Engineering of human vascular aortic tissue based on a xenogeneic starter matrix

Background. The goal for tissue engineering of vascular grafts is the repla cement of a diseased vessel with a functional and stable graft, We now intr oduce a new concept for the tissue engineering of vessels, The idea was to humanize a previously acellularized, but structurally intact, xenogeneic ve ssel by repopulation with human autologous cells. To this purpose, a gentle nondenaturing and nondeterging acellularization procedure for xenogeneic a ortas was developed, This structure was reseeded with pre-expanded peripher al vascular endothelial cells (EC) and myofibroblasts using specifically de signed bioreactors, Methods. Aortas from 15-30 kg female landrace pigs were acelullarized with a 0.1% trypsin solution for between 24 and 96 hr, Human vascular cells were harvested from saphenous vein biopsy specimens, Acellularized vessels were reseeded with EC and myofibroblasts. Cell viability after reseeding was as sayed by fluorescence staining. Morphologic features of the acellularized m atrix and tissue engineered vessel was assayed by transmission and scanning electron microscopy and histologic analysis. Nitric oxide-synthetase activ ity was investigated by mass spectrometric analysis of bioreactor supernata nts. The in vivo immune response was tested by subcutaneous implantation of acellularized porcine aortic tissue in a rat model. Results. The acellularization procedure resulted in an almost complete remo val of the original resident cells, and the 3-D matrix was loosened at inte rfibrillar zones, However, the 3-D arrangement of the matrix fibers was gro ssly maintained. The 3-D matrix was covered with a fully confluent human en dothelial cell layer obtained by continuous stress challenge in the bioreac tor, Myofibroblasts migrated into positions formerly occupied by the xenoge neic cells. Nitric oxide synthetase activity was maintained in the bioartif icial graft. T-lymphocyte and CD18 positive leukocyte infiltrate were great ly reduced after acellularization of porcine aortic specimens after implant ation in the rat. Conclusions, Porcine vessels were acellularized and consecutively fully rep opulated with human EC and myofibroblasts, This approach may eventually lea d to the engineering of vessels immunologically acceptable to the host usin g a relatively short preparation period of 2-3 weeks. We expect matrix turn over in vivo leading to a gradual assimilation of the matrix structure by t he host mediated by the hosts autologous cells.