Noa Slomka’s research while affiliated with Tel Aviv University and other places

Deep tissue injury (DTI) is a life-threatening type of pressure ulcer which initiates subdermally with muscle necrosis at weight-bearing anatomical locations, where localized elevated tissue strains exist. Though it has been suggested that excessive sustained soft tissue strains might compromise cell viability, which then initiates the DTI, there is no experimental evidence to describe how specifically such a process might take place. Here, we experimentally test the hypothesis that macroscopic tissue deformations translated to cell-level deformations and in particular, to localized tensile strains in the plasma membrane (PM) of cells, increase the permeability of the PM which could disrupt vital transport processes. In order to determine whether PM permeability changes can occur due to static stretching of cells we measured the uptake of fluorescein isothiocyanate (FITC)-labeled Dextran (molecular weight = 4 kDa) by deformed vs. undeformed myoblasts, using a fluorescence-activated cell sorting (FACS) method. These PM permeability changes were then correlated with tensile strains in the PM which correspond to the levels of substrate tensile strain (STS) that were applied in the experiments. The PM strains were evaluated by means of confocal-microscopy-based cell-specific finite element (FE) modeling. The FACS studies demonstrated a statistically significant rise in the uptake of the FITC-labeled Dextran with increasing STS levels in the STS ≤ 12% domain, which thereby indicates a rise in the permeability of the PM of the myoblasts with the extent of the applied cellular deformation. The cell-specific FE modeling simulating the experiments further demonstrated that applying average PM tensile strains which exceed 3%, or, applying peak PM tensile strains over 9%, substantially increases the permeability of the PM of myoblasts to the Dextran. Moreover, the permeability of the PM grew rapidly with any further increase in PM strains, though there were no significant changes in the uptake above average and peak PM tensile strain values of 9 and 26%, respectively. These results provide an experimental basis for studying the theory that cell-level deformation-diffusion relationships may be involved in determining the tolerance of soft tissues to sustained mechanical loading, as relevant to the etiology of DTI.

In the present study, we employ our recently developed confocal microscopy-based cell-specific finite element (FE) modeling method, which is suitable for large deformation analyses, to conduct inverse FE analyses aimed at determining the shear modulus of the cytoplasm of cultured skeletal myoblasts, G(cp), and its variation across a number of cells. We calibrate these cell-specific models against experimental data describing the force-deformation behavior of the same cell type, which were published by Peeters et al. (2005b) [J. Biomech.]. The G(cp) calculated for five different myoblasts were contained in the range of 0.8-2.4 kPa, with the median value being 1 kPa, the mean being 1.4 kPa, and the standard deviation being 0.7 kPa. The normalized sum of squared errors resulting from the fit between experimental and calculated force-deformation curves ranged between 0.12-0.73%, and Pearson correlations for all fits were greater than 0.99. Determining the mechanical properties of the cytoplasm through cell-specific FE will now allow calculation of cell stresses using cell-specific FE under various cell loading configurations, in support of experimental work in cellular mechanics.

Many biological consequences of external mechanical loads applied to cells depend on localized cell deformations rather than on average whole-cell-body deformations. Such localized intracellular deformations are likely to depend, in turn, on the individual geometrical features of each cell, e.g., the local surface curvatures or the size of the nucleus, which always vary from one cell to another, even within the same culture. Our goal here was to characterize cell-to-cell variabilities in magnitudes and distribution patterns of localized tensile strains that develop in the plasma membrane (PM) and nuclear surface area (NSA) of compressed myoblasts, in order to identify resemblance or differences in mechanical performances across the cells. For this purpose, we utilized our previously developed confocal microscopy-based three-dimensional cell-specific finite element modeling methodology. Five different C2C12 undifferentiated cells belonging to the same culture were scanned confocally and modeled, and were then subjected to compression in the simulation setting. We calculated the average and peak tensile strains in the PM and NSA, the percentage of PM area subjected to tensile strains above certain thresholds and the coefficient of variation (COV) in average and peak strains. We found considerable COV values in tensile strains developing at the PM and NSA (up to ~35%) but small external compressive deformations induced greater variabilities in intracellular strains across cells compared to large deformations. Interestingly, the external deformations needed to cause localized PM or NSA strains exceeding each threshold were very close across the different cells. Better understanding of variabilities in mechanical performances of cells-either of the same type or of different types-is important for interpreting experimental data in any experiments involving delivery of mechanical loads to cells.

Confocal-microscopy-based three-dimensional (3D) cell-specific finite element (FE) modeling has recently been introduced by our group as a method to simulate the structural behavior of realistic cell geometries under external loading, while considering details of intracellular organelle [1,2]. This method provides comprehensive knowledge regarding cellular mechanics problems, for example, it is useful in the context of understanding the aetiology of deep tissue injury (DTI) — a type of a serious pressure ulcer associated with sustained cellular deformations [3–6]. In this regard, we previously postulated that sustained deformations of soft tissues near bony prominences could cause cell death by a mechanism of locally stretching cells, the consequence of which being that the permeability of the plasma membrane and nuclear surface area (NSA) in the affected cells increases. This, in turn, pathologically changes cell-matrix and intracellular transport profiles and eventually disrupts cellular homeostasis [1,7]. We hypothesize that tensile strains in the plasma membrane and NSA might differ in magnitude and pattern across externally-loaded individual cells of the same cell type, due to cell-to-cell morphological differences. Hence, in this study, we utilize confocal-based cell-specific 3D modeling to analyze tensile strain states in the plasma membrane and NSA of 3 different skeletal muscle cells (myoblasts) subjected to compression. We were specifically interested in chacterizing cell-to-cell variability in magnitudes and patterns of the localized strains.

The mechanical and biological response of cells to various loading regimes is a subject of great interest in the research field of biomechanics. Extensive utilization of different cellular mechanics experimental designs has been made over the years in order to provide better insight regarding the mechanical behavior of cells, and the mechanisms underlying the transduction of the applied loads into biological reactions. These experimental protocols have limited ability in directly measuring different mechanical parameters (e.g. internal cellular strains and stresses). In addition, they are very costly and involve highly complex apparatuses and experimental designs. Thus, further understating of cellular response can be achieved by means of computational models, such as the finite element (FE) method. FE modeling of cells is an emerging direction in the research field of cellular mechanics. Its application has been rapidly growing over the last decade due to its ability to quantify deformations, strains and stresses in and around cells, thus providing basic understating of the mechanical state of cells and allowing identification of mechanical properties of cells and cellular organelles when coupled with appropriate experiments. In this chapter, we review the two-dimensional (2D) and three-dimensional (3D) reported cell models of various cell types, subjected to different applied mechanical stimuli, e.g. compression, micropipette aspiration, indentation.

This study introduces a new confocal microscopy-based three-dimensional cell-specific finite element (FE) modeling methodology for simulating cellular mechanics experiments involving large cell deformations. Three-dimensional FE models of undifferentiated skeletal muscle cells were developed by scanning C2C12 myoblasts using a confocal microscope, and then building FE model geometries from the z-stack images. Strain magnitudes and distributions in two cells were studied when the cells were subjected to compression and stretching, which are used in pressure ulcer and deep tissue injury research to induce large cell deformations. Localized plasma membrane and nuclear surface area (NSA) stretches were observed for both the cell compression and stretching simulation configurations. It was found that in order to induce large tensile strains (>5%) in the plasma membrane and NSA, one needs to apply more than approximately 15% of global cell deformation in cell compression tests, or more than approximately 3% of tensile strains in the elastic plate substrate in cell stretching experiments. Utilization of our modeling can substantially enrich experimental cellular mechanics studies in classic cell loading designs that typically involve large cell deformations, such as static and cyclic stretching, cell compression, micropipette aspiration, shear flow and hydrostatic pressure, by providing magnitudes and distributions of the localized cellular strains specific to each setup and cell type, which could then be associated with the applied stimuli.

Deep tissue injury (DTI) is a serious lesion typically involving necrosis of skeletal muscle tissue under intact skin. Currently, considerable research efforts are invested in understanding the mechanisms underlying the onset and progression of DTI. Recent studies indicated the involvement of deformation-related events at the cellular scale. Nevertheless, the specific processes at the cell level which ultimately lead to DTI formation are still unknown. We hypothesize that stretchinduced increase in the local permeability of plasma membranes may lead to intracellular cytotoxic concentrations of cell metabolites. A three-dimensional finite-element (FE) analysis of a compressed single skeletal muscle cell (from a murine C2C12 myoblast cell line) was conducted in order to study the aspects of localized plasma membrane stretches. Geometry of the cell was based on confocal microscopy images of an actin-stained cell, specifically stained with FITC-labeled phalloidin. The cell was compressed in the FE simulation by a rigid plate, up to a maximal global deformation of 70%. Large deformation strain analysis was preformed, and maximal local principal strains in the plasma membrane were obtained as function of the global deformation applied to the cell. It was found that platen compression causes substantial tensional strains in segments of the plasma membrane.

Deep tissue injury (DTI) is a serious pressure ulcer, involving a mass of necrotic soft tissue under bony prominences as a consequence of sustained tissue deformations. Though several processes are thought to participate in the onset and development of DTI (e.g., cellular deformation, ischemia, and ischemia-reperfusion), the specific mechanisms responsible for it are currently unknown. Recent work indicated that pathological processes at the cell level, which relate to cell deformation, are involved in the etiology. We hypothesized that sustained tissue deformations can lead to elevated intracellular concentration of cell metabolites, e.g., calcium ion (Ca2+), due to a stretch-induced increase in the local permeability of plasma membranes. This may ultimately lead to cell death due to intracellular cytotoxic concentrations of metabolites. In order to investigate this hypothesis, computational models were developed, for determining compression-induced membrane stretches and trends of times for reaching intracellular cytotoxic Ca2+ levels due to uncontrolled Ca2+ influx through stretched membranes. The simulations indicated that elevated compressive cell deformations exceeding 25% induce large tensional strains (>5%, and up to 11.5%) in membranes. These are likely to increase Ca2+ influx from the extracellular space into the cytosol through the stretched sites. Consistent with this assumption, the Ca2+ transport model showed high sensitivity of times for cell death to changes in membrane resistance. These results may open a new path in pressure ulcer research, by indicating how global tissue deformations are transformed to plasma membrane deformations, which in turn, affect transport properties and eventually, cell viability.

Deep tissue injury (DTI) is a serious lesion typically involving necrosis of skeletal muscle and fat tissues under intact skin. Currently, considerable research efforts are invested in understanding the mechanisms underlying the onset and progression of DTI. Recent studies indicated the involvement of deformation-related events at the cellular scale. Nevertheless, the specific processes at the cell level which ultimately lead to DTI are still unknown. We hypothesize that sustained deformations of soft tissues may lead to individual cell death, as a result of alteration in intracellular concentrations of cell metabolites that occur due to local plasma membrane stretches. A two-dimensional model of an adhered single generalized cell, three-dimensional models of adhered single myoblast and fibroblast, and a construct of cells embedded in ECM were developed. Finite-Element analyses of the compressed models were performed in order to study localized plasma membrane stretches. Models were compressed by a rigid plate, up to maximal global deformations of 65%, 35%, and 45%, respectively. Large deformation strain analysis was performed, and maximal local principal strains in the plasma membrane of the cells were obtained as function of the global deformation applied to the model. All models indicated that platen compression causes large tensional strains in segments of the plasma membrane. Three-Dimensional models of real cell geometry exhibited a maximal tensional strain of approximately 20%, at global cell deformation of 35%. These results support our above hypothesis, and may provide a new path in DTI research.

The purposes of the present study were to (1) determine the internal plantar mechanical stresses in diabetic and healthy subjects during everyday activities, and (2) identify stress parameters potentially capable of distinguishing between diabetic and healthy subjects. A self-designed, portable, real-time and subject-specific foot load monitor which employs the Hertz contact theory was utilized to determine the internal dynamic plantar tissue stresses in 10 diabetic patients and 6 healthy subjects during free walking and outdoors stair climbing. Internal stress parameters and average stress-doses were evaluated, and the results obtained from the two groups were compared. Internal plantar stresses and averaged stress-doses during free walking and outdoors stairs climbing in the diabetic group were 2.5-5.5-fold higher than in the healthy group (p<0.001; stair climbing comparisons incorporated data from five diabetic patients). The interfacial pressures measured during free walking were slightly higher ( approximately 1.5-fold) in the diabetic group (p<0.05), but there was no significant difference between the two groups during stairs climbing. We conclude that during walking and stair climbing, internal plantar tissue stresses are considerably higher than foot-shoe interface pressures, and in diabetic patients, internal stresses substantially exceed the levels in healthy. The proposed method can be used for rating performances or design of footwear for protecting sub-dermal plantar tissues in patients who are at risk for developing foot ulcers. It may also be helpful in providing biofeedback to neuropathic diabetic patients.