Experimental and theoretical analyses of compression induced muscle damage : aetiological factors in pressure ulcers

Pressure ulcers form a major problem in health care. They often occur when patients are bedridden, wheelchair bound or wearing prostheses. The ulcers can be very painful for the patient and often lead to prolonged hospitalization. In addition, the huge costs involved with treatment and prevention put a heavy burden on heath care budgets. Pressure ulcers occur often: between 14% and 33% of the patients in health care institutions develop an ulcer, ranging from discolouration of the skin to severe wounds involving necrosis of epidermis, extending to underlying bone, tendon and joints. It is clear that pressure ulcers are caused by prolonged mechanical loading, applied at the interface between skin and support surfaces. However, the aetiology of pressure ulcers is poorly understood. This forms an important obstacle in decreasing the unacceptably high prevalence figures. It is anticipated that a better understanding of the mechanobiological pathways leading to cell and tissue damage can lead to a breakthrough in reducing pressure ulcer prevalence. In addition, a solid scientific base may establish tools for objective risk assessment and judgement of preventive measures. The present study focuses on deep ulcers that initiate in skeletal muscle tissue, since deep ulcers are more extensive and often difficult to prevent. To obtain insight into the aetiology of these deep ulcers, it is necessary to understand the transfer from externally applied loads at the skin, to the local conditions that the cells experience within the tissue. In addition, the question which local conditions are harmful to the cell needs to be investigated. By combining knowledge on "what a cell feels" with knowledge on potentially harmful conditions, a better judgement of dangerous situations may be achieved. Although several causes of cell damage may play a role in the initiation of pressure ulcers, the present study focussed on the impact of cell deformations. To investigate the hypothesis that prolonged cell deformations lead to cell damage at clinically relevant strains, an experimental model system was developed. A key requirement of this experimental model is the possibility to study the role of cell deformation on cell damage independently of other possible causes of damage. To achieve this, in-vitro engineered muscle tissue constructs were developed. These constructs were compressed using a newly developed compression device. A custom made incubator system was developed to allow monitoring of the constructs for extended periods of time. In addition, a novel assay was developed to determine the viability of the cells during compression. This assay provides quantitative and spatial information on cell damage throughout a construct in a non-invasive manner, making use of fluorescent dyes which are visualized by confocal microscopy. The compression of the engineered muscle tissue constructs indicated that a significant increase in cell death occurs within 1-2 hours and that higher strain levels led to an earlier increase in damage. In addition, it was demonstrated that cell damage was uniformly distributed across the indented area of the construct, without a gradient in percentage dead cells between the centre and periphery of the constructs. The results strongly suggest that prolonged cell deformation was the predominant cause of cell damage in these experiments. This puts a new light on observations in literature which suggested that ischaemia is not the sole determinant for the onset of pressure ulcers. Nevertheless, more experiments are needed to clarify the role of prolonged cell deformations on cell damage. First, it is recommended that the actual local cell deformations are quantified during compression of the constructs. Furthermore, from the present experiments it could not be excluded that the compression of the constructs decreased the permeability of the construct and hence affected cellular metabolism. In future, measuring diffusion pathways of both small molecules and larger vital molecules, may indicate whether this change in permeability is significant. A numerical model was developed to predict local cell deformations, in response to tissue compression. Since the local cell deformations cannot be a-priori determined on the basis of homogenized tissue deformations, a multilevel finite element approach was adopted. In this approach, cell deformations are predicted from detailed nonlinear finite element analyses of the local microstructures of the tissue, which consist of an arrangement of cells embedded in a matrix material. To avoid unacceptably large computational times, the multilevel model was designed to run on a parallel computer system. Application of the multilevel model showed that the heterogeneity of the microstructure of the tissue has a profound impact on local cell deformations, which highly exceeded macroscopic tissue deformations. Moreover, microstructural heterogeneity led to complex cell shapes and caused non-uniform deformations within the cells. To investigate the evolution of compression induced damage in skeletal muscle tissue, the multilevel model was extended with a damage law, which was derived from the in-vitro experiments. With this model, the compression of muscle tissue against a bony prominence was simulated. The percentage of cell damage in the microstructure of the tissue was computed, which could be extrapolated to the bulk tissue level. In the present form, a schematic geometry was considered that intended to elucidate general patterns of tissue damage evolution. The simulations confirmed that it is not feasible to predict the onset of tissue damage on the basis of externally applied loading conditions at the skin surface alone, since these externally applied loads are not indicative of the local mechanical conditions that the cells experience within the tissue. In addition, the simulations showed that it is necessary to consider the local load history of the cells, and the tolerance of the tissue. These findings may explain why a strikingly large variability in load/time threshold values was found in animal studies, which attempted to relate external mechanical to tissue damage, thereby ignoring the local mechanical conditions within the tissue. At present, it is premature to utilize the models presented in this thesis in clinical practice, since the extrapolation towards human patients requires more research. Clearly, further extensions and validation of the numerical model with experimental animal models will be required. This should finally lead to the application in more realistic cases, involving patient data on geometry and tissue properties. Nevertheless, the present models provided an essential step towards evidence based risk assessment and prevention

Collagen type V modulates fibroblast behavior dependent on substrate stiffness

10 kPa), but not on stiffer substrates (68 kPa or glass). In sharp contrast, a pure collagen I coating does not impair cell spreading on soft substrates. The impairment of cell spreading by collagen V is accompanied by diffuse actin staining patterns and small focal adhesions. These observations suggest that collagen V plays an essential role in modifying cell behavior during development and remodeling, when very soft tissues are presen

In vitro models to study compressive strain-induced muscle cell damage

Skeletal muscle tissue is highly susceptible to sustained compressive straining, eventually leading to tissue breakdown in the form of pressure sores. This breakdown begins at the cellular level and is believed to be triggered by sustained cell deformation. To study the relationship between compressive strain-induced muscle cell deformation and damage, and to investigate the role of cell-cell interactions, cell-matrix interactions and tissue geometry in this process, in vitro models of single cells, monolayers and 3D tissue analogs under compression are being developed. Compression is induced using specially designed loading devices, while cell deformation is visualised with confocal microscopy. Cell damage is assessed from viability tests, vital microscopy and histological or biochemical analyses. Preliminary results from a 3D cell seeded agarose model indicate that cell deformation is indeed an important trigger for cell damage; sustained compression of the model at 20% strain results in a significant increase in cell damage with time of compression, whereas damage in unstrained controls remains constant over time

A theoretical analysis of damage evolution in skeletal muscle tissue with reference to pressure ulcer development

Soft tissues are sensitive to prolonged compressive loading, eventually leading to tissue necrosis in the form of pressure ulcers [1]. Pressure ulcers can occur in situations where people are subjected to sustained mechanical loads, such as when bedridden, sitting in a wheelchair or from wearing prostheses. Pressure ulcers severely affect the patient's quality of life, since the ulcers are painful, difficult to heal and often prolong hospitalization periods. Despite considerable attempts to prevent pressure ulcers, prevalence figures remain unacceptably high. In a prevalence study involving more than 16,000 patients in the Netherlands, a mean prevalence of 23.1% in health care institutions was reported [6]. Pressure ulcers are often classified in four different stages, ranging from discoloration of intact skin (Stage I) to full thickness skin loss involving tissue damage that extends to underlying bone involving both fat and muscle tissues (Stage IV). Although this classification is widely used in clinical practice, it does not necessarily relate to the origin of the ulcers. Depending on the nature of loading, pressure ulcers can either initiate superficially at the skin [15][33], or initiate in deeper layers such as muscle tissue [14][18][20][28]. The present study focuses on deep pressure ulcers that initiate in muscle tissue, since deeper ulcers are more harmful and show more extensive ulceration. In addition, muscle tissue is more susceptible to the development of pressure ulcers [2][23] and, hence, deep ulcers develop at a faster rate than superficial ulcers, making them particularly dangerous [2][14]. Yet, these deep ulcers are difficult to prevent and identify, since they are rarely visible at the skin surface at the time of initiation. As such, the four stage classification scheme can be misleading, since it does not represent an ordinal scale. In literature, a number of theories have been proposed to explain the pathophysiology of pressure ulcers. These theories suggest that compression of a tissue can lead to localized ischaemia, impaired interstitial fluid flow and/or insufficient lymphatic drainage. Furthermore, relieving the tissue after sustained compression may lead to reperfusion injury. Recently, it was demonstrated that the deformation of cells due to external loading of the tissue can directly induce cell damage [8]. The primary cause of deep pressure ulcers is the external load applied at the patient support interface, resulting in compression of the tissue between the skin surface and the bony prominences. However, the externally applied load is not indicative of the local mechanical conditions in underlying muscle tissue (for example expressed in terms of stresses and strains), and thus not directly related to tissue damage [12][24][30]. In particular, when a tissue is compressed against an irregularly shaped bony prominence, the local mechanical condition within the muscle tissue may well exceed the measured loading condition at the skin interface. Therefore, the local mechanical loading condition could prove a more reliable predictor for the onset of tissue damage in these deeper tissue layers. When considering the local mechanical tissue condition, it should be noticed that an averaged mechanical condition associated with a small volume within the tissue, is not representative of the mechanical condition experienced at the cellular level. This is due to the heterogeneity of the microstructure [10][17][31]. Since tissue damage initiates with local cellular damage [7][11][20][23], it is necessary to consider this microstructure. In addition, the microstructural behavior must be considered since it inevitably determines the macroscopic constitutive behavior of the tissue. For example, when cell damage occurs, the microstructure is likely to change, which will be reflected in the macroscopic behavior of the tissue. In this case, it is not possible to describe the macroscopic behavior with a conventional constitutive law [10][21]. Since local mechanical conditions are difficult to measure experimentally, theoretical and numerical models have been developed, which relate externally applied pressures to the local mechanical condition within a tissue. In a theoretical model based on dimensional analyses, Sacks [27] derived a relationship between tolerable external pressure and duration of pressure. The rationale behind this analysis was that there is a definable pressure that causes a pressure ulcer, which is a function of both tissue properties and the blood flow through the tissue. The model did not take into account the local geometry of, for example, a bony prominence, leading to load distributions in the tissues. Zhang and coworkers [33] performed a theoretical analysis on the stress distribution within the tissue after application of shear and normal forces at the skin. To allow calculation of an analytical solution, both skin and muscle tissue were assumed to behave as linear, isotropic elastic materials. This model demonstrated that the highest stress concentrations were found within deeper layers of the soft tissue. To analyze more complex geometries and material behavior, the use of computer models, in particular the finite-element (FE) approach, is indispensable. Chow and Odell [12] and Todd and Tacker [30] developed finite element models of the human buttocks. In the model of Todd and Tacker [30], seated positions were simulated, thereby manipulating boundary conditions of the model. The authors concluded that there is no clear correlation between interface pressure and the local mechanical conditions. Oomens and coworkers [24] created a FE model of a human subject sitting on a cushion, which incorporated three different tissue layers overlaying the human ischial tuberositas, muscle, fat and skin. These soft tissues were modeled as nonlinear viscoelastic materials. Despite the uncertainties in material properties, the high peak stresses were consistently found near the bone prominences and in the fat layer. Furthermore, models based on mixture theory have been proposed to examine the transient biomechanical response of a skin layer as a result of tissue fluid flow within the tissue [22][33]. The numerical models described in literature focus on determination of local mechanical conditions in skin and underlying tissues in terms of homogenized tissue stresses and strains [12][22][25][30][32][33]. However, from the previous discussion it is clear that a microstructural analysis is required to determine the mechanical conditions that a cell experiences. Moreover, knowledge of the local mechanical condition alone is not sufficient to predict tissue damage initiation and evolution. Also the loading history of the tissue is essential, since the time that a tissue is subjected to a sustained compression is a major determinant of tissue damage [26]. A second aspect that, to date, has received relatively little attention, is the tissue tolerance against compression. Different tissues, such as skin, fat and muscle, may have a different tolerance level, which in turn may be patient dependent. The objective of the present study is to illustrate that prediction of pressure ulcer initiation on the basis of external load measurements is inadequate. By considering local load-time threshold curves, it is illustrated which variables quantitatively influence the threshold curves and, therefore, should be considered for the prediction of damage initiation. To this end, a numerical model was developed, which is based on a multilevel FE approach. This model can relate external loads applied to the skin, to the local mechanical conditions at the cell level. With this model, simulations were performed in which muscle tissue is compressed against a bony prominence. As a starting point, the model focuses on the role of cell deformations on cell damage. The model incorporates both the time aspect and a tissue tolerance level for damage in the form of a damage law that was derived from in vitro experiments [9]. A parameter study was performed to illustrate the effect of the cell tolerance and cell stiffness on the macroscopic tissue damage evolution