Assembly and testing of stem cell-seeded layered collagen constructs for heart valve tissue engineering.

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.