Decellularization of Tissue Engineered Leaflets with Adult Stem Cell Recellularization

Introduction: Tissue engineered heart valves (TEHV) can revolutionize valve replacements, especially for juvenile patients. While much progress has been made toward implanting TEHV, a major limitation to date has been retraction of the leaflets by the cells transplanted with the TEHV, especially those developed from fibrin (Syedain et al. 2011, Flanagan et al. 2009). Here, we present a novel approach of decellularizing an engineered leaflet made from fibrin that is remodeled by dermal fibroblast, and subsequently recellularized with adult human mesenchymal stem cells (ahMSC). The approach has potential to deliver off-the-shelf engineered leaflet that can be recellularized with autologous stem cells.

Material and Methods: Fibrin-based engineered leaflets were cast with 4mg/ml fibrin gel and 1 million/ml neonatal human dermal fibroblast (nhDF). For decellularization, leaflets were incubated in 1% SDS over-night followed by 30 min rinse in 1% triton-X. Leaflets were characterized for mechanical and biochemical properties. For recellularization, leaflets were seeded with ahMSC. Reseeded samples were evaluated for cell proliferation and invasion up to 3 weeks.

Result and Discussion: Engineered leaflets had thickness, mechanical properties (UTS and Modulus), and anisotropy similar to native sheep pulmonary valve leaflets (the standard large animal model). No change in mechanical properties was seen after decellularization (Fig.1). Decellularization removed 99% of DNA with a drop in total protein (indicative of cellular protein loss) but no change in collagen concentration. Recellularized leaflets showed MSC proliferation over time with gradual invasion of MSC into the leaflet tissue (Fig.2). The mechanical properties after 3 weeks incubation with MSC showed no detrimental effects of MSC seeding and subsequent culture, and there was no evidence of tissue contraction (no change in thickness). MSC maintained positive staining for CD29, CD44, and CD90, and negative staining for CD45 and CD34 after 3 weeks.

Conclusion: We present here an approach to remove the tissue-forming but also tissue-contracting fibroblasts from TEHV, thereby creating a readily available biological scaffold for reseeding with autologous stem cells. Preliminary work with recellularization shows good penetration and increased cell density over 3 week without loss in tensile properties of the de/recellularized leaflets. References: Syedain et al. Cardio Vas Engr Tech 2011: in press Flanagan et al. Tissue Engr 2009:15(10) p2965-76

<>Acknowledgment: Funding from NIH R01 HL107572 (to R.T.T.)