Bioreactors and operating room centric protocols for clinical heart valve tissue engineering

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are the earliest tissue-engineered vascular grafts (TEVGs) to be used clinically. These TEVGs transform into living blood vessels in vivo, with an endothelial cell (EC) lining invested by smooth muscle cells (SMCs); however, the process by which this occurs is unclear. To test if the seeded BMCs differentiate into the mature vascular cells of the neovessel, we implanted an immunodeficient mouse recipient with human BMC (hBMC)-seeded scaffolds. As in humans, TEVGs implanted in a mouse host as venous interposition grafts gradually transformed into living blood vessels over a 6-month time course. Seeded hBMCs, however, were no longer detectable within a few days of implantation. Instead, scaffolds were initially repopulated by mouse monocytes and subsequently repopulated by mouse SMCs and ECs. Seeded BMCs secreted significant amounts of monocyte chemoattractant protein-1 and increased early monocyte recruitment. These findings suggest TEVGs transform into functional neovessels via an inflammatory process of vascular remodeling.

The potential for decellularized aortic heart valves (AVs) as heart valve replacements is based on the assumption that the major cellular immunogenic components have been removed, and that the remaining extracellular matrix (ECM) should retain the necessary mechanical properties and functional design. However, decellularization processes likely alter the ECM mechanical and structural properties, potentially affecting long-term durability. In the present study, we explored the effects of an anionic detergent (sodium dodecyl sulfate (SDS)), enzymatic agent (Trypsin), and a non-ionic detergent (Triton X-100) on the mechanical and structural properties of AV leaflets (AVLs) to provide greater insight into the initial functional state of the decellularized AVL. The overall extensibility represented by the areal strain under 60 N/m increased from 68.85% for the native AV to 139.95%, 137.51%, and 177.69% for SDS, Trypsin, and Triton X-100, respectively, after decellularization. In flexure, decellularized AVLs demonstrated a profound loss of stiffness overall, and also produced a nonlinear moment-curvature relation compared to the linear response of the native AVL. Effective flexural moduli decreased from 156.0+/-24.6 kPa for the native AV to 23.5+/-5.8, 15.6+/-4.8, and 19.4+/-8.9 kPa for SDS, Trypsin, and Triton X-100 treated leaflets, respectively. While the overall leaflet fiber architecture remained relatively unchanged, decellularization resulted in substantial microscopic disruption. In conclusion, changes in mechanical and structural properties of decellularized leaflets were likely associated with disruption of the ECM, which may impact the durability of the leaflets.

The first tissue engineered decellularized porcine heart valve, Synergraft (Cryolife Inc., USA) was introduced in Europe as an alternative to conventional biological valves. This is the first report of the rapid failure of these new grafts in a small series. In 2001, 2 model 500 and 2 model 700 Synergraft valves were implanted in four male children (age 2.5-11 years) in the right ventricular outflow tract as a root. Two patients had a Ross operation and two had a homograft replacement. The cryopreserved Synergraft valves appeared macroscopically unremarkable at implantation. Recovery from surgery was uneventful and good valve function was demonstrated postoperatively. Three children died, two suddenly with severely degenerated Synergraft valves 6 weeks and 1 year after implantation. The third child died on the 7th day due to Synergraft rupture. Subsequently the fourth graft was explanted prophylactically 2 days after implantation. Macroscopically all four grafts showed severe inflammation starting on the outside (day 2 explant) leading to structural failure (day 7 explant) and severe degeneration of the leaflets and wall (6 weeks and 1 year explant). Histology demonstrated severe foreign body type reaction dominated by neutrophil granulocytes and macrophages in the early explants and a lymphocytic reaction at 1 year. In addition significant calcific deposits were demonstrated at all stages. Surprisingly pre-implant samples of the Synergraft revealed incomplete decellularization and calcific deposits. No cell repopulation of the porcine matrix occurred. The xenogenic collagen matrix of the Synergraft valve elicits a strong inflammatory response in humans which is non-specific early on and is followed by a lymphocyte response. Structural failure or rapid degeneration of the graft occurred within 1 year. Calcific deposits before implantation and incomplete decellularization may indicate manufacturing problems. The porcine Synergraft treated heart valves should not be implanted at this stage and has been stopped.