult to detect and diagnose at an early stage, before the wound has severely progressed and becomes visible at the skin surface. At the present time, no such detection technique is available. To test the hypothesis that muscle damage biomarkers can be indicative of the development of deep tissue injury after sustained mechanical loading, an indentation test was performed for 2 h on the tibialis anterior muscle of rats. Myoglobin and troponin were analysed in blood plasma and urine over a period of 5 days. The damage as detected by the biomarkers was compared to damage as observed with T \u3csub\u3e2\u3c/sub\u3e MRI to validate the response. We found that myoglobin and troponin levels in blood increased due to the damage. Myoglobin was also increased in urine. The amount of damage observed with MRI immediately after loading had a strong correlation with the maximal biomarker levels: troponin in blood r \u3csub\u3es\u3c/sub\u3e = 0.94; myoglobin in blood r \u3csub\u3es\u3c/sub\u3e = 0.75; and myoglobin in urine r \u3csub\u3es\u3c/sub\u3e = 0.57. This study suggests that muscle damage markers measured in blood and urine could serve as early diagnosis for pressure induced deep tissue injury. \u3c/p\u3

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

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