Large strain stimulation promotes extracellular matrix production and stiffness in an elastomeric scaffold model.

Mechanical conditioning of engineered tissue constructs is widely recognized as one of the most relevant methods to enhance tissue accretion and microstructure, leading to improved mechanical behaviors. The understanding of the underlying mechanisms remains rather limited, restricting the development of in silico models of these phenomena, and the translation of engineered tissues into clinical application. In the present study, we examined the role of large strip-biaxial strains (up to 50%) on ECM synthesis by vascular smooth muscle cells (VSMCs) micro-integrated into electrospun polyester urethane urea (PEUU) constructs over the course of 3 weeks. Experimental results indicated that VSMC biosynthetic behavior was quite sensitive to tissue strain maximum level, and that collagen was the primary ECM component synthesized. Moreover, we found that while a 30% peak strain level achieved maximum ECM synthesis rate, further increases in strain level lead to a reduction in ECM biosynthesis. Subsequent mechanical analysis of the formed collagen fiber network was performed by removing the scaffold mechanical responses using a strain-energy based approach, showing that the denovo collagen also demonstrated mechanical behaviors substantially better than previously obtained with small strain training and comparable to mature collagenous tissues. We conclude that the application of large deformations can play a critical role not only in the quantity of ECM synthesis (i.e. the rate of mass production), but also on the modulation of the stiffness of the newly formed ECM constituents. The improved understanding of the process of growth and development of ECM in these mechano-sensitive cell-scaffold systems will lead to more rational design and manufacturing of engineered tissues operating under highly demanding mechanical environments.

Characterization of the complete fiber network topology of planar fibrous tissues and scaffolds.

Understanding how engineered tissue scaffold architecture affects cell morphology, metabolism, phenotypic expression, as well as predicting material mechanical behavior has 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. Micro-architectural 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.

On the biomechanical function of scaffolds for engineering load-bearing soft tissues.

Replacement or regeneration of load-bearing soft tissues has long been the impetus for the development of bioactive materials. While maturing, current efforts continue to be confounded by our lack of understanding of the intricate multi-scale hierarchical arrangements and interactions typically found in native tissues. The current state of the art in biomaterial processing enables a degree of controllable microstructure that can be used for the development of model systems to deduce fundamental biological implications of matrix morphologies on cell function. Furthermore, the development of computational frameworks which allow for the simulation of experimentally derived observations represents a positive departure from what has mostly been an empirically driven field, enabling a deeper understanding of the highly complex biological mechanisms we wish to ultimately emulate. Ongoing research is actively pursuing new materials and processing methods to control material structure down to the micro-scale to sustain or improve cell viability, guide tissue growth, and provide mechanical integrity, all while exhibiting the capacity to degrade in a controlled manner. The purpose of this review is not to focus solely on material processing but to assess the ability of these techniques to produce mechanically sound tissue surrogates, highlight the unique structural characteristics produced in these materials, and discuss how this translates to distinct macroscopic biomechanical behaviors.

Scale-dependent fiber kinematics of elastomeric electrospun scaffolds for soft tissue engineering.

Electrospun poly(ester urethane)urea (PEUU) scaffolds contain complex multiscale hierarchical structures that work simultaneously to produce unique macrolevel mechanical behaviors. In this study, we focused on quantifying key multiscale scaffold structural features to elucidate the mechanisms by which these scaffolds function to emulate native tissue tensile behavior. Fiber alignment was modulated via increasing rotational velocity of the collecting mandrel, and the resultant specimens were imaged using SEM under controlled biaxial strain. From the SEM images, fiber splay, tortuosity, and diameter were quantified in the unstrained and deformed configurations. Results indicated that not only fiber alignment increased with mandrel velocity but also, paradoxically, tortuosity increased concurrently with mandrel velocity and was highly correlated with fiber orientation. At microlevel scales (1-10 mum), local scaffold deformation behavior was observed to be highly heterogeneous, while increasing the scale resulted in an increasingly homogenous strain field. From our comprehensive measurements, we determined that the transition scale from heterogenous to homogeneous-like behavior to be approximately 1 mm. Moreover, while electrospun PEUU scaffolds exhibit complex deformations at the microscale, the larger scale structural features of the fibrous network allow them to behave as long-fiber composites that deform in an affine-like manner. This study underscores the importance of understanding the structure-function relationships in elastomeric fibrous scaffolds, and in particular allowed us to link microscale deformations with mechanisms that allow them to successfully simulate soft tissue mechanical behavior.

Tissue-to-cellular level deformation coupling in cell micro-integrated elastomeric scaffolds.

In engineered tissues we are challenged to reproduce extracellular matrix and cellular deformation coupling that occurs within native tissues, which is a meso-micro scale phenomenon that profoundly affects tissue growth and remodeling. With our ability to electrospin polymer fiber scaffolds while simultaneously electrospraying viable cells, we are provided with a unique platform to investigate cellular deformations within a three dimensional elastomeric fibrous scaffold. Scaffold specimens micro-integrated with vascular smooth muscle cells were subjected to controlled biaxial stretch with 3D cellular deformations and local fiber microarchitecture simultaneously quantified. We demonstrated that the local fiber geometry followed an affine behavior, so that it could be predicted by macro-scaffold deformations. However, local cellular deformations depended non-linearly on changes in fiber microarchitecture and ceased at large strains where the scaffold fibers completely straightened. Thus, local scaffold microstructural changes induced by macro-level applied strain dominated cellular deformations, so that monotonic increases in scaffold strain do not necessitate similar levels of cellular deformation. This result has fundamental implications when attempting to elucidate the events of de-novo tissue development and remodeling in engineered tissues, which are thought to depend substantially on cellular deformations.

On the biaxial mechanical properties of the layers of the aortic valve leaflet.

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.

Time-dependent biaxial mechanical behavior of the aortic heart valve leaflet.

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 for 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 ( approximately 17%). Stress relaxation and creep results indicated that while the AVL exhibited significant stress relaxation, it exhibited negligible creep over the 3h test duration. These results are all in accordance with our previous findings for the mitral valve anterior leaflet (MVAL) [Grashow, J.S., Sacks, M.S., Liao, J., Yoganathan, A.P., 2006a. Planar biaxial creep and stress relaxatin of the mitral valve anterior leaflet. Annals of Biomedical Engineering 34 (10), 1509-1518; Grashow, J.S., Yoganathan, A.P., Sacks, M.S., 2006b. Biaxial stress-stretch behavior of the mitral valve anterior leaflet at physiologic strain rates. Annals of Biomedical Engineering 34 (2), 315-325], 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, J., Yang, L., Grashow, J., Sacks, M.S., 2007. The relation between collagen fibril kinematics and mechanical properties in the mitral valve anterior leaflet. Journal of Biomechanical Engineering 129 (1), 78-87], we speculate that the mechanisms underlying this quasi-elastic behavior may be attributed to inter-fibrillar structures unique to valvular tissues. 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.