Assembly and Testing of Stem Cell-Seeded Layered Collagen Constructs for Heart Valve Tissue Engineering

Abstract

Tissue engineering holds great promise for treatment of valvular diseases. Despite excellent progress in the field, current approaches do not fully take into account each patient's valve anatomical uniqueness, the presence of a middle spongiosa cushion that allows shearing of external fibrous layers (fibrosa and ventricularis), and the need for autologous valvular interstitial cells. In this study we propose a novel approach to heart valve tissue engineering based on bioreactor conditioning of mesenchymal stem cell-seeded, valve-shaped constructs assembled from layered collagenous scaffolds. Fibrous scaffolds were prepared by decellularization of porcine pericardium and spongiosa scaffolds by decellularization and elastase treatment of porcine pulmonary arteries. To create anatomically correct constructs, we created silicone molds from native porcine aortic valves, dried two identical fibrous scaffolds onto the molds, and stabilized them with penta-galloyl-glucose a reversible collagen-binding polyphenol that reduces biodegradation. The layers were fused with a protein/aldehyde scaffold bio-adhesive and neutralized to reduce cytotoxicity. Spongiosa scaffolds, seeded with human bone marrow-derived stem cells, were inserted within the valve-shaped layered scaffolds and sutured inside the original aortic root. The final product was mounted in a heart valve bioreactor and cycled in cell culture conditions. Most cells were alive after 8 days, elongated significantly, and stained positive for vimentin, similar to native human valvular interstitial cells, indicating feasibility of our approach.

Functional Heart Valve Scaffolds Obtained by Complete Decellularization of Porcine Aortic Roots in a Novel Differential Pressure Gradient Perfusion System

“…9 To respond to these challenges, cusps have a trilayered (fibrosa, spongiosa, ventricularis) and anisotropic structure (stiffer in the circumferential axis vs. radial axis), which is essential for mechanical durability and stress distribution. 13,14 Currently, the most physiologic valve replacements are considered the homograft valves because of their outstanding valve function and hemodynamics. However, their durability is limited, showing 60% survival at 10 years and only 30% at 20 years, 15 probably because most cells are lost during the freeze-thaw cycle or shortly after implantation.…”

“…21 Other experimental approaches to building tissueengineered valves include use of cell-seeded biodegradable polymers (synthetic or fibrin-based, molded in the shape of an aortic valve root) to generate a homogeneous, isotropic collagenous structure lacking elastic fibers, before implantation, 22,23 implantation of stent-mounted acellular valves seeded with vascular fibroblasts or stem cells, 24 as well as use of polymers 22,25 and layered composites. 13,14,26 Creating structures as heterogeneous as that of the aortic roots using a bottom-up approach (such as three-dimensional printing 27 ) is quite difficult and requires major technological advances. Taken together, none of the above referenced approaches satisfied the design criteria.…”

Pulmonary heart valve replacement using stabilized acellular xenogeneic scaffolds; effects of seeding with autologous stem cells

“…According to the accepted paradigm, the two main TEHV components are the template, or scaffold and the cells (7). The scaffold, either biologic or synthetic, should provide an adequate support for cell attachment and an environment for cells which can remodel the scaffold as a response to physiological stimuli.…”

“…We also treat acellular scaffolds with penta-galloyl glucose (PGG) for tissue stabilization, to prevent premature scaffold degradation and to reduce calcification (13)(14)(15). We chose ADSCs because they are easy to obtain in large numbers and because of their plasticity and ability to differentiate into valve cells (7,16). The expectation for the stem cells was to differentiate into interstitial valvular cells in vivo and to provide viability and matrix remodeling before extracellular matrix deterioration.…”

Comparison of Mesenchymal Stem Cell Source Differentiation Toward Human Pediatric Aortic Valve Interstitial Cells within 3D Engineered Matrices

“…72 Tedder and his coworker dynamically conditioned decellularized porcine pericardium and spongiosa scaffolds seeded with BMMSC in a bioreactor and found that BMMSC were positive for vimentin, which was similar to HAVIC. 73,74 This indicates that biomechanical stimuli are likely also beneficial to promote MSC differentiation toward valve phenotypes.…”