Structural and biomechanical characterizations of acellular porcine mitral valve scaffolds: anterior leaflets, posterior leaflets, and chordae tendineae.

Mitral valve (MV) tissue engineering is still in its early stage, and one major challenge in MV tissue engineering is to identify appropriate scaffold materials. With the potential of acellular MV scaffolds being demonstrated recently, it is important to have a full understanding of the biomechanics of the native MV components and their acellular scaffolds. In this study, we have successfully characterized the structural and mechanical properties of porcine MV components, including anterior leaflet (AL), posterior leaflet (PL), strut chordae, and basal chordae, before and after decellularization. Quantitative DNA assay showed more than 90% reduction in DNA content, and Griffonia simplicifolia (GS) lectin immunohistochemistry confirmed the complete lack of porcine α-Gal antigen in the acellular MV components. In the acellular AL and PL, the atrialis, spongiosa, and fibrosa trilayered structure, along with its ECM constitutes, i.e., collagen fibers, elastin fibers, and portion of GAGs, were preserved. Nevertheless, the ECM of both AL and PL experienced a certain degree of disruption, exhibiting a less dense, porous ECM morphology. The overall anatomical morphology of the strut and basal chordae were also maintained after decellularization, with longitudinal morphology experiencing minimum disruption, but the cross-sectional morphology exhibiting evenly-distributed porous structure. In the acellular AL and PL, the nonlinear anisotropic biaxial mechanical behavior was overall preserved; however, uniaxial tensile tests showed that the removal of cellular content and the disruption of structural ECM did result in small decreases in maximum tensile modulus, tissue extensibility, failure stress, and failure strain for both MV leaflets and chordae.

Platform technologies for decellularization, tunic-specific cell seeding, and in vitro conditioning of extended length, small diameter vascular grafts.

The aim of this study was to generate extended length, small diameter vascular scaffolds that could serve as potential grafts for treatment of acute ischemia. Biological tissues are considered excellent scaffolds, which exhibit adequate biological, mechanical, and handling properties; however, they tend to degenerate, dilate, and calcify after implantation. We hypothesized that chemically stabilized acellular arteries would be ideal scaffolds for development of vascular grafts for peripheral surgery applications. Based on promising historical data from our laboratory and others, we chose to decellularize bovine mammary and femoral arteries and test them as scaffolds for vascular grafting. Decellularization of such long structures required development of a novel "bioprocessing" system and a sequence of detergents and enzymes that generated completely acellular, galactose-(α1,3)-galactose (α-Gal) xenoantigen-free scaffolds with preserved collagen, elastin, and basement membrane components. Acellular arteries exhibited excellent mechanical properties, including burst pressure, suture holding strength, and elastic recoil. To reduce elastin degeneration, we treated the scaffolds with penta-galloyl glucose and then revitalized them in vitro using a tunic-specific cell approach. A novel atraumatic endothelialization protocol using an external stent was also developed for the long grafts and cell-seeded constructs were conditioned in a flow bioreactor. Both decellularization and revitalization are feasible but cell retention in vitro continues to pose challenges. These studies support further efforts toward clinical use of small diameter acellular arteries as vascular grafts.

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 1 Hz 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.