All existing constitutive models for heart valve leaflet tissues either assume a uniform transmural stress distribution or utilize a membrane tension formulation. Both approaches ignore layer specific mechanical contributions and the implicit nonuniformity of the transmural stress distribution. To begin to address these limitations, we conducted novel studies to quantify the biaxial mechanical behavior of the two structurally distinct, load bearing aortic valve (AV) leaflet layers: the fibrosa and ventricularis. Strip biaxial tests, with extremely sensitive force sensing capabilities, were further utilized to determine the mechanical behavior of the separated ventricularis layer at very low stress levels. Results indicated that both layers exhibited very different nonlinear, highly anisotropic mechanical behaviors. While the leaflet tissue mechanical response was dominated by the fibrosa layer, the ventricularis contributed double the amount of the fibrosa to the total radial tension and experienced four times the stress level. The strip biaxial test results further indicated that the ventricularis exhibited substantial anisotropic mechanical properties at very low stress levels. This result suggested that for all strain levels, the ventricularis layer is dominated by circumferentially oriented collagen fibers, and the initial loading phase of this layer cannot be modeled as an isotropic material. Histological-based thickness measurements indicated that the fibrosa and ventricularis constitute 41% and 29% of the total layer thickness, respectively. Moreover, the extensive network of interlayer connections and identical strains under biaxial loading in the intact state suggests that these layers are tightly bonded. In addition to advancing our knowledge of the subtle but important mechanical properties of the AV leaflet, this study provided a comprehensive database required for the development of a true 3D stress constitutive model for the native AV leaflet.

Understanding how engineered tissue scaffold architecture affects cell morphology, metabolism, phenotypic expression, as well as predicting material mechanical behavior have recently received increased attention. In the present study, an image-based analysis approach that provides an automated tool to characterize engineered tissue fiber network topology is presented. Microarchitectural features that fully defined fiber network topology were detected and quantified, which include fiber orientation, connectivity, intersection spatial density, and diameter. Algorithm performance was tested using scanning electron microscopy (SEM) images of electrospun poly(ester urethane)urea (ES-PEUU) scaffolds. SEM images of rabbit mesenchymal stem cell (MSC) seeded collagen gel scaffolds and decellularized rat carotid arteries were also analyzed to further evaluate the ability of the algorithm to capture fiber network morphology regardless of scaffold type and the evaluated size scale. The image analysis procedure was validated qualitatively and quantitatively, comparing fiber network topology manually detected by human operators (n=5) with that automatically detected by the algorithm. Correlation values between manual detected and algorithm detected results for the fiber angle distribution and for the fiber connectivity distribution were 0.86 and 0.93 respectively. Algorithm detected fiber intersections and fiber diameter values were comparable (within the mean ± standard deviation) with those detected by human operators. This automated approach identifies and quantifies fiber network morphology as demonstrated for three relevant scaffold types and provides a means to: (1) guarantee objectivity, (2) significantly reduce analysis time, and (3) potentiate broader analysis of scaffold architecture effects on cell behavior and tissue development both in vitro and in vivo.

Despite continued progress in the treatment of aortic valve (AV) disease, current treatments continue to be challenged to consistently restore AV function for extended durations. Improved approaches toward AV repair and replacement rests upon our ability to more fully comprehend and simulate AV function. While the elastic behavior the AV leaflet (AVL) has been previously investigated, time dependent behaviors under physiological biaxial loading states have yet to be quantified. In the current study, we performed strain rate, creep, and stress relaxation experiments using porcine AVL under planar biaxial stretch and loaded to physiological levels (60 N/m equi-biaxial tension), with strain rates ranging from quasi-static to physiologic. The resulting stress-strain responses were found to be independent of strain rate, as was the observed low level of hysteresis (∼17%). Stress relaxation and creep results indicated that while the AVL exhibited significant stress relaxation, it exhibited negligible creep over the three hour test duration. These results are all in accordance with our previous findings for the mitral valve anterior leaflet (MVAL) (Grashow et al., 2006, ABME vol. 34, pp. 315-25; Grashow et al., ABME, Vol. 34, pp. 1509-18, 2006, and support our observations that valvular tissues are functionally anisotropic, quasi-elastic biological materials. These results appear to be unique to valvular tissues, and indicate an ability to withstand loading without time-dependent effects under physiologic loading conditions. Based on a recent study that suggested valvular collagen fibrils are not intrinsically viscoelastic (Liao, et al., JBME, vol. 129, 2007), we speculate that the mechanisms underlying this quasi-elastic behavior may be attributed to supra-fibrillar structure unique to valvular tissue. These mechanisms are an important functional aspect of native valvular tissues, and are likely critical to improve our understanding of valvular disease and help guide the development of valvular tissue engineering and surgical repair.