Kristen L. Billiar

Biaxial mechanical properties of the natural and glutaraldehyde treated aortic valve cusp--Part I: Experimental results.

To date, there are no constitutive models for either the natural or bioprosthetic aortic valve (AV), in part due to experimental complications related to the AVs small size and heterogeneous fibrous structure. In this study, we developed specialized biaxial testing techniques for the AV cusp, including a method to determine the local structure-strain relationship to assess the effects of boundary tethering forces. Natural and glutaraldehyde (GL) treated cusps were subjected to an extensive biaxial testing protocol in which the ratios of the axial tensions were held at constant values. Results indicated that the local fiber architecture clearly dominated cuspal deformation, and that the tethering effects at the specimen boundaries were negligible. Due to unique aspects of cuspal fiber architecture, the most uniform region of deformation was found at the lower portion as opposed to the center of the cuspal specimen. In general, the circumferential strains were much smaller than the radial strains, indicating a profound degree of mechanical anisotropy, and that natural cusps were significantly more extensible than the GL treated cusps. Strong mechanical coupling between biaxial stretch axes produced negative circumferential strains under equibiaxial tension. Further, the large radial strains observed could not be explained by uncrimping of the collagen fibers, but may be due to large rotations of the highly aligned, circumferential-oriented collagen fibers in the fibrosa. In conclusion, this study provides new insights into the AV cusps structure-function relationship in addition to requisite data for constitutive modeling.

Biaxial Mechanical Properties of the Native and Glutaraldehyde-Treated Aortic Valve Cusp: Part II—A Structural Constitutive Model

We have formulated the first constitutive model to describe the complete measured planar biaxial stress-strain relationship of the native and glutaraldehyde-treated aortic valve cusp using a structurally guided approach. When applied to native, zero-pressure fixed, and low-pressure fixed cusps, only three parameters were needed to simulate fully the highly anisotropic, and nonlinear in-plane biaxial mechanical behavior. Differences in the behavior of the native and zero- and low-pressure fixed cusps were found to be primarily due to changes in the effective fiber stress-strain behavior. Further, the model was able to account for the effects of small (< 10 deg) misalignments in the cuspal specimens with respect to the biaxial test axes that increased the accuracy of the model material parameters. Although based upon a simplified cuspal structure, the model underscored the role of the angular orientation of the fibers that completely accounted for extreme mechanical anisotropy and pronounced axial coupling. Knowledge of the mechanics of the aortic cusp derived from this model may aid in the understanding of fatigue damage in bioprosthetic heart valves and, potentially, lay the groundwork for the design of tissue-engineered scaffolds for replacement heart valves.

A method to quantify the fiber kinematics of planar tissues under biaxial stretch.

We have developed a method for measuring fiber kinematics in two-dimensional soft collagenous tissues. The technique combines small-angle light scattering (SALS) and biaxial stretch controlled by simultaneous optical strain measurement. Preliminary findings on porcine aortic valve leaflets and bovine pericardium indicate that fiber kinematics are highly tissue specific and are generally non-affine. The mobility of the fibers within each tissue seems to be specialized to perform a distinct physiological function. Quantitative knowledge of a tissues angular fiber distribution and its transformation during biaxial stretch is critical for microstructural modeling of planar tissues. Our results underscore the importance of measuring fiber kinematics for each specific tissue type that is to be modeled.

Evaluation of the porcine intestinal collagen layer as a biomaterial.

The submucosal layer of the small intestine has been investigated as a source of collagenous tissue with the potential to be used as a biomaterial because of its inherent strength and biocompatibility. In this study we utilized a novel method for processing the tissue to generate an acellular intestinal collagen layer (ICL). This nondetergent, nonenzymatic chemical cleaning protocol removes cells and cellular debris without damaging the native collagen structure. Multilayer laminates of ICL crosslinked with a water-soluble carbodiimide (EDC) were evaluated as a tissue repair material in a rabbit abdominal hernia model. The ICL laminates provided the requisite physical properties and did not lead to adhesion formation. No immune response to the porcine collagen was detectable, and this material did not show any calcification in either the rabbit model or in the juvenile rat model.

Effects of carbodiimide crosslinking conditions on the physical properties of laminated intestinal submucosa

Functional tissue engineering of load-bearing repair tissues requires the design and production of biomaterials that provide a remodelable scaffold for host infiltration and tissue regeneration while maintaining the repair function throughout the remodeling process. Layered constructs have been fabricated from chemically and mechanically cleaned porcine intestinal collagen using ethyl-3(3-dimethylamino) propyl carbodiimide (EDC) and an acetone solvent. By varying the concentration of the crosslinker from 1 to 10 mM and the solvent from 0 to 90% acetone, the strength, stiffness, maximum strain, thermal stability, lamination strength, and suture retention strength can be adjusted. These parameters have either functional importance or the potential to modify the remodeling kinetics, or they have both. This study investigates the interdependence of these parameters, the specific effects that variations in concentration can achieve, and how the two crosslinking variables interact. The results demonstrate that there is substantial latitude in the design of these constructs by these straightforward crosslinking modifications. These data provide the basis for studying the in vivo response to crosslinking conditions that will supply the requisite strength while still allowing host cell infiltration and remodeling.

Mechanical evaluation and design of a multilayered collagenous repair biomaterial

One method of fabricating implantable biomaterials is to utilize biologically derived, chemically modified tissues to form constructs that are both biocompatible and remodelable. Rigorous mechanical characterization is a necessary component in material evaluation to ensure that the constructs will withstand in vivo loading. In this study we performed an in-depth biaxial mechanical and quantitative structural analysis of GraftPatch (GP), a biomaterial constructed by assembling chemically treated layers of porcine small intestinal submucosa (SIS). The mechanical behavior of GP was compared to both native SIS and to glutaraldehyde-treated bovine pericardium (GLBP) as a reference biomaterial. Under biaxial loading, GP was found to be stiffer than native SIS and mechanically anisotropic, with the preferred fiber direction demonstrating greater stiffness. Quantitative structural analysis using small-angle light scattering indicated a uniform fiber structure similar to GLBP and SIS. To enable test-protocol-independent quantitative comparisons, the biaxial mechanical data were fit to an orthotropic constitutive model, which indicated a similar degree of mechanical anisotropy between the three groups. We also demonstrate how the constitutive model can be used to design layered biocomposite materials that can undergo large deformations.

Combining dynamic stretch and tunable stiffness to probe cell mechanobiology in vitro.

Cells have the ability to actively sense their mechanical environment and respond to both substrate stiffness and stretch by altering their adhesion, proliferation, locomotion, morphology, and synthetic profile. In order to elucidate the interrelated effects of different mechanical stimuli on cell phenotype in vitro, we have developed a method for culturing mammalian cells in a two-dimensional environment at a wide range of combined levels of substrate stiffness and dynamic stretch. Polyacrylamide gels were covalently bonded to flexible silicone culture plates and coated with monomeric collagen for cell adhesion. Substrate stiffness was adjusted from relatively soft (G′ = 0.3 kPa) to stiff (G′ = 50 kPa) by altering the ratio of acrylamide to bis-acrylamide, and the silicone membranes were stretched over circular loading posts by applying vacuum pressure to impart near-uniform stretch, as confirmed by strain field analysis. As a demonstration of the system, porcine aortic valve interstitial cells (VIC) and human mesenchymal stem cells (hMSC) were plated on soft and stiff substrates either statically cultured or exposed to 10% equibiaxial or pure uniaxial stretch at 1Hz for 6 hours. In all cases, cell attachment and cell viability were high. On soft substrates, VICs cultured statically exhibit a small rounded morphology, significantly smaller than on stiff substrates (p<0.05). Following equibiaxial cyclic stretch, VICs spread to the extent of cells cultured on stiff substrates, but did not reorient in response to uniaxial stretch to the extent of cells stretched on stiff substrates. hMSCs exhibited a less pronounced response than VICs, likely due to a lower stiffness threshold for spreading on static gels. These preliminary data demonstrate that inhibition of spreading due to a lack of matrix stiffness surrounding a cell may be overcome by externally applied stretch suggesting similar mechanotransduction mechanisms for sensing stiffness and stretch.

Effects of mechanical fatigue on the bending properties of the porcine bioprosthetic heart valve.

The mechanisms underlying the failure of porcine bioprosthetic aortic heart valves are not well understood. One possible explanation is that delaminations of the layered leaflet structure occur through flexion, leading to calcification and further delaminations, and finally resulting in valve failure. We investigated the changes in flexural rigidity of the belly of aortic valve cusps subjected to accelerated durability testing. We used three-point bending wherein a load was applied to the center of each specimen by a thin stainless steel bar calibrated to a known load-displacement relationship. Ten circumferential and 15 radial specimens from valves fatigued to 0, 50, 100, and 200 million cycles were flexed both with and against the curvature of the cusp. Linear beam theory was applied as a means to compare the relative bending stiffness between groups. Although specimens aligned to the circumferential direction were stiffer when bent against the cuspal curvature, the radial oriented specimens exhibited no bending directional dependence. Both the radial and circumferential specimens experienced a significant decrease in the bending stiffness with an increased number of accelerated test cycles. Overall, our results suggest that it is the fibrosa that experiences the greatest loss of stiffness with mechanically induced fatigue damage.

In Vivo IVUS-Based 3-D Fluid–Structure Interaction Models With Cyclic Bending and Anisotropic Vessel Properties for Human Atherosclerotic Coronary Plaque Mechanical Analysis

In this paper, a modeling approach combining in vivo intravascular ultrasound (IVUS) imaging, computational modeling, angiography, and mechanical testing is proposed to perform mechanical analysis for human coronary atherosclerotic plaques for potential more accurate plaque vulnerability assessment. A 44-slice in vivo IVUS dataset of a coronary plaque was acquired from one patient, and four 3-D models with fluid-structure interactions (FSIs) based on the data were constructed to quantify effects of anisotropic vessel properties and cyclic bending of the coronary plaque on flow and plaque stress/strain conditions. Compared to the isotropic model (model 1, no bending, no axial stretch), maximum stress-P1 (maximum principal stress) values on the cut surface with maximum bending (where applicable) from model 2 (anisotropic, no bending, no stretch), model 3 (anisotropic, with bending, no stretch), and model 4 (anisotropic with bending and stretch) were, respectively, 63%, 126%, and 345% higher than that from model 1. Effects of cyclic bending on flow behaviors were modest (5%-15%). Our preliminary results indicated that in vivo IVUS-based FSI models with cyclic bending and anisotropic material properties could improve the accuracies of plaque stress/strain predictions and plaque vulnerability assessment. Large-scale patient studies are needed to further validate our findings.

3D MRI-Based Anisotropic FSI Models With Cyclic Bending for Human Coronary Atherosclerotic Plaque Mechanical Analysis

Heart attack and stroke are often caused by atherosclerotic plaque rupture, which happens without warning most of the time. Magnetic resonance imaging (MRI)-based atherosclerotic plaque models with fluid-structure interactions (FSIs) have been introduced to perform flow and stress/strain analysis and identify possible mechanical and morphological indices for accurate plaque vulnerability assessment. For coronary arteries, cyclic bending associated with heart motion and anisotropy of the vessel walls may have significant influence on flow and stress/strain distributions in the plaque. FSI models with cyclic bending and anisotropic vessel properties for coronary plaques are lacking in the current literature. In this paper, cyclic bending and anisotropic vessel properties were added to 3D FSI coronary plaque models so that the models would be more realistic for more accurate computational flow and stress/strain predictions. Six computational models using one ex vivo MRI human coronary plaque specimen data were constructed to assess the effects of cyclic bending, anisotropic vessel properties, pulsating pressure, plaque structure, and axial stretch on plaque stress/strain distributions. Our results indicate that cyclic bending and anisotropic properties may cause 50-800% increase in maximum principal stress (Stress-P1) values at selected locations. The stress increase varies with location and is higher when bending is coupled with axial stretch, nonsmooth plaque structure, and resonant pressure conditions (zero phase angle shift). Effects of cyclic bending on flow behaviors are more modest (9.8% decrease in maximum velocity, 2.5% decrease in flow rate, 15% increase in maximum flow shear stress). Inclusion of cyclic bending, anisotropic vessel material properties, accurate plaque structure, and axial stretch in computational FSI models should lead to a considerable improvement of accuracy of computational stress/strain predictions for coronary plaque vulnerability assessment. Further studies incorporating additional mechanical property data and in vivo MRI data are needed to obtain more complete and accurate knowledge about flow and stress/strain behaviors in coronary plaques and to identify critical indicators for better plaque assessment and possible rupture predictions.