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Tissue-engineered AV based on decellularized valve allografts satisfy short-term requirements of the systemic circulation in sheep. Although results of long-term experiments are pending, the lack of degenerative traits thus far, makes these grafts a promising alternative for future aortic heart valve surgery.

Tissue-engineered (TE) grafts based on decellularized grafts have shown very promising results in preclinical and clinical studies. However, in animal models valves have either been tested in juvenile models or in the clinically less relevant pulmonary valve position. In this study, we tested the grafts in the aortic valve (AV) position of 6-year-old sheep, as geriatric patients in need of an AV substitute due to calcification are the largest patient group benefiting from TE grafts. Decellularized AV (DAV; n = 4) and DAV additionally re-endothelialized with autologous cells (n = 3) were implanted in the AV position of 6-year-old female sheep. Function was investigated at implantation and explantation 12 months later. Regeneration capacity was analyzed by the repopulation degree of the graft with recipient's cells, by the generation of a new endothelial layer and by intracellular staining against pro-collagen type I. DAV and re-endothelialized AV demonstrated excellent function with only two valves developing mild insufficiencies (1°). Of the repopulating cells only few cells were identified as inflammation cells, while the majority was found to be interstitial cells producing procollagen type I. Endothelial coverage was found, but seemed to be reduced. The regenerative capacity of decellularized matrix is not only a feature exhibited when implanted in juvenile individuals but also is evident when implanted in the high-pressure AV position of older sheep, revealing the potential of TE grafts in age-advanced patients.

Bioprosthetic heart valve replacement, recommended for patients older than 65 years of age, increases the quality of life, however grafts exhibit limited functionality due to degradation and calcification. Decellularized and tissue-engineered pulmonary valves (PV) get repopulated by autologous interstitial cells when implanted into juvenile sheep. Thus, in vivo matrix guided regeneration results in restored valve function. In this study, we investigated the regeneration capacity in elderly sheep. Sheep (Ø 7 yrs old, Ø weight of 88.5 kg) received pulmonary valve replacement. Decellularized PV (n=6), decellularized PV coated with proangiogenic CCN1 (n=6), and decellularized, CCN1 coated and reendothelized PV (n=6) were implanted in orthotopic position. For endothelialization PV were seeded with autologous endothelial cells (EC) differentiated from EPC from peripheral blood. Cells were flow adapted in a pulsatile bioreactor system prior implantation. PV functional analysis in vivo was realized by echocardiography, directly after implantation and prior explantation. Allografts were explanted after six and twelve months in vivo and investigated in respect to endothelium coverage, to the integrity of the extracellular matrix, and to the degree and cellular identity of invaded cells by histological and immunochemical stains. All allografts showed good to adequate function, no stenosis, low gradients and only occasional insufficiency without clinical symptoms. No signs of degradation of the extracellular matrix and minimal calcification restricted at the anastomosis of the grafts were found. All grafts were repopulated with cells but to various degree. Only one, explanted after one year, showed complete repopulation including the leaflets. In general, cell-density in the pulmonary artery was higher on the adventitial than on the luminal side and leaflets were better repopulated at the ventricular than at the arterial side. The majority of cells expressed sm-alpha-actin. Endothelial cells located on the luminal side appeared in some grafts as intact complete monolayer. Beside a slight tendency of better repopulation observed in the reendothelialized PV group, no significant difference of cell densities was found among the groups. Autologous repopulation of decellularized heart valve matrices implanted in the orthotopic position in the elderly sheep demonstrates retained regenerative capacity even in the older organism. This observation combined with the fact that no functional loss must have taken into account, implantation of decellularized heart valves matrices in patients over age 65 remains a therapy option to overcome the drawbacks of current used bioprosthesis.