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PURPOSE\nThe aim of this study was to investigate the depth-dependent intradermal immunogenicity of inactivated polio vaccine (IPV) delivered by depth-controlled microinjections via hollow microneedles (HMN) and to investigate antibody response enhancing effects of IPV immunization adjuvanted with CpG oligodeoxynucleotide 1826 (CpG) or cholera toxin (CT).\nMETHODS\nA novel applicator for HMN was designed to permit depth- and volume-controlled microinjections. The applicator was used to immunize rats intradermally with monovalent IPV serotype 1 (IPV1) at injection depths ranging from 50 to 550 μm, or at 400 μm for CpG and CT adjuvanted immunization, which were compared to intramuscular immunization.\nRESULTS\nThe applicator allowed accurate microinjections into rat skin at predetermined injection depths (50-900 μm), -volumes (1-100 μL) and -rates (up to 60 μL/min) with minimal volume loss (±1-2%). HMN-mediated intradermal immunization resulted in similar IgG and virus-neutralizing antibody titers as conventional intramuscular immunization. No differences in IgG titers were observed as function of injection depth, however IgG titers were significantly increased in the CpG and CT adjuvanted groups (7-fold).\nCONCLUSION\nIntradermal immunogenicity of IPV1 was not affected by injection depth. CpG and CT were potent adjuvants for both intradermal and intramuscular immunization, allowing effective vaccination upon a minimally-invasive single intradermal microinjection by HMN.

Background Repositioning of individuals with reduced mobility and at risk of pressure ulcers is an essential preventive step. Manual or automatic lateral tilting is a way of doing this and the international guidelines propose a 30° to 40° side lying position. The goal of the present study was to determine the internal strains in individuals lying in a supine position and during tilting. Methods Based on magnetic resonance imaging (MRI) of the sacral area of human volunteers, subject specific finite element models were developed. By comparing calculated contours of the skin, fat and muscle with MRI measurements on a flat surface the models were validated. A parameter study was performed to assess the sensitivity of the model for changes in material properties. Simulations were performed at tilting angles of volunteers between 0° and 45°. Findings Subjects in a supine position or tilted have the highest strains in the muscle and fat. Tilting does affect the strain distribution, taking away the highest peak strains. There seems to exist an optimal tilting angle between 20° and 30°, which may vary depending on factors such as BMI of the subject and is in the current paper investigated only for the sacrum. Interpretation The study shows that tilting indeed has a significant, positive influence on internal strains, which is important for the prevention of deep tissue injury. Additional studies are needed to draw conclusions about the greater trochanter area and the tissues around the shoulder.

Deep tissue injury (DTI), a type of pressure ulcer, arises in the muscle layers adjacent to bony prominences due to sustained mechanical loading. DTI presents a serious problem in the clinic, as it is often not visible until reaching an advanced stage. One of the causes can be direct mechanical deformation of the muscle tissue and cell. The mechanism of cell death induced by mechanical compression was studied using bio-artificial skeletal muscle tissues. Compression was applied by placing weights on top of the constructs. The morphological changes of the cytoskeleton and the phosphorylation of mitogen-activated protein kinases (MAPK) under compression were investigated. Moreover, inhibitors for each of the three major MAPK groups, p38, ERK, and JNK, were applied separately to look at their roles in the compression caused apoptosis and necrosis. The present study for the first time showed that direct mechanical compression activates MAPK phosphorylation. Compression also leads to a gradual destruction of the cytoskeleton. The percentage apoptosis is strongly reduced by p38 and JNK inhibitors down to the level of the unloaded group. This phenomenon could be observed up to 24. h after initiation of compression. Therefore, cell death in bio-artificial muscle tissue caused by mechanical compression is primarily caused by a physiological mechanism, rather than through a physical mechanism which kills the cell directly. These findings reveal insight of muscle cell death under mechanical compression. Moreover, the result indicates a potential clinical solution to prevent DTI by pre-treating with p38 or/and JNK inhibitors.

The skin is a promising location for vaccination with its abundant population ofantigen capturing and presenting cells. The development of new techniques, suchas the use of microneedles, can facilitate the delivery of vaccines into theskin. In recent years, many different types of microneedle arrays have beendesigned. However, their geometry and arrangement within an array may beoptimized to trigger sufficient antigen presenting cells. A computational modelcan support the rational design of microneedle arrays. Therefore, the aim of thecurrent study was to describe the distribution and kinetics of a deliveredantigen within the skin using a theoretical compartment model, which includedbinding of antigens to receptors and their uptake by cells, and to determinewhich parameters should be measured to validate the model for a specificapplication. Multiple simulations were performed using a high and low antigendelivery dose and a range of values for the rate constants. The resultsindicated that the cells were highly saturated when a high dose was applied,while for a low dose saturation was only reached in 5% of the simulations. Thiswas caused by the difference in the ratio between the administered dose and theavailable binding sites and suggests the dose should be adapted to the number ofcells and receptors for a specific compound. The sensitivity analysis of themodel parameters confirmed that the initial dose and receptor concentrationswere indeed the two parameters that had the largest influence on the variance inantigen concentrations within the cells and circulation at equilibrium. Hence,these parameters are important to be measured in vivo. The presentedpharmacokinetics model can be used in future computational models to predict theinfluence of microneedle array geometry to optimize their design.

Understanding collagen and stress fiber remodeling is essential for the development of engineered tissues with good functionality. These processes are complex, highly interrelated, and occur over different time scales. As a result, excessive computational costs are required to computationally predict the final organization of these fibers in response to dynamic mechanical conditions. In this study, an analytical approximation of a stress fiber remodeling evolution law was derived. A comparison of the developed technique with the direct numerical integration of the evolution law showed relatively small differences in results, and the proposed method is one to two orders of magnitude faster.

The anisotropic collagen architecture of an engineered cardiovascular tissue has a major impact on its in vivo mechanical performance. This evolving collagen architecture is determined by initial scaffold microstructure and mechanical loading. Here, we developed and validated a theoretical and computational microscale model to quantitatively understand the interplay between scaffold architecture and mechanical loading on collagen synthesis and degradation. Using input from experimental studies, we hypothesize that both the microstructure of the scaffold and the loading conditions influence collagen turnover. The evaluation of the mechanical and topological properties of in vitro engineered constructs reveals that the formation of extracellular matrix layers on top of the scaffold surface influences the mechanical anisotropy on the construct. Results show that the microscale model can successfully capture the collagen arrangement between the fibers of an electrospun scaffold under static and cyclic loading conditions. Contact guidance by the scaffold, and not applied load, dominates the collagen architecture. Therefore, when the collagen grows inside the pores of the scaffold, pronounced scaffold anisotropy guarantees the development of a construct that mimics the mechanical anisotropy of the native cardiovascular tissue.

In order to maintain tissue homeostasis and functionality, adherent cells need to sense and respond to environmental mechanical stimuli. An important ability that adherent cells need in order to properly sense and respond to mechanical stimuli is the ability to exert contractile stress onto the environment via actin stress fibers. The actin stress fibers form a structural chain between the cells' environment via focal adhesions and the nucleus via the nuclear lamina. In case one of the links in this chain is missing or aberrant, contractile stress generation will be affected. This is especially the case in laminopathic cells, which have a missing or mutated form of the LMNA gene encoding for part of the nuclear lamina. Using the thin film method combined with sample specific finite element modeling, we quantitatively showed a fivefold lower contractile stress generation of Lmna knockout mouse embryonic fibroblasts (MEFs) as compared to wild-type MEFs. Via fluorescence microscopy it was demonstrated that the lower contractile stress generation was associated with an impaired actin stress fiber organization with thinner actin fibers and smaller focal adhesions. Similar experiments with wild-type MEFs with chemically disrupted actin stress fibers verified these findings. These data illustrate the importance of an organized actin stress fiber network for contractile stress generation and demonstrate the devastating effect of an impaired stress fiber organization in laminopathic fibroblasts. Next to this, the thin film method is expected to be a promising tool in unraveling contractility differences between fibroblasts with different types of laminopathic mutations.

Dermal immunization using antigen-coated microneedle arrays is a promising vaccination strategy. However, reduction of microneedle sharpness and the available surface area for antigen coating is a limiting factor. To overcome these obstacles, a layer-by-layer coating approach can be applied onto pH-sensitive microneedles. Following this approach, pH-sensitive microneedle arrays (positively charged at coating pH 5.8 and nearly uncharged at pH 7.4) were alternatingly coated with negatively charged diphtheria toxoid (DT) and N-trimethyl chitosan (TMC), a cationic adjuvant. First, the optimal DT dose for intradermal immunization was determined in a dose-response study, which revealed that low-dose intradermal immunization was more efficient than subcutaneous immunization and that the EC50 dose of DT upon intradermal immunization is 3-fold lower, as compared to subcutaneous immunization. In a subsequent immunization study, microneedle arrays coated with an increasing number (2, 5, and 10) of DT/TMC bilayers resulted in step-wise increasing DT-specific immune responses. Dermal immunization with microneedle arrays coated with 10 bilayers of DT/TMC (corresponding with ± 0.6 μg DT delivered intradermally) resulted in similar DT-specific immune responses as subcutaneous immunization with 5 μg of DT adjuvanted with aluminum phosphate (8-fold dose reduction). Summarizing, the layer-by-layer coating approach onto pH-sensitive microneedles is a versatile method to precisely control the amount of coated and dermally-delivered antigen that is highly suitable for dermal immunization.

Collagen provides cardiovascular tissues with the ability to withstand haemodynamic loads. A similar network is essential to obtain in tissue-engineered (TE) samples of the same nature. Yet, the mechanism of collagen orientation is not fully understood. Typically collagen remodelling is linked to mechanical loading. However, TE constructs also show an oriented collagen network when developed under static culture. Experiments under these conditions also indicate that the tissue gradually compacts due to contractile stresses developed in the α-actin fibres of the cells. Therefore, it is hypothesised that cellular contractile stresses are responsible for collagen orientation. A model describing the cellular α-actin turnover and the stresses developed by them is integrated in a structural constitutive model describing the mechanical behaviour of collagen fibres. Results show that the model can successfully capture the sample compaction, tissue stress generation and its heterogeneous collagen arrangement.

Accurate material models and associated parameters of atherosclerotic plaques are crucial for reliable biomechanical plaque prediction models. These biomechanical models have the potential to increase our understanding of plaque progression and failure, possibly improving risk assessment of plaque rupture, which is the main cause of ischaemic strokes and myocardial infarction. However, experimental biomechanical data on atherosclerotic plaque tissue is scarce and shows a high variability. In addition, most of the biomechanical models assume isotropic behaviour of plaque tissue, which is a general over-simplification. This review discusses the past and the current literature that focus on mechanical properties of plaque derived from compression experiments, using unconfined compression, micro-indentation or nano-indentation. Results will be discussed and the techniques will be mutually compared. Thereafter, an in-house developed indentation method combined with an inverse finite element method is introduced, allowing analysis of the local anisotropic mechanical properties of atherosclerotic plaques. The advantages and limitations of this method will be evaluated and compared to other methods reported in literature.

Electrospinning has proven to be a promising method to produce scaffolds for tissue engineering despite the frequently encountered limitations in 3-dimensional tissue formation due to a lack of cell infiltration. To fully unlock the potential of electrospun scaffolds for tissue engineering, the void space within the fibrous network needs to be increased substantially and in a controlled manner. Low-temperature electrospinning (LTE) increases the fiber to fiber distance by embedding ice particles as void spacers during fiber deposition. Scaffold porosities up to 99.5% can be reached and in line with the increase in void space, the mechanical properties of the scaffolds shift towards the range for native biological tissue. While both the physiological mechanical properties and high porosity were promising for tissue engineering applications, control of the porosity in three dimensions was still limited when using LTE methods. Based on a range of LTE spun scaffolds made of poly(lactic acid) and poly(ε-caprolactone), we found that changing the ratio between the rate of ice crystal formation and polymer fiber deposition only had a small effect on the 3D-porosity of the final scaffold architecture. Varying the fiber stiffness, however, offers considerable control over the scaffold void space.

To build anisotropic, mechanically functioning tissue, it is essential to understand how cells orient in response to mechanical stimuli. Therefore, a computational model was developed which predicts cell orientation, based on the actin stress fiber distribution inside the cell. In the model, the stress fiber distribution evolves dynamically according to the following: (1) Stress fibers contain polymerized actin. The total amount of depolymerized plus polymerized actin is constant. (2) Stress fibers apply tension to their environment. This active tension is maximal when strain rate and absolute strain are zero and reduces with increasing shortening rate and absolute strain. (3) A high active fiber stress in a direction leads to a large amount of fibers in this direction. (4) The cell is attached to a substrate; all fiber stresses are homogenized into a total cell stress, which is in equilibrium with substrate stress. This model predicts that on a substrate of anisotropic stiffness, fibers align in the stiffest direction. Under cyclic strain when the cellular environment is so stiff that no compaction occurs (1 MPa), the model predicts strain avoidance, which is more pronounced with increasing strain frequency or amplitude. Under cyclic strain when the cellular environment is so soft that cells can compact it (10 kPa), the model predicts a preference for the cyclically strained compared to the compacting direction. These model predictions all agree with experimental evidence. For the first time, a computational model predicts cell orientation in response to this range of mechanical stimuli using a single set of parameters. © 2013 Springer-Verlag Berlin Heidelberg.

In tissue-engineered (TE) heart valves, cell-mediated processes cause tissue compaction during culture and leaflet retraction at time of implantation. We have quantified and correlated stress generation, compaction, retraction, and tissue quality during a prolonged culture period of 8 weeks. Polyglycolic acid /poly-4-hydroxybutyrate strips were seeded with vascular-derived cells and cultured for 4-8 weeks. Compaction in width, generated force, and stress was measured during culture. Retraction in length, generated force, and stress was measured after release of constraints at weeks 4, 6, and 8. Further, the amount of DNA, glycosaminoglycans (GAGs), collagen, and collagen cross-links was assessed. During culture, compaction and force generation increased to, respectively, 63.9% +/- 0.8% and 43.7 +/- 4.3mN at week 4, after which they remained stable. Stress generation reached 27.7 +/- 3.2 kPa at week 4, after which it decreased to similar to 8.5 kPa. At release of constraints, tissue retraction was 44.0% +/- 3.7% at week 4 and decreased to 29.2% +/- 2.8% and 26.1% +/- 2.2% at, respectively, 6 and 8 weeks. Generated force (8-16mN) was lower at week 6 than at weeks 4 and 8. Generated stress decreased from 11.8 +/- 0.9 kPa at week 4 to 1.4 +/- 0.3 and 2.4 +/- 0.4 kPa at, respectively, weeks 6 and 8. The amount of GAGs increased at weeks 6 and 8 compared to week 4 and correlated to the reduced stress and retraction. In summary, prolonged culture resulted in decreased stress generation and retraction, likely as a result of the increased amount of GAGs. These results demonstrate the potential of prolonged tissue culture in developing functional, nonretracting, TE heart valves.

Background: Spine boards are used to immobilise accident victims suspected of having spinal injury. Guidelines about the maximum time patients remain on the board are often exceeded and on occasions may lead to pressure ulcers. Etiological research has shown that two processes ultimately lead to pressure ulcers:"Ischemic damage" which takes several hours to initiate and "deformation damage" at high strains. The latter process is very quick and the first signs of cell damage are already evident within minutes. Thus in order to minimise the risk of pressure ulcer development during prolonged loading, a new soft-layered long spine board has been designed. Methods: A subject specific numerical approach has been adopted to evaluate the prototype spine board in comparison to a conventional spine board, with reference to the estimated strains in the soft tissues adjacent to the sacrum in the supine position. The model geometry is derived from magnetic resonance images of three human volunteers in an unloaded situation. The loaded images are used to "tune" the material parameters of skin, fat and muscle. The prediction of the deformed contours on the soft-layered board is used to validate the model. Findings: Comparison of the internal strains in muscle tissue near the spine showed that internal strains on the soft-layered board are reduced and maximum strains are considerably less than the threshold at which deformation damage is possible. By contrast, on the rigid spine board this threshold is exceeded in all cases. Interpretation: The prototype comfort board is able to reduce the risk for deformation damage and thus reduces the risk of developing pressure ulcers. (C) 2013 Elsevier Ltd. All rights reserved.

Deep tissue injury (DTI) is a severe form of pressure ulcer where tissue damage starts in deep tissues underneath intact skin. Tissue deformation may play an important role in the aetiology, which can be investigated using an experimental–numerical approach. Recently, an animal-specific finite element model has been developed to simulate experiments in which muscle tissue was compressed with an indenter. In this study, the material behaviour and boundary conditions were adapted to improve the agreement between model and experiment and to investigate the influence of these adaptations on the predicted strain distribution. The use of a highly nonlinear material law and including friction between the indenter and the muscle both improved the quality of the model and considerably influenced the estimated strain distribution. With the improved model, the required sample size to detect significant differences between loading conditions can be diminished, which is clearly relevant in experiments involving animals.

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.

The purpose of this study was to investigate the effect of various repeated fractional intradermal dosing schedules of inactivated polio vaccine serotype 1 (IPV1) on IPV1-specific IgG responses in rats. By utilizing an applicator that allowed for precisely controlled intradermal microinjections by using a single hollow microneedle, rats were immunized intradermally with 5 D-antigen units (DU) of IPV1 at 150μm skin depth. This dose was administered as a bolus, or in a repeated fractional dosing schedule: 4 doses of 1.25 DU (1/4th of total dose) were administered on four consecutive days or every other day; 8 doses of 0.625 DU (1/8th of total dose) were administered on eight consecutive days; or 4 exponentially increasing doses (0.04, 0.16, 0.8 and 4 DU), either with or without an exponentially increasing CpG oligodeoxynucleotide 1826 (CpG) dose, were administered on four consecutive days. All of these fractional dosing schedules resulted in up to ca. 10-fold higher IPV1-specific IgG responses than intradermal and intramuscular bolus dosing. IPV1 combined with adjuvant CpG in exponential dosing did not significantly increase the IPV1-specific IgG responses further, which demonstrated that maximal responses were achieved by fractional dosing. In conclusion, repeated fractional intradermal IPV1 dosing leads to superior IPV1-specific IgG responses without the use of adjuvants. These results indicate that a controlled release delivery system for intradermal IPV1 delivery can potentiate IPV1-specific IgG responses.

Articular cartilage experiences significant mechanical loads during daily activities. Healthy cartilage provides the capacity for load bearing and regulates the mechanobiological processes for tissue development, maintenance, and repair. Experimental studies at multiple scales have provided a fundamental understanding of macroscopic mechanical function, evaluation of the micromechanical environment of chondrocytes, and the foundations for mechanobiological response. In addition, computational models of cartilage have offered a concise description of experimental data at many spatial levels under healthy and diseased conditions, and have served to generate hypotheses for the mechanical and biological function. Further, modeling and simulation provides a platform for predictive risk assessment, management of dysfunction, as well as a means to relate multiple spatial scales. Simulation-based investigation of cartilage comes with many challenges including both the computational burden and often insufficient availability of data for model development and validation. This review outlines recent modeling and simulation approaches to understand cartilage function from a mechanical systems perspective, and illustrates pathways to associate mechanics with biological function. Computational representations at single scales are provided from the body down to the microstructure, along with attempts to explore multiscale mechanisms of load sharing that dictate the mechanical environment of the cartilage and chondrocytes. © 2012 Biomedical Engineering Society.

Electrospinning is a promising technology to produce scaffolds for cardiovascular tissue engineering. Each electrospun scaffold is characterized by a complex micro-scale structure that is responsible for its macroscopic mechanical behavior. In this study, we focus on the development and the validation of a computational micro-scale model that takes into account the structural features of the electrospun material, and is suitable for studying the multi-scale scaffold mechanics. We show that the computational tool developed is able to describe and predict the mechanical behavior of electrospun scaffolds characterized by different microstructures. Moreover, we explore the global mechanical properties of valve-shaped scaffolds with different microstructural features, and compare the deformation of these scaffolds when submitted to diastolic pressures with a tissue engineered and a native valve. It is shown that a pronounced degree of anisotropy is necessary to reproduce the deformation patterns observed in the native heart valve.

Autologous heart valve tissue engineering relies on extracellular matrix production by cells seeded into a degrading scaffold material. The cells naturally exert traction forces to their surroundings, and due to an imbalance between scaffold, tissue, and these traction forces, stress is generated within the tissue. This stress results in compaction during culture and retraction of the leaflets at release of constraints, causing shape loss of the heart valve leaflets. In the present study, an in vitro model system has been developed to quantify stress generation, compaction, and retraction during culture and after release of constraints. Tissue-engineered (TE) constructs based on polyglycolic acid/poly-4-hydroxybutyrate scaffolds seeded with human vascular-derived cells were cultured for 4 weeks. Compaction in width was measured during culture, stress generation was measured during culture and after release of constraints at week 4, and contraction was measured after release of constraints at week 4. Both compaction and stress generation started after 2 weeks of culture and continued up to week 4. TE constructs compacted up to half of their original width and reached an internal stress of 6-8kPa at week 4, which resulted in a retraction of 36%. The model system has provided a useful tool to unravel and optimize the balance between the different aspects of TE constructs to develop functional TE leaflets.

Keywords:human skin;fibroblasts;heat shock;procollagen;rejuvenation Background: The well-known characteristics of aging skin are the development of fine lines and wrinkles, but changes in skin tone, skin texture, thickness and moisture content are also aspects of aging. Rejuvenation of the skin aims at reversing the signs of aging and can be established in the epidermis as well as in the dermis. Aged dermis, in fact, has a degenerated collagen matrix. To regenerate this matrix, fibroblasts need to be stimulated into synthesizing new collagen. Aims: In this study, the effects of heat shocks of different temperatures on human dermal fibroblasts in ex vivo skin on the expression of procollagen 1, procollagen 3, heat shock protein (hsp)27, hsp47, and hsp70 are investigated. Materials and methods: The heat shocks were applied on ex vivo skin samples by immersing the samples in heated phosphate-buffered saline of 45 °C or 60 °C. Metabolic activity was measured and at similar time points propidium–iodide–calceine staining was performed to establish cell viability. Quantitative polymerase chain reaction (qPCR) was performed after the heat shock to determine gene expression levels relative to the reference temperature. Furthermore, PicroSirius Red and hematoxylin stainings were performed to visualize the collagen network and the cells. Results: The skin samples were shown to be viable and metabolically active. Histology indicated that the heat shocks did not influence the structure of the collagen network or cell appearance. qPCR results showed that in contrast to the 45 °C heat shock the 60 °C heat shock resulted in significant upregulations of procollagen type I and III, hsp70 and hsp47. Conclusion: A 60 °C, heat shock stimulates the human dermal fibroblasts in ex vivo skin to upregulate their procollagen type I and type III expression.

This study presents an in vitro experimental method to determine shear properties of the epidermis. Shear tests were performed with a parallel plate rheometer on samples of stratum corneum and the viable epidermis. The method was validated on very thin silicon sheets. Preliminary test were performed to determine the linear viscoelastic range, the effect of normal loading on the sample and the time to reach equilibrium after changes of temperature and relative humidity. The study shows that reproducible results can be obtained for the shear properties of epidermis in an in vitro set up. The dynamic shear modulus for stratum corneum roughly ranges from 4 to 12 kPa, decreasing with increasing relative humidity. The values are considerably lower than the shear modulus value based on tensile Young’s moduli in literature, indicating a considerable anisotropic material behavior. Results for the epidermis were of the same order of magnitude, but were less consistent possibly due to a less well-defined tissue composition.

Extracellular matrix (ECM) in chondrocytes-seeded agarose aggregates to form islands of matrix. These islands need to coalesce to develop functional cartilage. Hence, macroscopic properties are determined by transport and aggregation of macromolecules at the microscale, which varies temporally and spatially. This study evaluates the importance of the mutual interaction between matrix components and matrix development. Fluorescence recovery after photobleaching measurements demonstrates that diffusivity depends on the presence and density of ECM. A reaction-diffusion model describing synthesis, transport and immobilisation of ECM predicts steep gradients in ECM around chondrocytes, resembling histology. Steric hindrance of diffusion by ECM is essential for the formation of these gradients. Finally, microscopic ECM concentration is linked with macroscopic mechanical properties. Construct softening is predicted when temporal and spatial variations in diffusivity are considered. In conclusion, non-constant diffusion renders significant effects on both the microscopic ECM development and the macroscopic mechanical properties of developing tissue-engineered cartilage.

For pressure ulcer prevention an ambitious goal would be the establishment of a mechanical threshold for tissue damage. In the past, several researchers have sought to establish such a threshold often involving the loading time. However, they have not resulted in a unique reliable value that could be used in practice. This limitation is probably due to the focus on interface pressure. The objective of this paper is to clarify to an audience with no conventional background in mechanics, why interface pressure is not the appropriate parameter to define a damage threshold, whereas internal local deformations (strains) may prove more suitable. The paper reveals that it may be possible to identify a damage threshold for healthy skeletal muscle tissue based on local internal deformations.

Pressure-related deep tissue injury may develop in skeletal muscle tissue which is subjected to prolonged compression. For early detection, it is important to understand the underlying damage processes. Gawlitta et al. [Gawlitta, D., C. W. J. Oomens, D. L. Bader, F. P. T. Baaijens, and C. V. C. Bouten. Temporal differences in the influence of ischemic factors and deformation on the metabolism of engineered skeletal muscle. J. Appl. Physiol. 103(2):464-473, 2007b] subjected tissue-engineered muscle constructs to ischemia and deformation to study their effects on viability. Contrary to previous findings, no decrease in viability was found due to compression. However, the nature of their measurement method complicated interpretation of the results, particulary when deformation was involved. Changes in the constructs were assessed by measurements in the surrounding medium. The theoretical model developed in the present study describes metabolism, diffusion, and cell death in the experiments, and accounts for reduced diffusion due to compression. It was demonstrated that the lack of effect of compression on tissue viability, as measured in the experiments, could be explained by the compression-induced decrease in diffusivity. Compression did lead to considerable cell death but this could not be measured by Gawlitta et al. [Gawlitta, D., C. W. J. Oomens, D. L. Bader, F. P. T. Baaijens, and C. V. C. Bouten. Temporal differences in the influence of ischemic factors and deformation on the metabolism of engineered skeletal muscle. J. Appl. Physiol. 103(2):464-473, 2007b] because diffusion of the cell death marker to the medium was limited. This study shows that a proper description of transport processes is essential for a correct interpretation of experiments in which indirect measurement methods are used. © 2009 The Author(s).

The mechanical properties of soft biological tissues in general and early stage engineered tissues in particular limit the feasibility of conventional tensile tests for their mechanical characterisation. Furthermore, the most important mode in development of deep tissue injury (DTI) is compression. Therefore, an inverse numerical-experimental approach using a finite spherical indentation test is proposed. To demonstrate the feasibility of the approach indentation tests are applied to bio-artificial muscle (BAM) tissue. BAMs are cultured in vitro with (n = 20) or without (n = 12) myoblast cells to quantify the effect of the cells on the passive transverse mechanical properties. Indentation tests are applied up to 80% of the tissue thickness. A non-linear Neo-Hookean constitutive model is fitted to the experimental results for parameter estimation. BAMs with cells demonstrated both stiffer and more non-linear material behaviour than BAMs without cells.

Background/purpose: Pressure ulcers are areas of soft tissue breakdown, resulting from sustained mechanical loading of the skin and underlying tissues. Measuring biochemical markers that are released upon mechanical loading by the epidermis seems a promising method for objective risk assessment of the development of pressure ulcers. This risk assessment method will better determine the risk of a patient to develop pressure ulcers than the risk score lists currently used. So far, experimental studies have been performed that measure the tissue response in the culture supernatant. To elucidate the transport of the biochemical markers within the epidermis, the diffusion coefficient needs to be established. Methods: In the current study, fluorescent recovery after photobleaching (FRAP) is used to determine the diffusion coefficient of fluorescent-labeled dextran molecules in human epidermis, porcine epidermis and engineered epidermal equivalents. These dextran molecules have a similar weight to the biochemical markers. Results: Similar diffusion coefficients were found for human and porcine epidermal samples (6.2 × 10−5±1.2 × 10−5 and 5.9 × 10−5±6.1 × 10−6 mm2/s, respectively), whereas the diffusion coefficient of the engineered epidermal equivalent was significantly lower (2.3 × 10−5±1.0 × 10−5 mm2/s). Conclusion: The diffusion could be measured in epidermal tissues using FRAP. In the future, the diffusion coefficients obtained in the current study will be used to study the difference between the transport in EpiDerm cultures and in human epidermis.

Subcutaneous adipose tissue contributes to the overall mechanical behavior of the skin. Until today, however, no thorough constitutive model is available for this layer of tissue. As a start to the development of such a model, the objective of this study was to measure and describe the linear viscoelastic behavior of subcutaneous adipose tissue. Although large strains occur in vivo, this work only focuses on the linear behavior to show the applicability of the described methods to adipose tissue. Shear experiments are performed on porcine samples on a rotational rheometer using parallel plate geometry. In the linear viscoelastic regime, up to 0.1% strain, the storage and loss modulus showed a frequency- and temperature-dependent behavior. The ratio between the two moduli, the phase angle, did not show any dependency on temperature and frequency. The shear modulus was found to be 7.5 kPa at 10 rad/s and 37 degrees C. Time-temperature superposition was applicable through shifting the shear modulus horizontally. A power-law function model was introduced to describe both the frequency dependent behavior at constant temperature and the stress relaxation behavior. In addition, the effect of snap freezing as a preservation method was analyzed. Histological examination demonstrated possible tissue damage after freezing, but the mechanical properties did not change. Since results were reproducible, it is concluded that the methods we used are most probably suited to explore the non-linear behavior of subcutaneous adipose tissue.

Pressure-related deep tissue injury (DTI) represents a severe pressure ulcer, which initiates in compressed muscle tissue overlying a bony prominence and progresses to more superficial tissues until penetrating the skin. Individual subjects with impaired motor and/or sensory capacities are at high risk of developing DTI. Impaired diffusion of critical metabolites in compressed muscle tissue may contribute to DTI, and impaired diffusion of tissue damage biomarkers may further impose a problem in developing early detection blood tests. We hypothesize that compression of muscle tissue between a bony prominence and a supporting surface locally influences the diffusion capacity of muscle. The objective of this study was therefore, to determine the effects of large compression strains on free diffusion in a tissue-engineered skeletal muscle model. Diffusion was measured with a range of fluorescently labeled dextran molecules (10, 20, 150kDa) whose sizes were representative of both hormones and damage biomarkers. We used fluorescence recovery after photobleaching (FRAP) to compare diffusion coefficients (D) of the different dextrans between the uncompressed and compressed (48-60% strain) states. In a separate experiment, we simulated the effects of local partial muscle ischemia in vivo, by reducing the temperature of compressed specimens from 37 to 34 degrees C. Compared to the D in the uncompressed model system, values in the compressed state were significantly reduced by 47+/-22% (p0.02). A 3 degrees C temperature decrease further reduced D in the compressed specimens by 10+/-6% (p0.05). In vivo, the effects of large strains and ischemia are likely to be summative, and hence, the present findings suggest an important role of impaired diffusion in the etiology of DTI, and should also be considered when developing biochemical screening methods for early detection of DTI.

Deep tissue injury (DTI) is a severe pressure ulcer that results from sustained deformation of muscle tissue overlying bony prominences. In order to understand the etiology of DTI, it is essential to determine the tolerance of muscle cells to large mechanical strains. In this study, a new experimental method of determining the time-dependent critical compressive strains for necrotic cell death (E(zz)(c)(t)) in a planar tissue-engineered construct under static loading was developed. A half-spherical indentor is used to induce a non-uniform, concentric distribution of strains in the construct, and E(zz)(c)(t) is calculated from the radius of the damage region in the construct versus time. The method was employed to obtain E(zz)(c)(t) for bio-artificial muscles (BAMs) cultured from C2C12 murine cells, as a model system for DTI. Specifically, propidium iodine was used to fluorescently stain the development of necrosis in BAMs subjected to strains up to 80%. Two groups of BAMs were tested at an extracellular pH of 7.4 (n=10) and pH 6.5 (n=5). The lowest strain levels causing cell death in the BAMs were determined every 15min, during 285-min-long trials, from confocal microscopy fluorescent images of the size of the damage regions. The experimental E(zz)(c)(t) data fitted a decreasing single-step sigmoid of the Boltzmann type. Analysis of the parameters of this sigmoid function indicated a 95% likelihood that cells could tolerate engineering strains below 65% for 1h, whereas the cells could endure strains below 40% over a 285min trial period. The decrease in endurance of the cells to compressive strains occurred between 1-3h post-loading. The method developed in this paper is generic and suitable for studying E(zz)(c)(t) in virtually any planar tissue-engineered construct. The specific E(zz)(c)(t) curve obtained herein is necessary for extrapolating biological damage from muscle-strain data in biomechanical studies of pressure ulcers and DTI.

In recent years, damage directly due to tissue deformation has gained interest in deep pressure ulcer aetiology research. It has been shown that deformation causes muscle cell damage, though the pathway is unclear. Mechanically induced skeletal muscle damage has often been associated with an increased intracellular Ca2+ concentration, e.g. in eccentric exercise (Allen et al., J Physiol 567(3):723–735, 2005). Therefore, the hypothesis was that compression leads to membrane disruptions, causing an increased Ca2+-influx, eventually leading to Ca2+ overload and cell death. Monolayers of differentiated C2C12 myocytes, stained with a calcium-sensitive probe (fluo-4), were individually subjected to compression while monitoring the fluo-4 intensity. Approximately 50% of the cells exhibited brief calcium transients in response to compression, while the rest did not react. However, all cells demonstrated a prolonged Ca2+ up-regulation upon necrosis, which induced similar up-regulations in some of the surrounding cells. Population heterogeneity is a possible explanation for the observed differences in response, and it might also become important in tissue damage development. It did not become clear however whether Ca2+-influxes were the initiators of damage.

Deep pressure ulcers are caused by sustained mechanical loading and involve skeletal muscle tissue injury. The exact underlying mechanisms are unclear, and the prevalence is high. Our hypothesis is that the aetiology is dominated by cellular deformation (Bouten et al. in Ann Biomed Eng 29:153-163, 2001; Breuls et al. in Ann Biomed Eng 31:1357-1364, 2003; Stekelenburg et al. in J App Physiol 100(6):1946-1954, 2006) and deformation-induced ischaemia. The experimental observation that mechanical compression induced a pattern of interspersed healthy and dead cells in skeletal muscle (Stekelenburg et al. in J App Physiol 100(6):1946-1954, 2006) strongly suggests to take into account the muscle microstructure in studying damage development. The present paper describes a computational model for deformation-induced hypoxic damage in skeletal muscle tissue. Dead cells stop consuming oxygen and are assumed to decrease in stiffness due to loss of structure. The questions addressed are if these two consequences of cell death influence the development of cell injury in the remaining cells. The results show that weakening of dead cells indeed affects the damage accumulation in other cells. Further, the fact that cells stop consuming oxygen after they have died, delays cell death of other cells.

Delivering a drug into and through the skin is of interest as the skin can act as an alternative drug administration route for oral delivery. The development of new delivery methods, such as microneedles, makes it possible to not only deliver small molecules into the skin, which are able to pass the outer layer of the skin in therapeutic amounts, but also macromolecules. To provide insight into the administration of these molecules into the skin, the aim of this study was to assess the transport of macromolecules within and between its various layers. The diffusion coefficients in the epidermis and several locations in the papillary and reticular dermis were determined for fluorescein dextran of 40 and 500 kDa using a combination of fluorescent recovery after photobleaching experiments and finite element analysis. The diffusion coefficient was significantly higher for 40 kDa than 500 kDa dextran, with median values of 23 and 9 µm(2)/s in the dermis, respectively. The values only marginally varied within and between papillary and reticular dermis. For the 40 kDa dextran, the diffusion coefficient in the epidermis was twice as low as in the dermis layers. The adopted method may be used for other macromolecules, which are of interest for dermal and transdermal drug delivery. The knowledge about diffusion in the skin is useful to optimize (trans)dermal drug delivery systems to target specific layers or cells in the human skin.

The paper describes the current views on the cause of a sub-class of pressure ulcers known as pressure induced deep tissue injury (DTI). A multi-scale approach was adopted using model systems ranging from single cells in culture, tissue engineered muscle to animal studies with small animals. This has led to a clear understanding on two damage mechanisms associated with the development of DTI. Direct deformation results from high, but physiologically relevant, strains and is a process that leads to the first signs of cell damage within minutes. Ischaemic damage is caused by occlusion of blood vessels, but takes several hours to develop. The paper ends with some clinical consequences.

Although the mechanical behavior of the top layer of the skin, the epidermis, is an important consideration in several clinical and cosmetic applications, there are few reported studies on this layer. The in vivo mechanical behavior of the upper skin layer (here defined as epidermis and papillar dermis) was characterized using a combined experimental and modeling approach. The work was based on the hypothesis that experiments with different length scales represent the mechanical behavior of different skin layers. Suction measurements with aperture diameters of 1, 2 and 6 mm were combined with ultrasound and optical coherence tomography to study the deformation of the skin layers. The experiments were simulated for small displacements with a two-layered finite element model representing the upper layer and the reticular dermis. An identification method compared the experimental and numerical results to identify the material parameters of the model. For one subject the whole parameter estimation procedure was completed, leading to a stiffness of C(10,ul) = 0.11 kPa for the top-layer and C(10,rd) = 0.16 MPa for the reticular dermis. This unexpected, extreme stiffness ratio of the material parameters let to convergence problems of the finite element software for most of the individuals.

Based on previously published experimental work, computational models were developed to simulate the effect of different dynamic compression regimens on the activity of chondrocytes seeded in agarose constructs. In particular, the balance between proliferation and matrix synthesis can be adjusted by applying different intervals of continuous or intermittent mechanical compression. A phenomenological compartment based-modeling approach was used as first model. A more mechanistic cell cycle model was used as the second model. The compartment-based modeling approach was found to be useful in representing a balance between proliferation and proteoglycan synthesis, when the effect of a certain stimulation protocol is known. In order to predict the response to different intervals of mechanical stimulation, however, a more mechanistic cell cycle-based approach is required. The cell cycle model supports an important role of the onset of loading. In addition, an inhibitory effect of further loading is required, which is more likely to be related to cell cycle progression velocity than to a decreased probability of commitment to the cell cycle. The mechanisms behind this inhibitory effect and the computational implementation, however, require further investigation.

Eukaryotic cells are continuously subjected to mechanical forces under normal physiological conditions. These forces and associated cellular deformations induce a variety of biological processes. The degree of deformation depends on the mechanical properties of the cell. As most cells are anchorage dependent for normal functioning, it is important to study the mechanical properties of cells in their attached configuration. The goal of the present study was to obtain the mechanical and failure properties of attached cells. Individual, attached C2C12 mouse myoblasts were subjected to unconfined compression experiments using a recently developed loading device. The device allows global compression of the cell until cell rupture and simultaneously measures the associated forces. Cell bursting was characterized by a typical reduction in the force, referred to as the bursting force. Mean bursting forces were calculated as 8.7 ± 2.5 μ N at an axial strain of 72 ± 4 %. Visualization of the cell using confocal microscopy revealed that cell bursting was preceded by the formation of bulges at the cell membrane, which eventually led to rupturing of the cell membrane. Finite element calculations were performed to simulate the obtained force–deformation curves. A finite element mesh was built for each cell to account for its specific geometrical features. Using an axisymmetric approximation of the cell geometry, and a Neo–Hookean constitutive model, excellent agreement between predicted and measured force–deformation curves was obtained, yielding an average Young's modulus of 1.14 ± 0.32 kPa .

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.

Backgrounds/aims Human skin is a complex tissue consisting of different layers. To gain better insight into the mechanical behaviour of different skin layers, the mechanical response was studied with experiments of various length scales. Also, the influence of (superficial) hydration on the mechanical response is studied. The work is based on the hypothesis that experiments with different length scales represent the mechanical behaviour of different skin layers. For suction, this means that a large aperture diameter reflects the behaviour of mainly dermis, whereas a very small diameter reflects the behaviour of only the top layer of the skin. Methods Suction measurements at varying pressures and aperture sizes were performed on the volar forearm of 13 subjects aged 29-47 years. The deformation of the skin was visualized using ultrasound (US) (dermis) and optical coherence tomography (OCT) (epidermis and dermis). US measurements were performed on hydrated skin, OCT measurements on dry and hydrated skin. The experiment was simulated by a finite element model (FEM) exhibiting extended Mooney material behaviour. An identification method was used to compare the experimental and numerical results to identify the parameters of the material. Results The material parameters C(10) and C(11) were calculated for four subjects: C(10)=29.6+/-21.1 kPa and C(11)=493+/-613 kPa for 6 mm aperture diameter, C(10)=11.5+/-8.7 kPa and C(11)=18.3+/-12.6 kPa for 2 mm aperture diameter and C(10)=10.8+/-9.5 kPa and C(11)=9.3+/-7.7 kPa for 1 mm aperture diameter. Skin hydration caused ambiguous effects on the mechanical response. Conclusions US and OCT, combined with suction, using varying apertures sizes, proved to be a valuable tool to study the mechanical behaviour of different skin layers. With increasing experimental length scale, increasing values for the parameters of the material model were found. This indicates the need of a multi-layered material layer FEM, which can be used to identify mechanical behaviour of epidermis and dermis.

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.

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.

Background/aims: Human skin is a complex tissue consisting of several distinct layers. Each layer consists of various components with a specific structure. To gain a better insight into the overall mechanical behaviour of the skin, we wish to study the mechanical properties of the different layers. A numerical-experimental method was developed to characterize the non-linear mechanical behaviour of human dermis. Methods: Suction measurements at varying pressures were performed on the volar forearm skin of 10 subjects aged 19–24 years old. Deformation of dermis and fat during suction was measured using ultrasound. The experiment was simulated by a finite element model exhibiting extended Mooney material behaviour to account for the non-linear stress–strain relationship. An identification method is used to compare the experimental and numerical results to identify the parameters of the material model. Results:C10, dermis was found to be 9.4 ± 3.6 kPa and C11, dermis to be 82 ± 60 kPa. A first rough estimate of C10, fat was 0.02 kPa. Conclusions: The resulting finite element model demonstrated its ability to describe the response of the skin to suction at various pressures. In the future, this method can be used to characterize the mechanical behaviour of different skin layers using various aperture sizes and to characterize the skin behaviour under various loading conditions.

Pressure ulcers are areas of soft tissue breakdown that result from sustained mechanical loading of the skin and underlying tissues; they can affect the quality of life of many individuals. Despite considerable efforts to prevent pressure ulcers, data on prevalence are unacceptably high. This can at least partly be attributed to limited knowledge of the etiology of the clinical condition and the fact that identification and prevention of pressure ulcers mainly focus on skin tissue, even though the underlying muscle tissue may be more susceptible to mechanical loading. The present article proposes a new, hierarchical research approach to obtain improved insights into the basic pathways whereby mechanical loading leads to soft tissue breakdown. This approach investigates the relationships between (1) global mechanical loading at skin level, (2) the resulting local internal mechanical conditions within the soft tissue layers extending from skin to muscle tissue, and (3) the pathophysiologic response to loading. The latter response should be assessed from the various functional tissue units involved in soft-tissue breakdown-the cells, the interstitial space, and blood and lymph vessels. We predict that the proposed strategy will provide new fundamental knowledge about the etiology of pressure ulcers that can serve as a sound basis for effective clinical identification and prevention.

Skeletal muscle cells are sensitive to sustained compression, which can lead to the development of pressure sores. Although it is known that this type of tissue breakdown depends on the magnitude and duration of the applied load, the exact relationship between cell deformation and damage remains unclear. To gain more insight into this process, a method has been developed, that incorporates the use of a new loading device and confocal microscopy. The loading device is able to compress individual cells, either statically or dynamically, while measuring the resulting forces. Experiments can be performed under ideal environmental conditions, comparable with those of a CO2 incubator. First compression experiments on C2C12 mouse myoblasts showed the shape changes that cells undergo during static compression by the loading device. Calculations using the three-dimensional confocal images showed no change in volume and an increase in the surface area of the cell as a result of compression. The device presented here provides a useful way to monitor the biomechanical response of skeletal muscle cells during long-term compression experiments. Therefore it will contribute to the knowledge about strain-induced cell damage, as seen in pressure sores and other mechanically induced clinical conditions.

Spatial and temporal heterogeneity of superficial muscle strain during in situ fixed-end contractions.Drost MR, Maenhout M, Willems PJ, Oomens CW, Baaijens FP, Hesselink MK.Department of Movement Sciences, Nutrition and Toxicology Research Institute Maastricht, Maastricht University, P.O. Box 616, 6200, Maastricht, The Netherlands. maarten.drost@bw.unimaas.nlNumerical models of contracting muscle offer a powerful tool to study local mechanical load. For validation of these models, the spatial and temporal distribution of strain was quantified in fixed-end contracting rat tibialis anterior muscle in situ at optimal muscle length (L(o)) and at 120 degrees plantar flexion as well as at 125 and 33Hz stimulation frequency. We studied the hypothesis that after termination of stimulation in situ muscle segments near the motor endplates elongate while segments away from the endplates shorten. We show that both spatial and temporal inhomogeneities in muscle deformation occurred during contraction. Muscle plateau shortening strain equalled 4.1%. Maximal plateau shortening of a muscle segment was much larger (9.6%) and occurred distally (at 0.26 of the scaled length of the muscle). Manipulating torque levels by decreasing the stimulation frequency at the same muscle length induced a decrease in torque ( approximately 20%) with a smaller effect on the level and no effect on the pattern of muscle deformation. During relaxation, distal segments actively shortened at the expense of proximal muscle segments, which elongated. The segments undergoing lengthening were nearer to motor endplates than segments undergoing shortening.In conclusion, the present study provides experimental data on magnitude of contraction-induced deformation needed for validation of numerical models. Local muscle deformation is heterogeneous both temporally and spatially and may be related to proximity to the motor endplates

A multilevel finite element approach is applied to predict local cell deformations in engineered tissue constructs. Cell deformations are predicted from detailed nonlinear FE analysis of the microstructure, consisting of an arrangement of cells embedded in matrix material. Effective macroscopic tissue behavior is derived by a computational homogenization procedure. To illustrate this approach, we simulated the compression of a skeletal muscle tissue construct and studied the influence of microstructural heterogeneity on local cell deformations. Results show that heterogeneity has a profound impact on local cell deformations, which highly exceed macroscopic deformations. Moreover, microstructural heterogeneity and the presence of neighboring cells leads to complex cell shapes and causes non-uniform deformations within a cell.

Microneedles represent promising tools for delivery of drugs to the skin. However, before these microneedles can be used in clinical practice, it is essential to understand the process of skin penetration by these microneedles. The present study was designed to monitor both penetration depth and force of single solid microneedles with various tip diameters ranging from 5 to 37 µm to provide insight into the penetration process into the skin of these sharp microneedles. To determine the microneedle penetration depth, single microneedles were inserted in human ex vivo skin while monitoring the surface of the skin. Simultaneously, the force on the microneedles was measured. The average penetration depth at 1.5 mm displacement was similar for all tip diameters. However, the process of penetration depth was significantly different for the various microneedles. Microneedles with a tip diameter of 5 µm were smoothly inserted into the skin, while the penetration depth of microneedles with a larger tip diameter suddenly increased after initial superficial penetration. In addition, the force at insertion (defined as the force at a sudden decrease in measured force) linearly increased with tip diameter ranging from 20 to 167 mN. The force drop at insertion was associated with a measured penetration depth of approximately 160 μm for all tip diameters, suggesting that the drop in force was due to the penetration of a deeper skin layer. This study showed that sharp microneedles are essential to insert microneedles in a well-controlled way to a desired depth.

Med Eng Phys 2001 Apr;23(3):195-200 Related Articles, Books, LinkOut Quantification and localisation of damage in rat muscles after controlled loading; a new approach to study the aetiology of pressure sores.Bosboom EM, Bouten CV, Oomens CW, van Straaten HW, Baaijens FP, Kuipers H.Department of Materials Technology, Eindhoven University of Technology, P.O. Box 513, 5600 MB, Eindhoven, The Netherlands. e.m.h.bosboom@tue.nlTo obtain more insight in the aetiology of deep pressure sores, an animal model was developed to relate controlled external loading to local muscle damage. The tibialis anterior muscle (TA) and overlying skin of a rat were compressed between indentor and tibia. Loads of 10, 70 and 250kPa at skin surface were applied for 2 or 6h. During half of the 10 and 250kPa experiments interstitial fluid pressure (IFP) in the TA was measured. The TAs were excised 24h after load application. Both amount and location of damage were assessed by histological examination using a semi-automated image-processing program. In six of eleven loaded muscles damage was found. The damage was located from superficial to deep muscle tissue in a zone never exceeding the diameter of the indentor. The IFP measurements interfered with the occurrence of damage; application of 10 and 70kPa loads only caused damage when combined with IFP measurements, whereas IFP measurements increased damage at 250kPa loads. The results showed that the developed animal model can be used to provoke local damage by applying a controlled load and that the amount and location of damage can be assessed using the newly developed techniques.

J Biomech 2001 Oct;34(10):1365-8 Related Articles, Books, LinkOut Passive transverse mechanical properties of skeletal muscle under in vivo compression.Bosboom EM, Hesselink MK, Oomens CW, Bouten CV, Drost MR, Baaijens FP.Department of Materials Technology, Eindhoven University of Technology, P.O. Box 513, 5600 MB, Eindhoven, The Netherlands. e.m.h.bosboom@tue.nlThe objective of the present study is to determine the passive transverse mechanical properties of skeletal muscle. Compression experiments were performed on four rat tibialis anterior muscles. To assess the stress- and strain-distributions in the muscle during the experiment, a plane stress model of the cross section was developed for each muscle. The incompressible viscoelastic Ogden model was used to describe the passive muscle behaviour. The four material parameters were determined by fitting calculated indentation forces on measured indentation forces. The elastic parameters, mu and alpha, were 15.6+/-5.4 kPa and 21.4+/-5.7, respectively. The viscoelastic parameters, delta and tau, were 0.549+/-0.056 and 6.01+/-0.42 s. When applying the estimated material parameters in a three-dimensional finite element model, the measured behaviour can be accurately simulated.