To determine the most effective method of producing the acellularized xenograft heart valve leaflets, we compared pathological findings of the xenograft heart valve leaflets produced by three methods; freeze-thawing, Triton and NaCl-SDS treatment and further analyzed the pattern of endothelial cells seeded onto them. Materials and methods Two pigs were sacrificed and three pulmonary valve leaflets were harvested from each animal. They were immediately stored in a tissue preservation solution and assigned in one of the three preparation methods for acellularization. Endothelial cells from the jugular vein of a goat were isolated and seeded onto the acellularized xenograft heart valve leaflets. Light and Electron microscopic analyses were performed. Result and conclusion H & E stain showed that cells were almost absent in the leaflet treated with NaCl-SDS, while cells were partly present in the leaflets treated, one with Triton and the other Freeze-thawing. Transmission microscopic analyses showed cell-free matrix with well preserved collagen architecture under the seeded endothelial cells in the leaflets treated with NaCl-SDS. In conclusion, the valve leaflets treated with NaCl-SDS among three representative methods of acellularization of tissues (freeze-thawing, Triton X-l00, and NaCl-SDS) showed the better results than the others in terms of the efficacy of decellularization and response to endothelial cell seeding.

Background Acellularized valve xenografts are considered a promising way of overcoming the inherent limitations of current prosthetic valves. The aim of this study was to compare the biological responses of an autologous endothelial cell seeded acellularized xenograft (AAX) and a plain acellularized xenograft (PAX) implanted in the pulmonary valve leaflet in the same animal. Methods Endothelial cells were isolated and cultured from the jugular vein of goats. Porcine valve leaflets were acellularized with Nacl-SDS, and for AAX, leaflets were then seeded with autologous endothelial cells. A PAX and an AAX were implanted as double pulmonary valve leaflet replacement in the same animal in a goat model (n=6). After sacrifice, the implanted valve leaflet tissues were retrieved and analyzed visually and under a light microscope. Results and Conclusions Six animals were sacrificed as scheduled during the short-term (6 and 24 hours), mid-term (1 week and 1 month) and long-term (3 and 6 months). Gross and ultrasonographic examinations revealed good valve function with no thrombosis but with slight thickening. Microscopic analysis of the leaflets showed abundant cellular ingrowth into the acellularized leaflets over time. The role of endothelial cell seeding remains controversial. This animal experiment demonstrates the practical feasibility of using acellularized valve xenografts.