Cellular strain avoidance is mediated by a functional actin cap - observations in an Lmna-deficient cell model.

In adherent cells, the relevance of a physical mechanotransduction pathway provided by the perinuclear actin cap stress fibers has recently emerged. Here, we investigate the impact of a functional actin cap on the cellular adaptive response to topographical cues and uniaxial cyclic strain. Lmna-deficient fibroblasts are used as a model system because they do not develop an intact actin cap, but predominantly form a basal layer of actin stress fibers underneath the nucleus. We observe that topographical cues induce alignment in both normal and Lmna-deficient fibroblasts, suggesting that the topographical signal transmission occurs independently of the integrity of the actin cap. By contrast, in response to cyclic uniaxial strain, Lmna-deficient cells show a compromised strain avoidance response, which is completely abolished when topographical cues and uniaxial strain are applied along the same direction. These findings point to the importance of an intact and functional actin cap in mediating cellular strain avoidance.

3D Fiber Orientation in Atherosclerotic Carotid Plaques.

Atherosclerotic plaque rupture is the primary trigger of fatal cardiovascular events. Fibrillar collagen in atherosclerotic plaques and their directionality are anticipated to play a crucial role in plaque rupture. This study aimed assessing 3D fiber orientations and architecture in atherosclerotic plaques for the first time. Seven carotid plaques were imaged ex-vivo with a state-of-the-art Diffusion Tensor Imaging (DTI) technique, using a high magnetic field (9.4Tesla) MRI scanner. A 3D spin-echo sequence with uni-polar diffusion sensitizing pulsed field gradients was utilized for DTI and fiber directions were assessed from diffusion tensor measurements. The distribution of the 3D fiber orientations in atherosclerotic plaques were quantified and the principal fiber orientations (circumferential, longitudinal or radial) were determined. Overall, 52% of the fiber orientations in the carotid plaque specimens were closest to the circumferential direction, 34% to the longitudinal direction, and 14% to the radial direction. Statistically no significant difference was measured in the amount of the fiber orientations between the concentric and eccentric plaque sites. However, concentric plaque sites showed a distinct structural organization, where the principally longitudinally oriented fibers were closer to the luminal side and the principally circumferentially oriented fibers were located more abluminally. The acquired unique information on 3D plaque fiber direction will help understanding pathobiological mechanisms of atherosclerotic plaque progression and pave the road to more realistic biomechanical plaque modeling for rupture assessment.

Prediction of Cell Alignment on Cyclically Strained Grooved Substrates.

Cells respond to both mechanical and topographical stimuli by reorienting and reorganizing their cytoskeleton. Under certain conditions, such as for cells on cyclically stretched grooved substrates, the effects of these stimuli can be antagonistic. The biophysical processes that lead to the cellular reorientation resulting from such a competition are not clear yet. In this study, we hypothesized that mechanical cues and the diffusion of the intracellular signal produced by focal adhesions are determinants of the final cellular alignment. This hypothesis was investigated by means of a computational model, with the aim to simulate the (re)orientation of cells cultured on cyclically stretched grooved substrates. The computational results qualitatively agree with previous experimental studies, thereby supporting our hypothesis. Furthermore, cellular behavior resulting from experimental conditions different from the ones reported in the literature was simulated, which can contribute to the development of new experimental designs.

Collagen Matrix Remodeling in Stented Pulmonary Arteries after Transapical Heart Valve Replacement.

The use of valved stents for minimally invasive replacement of semilunar heart valves is expected to change the extracellular matrix and mechanical function of the native artery and may thus impair long-term functionality of the implant. Here we investigate the impact of the stent on matrix remodeling of the pulmonary artery in a sheep model, focusing on matrix composition and collagen (re)orientation of the host tissue. Ovine native pulmonary arteries were harvested 8 (n = 2), 16 (n = 4) and 24 (n = 2) weeks after transapical implantation of self-expandable stented heart valves. Second harmonic generation (SHG) microscopy was used to assess the collagen (re)orientation of fresh tissue samples. The collagen and elastin content was quantified using biochemical assays. SHG microscopy revealed regional differences in collagen organization in all explants. In the adventitial layer of the arterial wall far distal to the stent (considered as the control tissue), we observed wavy collagen fibers oriented in the circumferential direction. These circumferential fibers were more straightened in the adventitial layer located behind the stent. On the luminal side of the wall behind the stent, collagen fibers were aligned along the stent struts and randomly oriented between the struts. Immediately distal to the stent, however, fibers on both the luminal and the adventitial side of the wall were oriented in the axial direction, demonstrating the stent impact on the collagen structure of surrounding arterial tissues. Collagen orientation patterns did not change with implantation time, and biochemical analyses showed no changes in the trend of collagen and elastin content with implantation time or location of the vascular wall. We hypothesize that the collagen fibers on the adventitial side of the arterial wall and behind the stent straighten in response to the arterial stretch caused by oversizing of the stent. However, the collagen organization on the luminal side suggests that stent-induced remodeling is dominated by contact guidance.

Shear flow affects selective monocyte recruitment into MCP-1-loaded scaffolds.

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.

Differential response of endothelial and endothelial colony forming cells on electrospun scaffolds with distinct microfiber diameters.

Electrospun scaffolds for in situ tissue engineering can be prepared with different fiber diameters to influence cell recruitment, adhesion, and differentiation. For cardiovascular applications, we investigated the impact of different fiber diameters (2, 5, 8, and 11 µm) in electrospun poly(ε-caprolactone) scaffolds on endothelial colony forming cells (ECFCs) in comparison to mature endothelial cells (HUVECs). In 2D cultures and on 2 µm fiber scaffolds, ECFC morphology and phenotype resemble those of HUVECs. When cultured on scaffolds with 5-11 µm fibers, a different behavior was detected. HUVECs developed a cytoskeleton organized circumferentially around the fibers, with collagen alignment in the same direction. ECFCs, instead, aligned the cytoskeleton along the scaffold fiber axis and deposited a homogeneous layer of collagen over the fibers; moreover, a subpopulation of ECFCs gained the αSMA marker. These results showed that ECFCs do not behave like mature endothelial cells in a 3D fibrous environment.

Understanding strain-induced collagen matrix development in engineered cardiovascular tissues from gene expression profiles.

Mechanical conditioning is often used to enhance collagen synthesis, remodeling and maturation and, hence, the structural and mechanical properties of engineered cardiovascular tissues. Intermittent straining, i.e., alternating periods of cyclic and static strain, has previously been shown to result in more mature tissue compared with continuous cyclic straining. Nevertheless, the underlying mechanism is unknown. We have determined the short-term effects of continuous cyclic strain and of cyclic strain followed by static strain at the gene expression level to improve insight into the mechano-regulatory mechanism of intermittent conditioning on collagen synthesis, remodeling and maturation. Tissue-engineered constructs, consisting of human vascular-derived cells seeded onto rapidly degrading PGA/P4HB scaffolds, were conditioned with 4% strain at 1 Hz for 3 h in order to study the immediate effects of cyclic strain (n=18). Next, the constructs were either subjected to ongoing cyclic strain (4% at 1 Hz; n=9) or to static strain (n=9). Expression levels of genes involved in collagen synthesis, remodeling and maturation were studied at various time points up to 24 h within each straining protocol. The results indicate that a period of static strain following cyclic strain favors collagen synthesis and remodeling, whereas ongoing cyclic strain shifts this balance toward collagen remodeling and maturation. The data suggest that, with prolonged culture, the conditioning protocol should be changed from intermittent straining to continuous cyclic straining to improve collagen maturation after its synthesis and, hence, the tissue (mechanical) properties.

A multiscale computational model of arterial growth and remodeling including Notch signaling.

Blood vessels grow and remodel in response to mechanical stimuli. Many computational models capture this process phenomenologically, by assuming stress homeostasis, but this approach cannot unravel the underlying cellular mechanisms. Mechano-sensitive Notch signaling is well-known to be key in vascular development and homeostasis. Here, we present a multiscale framework coupling a constrained mixture model, capturing the mechanics and turnover of arterial constituents, to a cell-cell signaling model, describing Notch signaling dynamics among vascular smooth muscle cells (SMCs) as influenced by mechanical stimuli. Tissue turnover was regulated by both Notch activity, informed by in vitro data, and a phenomenological contribution, accounting for mechanisms other than Notch. This novel framework predicted changes in wall thickness and arterial composition in response to hypertension similar to previous in vivo data. The simulations suggested that Notch contributes to arterial growth in hypertension mainly by promoting SMC proliferation, while other mechanisms are needed to fully capture remodeling. The results also indicated that interventions to Notch, such as external Jagged ligands, can alter both the geometry and composition of hypertensive vessels, especially in the short term. Overall, our model enables a deeper analysis of the role of Notch and Notch interventions in arterial growth and remodeling and could be adopted to investigate therapeutic strategies and optimize vascular regeneration protocols.

Trans-apical versus surgical implantation of autologous ovine tissue-engineered heart valves.

BACKGROUND AND AIM OF THE STUDY: Living tissue-engineered heart valves (TEHVs) based on rapidly degrading scaffolds and autologous cells might overcome the limitations of today's valve substitutes. Following minimally invasive trans-apical implantation into an ovine model, TEHVs showed adequate in-vivo functionality, but a thickening of the leaflets was observed. In order to evaluate the impact of the substantial tissue deformations of TEHVs associated with the crimping procedure during minimally invasive delivery, trans-apical and conventional implantation technologies were compared in an ovine model. METHODS: Trileaflet heart valves (n=11) based on PGA/P4HB-scaffolds, integrated into self-expandable stents, were engineered from autologous ovine vascular-derived cells. After in-vitro culture, the TEHVs were either implanted surgically (n=5), replacing the native pulmonary valve, or delivered trans-apically (n=6) into the orthotopic pulmonary valve position. In-vivo functionality was assessed by echocardiography and by angiography for up to eight weeks. The tissue compositions of the explanted TEHVs and corresponding control valves were analyzed. RESULTS: TEHV implantations were successful in all cases. Independent of the implantation method, the explants demonstrated a comparable layered tissue formation with thickening and deposited fibrous layers. Active remodeling of these layers was evident in the explants, as indicated by vascularization of the walls, invasion of the host cells, and the formation of a luminal endothelial layer on the TEHV leaflets. CONCLUSION: This direct comparison of trans-apical and conventional surgical implantation techniques showed that crimping had no adverse effect on the integrity or functional outcome of TEHVs. This suggests that a thickening of TEHVs in vivo is neither caused by nor enhanced by the crimping procedure, but represents a functional tissue remodeling process.

Computational analysis of the role of mechanosensitive Notch signaling in arterial adaptation to hypertension.

Arteries grow and remodel in response to mechanical stimuli. Hypertension, for example, results in arterial wall thickening. Cell-cell Notch signaling between vascular smooth muscle cells (VSMCs) is known to be involved in this process, but the underlying mechanisms are still unclear. Here, we investigated whether Notch mechanosensitivity to strain may regulate arterial thickening in hypertension. We developed a multiscale computational framework by coupling a finite element model of arterial mechanics, including residual stress, to an agent-based model of mechanosensitive Notch signaling, to predict VSMC phenotypes as an indicator of growth and remodeling. Our simulations revealed that the sensitivity of Notch to strain at mean blood pressure may be a key mediator of arterial thickening in hypertensive arteries. Further simulations showed that loss of residual stress can have synergistic effects with hypertension, and that changes in the expression of Notch receptors, but not Jagged ligands, may be used to control arterial growth and remodeling and to intensify or counteract hypertensive thickening. Overall, we identify Notch mechanosensitivity as a potential mediator of vascular adaptation, and we present a computational framework that can facilitate the testing of new therapeutic and regenerative strategies.

Remodeling of the collagen fiber architecture due to compaction in small vessels under tissue engineered conditions.

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.

Next-generation tissue-engineered heart valves with repair, remodelling and regeneration capacity.

Valvular heart disease is a major cause of morbidity and mortality worldwide. Surgical valve repair or replacement has been the standard of care for patients with valvular heart disease for many decades, but transcatheter heart valve therapy has revolutionized the field in the past 15 years. However, despite the tremendous technical evolution of transcatheter heart valves, to date, the clinically available heart valve prostheses for surgical and transcatheter replacement have considerable limitations. The design of next-generation tissue-engineered heart valves (TEHVs) with repair, remodelling and regenerative capacity can address these limitations, and TEHVs could become a promising therapeutic alternative for patients with valvular disease. In this Review, we present a comprehensive overview of current clinically adopted heart valve replacement options, with a focus on transcatheter prostheses. We discuss the various concepts of heart valve tissue engineering underlying the design of next-generation TEHVs, focusing on off-the-shelf technologies. We also summarize the latest preclinical and clinical evidence for the use of these TEHVs and describe the current scientific, regulatory and clinical challenges associated with the safe and broad clinical translation of this technology.

Hypoxia induces near-native mechanical properties in engineered heart valve tissue.

BACKGROUND: Previous attempts in heart valve tissue engineering (TE) failed to produce autologous valve replacements with native-like mechanical behavior to allow for systemic pressure applications. Because hypoxia and insulin are known to promote protein synthesis by adaptive cellular responses, a physiologically relevant oxygen tension and insulin supplements were applied to the growing heart valve tissues to enhance their mechanical properties. METHODS AND RESULTS: Scaffolds of rapid-degrading polyglycolic acid meshes coated with poly-4-hydroxybutyrate were seeded with human saphenous vein myofibroblasts. The tissue-engineered constructs were cultured under normal oxygen tension (normoxia) or hypoxia (7% O(2)) and incubated with or without insulin. Glycosaminoglycan production in the constructs approached that of native values under the influence of hypoxia and under the influence of insulin. Both insulin and hypoxia were associated with enhanced matrix production and improved mechanical properties; however, a synergistic effect was not observed. Although the amount of collagen and cross-links in the engineered tissues was still lower than that in native adult human aortic valves, constructs cultured under hypoxic conditions reached native human aortic valve levels of tissue strength and stiffness after 4 weeks of culturing. CONCLUSIONS: These results indicate that oxygen tension may be a key parameter for the achievement of sufficient tissue quality and mechanical integrity in tissue-engineered heart valves. Engineered tissues of such strength, based on rapid-degrading polymers, have not been achieved to date. These findings bring the potential use of tissue-engineered heart valves for systemic applications a step closer and represent an important improvement in heart valve tissue engineering.

Transcatheter-Delivered Expandable Bioresorbable Polymeric Graft With Stenting Capacity Induces Vascular Regeneration.

As the next step in the translation of vascular tissue engineering, this study uniquely combines transcatheter delivery and in situ tissue regeneration using a novel bioresorbable electrospun polymer graft that can be implanted minimally invasively. Once delivered inside a small-diameter vessel, the electrospun microstructure supports the vessel wall, facilitates cellular infiltration, and guides organized tissue formation.

Geometry influences inflammatory host cell response and remodeling in tissue-engineered heart valves in-vivo.

Regenerative tissue-engineered matrix-based heart valves (TEM-based TEHVs) may become an alternative to currently-used bioprostheses for transcatheter valve replacement. We recently identified TEM-based TEHVs-geometry as one key-factor guiding their remodeling towards successful long-term performance or failure. While our first-generation TEHVs, with a simple, non-physiological valve-geometry, failed over time due to leaflet-wall fusion phenomena, our second-generation TEHVs, with a computational modeling-inspired design, showed native-like remodeling resulting in long-term performance. However, a thorough understanding on how TEHV-geometry impacts the underlying host cell response, which in return determines tissue remodeling, is not yet fully understood. To assess that, we here present a comparative samples evaluation derived from our first- and second-generation TEHVs. We performed an in-depth qualitative and quantitative (immuno-)histological analysis focusing on key-players of the inflammatory and remodeling cascades (M1/M2 macrophages, α-SMA+- and endothelial cells). First-generation TEHVs were prone to chronic inflammation, showing a high presence of macrophages and α-SMA+-cells, hinge-area thickening, and delayed endothelialization. Second-generation TEHVs presented with negligible amounts of macrophages and α-SMA+-cells, absence of hinge-area thickening, and early endothelialization. Our results suggest that TEHV-geometry can significantly influence the host cell response by determining the infiltration and presence of macrophages and α-SMA+-cells, which play a crucial role in orchestrating TEHV remodeling.

Mechanical characterization of anisotropic planar biological soft tissues using finite indentation: experimental feasibility.

Heart valve tissue engineering offers a promising alternative for current treatment and replacement strategies, e.g., synthetic or bioprosthetic heart valves. In vitro mechanical conditioning is an important tool for engineering strong, implantable heart valves. Detailed knowledge of the mechanical properties of the native tissue as well as the developing tissue construct is vital for a better understanding and control of the remodeling processes induced by mechanical conditioning. The nonlinear, anisotropic and inhomogeneous mechanical behavior of heart valve tissue necessitates a mechanical characterization method that is capable of dealing with these complexities. In a recent computational study we showed that one single indentation test, combining force and deformation gradient data, provides sufficient information for local characterization of nonlinear soft anisotropic tissue properties. In the current study this approach is validated in two steps. First, indentation tests with varying indenter sizes are performed on linear elastic PDMS rubbers and compared to tensile tests on the same specimen. For the second step, tissue constructs are engineered using uniaxial or equibiaxial static constrained culture conditions. Digital image correlation (DIC) is used to quantify the anisotropy in the tissue constructs. For both validation steps, material parameters are estimated by inverse fitting of a computational model to the experimental results.

Stress related collagen ultrastructure in human aortic valves--implications for tissue engineering.

Understanding the response of tissue structures to mechanical stress is crucial for optimization of mechanical conditioning protocols in the field of heart valve tissue engineering. In heart valve tissue, it is unclear to what extent mechanical loading affects the collagen fibril morphology. To determine if local stress affects the collagen fibril morphology, in terms of fibril diameter, its distribution, and the fibril density, this was investigated in adult native human aortic valve leaflets. Transmission electron microscopy images of collagen fibrils were analyzed at three locations: the commissures, the belly, and the fixed edge of the leaflets. Subsequently, the mechanical behavior of human aortic valves was used in a computational model to predict the stress distribution in the valve leaflet during the diastolic phase of the cardiac cycle. The local stresses at the three locations were related to the collagen fibril morphology. The fibril diameter and density varied significantly between the measured locations, and appeared inversely related. The average fibril diameter increased from the fixed edge, to the belly, and to the commissures of the leaflets, while fibril density decreased. Interestingly, these differences corresponded well with the level of stress at the locations. The presented data showed that large tissue stress is associated with greater average fibril diameter, lower fibril density, and wider fibril size distribution compared with low stress locations in the leaflets. The findings here provide insight in the effect of mechanical loading on the collagen ultrastructure, and are valuable to improve conditioning protocols for tissue engineering.

Diffusion measurements in epidermal tissues with fluorescent recovery after photobleaching.

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 x 10(-5)+/-1.2 x 10(-5) and 5.9 x 10(-5)+/-6.1 x 10(-6) mm2/s, respectively), whereas the diffusion coefficient of the engineered epidermal equivalent was significantly lower (2.3 x 10(-5)+/-1.0 x 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.

Remodelling of the angular collagen fiber distribution in cardiovascular tissues.

Understanding collagen fiber remodelling is desired to optimize the mechanical conditioning protocols in tissue-engineering of load-bearing cardiovascular structures. Mathematical models offer strong possibilities to gain insight into the mechanisms and mechanical stimuli involved in these remodelling processes. In this study, a framework is proposed to investigate remodelling of angular collagen fiber distribution in cardiovascular tissues. A structurally based model for collagenous cardiovascular tissues is extended with remodelling laws for the collagen architecture, and the model is subsequently applied to the arterial wall and aortic valve. For the arterial wall, the model predicts the presence of two helically arranged families of collagen fibers. A branching, diverging hammock-type fiber architecture is predicted for the aortic valve. It is expected that the proposed model may be of great potential for the design of improved tissue engineering protocols and may give further insight into the pathophysiology of cardiovascular diseases.

Linear viscoelastic behavior of subcutaneous adipose tissue.

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.