Introduction: Decellularized heart valves (DHV) have emerged as truly regenerative material that can be actively repopulated with cells and remodeled upon orthotopic implantation by the recipient. However, the limited availability of small sized human DHV for pediatric clinical application has prompted research for alternatives such as the development of pig derived DHV. To overcome the immunogenic hurdles, here we used DHVs from genetically modified donor pigs in combination with additional chemical glycan antigen modifications.

Methods: Pulmonary heart valves from wildtype (wt) and GGTA1-KO pigs were decellularized by Trypsin/EDTA and Triton X-100 treatment. The resulting acellular heart valves matrices were treated with sodium periodate which specifically oxidizes glycan epitopes that could be subsequently conjugated with either aminooxy-biotin or PEG-hydrazide (40kDA). We assessed biomechanical and biochemical properties of the glycan-modified wt DHVs in vitro. In an additional in vivo study, we orthotopically implanted DHVs from GGTA1-KO pigs with or without chemical glycan-modifications into baboons and followed-up the animals for 6 months. Finally, explanted grafts and sera of the baboons were analyzed for graft recipient interactions, focusing on immune reaction and remodeling.

Results: We could show that oxidation of glycan epitopes and their subsequent conjugation with biotin or PEG significantly reduced binding of glycan-specific antibodies or lectins. Biomechanical properties remained unchanged by the applied chemical treatment. In vivo, all implanted DHVs exhibited very good functionality for up to 3 months and five out of six baboons finished the observation period of 6 months, exhibiting moderate graft stenosis and regurgitation at the time of explantation. Mural as well as immunological recipient cells, mainly foreign body giant cells and B-cell clusters, repopulated the implanted acellular valvular matrix. Analysis of sera revealed newly formed antibodies against the implants in all baboons. Notably, antibodies against the biotin moiety rapidly emerged in the baboons that had received biotinylated DHVs.

Conclusion:  Glycans of DHVs can be efficiently masked by chemical treatment. However, the masking did not prevent an immune reaction against the material upon transplantation and even induced the formation of antibodies against the masking reagent. Long-term functionality of the graft in vivo was not improved.