Finite element model of the testing procedure to investigate the stresses applied to the tissue and whether they are physiological.

Continuum elements are in blue and rigid bodies are in grey. The dashed line indicates a plane of symmetry and the red markers indicate the region where displacement boundary conditions were applied. The simulation consisted of two steps; the first used the larger spherical rigid body to deform the sample to the radius of curvature at the start of the in vitro testing. The elements on the left edge highlighted were fixed in the y direction but could move in the x direction to allow the tissue to be pulled over the larger contact body. The second step removed this contact body then applied the maximum pressure in the in vitro testing (260 mmHg) to the base of the in silico sample. Here the highlighted elements were then held in both directions to mimic the sample being pinned.</p

Nominal stress versus strain curve of native tissue, and tissue that has undergone 10 million (10m) or 20 million (20m) cycles of loading in the heart valve tester.

Tissue types were porcine pericardium (PP), bovine pericardium (BP), or glutaraldehyde fixed bovine pericardium (GFBP). Overall despite the differences between tissue types and fatigue the curves show a similar trend of initial strain-stiffening followed by a linear region and then a decrease in gradient to breaking. This may be interpreted as collagen fibers unfurling then becoming stretched before gradually damaging and ultimate failure.</p

Cyclic deformation alters mechanical properties of glutaraldehyde-fixed bovine pericardium, but leaves porcine pericardium and bovine pericardium unchanged.

Tangent modulus (A) and ultimate tensile strength (B) of glutaraldehyde-fixed bovine pericardium decreased following 10 million cycles of deformation but returned to baseline levels at 20 million cycles. No alteration in tangent modulus or ultimate tensile strength of either porcine or bovine pericardium was found following cyclic deformation. Tangent modulus and ultimate tensile strength were assessed using a two-way analysis of variance, comparing means between tissue types and cycle number. n = 6 per group, per cycle number. Groups not connected by the same lower case letter (porcine pericardium), lower case double apostrophe (bovine pericardium), or lower case single apostrophe (glutaraldehyde-fixed bovine pericardium) are statistically significantly different. Data represent the mean ± s.d.</p

Flow diagram depicting how strips of pericardia were loaded to undergo consistent cyclic deformation.

Tissues were harvested and cut into strips, marked and imaged. Strips were then loaded to maintain a standard radius of curvature in cyclic deformation and ran in Dynatek’s M6 Heart Valve Tester.</p

Biochemical composition of glutaraldehyde-fixed bovine pericardium is altered following cyclic deformation, but maintained in porcine pericardium and bovine pericardium.

Both collagen (A) and elastin (B) content of glutaraldehyde-fixed bovine pericardium decreased following 20 million cycles. Biochemical composition of both porcine and bovine pericardium was unaltered by cyclic deformation. There was no change in tissue hydration with increased cycle number for any of the tissues (C). Biochemical composition was assessed using a two-way analysis of variance and comparing means between tissue types and cycle number. n = 6 per group, per cycle number. Groups not connected by the same lower case letter (porcine pericardium), lower case double apostrophe (bovine pericardium), or lower case single apostrophe (glutaraldehyde-fixed bovine pericardium) are statistically significantly different. Data represent the mean ± s.d.</p

Effect of cyclic deformation on xenogeneic heart valve biomaterials - Fig 3

Finite element model of the testing procedure at the end of the first step (A) and the end of the second step (B). For A, the tissue deformed around the two contact bodies with the profile of the tissue resembling that in the in vitro test. While for B, the tissue deformation exceeds that originally applied by the larger contact body and the maximum principal stress is also higher than at the end of the first step. As expected, the maximum principal stress is far lower in A than B. In the second step (B), the maximum principal stress in the central region of the model where the largest deflection occurred was 1.2 MPa and was located on the upper side of the tissue. The lower maximum principal stress on the underside of the tissue was approximately 0.42 MPa. The stress distribution is relatively consistent along the tissue likely because of the uniform loading applied.</p

Qualitative histological assessment of fixed and unfixed tissue biomaterials following cyclic deformation.

Nuclei density was maintained across all groups regardless of cycle number (hematoxylin and eosin (H&E)) (A). Elastin content decreased with increasing number of cycles for glutaraldehyde-fixed bovine pericardium, whereas both porcine and bovine pericardium maintained elastin content (Verhoeff-Van Gieson (VVG)) (A). However, elastin in porcine pericardium appeared fragmented with increasing number of cycles. All three tissue types underwent some cycle dependent separation of collagen bundles, although this was more dramatic in porcine pericardium and glutaraldehyde-fixed bovine pericardium than in bovine pericardium. n = 6 per group, per cycle number. Scale bar represents 50 μm (A). Collagen birefringence (B) of glutaraldehyde-fixed bovine pericardium at baseline is less than that of either of the non-fixed tissues (C). Collagen birefringence was significantly reduced following 20 million cycles for bovine pericardium (C). Glutaraldehyde-fixed bovine pericardium exhibited a trend towards increased collagen birefringence with increased cycle number, although this finding failed to reach significance (C). Percentage of birefringence was assessed using a two-way analysis of variance, comparing means between tissue types and cycle number. Scale bar represents 10 μm. n = 6 per group, per cycle number. Groups not connected by the same lower-case letter (porcine pericardium), lower-case double apostrophe (bovine pericardium), or lower-case single apostrophe (glutaraldehyde-fixed bovine pericardium) are statistically significantly different. Data represent the mean ± s.d.</p

Cyclic dependent strain is dependent on tissue source and fixation.

Longitudinal cycle dependent strain did not occur in unfixed bovine pericardium (A). Conversely, glutaraldehyde-fixed bovine pericardium exhibited negative longitudinal strain with increased cycle number (A). Porcine pericardium exhibited positive cycle dependent longitudinal strain which plateaued by 10 million cycles (A). Both bovine and porcine pericardium underwent negative cycle dependent perpendicular strain with increased cycle number (B). Glutaraldehyde-fixed bovine pericardium underwent positive cycle dependent perpendicular strain at 20 million cycles (B). Samples were assessed using a two-way analysis of variance, comparing means between tissue types and cycle number. n = 6 per group, per cycle number. Groups not connected by the same lower case letter (porcine pericardium), lower case double apostrophe (bovine pericardium), or lower case single apostrophe (glutaraldehyde-fixed bovine pericardium) are statistically significantly different. Data represent the mean ± s.d.</p

DataSheet1_Basement Membrane of Tissue Engineered Extracellular Matrix Scaffolds Modulates Rapid Human Endothelial Cell Recellularization and Promote Quiescent Behavior After Monolayer Formation.PDF

Off-the-shelf small diameter vascular grafts are an attractive alternative to eliminate the shortcomings of autologous tissues for vascular grafting. Bovine saphenous vein (SV) extracellular matrix (ECM) scaffolds are potentially ideal small diameter vascular grafts, due to their inherent architecture and signaling molecules capable of driving repopulating cell behavior and regeneration. However, harnessing this potential is predicated on the ability of the scaffold generation technique to maintain the delicate structure, composition, and associated functions of native vascular ECM. Previous de-cellularization methods have been uniformly demonstrated to disrupt the delicate basement membrane components of native vascular ECM. The antigen removal (AR) tissue processing method utilizes the protein chemistry principle of differential solubility to achieve a step-wise removal of antigens with similar physiochemical properties. Briefly, the cellular components of SV are permeabilized and the actomyosin crossbridges are relaxed, followed by lipophilic antigen removal, sarcomeric disassembly, hydrophilic antigen removal, nuclease digestion, and washout. Here, we demonstrate that bovine SV ECM scaffolds generated using the novel AR approach results in the retention of native basement membrane protein structure, composition (e.g., Collagen IV and laminin), and associated cell modulatory function. Presence of basement membrane proteins in AR vascular ECM scaffolds increases the rate of endothelial cell monolayer formation by enhancing cell migration and proliferation. Following monolayer formation, basement membrane proteins promote appropriate formation of adherence junction and apicobasal polarization, increasing the secretion of nitric oxide, and driving repopulating endothelial cells toward a quiescent phenotype. We conclude that the presence of an intact native vascular basement membrane in the AR SV ECM scaffolds modulates human endothelial cell quiescent monolayer formation which is essential for vessel homeostasis.</p

Mesenchymal Stem Cell Seeding of Porcine Small Intestinal Submucosal Extracellular Matrix for Cardiovascular Applications

<div><p>In this study, we investigate the translational potential of a novel combined construct using an FDA-approved decellularized porcine small intestinal submucosa extracellular matrix (SIS-ECM) seeded with human or porcine mesenchymal stem cells (MSCs) for cardiovascular indications. With the emerging success of individual component in various clinical applications, the combination of SIS-ECM with MSCs could provide additional therapeutic potential compared to individual components alone for cardiovascular repair. We tested the <i>in vitro</i> effects of MSC-seeding on SIS-ECM on resultant construct structure/function properties and MSC phenotypes. Additionally, we evaluated the ability of porcine MSCs to modulate recipient graft-specific response towards SIS-ECM in a porcine cardiac patch <i>in vivo</i> model. Specifically, we determined: 1) <i>in vitro</i> loading-capacity of human MSCs on SIS-ECM, 2) effect of cell seeding on SIS-ECM structure, compositions and mechanical properties, 3) effect of SIS-ECM seeding on human MSC phenotypes and differentiation potential, and 4) optimal orientation and dose of porcine MSCs seeded SIS-ECM for an <i>in vivo</i> cardiac application. In this study, histological structure, biochemical compositions and mechanical properties of the FDA-approved SIS-ECM biomaterial were retained following MSCs repopulation <i>in vitro</i>. Similarly, the cellular phenotypes and differentiation potential of MSCs were preserved following seeding on SIS-ECM. In a porcine <i>in vivo</i> patch study, the presence of porcine MSCs on SIS-ECM significantly reduced adaptive T cell response regardless of cell dose and orientation compared to SIS-ECM alone. These findings substantiate the clinical translational potential of combined SIS-ECM seeded with MSCs as a promising therapeutic candidate for cardiac applications.</p></div