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We present a model for stress-fiber reorganization and the associated contractility that includes both the kinetics of stress-fiber formation and dissociation as well as the kinetics of stress-fiber remodeling. These kinetics are motivated by considering the enthalpies of the actin/myosin functional units that constitute the stress fibers. The stress, strain and strain rate dependence of the stress-fiber dynamics are natural outcomes of the approach. The model is presented in a general 3D framework and includes the transport of the unbound stress-fiber proteins. Predictions of the model for a range of cyclic loadings are illustrated to rationalize hitherto apparently contrasting observations. These observations include: (1) For strain amplitudes around 10 % and cyclic frequencies of about 1 Hz, stress fibers align perpendicular to the straining direction in cells subjected to cyclic straining on a 2D substrate while the stress fibers align parallel with the straining direction in cells constrained in a 3D tissue. (2) At lower applied cyclic frequencies, stress fibers in cells on 2D substrates display no sensitivity to symmetric applied strain versus time waveforms but realign in response to applied loadings with a fast lengthening rate and slow shortening. (3) At very low applied cyclic frequencies (on the order of mHz) with symmetric strain versus time waveforms, cells on 2D substrates orient perpendicular to the direction of cyclic straining above a critical strain amplitude. A.V. and V.S.D. acknowledge the Royal Society for supporting A.V. through a Newton International Fellowship. peer-reviewed 2016-09-04

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 aim of cardiovascular regeneration is to mimic the biological and mechanical functioning of tissues. For this it is crucial to recapitulate the in vivo cellular organization, which is the result of controlled cellular orientation. Cellular orientation response stems from the interaction between the cell and its complex biophysical environment. Environmental biophysical cues are continuously detected and transduced to the nucleus through entwined mechanotransduction pathways. Next to the biochemical cascades invoked by the mechanical stimuli, the structural mechanotransduction pathway made of focal adhesions and the actin cytoskeleton can quickly transduce the biophysical signals directly to the nucleus. Observations linking cellular orientation response to biophysical cues have pointed out that the anisotropy and cyclic straining of the substrate influence cellular orientation. Yet, little is known about the mechanisms governing cellular orientation responses in case of cues applied separately and in combination. This review provides the state-of-the-art knowledge on the structural mechanotransduction pathway of adhesive cells, followed by an overview of the current understanding of cellular orientation responses to substrate anisotropy and uniaxial cyclic strain. Finally, we argue that comprehensive understanding of cellular orientation in complex biophysical environments requires systematic approaches based on the dissection of (sub)cellular responses to the individual cues composing the biophysical niche.

Inflammation is a natural phase of the wound healing response, which can be harnessed for the in situ tissue engineering of small-diameter blood vessels using instructive, bioresorbable synthetic grafts. This process is dependent on colonization of the graft by host circulating cells and subsequent matrix formation. Typically, vascular regeneration in small animals is governed by transanastomotic cell ingrowth. However, this process is very rare in humans and hence less relevant for clinical translation. Therefore, a novel rat model was developed, in which cell ingrowth from the adjacent tissue is inhibited using Gore-Tex sheathing. Using this model, our aim here was to prove that functional blood vessels can be formed in situ through the host inflammatory response, specifically by blood-borne cells. The model was validated by implanting sex-mismatched aortic segments on either anastomoses of an electrospun poly(ɛ-caprolactone) (PCL) graft, filled with fibrin gel, into the rat abdominal aorta. Fluorescent in situ hybridization analysis revealed that after 1 and 3 months in vivo, over 90% of infiltrating cells originated from the bloodstream, confirming the effective shielding of transanastomotic cell ingrowth. Using the validated model, PCL/fibrin grafts were implanted, either or not loaded with monocyte chemotactic protein-1 (MCP-1), and cell infiltration and tissue development were investigated at various key time points in the healing cascade. A phased healing response was observed, initiated by a rapid influx of inflammatory cells, mediated by the local release of MCP-1. After 3 months in vivo, the grafts consisted of a medial layer with smooth muscle cells in an oriented collagen matrix, an intimal layer with elastin fibers, and confluent endothelium. This study proves the regenerative potential of cells in the circulatory system in the setting of in situ vascular tissue engineering.

Background: Decellularized tissue-engineered heart valves (TEHVs) are under investigation as alternative for current heart valve prostheses with the potential to rapidly repopulate with cells within the body. Ideally, these valves are stented for transapical or minimally invasive delivery. It is unclear if and how the matrix of these valves remodels under in vivo hemodynamic loading conditions and in the presence of a stent. Here, we study the evolution of collagen orientation and tissue maturation in the wall of stented decellularized TEHVs with time after implantation. Methods and Results: In a previous study, stented TEHVs based on rapidly degrading scaffolds were cultured in bioreactors, decellularized, and transapically implanted as pulmonary valve replacement in sheep. In the present study, collagen (re)orientation in the initially isotropic valvular wall was assessed using a fluorescent collagen probe combined with confocal imaging and image analysis of explanted tissue at 8, 16, and 24 weeks following implantation. Collagen tortuosity or waviness in the explants, as a measure of matrix maturity, was quantified using a Gabor wavelet method and compared with tortuosity in native sheep vascular wall tissue. Results indicate that on the luminal side of the valvular wall, fibers became aligned in circumferential direction, while tortuosity increased with implantation time, showing striking similarities with the native collagen structure after 24 weeks. On the outside of the wall, where the engineered tissue touches the stent, collagen fibers in the vicinity of the struts aligned along the struts, whereas collagen fibers in between struts were randomly oriented. Immunohistochemistry was performed to evaluate the presence of elastin and collagen type I and III. After 8 weeks, collagen types I and III were mostly present at the luminal side of the wall, whereas at 16 and 24 weeks, a homogenous distribution of collagen I and III was observed throughout the wall. Elastin was mostly expressed at the luminal side after 24 weeks. Biochemical assays showed that the amount of DNA (as a measure of cell number) increased significantly after 8 and 24 weeks, glycosaminoglycans increased significantly after 8, 16, and 24 weeks, and hydroxyproline, as a measure of collagen amount, increased significantly after 24 weeks compared to the controls. Conclusions: The collagen matrix in the wall of decellularized TEHVs shows clear structural remodeling and maturation with time. While collagen orientation rapidly remodels toward a native anisotropic architecture on the luminal side of the engineered valvular wall, it is dominated and guided by stent geometry on the outer side of the wall. Collagen tortuosity was increased with implantation time and was accompanied by an increase in elastin, especially on the luminal side of the vessel.

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 vivo, adhesive cells continuously respond to a complex range of physical cues coming from the surrounding microenvironment by remodeling their cytoskeleton. Topographical and mechanical cues applied separately have been shown to affect the orientation of the actin stress fibers. Here we investigated the combined effects of contact guidance by topographical cues and uniaxial cyclic strain on actin cytoskeleton orientation of vascular derived cells. We devised a modular setup of stretchable circular and elliptic elastomeric microposts, capable to expose the cells to both contact guidance and uniaxial cyclic strain. A competition occurs between these cues when both contact guidance and strain are oriented along the same direction. For the first time we show that this competition originates from the distinct response of perinuclear basal and actin cap fibers: While basal fibers follow the contact guidance cue, actin cap fibers respond to the cyclic strain by strain avoidance. We also show that nuclear orientation follows actin cap fiber orientation, suggesting that actin cap fibers are responsible for cellular reorientation. Taken together, these findings may have broad implications in understanding the response of cells to combined topographical and mechanical cues.

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.

Collagen is the main load-bearing component of many soft tissues and has a large influence on the mechanical behavior of tissues when exposed to mechanical loading. Therefore, it is important to increase our understanding of collagen remodeling in soft tissues to understand the mechanisms behind pathologies and to control the development of the collagen network in engineered tissues. In the present study, a constitutive model was developed by coupling a recently developed model describing the orientation and contractile stresses exerted by cells in response to mechanical stimuli to physically motivated collagen remodeling laws. In addition, cell-mediated contraction of the collagen fibers was included as a mechanism for tissue compaction. The model appeared to be successful in predicting a range of experimental observations, which are (1) the change in transition stretch of periosteum after remodeling at different applied stretches, (2) the compaction and alignment of collagen fibers in tissue-engineered strips, (3) the fiber alignment in cruciform gels with different arm widths, and (4) the alignment of collagen fibers in engineered vascular grafts. Moreover, by changing the boundary conditions, the model was able to predict a helical architecture in the vascular graft without assuming the presence of two helical fiber families a priori. Ultimately, this model may help to increase our understanding of collagen remodeling in physiological and pathological conditions, and it may provide a tool for determining the optimal experimental conditions for obtaining native-like collagen architectures in engineered tissues.

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.

Tissue-engineered heart valves (TEHVs), based on polyglycolic acid (PGA) scaffolds coated with poly-4-hydroxybutyrate (P4HB), have shown promising in vivo results in terms of tissue formation. However, a major drawback of these TEHVs is compaction and retraction of the leaflets, causing regurgitation. To overcome this problem, the aim of this study was to investigate: (a) the use of the slowly degrading poly-e-caprolactone (PCL) scaffold for prolonged mechanical integrity; and (b) the use of lower passage cells for enhanced tissue formation. Passage 3, 5 and 7 (P3, P5 and P7) human and ovine vascular-derived cells were seeded onto both PGA-P4HB and PCL scaffold strips. After 4 weeks of culture, compaction, tissue formation, mechanical properties and cell phenotypes were compared. TEHVs were cultured to observe retraction of the leaflets in the native-like geometry. After culture, tissues based on PGA-P4HB scaffold showed 50-60% compaction, while PCL-based tissues showed compaction of 0-10%. Tissue formation, stiffness and strength were increased with decreasing passage number; however, this did not influence compaction. Ovine PCL-based tissues did render less strong tissues compared to PGA-P4HB-based tissues. No differences in cell phenotype between the scaffold materials, species or cell passage numbers were observed. This study shows that PCL scaffolds may serve as alternative scaffold materials for human TEHVs with minimal compaction and without compromising tissue composition and properties, while further optimization of ovine TEHVs is needed. Reducing cell expansion time will result in faster generation of TEHVs, providing more rapid treatment for patients.

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.

Matrix anisotropy is important for long term in vivo functionality. However, it is not fully understood how to guide matrix anisotropy in vitro. Experiments suggest actin-mediated cell traction contributes. Although F-actin in 2D displays a stretch-avoidance response, 3D data are lacking. We questioned how cyclic stretch influences F-actin and collagen orientation in 3D. Small-scale cell-populated fibrous tissues were statically constrained and/or cyclically stretched with or without biochemical agents. A rectangular array of silicone posts attached to flexible membranes constrained a mixture of cells, collagen I and matrigel. F-actin orientation was quantified using fiber-tracking software, fitted using a bi-model distribution function. F-actin was biaxially distributed with static constraint. Surprisingly, uniaxial cyclic stretch, only induced a strong stretch-avoidance response (alignment perpendicular to stretching) at tissue surfaces and not in the core. Surface alignment was absent when a ROCK-inhibitor was added, but also when tissues were only statically constrained. Stretch-avoidance was also observed in the tissue core upon MMP1-induced matrix perturbation. Further, a strong stretch-avoidance response was obtained for F-actin and collagen, for immediate cyclic stretching, i.e. stretching before polymerization of the collagen. Results suggest that F-actin stress-fibers avoid cyclic stretch in 3D, unless collagen contact guidance dictates otherwise.

Collagen is the main load-bearing component of the artery. The 3D arrangement of the collagen fibers is crucial to understand the mechanical behavior of such tissues. We compared collagen fiber alignment obtained by second harmonic generation (SHG) microscopy with the alignment obtained by diffusion tensor imaging (DTI) throughout the wall of a porcine carotid artery to check the feasibility of using DTI as a fast and non-destructive method instead of SHG. The middle part of the artery was cut into two segments: one for DTI and one for the SHG measurements. The tissue for SHG measurements was cut into 30 mu m tangential sections. After scanning all sections, they were registered together and the fiber orientation was quantified by an in-house algorithm. The tissue for DTI measurement was embedded in type VII agarose and scanned with an MRI-scanner. Fiber tractography was performed on the DTI images. Both methods showed a layered structure of the wall. The fibers were mainly oriented circumferentially in the outer adventitia and media. DTI revealed the predominant layers of the arterial wall. This study showed the feasibility of using DTI for evaluating the collagen orientation in native artery as a fast and non-destructive method. (C) 2012 Elsevier Inc. All rights reserved.

Recently, in situ tissue engineering has emerged as a new approach to obtain autologous, living replacement tissues with off-the-shelf availability. The method is based on the use of an instructive biodegradable scaffold that is capable of repopulation with host cells in situ and subsequent tissue formation. This approach imposes high demands on scaffold properties. For cardiovascular grafts, the repopulation with endogenous cells from the circulation is further hypothesized to be influenced by the hemodynamic environment of the scaffold. To systematically study the effect of scaffold properties on the response of circulating cells, we aimed to develop a mesofluidics-based in vitro test platform that enables on-stage investigation of the interaction of circulating cells with three-dimensional (3D) synthetic scaffolds under physiologic hemodynamic conditions. The test platform consists of a custom-developed cross-flow chamber that houses small-scale 3D scaffolds. The cross-flow chamber is incorporated into a flow-loop to drive a cell suspension along the scaffold with physiological wall shear stress and perfusion pressure. The fluidics system is validated numerically and experimentally using a computational fluid dynamics model and real-time microbead tracing studies, demonstrating a fully developed flow profile with a homogeneous shear stress distribution over the scaffold. Wall shear stresses and pressure can be controlled independently, well within the target physiological range (0-8 Pa and 0-100 mmHg, respectively). Bench-top evaluation is performed using electrospun poly(¿-caprolactone) scaffolds with varying fiber diameter, exposed to a suspension of human peripheral blood mononuclear cells in pulsatile flow for 72¿h. Cell adhesion and infiltration are monitored using time-lapsed confocal laser scanning microscopy. In conclusion, we have successfully developed a mesofluidics platform to study cell-scaffold interactions under hemodynamic conditions in vitro. This platform not only enables us to systematically screen and develop potential scaffolds for future in situ cardiovascular tissue engineering approaches, but also acts as a tool to further elucidate processes as observed in vivo.

Decellularized xenogenic or allogenic heart valves have been used as starter matrix for tissue-engineering of valve replacements with (pre-)clinical promising results. However, xenografts are associated with the risk of immunogenic reactions or disease transmission and availability of homografts is limited. Alternatively, biodegradable synthetic materials have been used to successfully create tissue-engineered heart valves (TEHV). However, such TEHV are associated with substantial technological and logistical complexity and have not yet entered clinical use. Here, decellularized TEHV, based on biodegradable synthetic materials and homologous cells, are introduced as an alternative starter matrix for guided tissue regeneration. Decellularization of TEHV did not alter the collagen structure or tissue strength and favored valve performance when compared to their cell-populated counterparts. Storage of the decellularized TEHV up to 18 months did not alter valve tissue properties. Reseeding the decellularized valves with mesenchymal stem cells was demonstrated feasible with minimal damage to the reseeded valve when trans-apical valve delivery was simulated. In conclusion, decellularization of in-vitro grown TEHV provides largely available off-the-shelf homologous scaffolds suitable for reseeding with autologous cells and trans-apical valve delivery.

In situ vascular tissue engineering (TE) aims at regenerating vessels using implanted synthetic scaffolds. An envisioned strategy is to capture and differentiate progenitor cells from the bloodstream into the porous scaffold to initiate tissue formation. Among these cells are the endothelial colonies forming cells (ECFCs) that can differentiate into endothelial cells and transdifferentiate into smooth muscle cells under biochemical stimulation. The influence of mechanical stimulation is unknown, but relevant for in situ vascular TE because the cells perceive a change in mechanical environment when captured inside the scaffold, where they are shielded from blood flow induced shear stresses. Here we investigate the effects of substrate stiffness as one of the environmental mechanical cues to control ECFC fate within scaffolds. ECFCs were seeded on soft (3.58±0.90. kPa), intermediate (21.59±2.91. kPa), and stiff (93.75±18.36. kPa) fibronectin-coated polyacrylamide gels, as well as on glass controls, and compared to peripheral blood mononuclear cells (PBMC). Cell behavior was analyzed in terms of adhesion (vinculin staining), proliferation (BrdU), phenotype (CD31, aSMA staining, and flow cytometry), and collagen production (col I, III, and IV). While ECFCs adhesion and proliferation increased with substrate stiffness, no change in phenotype was observed. The cells produced no collagen type I, but abundant amounts of collagen type III and IV, albeit in a stiffness-dependent organization. PBMCs did not adhere to the gels, but they did adhere to glass, where they expressed CD31 and collagen type III. Addition mechanical cues, such as cyclic strains, should be studied to further investigate the effect of the mechanical environment on captured circulating cells for in situ TE purposes.

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.

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.

© 2014, Biomedical Engineering Society. The engineering of technologies for heart valve replacement (i.e., heart valve engineering) is an exciting and evolving field. Since the first valve replacement, technology has progressed by leaps and bounds. Innovations emerge frequently and supply patients and physicians with new, increasingly efficacious and less invasive treatment options. As much as any other field in medicine the treatment of heart valve disease has experienced a renaissance in the last 10 years. Here we review the currently available technologies and future options in the surgical and transcatheter treatment of aortic valve disease. Different valves from major manufacturers are described in details with their applications.

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.

The collagen architecture is the major determinant of the function and mechanical behavior of cardiovascular tissues. In order to engineer a functional and load-bearing cardiovascular tissue with a structure thatmimics the native tissue to meet in vivo mechanical demands, a complete understanding of the collagen orientation mechanism is required. Several methods have been used to visualize collagen architecture in tissue-engineered (TE) constructs, but they either have a limited imaging depth or have a complicated set up. In this study, Diffusion Tensor Imaging (DTI) is explored as a fast and reliable method to visualize collagen arrangement, and Confocal Laser Scanning Microscopy (CLSM) was used as a validation technique. Uniaxially constrained TE strips were cultured for 2 days, 10 days, 3 and 6 weeks to investigate the evolution of the collagen orientation with time. Moreover, a comparison of the collagen orientation in high and low aspect ratio (length/ width) TE constructs was made with both methods. Both methods showed similar fiber orientation in TE constructs. Collagen fibers in the high aspect ratio samples were mostly aligned in the constrained direction, while the collagen fibers in low aspect ratio strips were mainly oriented in the oblique direction. The orientation changed to the oblique direction by extending culture time and could also be visualized. DTI captured the collagen orientation differences between low and high aspect ratio samples and with time. Therefore, it can be used as a fast, non-destructive and reliable tool to study the evolution of the collagen orientation in TE constructs.

Two types of scaffolds were developed for tissue engineering of the aortic valve; an electrospun valvular scaffold and a knitted valvular scaffold. These scaffolds were compared in a physiologic flow system and in a tissue-engineering process. In fibrin gel enclosed human myofibroblasts were seeded onto both types of scaffolds and cultured for 23 days under continuous medium perfusion. Tissue formation was evaluated by confocal laser scanning microscopy, histology and DNA quantification. Collagen formation was quantified by a hydroxyproline assay. When subjected to physiologic flow, the spun scaffold tore within 6 h, whereas the knitted scaffold remained intact. Cells proliferated well on both types of scaffolds, although the cellular penetration into the spun scaffold was poor. Collagen production, normalized to DNA content, was not significantly different for the two types of scaffolds, but seeding efficiency was higher for the spun scaffold, because it acted as a cell impermeable filter. The knitted tissue constructs showed complete cellular in-growth into the pores. An optimal scaffold seems to be a combination of the strength of the knitted structure and the cell-filtering ability of the spun structure.

Mesenchymal stromal cells (MSC) play an important role in natural wound healing via paracrine and juxtacrine signaling to immune cells. The aim of this study was to identify the signaling factors secreted by preseeded cells in a biomaterial and their interaction with circulating leukocytes, in the presence of physiological biomechanical stimuli exerted by the hemodynamic environment (i.e. strain and shear flow). Electrospun poly(e-caprolactone)-based scaffolds were seeded with human peripheral blood mononuclear cells (PBMC) or MSC. Protein secretion was analyzed under static conditions and cyclic strain. Subsequently, the cross-talk between preseeded cells and circulating leukocytes was addressed by exposing the scaffolds to a suspension of PBMC in static transwells and in pulsatile flow. Our results revealed that PBMC exposed to the scaffold consistently secreted a cocktail of immunomodulatory proteins under all conditions tested. Preseeded MSC, on the other hand, secreted the trophic factors MCP-1, VEGF and bFGF. Furthermore, we observed a synergistic upregulation of CXCL12 gene expression and a synergistic increase in bFGF protein production by preseeded MSC exposed to PBMC in pulsatile flow. These findings identify CXCL12 and bFGF as valuable targets for the development of safe and effective acellular instructive grafts for application in in situ cardiovascular regenerative therapies. © 2014 Elsevier Ltd.

Implanted synthetic substrates for the regeneration of cardiovascular tissues are exposed to mechanical forces that induce local deformation. Circulating inflammatory cells, actively participating in the healing process, will be subjected to strain once recruited. We investigated the effect of deformation on human peripheral blood mononuclear cells (hPBMCs) adherent onto a scaffold, with respect to macrophage polarization towards an inflammatory (M1) and reparative (M2) phenotype and to early tissue formation. HPBMCs were seeded onto poly-e-caprolactone bisurea strips and subjected to 0%, 7% and 12% cyclic strain for up to one week. After 1 day, cells subjected to 7% deformation showed upregulated expression of pro and anti-inflammatory chemokines, such as MCP-1 and IL10. Immunostaining revealed presence of inflammatory macrophages in all groups, while immunoregulatory macrophages were detected mainly in the 0 and 7% groups and increased significantly over time. Biochemical assays indicated deposition of sulphated glycosaminoglycans and collagen after 7 days in both strained and unstrained samples. These results suggest that 7% cyclic strain applied to hPBMCs adherent on a scaffold modulates their polarization towards reparative macrophages and allows for early synthesis of extracellular matrix components, required to promote further cell adhesion and proliferation and to bind immunoregulatory cytokines. © 2014 Elsevier Ltd.

Novel cardiovascular replacements are being developed by using degradable synthetic scaffolds, which function as a temporary guide to induce neotissue formation directly in situ. Priming of such scaffolds with fast-releasing monocyte chemoattractant protein-1 (MCP-1) was shown to improve the formation of functional neoarteries in rats. However, the underlying mechanism has not been clarified. Therefore, the goal of this study was to investigate the effect of a burst-release of MCP-1 from a synthetic scaffold on the local recruitment of circulating leucocytes under haemodynamic conditions. Herein, we hypothesized that MCP-1 initiates a desired healing cascade by recruiting favourable monocyte subpopulations into the implanted scaffold. Electrospun poly(ε-caprolactone) scaffolds were loaded with fibrin gel containing various doses of MCP-1 and exposed to a suspension of human peripheral blood mononuclear cells in static or dynamic conditions. In standard migration assay, a dose-dependent migration of specific CD14(+) monocyte subsets was observed, as measured by flow cytometry. In conditions of pulsatile flow, on the other hand, a marked increase in immediate monocyte recruitment was observed, but without evident selectivity in monocyte subsets. This suggests that the selectivity was dependent on the release kinetics of the MCP-1, as it was overruled by the effect of shear stress after the initial burst-release. Furthermore, these findings demonstrate that local recruitment of specific MCP-1-responsive monocytes is not the fundamental principle behind the improved neotissue formation observed in long-term in vivo studies, and mobilization of MCP-1-responsive cells from the bone marrow into the bloodstream is suggested to play a predominant role in vivo.

This study is focused on the description of ductile fracture initiation, which is needed to predict product shapes in the blanking process. Two approaches are elaborated using a local ductile fracture model. According to literature, characterization of such a model should take place under loading conditions, comparable to the application. Therefore, the first approach incorporates the characterization of a ductile fracture model in a blanking experiment. The second approach is more favorable for industry. In this approach a tensile test is used to characterize the fracture model, instead of a complex and elaborate blanking experiment. Finite element simulations and blanking experiments are performed for five different clearances to validate both approaches. In conclusion it can be stated that for the investigated material, the first approach gives very good results within the experimental error. The second approach, the more favorable one for industry, yields results within 6 percent of the experiments over a wide, industrial range of clearances, when a newly proposed criterion is used. [S1087-1357(00)02202-4]

Tissue engineering represents a promising technique to overcome the limitations of the current valve replacements, since it allows for creating living autologous heart valves that have the potential to grow and remodel. However, also this approach still faces a number of challenges. One particular problem is regurgitation, caused by cell-mediated tissue retraction or the mismatch in geometrical and material properties between tissue-engineered heart valves (TEHVs) and their native counterparts. The goal of the present study was to assess the influence of valve geometry and tissue anisotropy on the deformation profile and closed configuration of TEHVs. To achieve this aim, a range of finite element models incorporating different valve shapes was developed, and the constitutive behavior of the tissue was modeled using an established computational framework, where the degree of anisotropy was varied between values representative of TEHVs and native valves. The results of this study suggest that valve geometry and tissue anisotropy are both important to maximize the radial strains and thereby the coaptation area. Additionally, the minimum degree of anisotropy that is required to obtain positive radial strains was shown to depend on the valve shape and the pressure to which the valves are exposed. Exposure to pulmonary diastolic pressure only yielded positive radial strains if the anisotropy was comparable to the native situation, whereas considerably less anisotropy was required if the valves were exposed to aortic diastolic pressure.

Mathematical models can provide valuable information to assess and evaluate the mechanical behavior and remodeling of native tissue. A relevant example when studying collagen remodeling is the Ross procedure because it involves placing the pulmonary autograft in the more demanding aortic valve mechanical environment. The objective of this study was therefore to assess and evaluate the mechanical differences between the aortic valve and pulmonary valve and the remodeling that may occur in the pulmonary valve when placed in the aortic position. The results from biaxial tensile tests of pairs of human aortic and pulmonary valves were compared and used to determine the parameters of a structurally based constitutive model. Finite element analyzes were then performed to simulate the mechanical response of both valves to the aortic diastolic load. Additionally, remodeling laws were applied to assess the remodeling of the pulmonary valve leaflet to the new environment. The pulmonary valve showed to be more extensible and less anisotropic than the aortic valve. When exposed to aortic pressure, the pulmonary leaflet appeared to remodel by increasing its thickness and reorganizing its collagen fibers, rotating them toward the circumferential direction. © 2013 Elsevier Ltd.

Mechanical loading of soft tissues covering bony prominences can cause skeletal muscle damage, ultimately resulting in a severe pressure ulcer termed deep tissue injury (DTI). Deformation plays an important role in the aetiology of DTI. Therefore, it is essential to minimise internal muscle deformations in subjects at risk of DTI. As an example, spinal cord-injured (SCI) individuals exhibit structural changes leading to a decrease in muscle thickness and stiffness, which subsequently increase the tissue deformations. In the present study, an animal-specific finite element model, where the geometry and boundary conditions were derived from magnetic resonance images, was developed. It was used to investigate the internal deformations in the muscle, fat and skin layers of the porcine buttocks during loading. The model indicated the presence of large deformations in both the muscle and the fat layers, with maximum shear strains up to 0.65 in muscle tissue and 0.63 in fat. Furthermore, a sensitivity analysis showed that the tissue deformations depend considerably on the relative stiffness values of the different tissues. For example, a change in muscle stiffness had a large effect on the muscle deformations. A 50% decrease in stiffness caused an increase in maximum shear strain from 0.65 to 0.99, whereas a 50% increase in stiffness resulted in a decrease in maximum shear strain from 0.65 to 0.49. These results indicate the importance of restoring tissue properties after SCI, with the use of, for example, electrical stimulation, to prevent the development of DTI.

Skin is a multilayer composite and exhibits highly non-linear, viscoelastic, anisotropic material properties. In many consumer product and medical applications (e.g. during shaving, needle insertion, patient re-positioning), large tissue displacements and deformations are involved; consequently large local strains in the skin tissue can occur. Here, we present a novel imaging-based method to study skin deformations and the mechanics of interacting skin layers of full-thickness skin. Shear experiments and real-time video recording were combined with digital image correlation and strain field analysis to visualise and quantify skin layer deformations during dynamic mechanical testing. A global shear strain of 10% was applied to airbrush-patterned porcine skin (thickness: 1.2–1.6 mm) using a rotational rheometer. The recordings were analysed with ARAMIS image correlation software, and local skin displacement, strain and stiffness profiles through the skin layers determined. The results of this pilot study revealed inhomogeneous skin deformation, characterised by a gradual transition from a low (2.0–5.0%; epidermis) to high (10–22%; dermis) shear strain regime. Shear moduli ranged from 20 to 130 kPa. The herein presented method will be used for more extended studies on viable human skin, and is considered a valuable foundation for further development of constitutive models which can be used in advanced finite element analyses of skin

Since the implantation of the first artificial aortic heart valve by Hufnagel et al., the design and construction of heart valve prostheses has undergone continuous and drastic changes to improve t...

Mechanical loading protocols in tissue engineering (TE) aim to improve the deposition of a properly organized collagen fiber network. In addition to collagen remodeling, these conditioning protocols can result in tissue compaction. Tissue compaction is beneficial to tissue collagen alignment, yet it may lead to a loss of functionality of the TE construct due to changes in geometry after culture. Here, a mathematical model is presented to relate the changes in collagen architecture to the local compaction within a TE small blood vessel, assuming that under static conditions, compaction is the main factor responsible for collagen fiber organization. An existing structurally based model is extended to incorporate volumetric tissue compaction. Subsequently, the model is applied to describe the collagen architecture of TE constructs under either strain based or stress based stimulus functions. Our computations indicate that stress based simulations result in a helical collagen fiber distribution along the vessel wall. The helix pitch angle increases from a circumferential direction in the inner wall, over about 45 deg in the middle vessel layer, to a longitudinal direction in the outer wall. These results are consistent with experimental data from TE small diameter blood vessels. In addition, our results suggest a stress dependent remodeling of the collagen, suggesting that cell traction is responsible for collagen orientation. These findings may be of value to design improved mechanical conditioning protocols to optimize the collagen architecture in engineered tissues.

Mechanical behavior of tissue-engineered heart valves still needs improvement when native aortic valves are considered as a benchmark [1]. Research showed that mechanical stimulation of tissue-engineered heart valves improved tissue properties [2], although a complete understanding of the effect of loading on tissue development has not been established yet.Copyright © 2009 by ASME

In cardiovascular tissue engineering, mechanical stimulation of tissue-engineered constructs is known to improve tissue properties. During tissue culture, the mechanical properties of the tissue construct change. To impose a predefined deformation protocol and to avoid negative effects of excessive strain, it is desired to monitor and control deformations during load application. In a previous study, load application and resulting deformation of tissue-engineered heart valve leaflets were monitored during culture inside a bioreactor in real time and noninvasively. A combined experimental-numerical approach was applied to assess volumetric and local leaflet deformation of the cultured heart valve in a diastolic configuration. In this study, this approach was further developed and a feedback controller to regulate deformation was incorporated into the bioreactor system. Functionality of this technique was demonstrated in two tissue engineering experiments in which a total of eight heart valves were cultured by application of two different deformation protocols. Results indicated a good correspondence between the measured and the prescribed deformation values in both experiments. In addition, the cultured heart valves showed mechanical properties in the range of previous tissue engineering studies. The bioreactor system including the deformation measurement and control features has promising possibilities of systematically elucidating the effects of loading protocols on tissue properties. In conclusion, it facilitates the development of an optimal conditioning protocol for tissue engineering of aortic heart valves. © 2009 Mary Ann Liebert, Inc.

Pressure ulcers are areas of soft tissue breakdown resulting from sustained mechanical loading of the skin and underlying tissues. These ulcers are painful, difficult to treat, and represent a burden to the community in terms of health care and money. Currently, pressure ulcer risk assessment is dominated by subjective measures and does not predict pressure ulcer development satisfactorily [1]. Objective measures are therefore needed for early, non-invasive detection.

A finite-element approach was formulated, aimed at enabling an integrated study of mechanical and biochemical factors that control the functional development of tissue engineered constructs. A nonlinear biphasic displacement-velocity-pressure description was combined with adjective and diffusive solute transport, uptake and biosynthesis. To illustrate the approach we focused on the synthesis and transport of macromolecules under influence of fluid flow induced by cyclic compression. In order to produce net transport the effect of dispersion was investigated. An abstract representation of biosynthesis was employed, three cases were distinguished: Synthesis dependent on a limited small solute, synthesis dependent on a limited large solute and synthesis independent of solute transport. Results show that a dispersion model can account for augmented solute transport by cyclic compression and indicate the different sensitivity to loading that can be expected depending on the size of the limiting solute.

Transcatheter heart valve replacement is an attractive and promising technique for congenital as well as acquired heart valve disease. In this procedure, the replacement valve is mounted in a stent that is expanded at the aimed valve position and fixated by clamping. However, for this technique to be appropriate for pediatric patients, the material properties of the host tissue need to be determined to design stents that can be optimized for this particular application. In this study we performed equibiaxial tensile tests on four adult ovine pulmonary artery walls and compared the outcomes with one pediatric pulmonary artery. Results show that the pediatric pulmonary artery was significantly thinner (1.06±0.36. mm (mean±SD)) than ovine tissue (2.85±0.40. mm), considerably stiffer for strain values that exceed the physiological conditions (beyond 50% strain in the circumferential and 60% in the longitudinal direction), more anisotropic (with a significant difference in stiffness between the longitudinal and circumferential directions beyond 60% strain) and presented stronger non-linear stress-strain behavior at equivalent strains (beyond 26% strain) compared to ovine tissue. These discrepancies suggest that stents validated and optimized using the ovine pre-clinical model might not perform satisfactorily in pediatric patients. The material parameters derived from this study may be used to develop stent designs for both applications using computational models. © 2013 Elsevier Ltd.

One of the most critical problems in heart valve tissue engineering is the progressive development of valvular insufficiency due to leaflet retraction. Understanding the underlying mechanisms of this process is crucial for developing tissue-engineered heart valves (TEHVs) that maintain their functionality in the long term. In the present study, we adopted a computational approach to predict the remodeling process in TEHVs subjected to dynamic pulmonary and aortic pressure conditions, and to assess the risk of valvular insufficiency. In addition, we investigated the importance of the intrinsic cell contractility on the final outcome of the remodeling process. For valves implanted in the aortic position, the model predictions suggest that valvular insufficiency is not likely to occur as the blood pressure is high enough to prevent the development of leaflet retraction. In addition, the collagen network was always predicted to remodel towards a circumferentially aligned network, which is corresponding to the native situation. In contrast, for valves implanted in the pulmonary position, our model predicted that there is a high risk for the development of valvular insufficiency, unless the cell contractility is very low. Conversely, the development of a circumferential collagen network was only predicted at these pressure conditions when cell contractility was high. Overall, these results, therefore, suggest that tissue remodeling at aortic pressure conditions is much more stable and favorable compared to tissue remodeling at pulmonary pressure conditions.