Initial scaffold thickness affects the emergence of a geometrical and mechanical equilibrium in engineered cardiovascular tissues.

In situ cardiovascular tissue-engineering can potentially address the shortcomings of the current replacement therapies, in particular, their inability to grow and remodel. In native tissues, it is widely accepted that physiological growth and remodelling occur to maintain a homeostatic mechanical state to conserve its function, regardless of changes in the mechanical environment. A similar homeostatic state should be reached for tissue-engineered (TE) prostheses to ensure proper functioning. For in situ tissue-engineering approaches obtaining such a state greatly relies on the initial scaffold design parameters. In this study, it is investigated if the simple scaffold design parameter initial thickness, influences the emergence of a mechanical and geometrical equilibrium state in in vitro TE constructs, which resemble thin cardiovascular tissues such as heart valves and arteries. Towards this end, two sample groups with different initial thicknesses of myofibroblast-seeded polycaprolactone-bisurea constructs were cultured for three weeks under dynamic loading conditions, while tracking geometrical and mechanical changes temporally using non-destructive ultrasound imaging. A mechanical equilibrium was reached in both groups, although at different magnitudes of the investigated mechanical quantities. Interestingly, a geometrically stable state was only established in the thicker constructs, while the thinner constructs length continuously increased. This demonstrates that reaching geometrical and mechanical stability in TE constructs is highly dependent on functional scaffold design

Intrinsic cell stress is independent of organization in engineered cell sheets

Understanding cell contractility is of fundamental importance for cardiovascular tissue engineering, due to its major impact on the tissue’s mechanical properties as well as the development of permanent dimensional changes, e.g., by contraction or dilatation of the tissue. Previous attempts to quantify contractile cellular stresses mostly used strongly aligned monolayers of cells, which might not represent the actual organization in engineered cardiovascular tissues such as heart valves. In the present study, therefore, we investigated whether differences in organization affect the magnitude of intrinsic stress generated by individual myofibroblasts, a frequently used cell source for in vitro engineered heart valves. Four different monolayer organizations were created via micro-contact printing of fibronectin lines on thin PDMS films, ranging from strongly anisotropic to isotropic. Thin film curvature, cell density, and actin stress fiber distribution were quantified, and subsequently, intrinsic stress and contractility of the monolayers were determined by incorporating these data into sample-specific finite element models. Our data indicate that the intrinsic stress exerted by the monolayers in each group correlates with cell density. Additionally, after normalizing for cell density and accounting for differences in alignment, no consistent differences in intrinsic contractility were found between the different monolayer organizations, suggesting that the intrinsic stress exerted by individual myofibroblasts is independent of the organization. Consequently, this study emphasizes the importance of choosing proper architectural properties for scaffolds in cardiovascular tissue engineering, as these directly affect the stresses in the tissue, which play a crucial role in both the functionality and remodeling of (engineered) cardiovascular tissues

Intrinsic cell stress is independent of organization in engineered cell sheets

\u3cp\u3eUnderstanding cell contractility is of fundamental importance for cardiovascular tissue engineering, due to its major impact on the tissue’s mechanical properties as well as the development of permanent dimensional changes, e.g., by contraction or dilatation of the tissue. Previous attempts to quantify contractile cellular stresses mostly used strongly aligned monolayers of cells, which might not represent the actual organization in engineered cardiovascular tissues such as heart valves. In the present study, therefore, we investigated whether differences in organization affect the magnitude of intrinsic stress generated by individual myofibroblasts, a frequently used cell source for in vitro engineered heart valves. Four different monolayer organizations were created via micro-contact printing of fibronectin lines on thin PDMS films, ranging from strongly anisotropic to isotropic. Thin film curvature, cell density, and actin stress fiber distribution were quantified, and subsequently, intrinsic stress and contractility of the monolayers were determined by incorporating these data into sample-specific finite element models. Our data indicate that the intrinsic stress exerted by the monolayers in each group correlates with cell density. Additionally, after normalizing for cell density and accounting for differences in alignment, no consistent differences in intrinsic contractility were found between the different monolayer organizations, suggesting that the intrinsic stress exerted by individual myofibroblasts is independent of the organization. Consequently, this study emphasizes the importance of choosing proper architectural properties for scaffolds in cardiovascular tissue engineering, as these directly affect the stresses in the tissue, which play a crucial role in both the functionality and remodeling of (engineered) cardiovascular tissues.\u3c/p\u3

Diffusion of water in skeletal muscle tissue is not influenced by compression in a rat model of deep tissue injury

Sustained mechanical loading of skeletal muscle may result in the development of a severe type of pressure ulcer, referred to as deep tissue injury. Recently it was shown that the diffusion of large molecules (10–150kDa) is impaired during deformation of tissue-engineered skeletal muscle, suggesting a role for impaired diffusion in the aetiology of deep tissue injury. However, the influence of deformation on diffusion of smaller molecules on its aetiology is less clear. This motivated the present study designed to investigate the influence of deformation of skeletal muscle on the diffusion of water, which can be measured with diffusion tensor magnetic resonance imaging (MRI). It could be predicted that this approach will provide valuable information on the diffusion of small molecules. Additionally the relationship between muscle temperature and diffusion was investigated. During deformation of the tibialis anterior a decrease of the apparent diffusion coefficient (ADC) was observed (7.2±3.9%). The use of a finite element model showed that no correlation existed between the maximum shear strain and the decrease of the ADC. The ADC in the uncompressed gastrocnemius muscle decreased with 5.9±3.7%. In an additional experiment a clear correlation was obtained between the decrease of the ADC and the relative temperature change of skeletal muscle tissue as measured by MRI. Taken together, it was concluded that (1) the decreased diffusion of water was not a direct effect of tissue deformation and (2) that it is likely that the observed decreased ADC during deformation was a result of a decreased muscle temperature. The present study therefore provides evidence that diffusion of small molecules, particularly oxygen and carbon dioxide, is not impaired during deformation of skeletal muscle tissue.<br/

Temporal effects of mechanical loading on deformation-induced damage in skeletal muscle tissue

Mechanical loading of soft tissues covering bony prominences can cause skeletal muscle damage, ultimately resulting in a severe pressure ulcer termed deep tissue injury. Recently, by means of an experimental-numerical approach, it was shown that local tissue deformations cause tissue damage once a deformation threshold is exceeded. In the present study, the effects of load exposure time and intermittent load relief on the development of deformation-induced muscle damage were investigated. The data showed that a 2 h loading period caused more damage than 10 min loading. Intermittent load reliefs of 2 min during a 2 h loading period had minimal effect on the evolution of skeletal muscle damage. In addition, a local deformation threshold for damage was found, which was similar for each of the loading regimes applied in this study. For short loading periods, these results imply that local tissue deformations determine whether muscle damage will develop and the exposure time influences the amount of tissue damage. Temporary load reliefs were inefficient in reducing deformation-induced damage, but may still influence the development of ischemia-induced damage during longer loading period

Initial scaffold thickness affects the emergence of a geometrical and mechanical equilibrium in engineered cardiovascular tissues

\u3cp\u3eIn situ cardiovascular tissue-engineering can potentially address the shortcomings of the current replacement therapies, in particular, their inability to grow and remodel. In native tissues, it is widely accepted that physiological growth and remodelling occur to maintain a homeostatic mechanical state to conserve its function, regardless of changes in the mechanical environment. A similar homeostatic state should be reached for tissue-engineered (TE) prostheses to ensure proper functioning. For in situ tissue-engineering approaches obtaining such a state greatly relies on the initial scaffold design parameters. In this study, it is investigated if the simple scaffold design parameter initial thickness, influences the emergence of a mechanical and geometrical equilibrium state in in vitro TE constructs, which resemble thin cardiovascular tissues such as heart valves and arteries. Towards this end, two sample groups with different initial thicknesses of myofibroblast-seeded polycaprolactone-bisurea constructs were cultured for three weeks under dynamic loading conditions, while tracking geometrical and mechanical changes temporally using non-destructive ultrasound imaging. A mechanical equilibrium was reached in both groups, although at different magnitudes of the investigated mechanical quantities. Interestingly, a geometrically stable state was only established in the thicker constructs, while the thinner constructs' length continuously increased. This demonstrates that reaching geometrical and mechanical stability in TE constructs is highly dependent on functional scaffold design.\u3c/p\u3

Lmna knockout mouse embryonic fibroblasts are less contractile than their wild-type counterparts

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

Initial scaffold thickness affects the emergence of a geometrical and mechanical equilibrium in engineered cardiovascular tissues

In situ cardiovascular tissue-engineering can potentially address the shortcomings of the current replacement therapies, in particular, their inability to grow and remodel. In native tissues, it is widely accepted that physiological growth and remodelling occur to maintain a homeostatic mechanical state to conserve its function, regardless of changes in the mechanical environment. A similar homeostatic state should be reached for tissue-engineered (TE) prostheses to ensure proper functioning. For in situ tissue-engineering approaches obtaining such a state greatly relies on the initial scaffold design parameters. In this study, it is investigated if the simple scaffold design parameter initial thickness, influences the emergence of a mechanical and geometrical equilibrium state in in vitro TE constructs, which resemble thin cardiovascular tissues such as heart valves and arteries. Towards this end, two sample groups with different initial thicknesses of myofibroblast-seeded polycaprolactone-bisurea constructs were cultured for three weeks under dynamic loading conditions, while tracking geometrical and mechanical changes temporally using non-destructive ultrasound imaging. A mechanical equilibrium was reached in both groups, although at different magnitudes of the investigated mechanical quantities. Interestingly, a geometrically stable state was only established in the thicker constructs, while the thinner constructs' length continuously increased. This demonstrates that reaching geometrical and mechanical stability in TE constructs is highly dependent on functional scaffold design

Ischemia-reperfusion injury in rat skeletal muscle assessed with T2-weighted and dynamic contrast-enhanced MRI

Pressure ulcers are localized areas of soft tissue breakdown due to mechanical loading. Susceptible individuals are subjected to pressure relief strategies to prevent long loading periods. Therefore, ischemia-reperfusion injury may play an important role in the etiology of pressure ulcers. To investigate the inter-relation between postischemic perfusion and changes in skeletal muscle integrity, the hindlimbs of Brown Norway rats were subjected to 4-h ischemia followed by 2-h reperfusion. Dynamic contrast-enhanced MRI was used to examine perfusion, and changes in skeletal muscle integrity were monitored with T2-weighted MRI. The dynamic contrast-enhanced MRI data showed a heterogeneous postischemic profile in the hindlimb, consisting of areas with increased contrast enhancement (14-76% of the hindlimb) and regions with no-reflow (5-77%). For T2, a gradual increase in the complete leg was observed during the 4-h ischemic period (from 34 to 41 msec). During the reperfusion phase, a heterogeneous distribution of T2 was observed. Areas with increased contrast enhancement were associated with a decrease in T2 (to 38 msec) toward preischemic levels, whereas no-reflow areas exhibited a further increase in T2 (to 42 msec). These results show that reperfusion after prolonged ischemia may not be complete, thereby continuing the ischemic condition and aggravating tissue damag

The effects of deformation, ischemia, and reperfusion on the development of muscle damage during prolonged loading

Deep tissue injury (DTI) is a severe form of pressure ulcer where tissue damage starts in deep tissues underneath intact skin. In the present study, the contributions of deformation, ischemia, and reperfusion to skeletal muscle damage development were examined in a rat model during a 6-h period. Magnetic resonance imaging (MRI) was used to study perfusion (contrast-enhanced MRI) and tissue integrity (T2-weighted MRI). The levels of tissue deformation were estimated using finite element models. Complete ischemia caused a gradual homogeneous increase in T2 (?20% during the 6-h period). The effect of reperfusion on T2 was highly variable, depending on the anatomical location. In experiments involving deformation, inevitably associated with partial ischemia, a variable T2 increase (17–66% during the 6-h period) was observed reflecting the significant variation in deformation (with two-dimensional strain energies of 0.60–1.51 J/mm) and ischemia (50.8–99.8% of the leg) between experiments. These results imply that deformation, ischemia, and reperfusion all contribute to the damage process during prolonged loading, although their importance varies with time. The critical deformation threshold and period of ischemia that cause muscle damage will certainly vary between individuals. These variations are related to intrinsic factors, such as pathological state, which partly explain the individual susceptibility to the development of DTI and highlight the need for regular assessments of individual subjects