Decellularization does not eliminate thrombogenicity and inflammatory stimulation in tissue-engineered porcine heart valves.
OBJECTIVE
In tissue engineering of heart valves using decellularized xenogenic valves, it has been suggested that cell elimination would result in a biologically inert matrix. The aim of this in-vitro investigation was to evaluate different decellularization methods in regard to the completeness of cell removal, inflammatory response, and thrombocyte activation.


METHODS
Decellularized porcine Synergraft valves were compared with porcine pulmonary conduits decellularized with Triton X-100, sodium deoxycholate, Igepal CA-630 and ribonuclease. Completeness of decellularization was evaluated with staining for nuclei and alpha-Gal epitope. Decellularized heart valves with and without seeding with endothelial cells (ECs) were incubated with human platelet-rich plasma and stained for CD41 and PAC-1 to evaluate thrombocyte activation. Samples were processed for laser scanning microscopy (LSM) and scanning electron microscopy (SEM). Migration of human monocytic cells towards extracted valve proteins was tested.


RESULTS
In contrast to the Synergraft, complete cell removal and elimination of the alpha-gal epitope was achieved with the new decellularization method. Numerous adherent and activated platelets were found on the decellularized matrix. This was inhibited by seeding with ECs. Even in completely cell-free valve tissue extracellular matrix proteins attracted human monocytic cells as in early inflammation, depending on whether porcine or human tissue was used.


CONCLUSIONS
Important differences were found in the decellularization efficacy of treatment methods. However, even complete elimination of cells and their remnants did not result in a biologically inert matrix. The decellularized porcine heart valve matrix has the potential to attract inflammatory cells and to induce platelet activation. These findings suggest that it will be important to control the different inflammation-stimulating factors if porcine tissues are to be used successfully in tissue engineering.
Kasimir
Marie-Theres
MT
Department of Cardiothoracic Surgery, Medical University of Vienna, Vienna, Austria.
Rieder
Erwin
E
Seebacher
Gernot
G
Nigisch
Anneliese
A
Dekan
Barbara
B
Wolner
Ernst
E
Weigel
Guenter
G
Simon
Paul
P
D000818:Animals; D002465:Cell Movement / Q000187:drug effects; D003840:Deoxycholic Acid / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004195:Disease Models, Animal; D006349:Heart Valve Diseases / Q000473:pathology / Q000601:surgery; D006350:Heart Valve Prosthesis; D019918:Heart Valve Prosthesis Implantation / Q000009:adverse effects*; D018613:Microscopy, Confocal; D017830:Octoxynol / Q000494:pharmacology; D015539:Platelet Activation / Q000187:drug effects; D011092:Polyethylene Glycols / Q000494:pharmacology; D011183:Postoperative Complications / Q000209:etiology / Q000517:prevention & control; D011474:Prosthesis Design; D011664:Pulmonary Valve / Q000187:drug effects / Q000601:surgery / Q000648:ultrastructure; D012260:Ribonucleases / Q000494:pharmacology; D013552:Swine; D013927:Thrombosis / Q000209:etiology / Q000517:prevention & control*; D016610:Tissue Embedding / Q000379:methods*
D003902:Detergents; D003840:Deoxycholic Acid; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D012260:Ribonucleases