Preclinical testing of tissue-engineered heart valves re-endothelialized under simulated physiological conditions.
BACKGROUND
The in vivo regeneration capacity of decellularized heart valve grafts is still controversial. The aim of this study was to evaluate function, morphological changes, and cellular composition of decellularized versus re-endothelialized ovine pulmonary valves (PV) after implantation into lambs for 1 or 3 months.


RESULTS
PV (n=21) were decellularized using detergents. Twelve PV were repopulated with autologous jugular veins endothelial cells (ECs) in a dynamic pulsatile bioreactor under simulated physiological conditions. Morphological evaluation before implantation included histological stainings (H&E, Movat-pentachrome, von-Kossa, DAPI), immunostainings (anti-perlecan, anti-eNOS, anti-procollagen-I, anti-SM-alpha-actin), electron microscopy (EM), and DNA extraction. Decellularization led to cell-free scaffolds with preserved extracellular matrix (ECM) including basement membrane. Reseeded PV (n=5) were completely covered with ECs expressing endothelial nitric oxide synthase (eNOS) and von Willebrand factor (vWF). The function of orthotopically implanted decellularized and re-endothelialized PV (n=7, each) was analyzed after 1 and 3 months by echocardiography and revealed no differences in competence between both groups. A confluent EC monolayer expressing eNOS/vWF was only found in re-endothelialized PV but not in decellularized PV, whereas the valve matrices were comparable repopulated with interstitial cells expressing SM-alpha-actin and procollagen-I. More thrombotic and neointima formations were observed in decellularized PV. No signs of calcification were detected in both PV types.


CONCLUSIONS
In vitro re-endothelialization of detergent-decellularized valves with autologous ECs under simulated physiological conditions significantly improves total EC valve coverage 3 months after implantation, whereas the valve repopulation with interstitial cells in vivo occurs most likely by cell migration inside the scaffold.
Lichtenberg
Artur
A
Department of Thoracic and Cardiovascular Surgery, Medical School Hannover, Hannover, Germany. lichtenberg.artur@mh-hannover.de
Tudorache
Igor
I
Cebotari
Serghei
S
Suprunov
Mark
M
Tudorache
Greta
G
Goerler
Heidi
H
Park
Joon-Keun
JK
Hilfiker-Kleiner
Denise
D
Ringes-Lichtenberg
Stefanie
S
Karck
Matthias
M
Brandes
Gudrun
G
Hilfiker
Andres
A
Haverich
Axel
A
D000818:Animals; D001705:Bioprosthesis; D019149:Bioreactors; D002114:Calcinosis / Q000209:etiology; D003902:Detergents / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology*; D004730:Endothelium, Vascular / Q000166:cytology; D005109:Extracellular Matrix / Q000637:transplantation*; D006350:Heart Valve Prosthesis; D019918:Heart Valve Prosthesis Implantation; D006965:Hyperplasia; D020007:Implants, Experimental; D007601:Jugular Veins / Q000166:cytology; D008855:Microscopy, Electron, Scanning; D011183:Postoperative Complications / Q000209:etiology; D011312:Pressure; D011664:Pulmonary Valve / Q000000981:diagnostic imaging / Q000601:surgery*; D012756:Sheep; D013927:Thrombosis / Q000209:etiology; D023822:Tissue Engineering / Q000295:instrumentation / Q000379:methods*; D014182:Transplantation, Autologous; D017539:Tunica Intima / Q000473:pathology; D014463:Ultrasonography
D003902:Detergents