Scaffold Geometry-Imposed Anisotropic Mechanical Loading Guides the Evolution of the Mechanical State of Engineered Cardiovascular Tissues in vitro.

Cardiovascular tissue engineering is a promising approach to develop grafts that, in contrast to current replacement grafts, have the capacity to grow and remodel like native tissues. This approach largely depends on cell-driven tissue growth and remodeling, which are highly complex processes that are difficult to control inside the scaffolds used for tissue engineering. For several tissue engineering approaches, adverse tissue growth and remodeling outcomes were reported, such as aneurysm formation in vascular grafts, and leaflet retraction in heart valve grafts. It is increasingly recognized that the outcome of tissue growth and remodeling, either physiological or pathological, depends at least partly on the establishment of a homeostatic mechanical state, where one or more mechanical quantities in a tissue are maintained in equilibrium. To design long-term functioning tissue engineering strategies, understanding how scaffold parameters such as geometry affect the mechanical state of a construct, and how this state guides tissue growth and remodeling, is therefore crucial. Here, we studied how anisotropic versus isotropic mechanical loading-as imposed by initial scaffold geometry-influences tissue growth, remodeling, and the evolution of the mechanical state and geometry of tissue-engineered cardiovascular constructs in vitro. Using a custom-built bioreactor platform and nondestructive mechanical testing, we monitored the mechanical and geometric changes of elliptical and circular, vascular cell-seeded, polycaprolactone-bisurea scaffolds during 14 days of dynamic loading. The elliptical and circular scaffold geometries were designed using finite element analysis, to induce anisotropic and isotropic dynamic loading, respectively, with similar maximum stretch when cultured in the bioreactor platform. We found that the initial scaffold geometry-induced (an)isotropic loading of the engineered constructs differentially dictated the evolution of their mechanical state and geometry over time, as well as their final structural organization. These findings demonstrate that controlling the initial mechanical state of tissue-engineered constructs via scaffold geometry can be used to influence tissue growth and remodeling and determine tissue outcomes.

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.

Nondestructive mechanical characterization of developing biological tissues using inflation testing.

One of the hallmarks of biological soft tissues is their capacity to grow and remodel in response to changes in their environment. Although it is well-accepted that these processes occur at least partly to maintain a mechanical homeostasis, it remains unclear which mechanical constituent(s) determine(s) mechanical homeostasis. In the current study a nondestructive mechanical test and a two-step inverse analysis method were developed and validated to nondestructively estimate the mechanical properties of biological tissue during tissue culture. Nondestructive mechanical testing was achieved by performing an inflation test on tissues that were cultured inside a bioreactor, while the tissue displacement and thickness were nondestructively measured using ultrasound. The material parameters were estimated by an inverse finite element scheme, which was preceded by an analytical estimation step to rapidly obtain an initial estimate that already approximated the final solution. The efficiency and accuracy of the two-step inverse method was demonstrated on virtual experiments of several material types with known parameters. PDMS samples were used to demonstrate the method's feasibility, where it was shown that the proposed method yielded similar results to tensile testing. Finally, the method was applied to estimate the material properties of tissue-engineered constructs. Via this method, the evolution of mechanical properties during tissue growth and remodeling can now be monitored in a well-controlled system. The outcomes can be used to determine various mechanical constituents and to assess their contribution to mechanical homeostasis.

Age-dependent changes of stress and strain in the human heart valve and their relation with collagen remodeling.

In order to create tissue-engineered heart valves with long-term functionality, it is essential to fully understand collagen remodeling during neo-tissue formation. Collagen remodeling is thought to maintain mechanical tissue homeostasis. Yet, the driving factor of collagen remodeling remains unidentified. In this study, we determined the collagen architecture and the geometric and mechanical properties of human native semilunar heart valves of fetal to adult age using confocal microscopy, micro-indentation and inverse finite element analysis. The outcomes were used to predict age-dependent changes in stress and stretch in the heart valves via finite element modeling. The results indicated that the circumferential stresses are different between the aortic and pulmonary valve, and, moreover, that the stress increases considerably over time in the aortic valve. Strikingly, relatively small differences were found in stretch with time and between the aortic and pulmonary valve, particularly in the circumferential direction, which is the main determinant of the collagen fiber stretch. Therefore, we suggest that collagen remodeling in the human heart valve maintains a stretch-driven homeostasis. Next to these novel insights, the unique human data set created in this study provides valuable input for the development of numerical models of collagen remodeling and optimization of tissue engineering. STATEMENT OF SIGNIFICANCE: Annually, over 280,000 heart valve replacements are performed worldwide. Tissue engineering has the potential to provide valvular disease patients with living valve substitutes that can last a lifetime. Valve functionality is mainly determined by the collagen architecture. Hence, understanding collagen remodeling is crucial for creating tissue-engineered valves with long-term functionality. In this study, we determined the structural and material properties of human native heart valves of fetal to adult age to gain insight into the mechanical stimuli responsible for collagen remodeling. The age-dependent evolutionary changes in mechanical state of the native valve suggest that collagen remodeling in heart valves is a stretch-driven process.