Decellularization combined with enzymatic removal of N‐linked glycans and residual DNA reduces inflammatory response and improves performance of porcine xenogeneic pulmonary heart valves in an ovine in vivo model

Abstract

Background Limited availability of decellularized allogeneic heart valve substitutes restricts the clinical application thereof. Decellularized xenogeneic valves might constitute an attractive alternative; however, increased immunological hurdles have to be overcome. This study aims for the in vivo effect in sheep of decellularized porcine pulmonary heart valves (dpPHV) enzymatically treated for N‐glycan and DNA removal. Methods dpPHV generated by nine different decelluarization methods were characterized in respect of DNA, hydroxyproline, GAGs, and SDS content. Orthotopic implantation in sheep for six months of five groups of dpPHV (n = 3 each; 3 different decellularization protocols w/o PNGase F and DNase I treatment) allowed the analysis of function and immunological reaction in the ovine host. Allogenic doPHV implantations (n = 3) from a previous study served as control. Results Among the decellularization procedures, Triton X‐100 & SDS as well as trypsin & Triton X‐100 resulted in highly efficient removal of cellular components, while the extracellular matrix remained intact. In vivo, the functional performance of dpPHV was comparable to that of allogeneic controls. Removal of N‐linked glycans and DNA by enzymatic PNGase F and DNase I treatment had positive effects on the clinical performance of Triton X‐100 & SDS dpPHV, whereas this treatment of trypsin & Triton X‐100 dpPHV induced the lowest degree of inflammation of all tested xenogeneic implants. Conclusion Functional xenogeneic heart valve substitutes with a low immunologic load can be produced by decellularization combined with enzymatic removal of DNA and partial deglycosylation of dpPHV.

Toward acellular xenogeneic heart valve prostheses: Histological and biomechanical characterization of decellularized and enzymatically deglycosylated porcine pulmonary heart valve matrices

“…Mechanical stability and decreased immunogenicity of PNGaseF-treated porcine pulmonary valves have been demonstrated by our recently published in vivo study. 33 Indeed, PNGaseF-treated pulmonary valves were…”

“…All reagents were purchased from Carl Roth, with the exception of trypsin, which was purchased from PAN-Biotech. Decellularization was carried out as described by Ramm et al 33 A description of the various steps of the decellularization protocols is reported in Table 1. A volume of 100 mL per heart valve was used for treatment in trypsin, whereas 150 mL was used per heart valve for all the other treatments and washing steps.…”

“…One third of dissected longitudinally native, decellularized, and decellularized and enzymatic treated pulmonary trunk samples (n = 3, each) were fixed in 10% (v/v) formalin, and dehydrated and embedded in paraffin. Histological sections (6 μm) were stained for hematoxylin and eosin (H&E) and Movat pentachrome, as described by Ref.,33,34 to investigate cell removal and ECM morphology.…”

“…: nonsignificant functional and exhibited very good haemodynamic performance and lower inflammation than decellularized and non-enzymatic treated valves in vivo. 33 Decellularization alone leads to the removal of cells and, thus, the decrease in immunogenic epitopes, but still results in rejection reaction upon transplantation into humans, due to pig-to-human species barrier, as shown by transplantation in human of the xenogeneic SynerGraft and Matrix P valves 15,16 ; the α-Gal epitope has been identified as key antigen in the rejection reaction of xenografts. 28 The decellularization protocols investigated here were developed in house, and they were chosen mostly on the basis of previous studies carried out in our research group.…”

“…Indeed, a beneficial effect in terms of decreased immunogenicity was observed in our in vivo study in sheep, when transplanting decellularized porcine pulmonary valves treated with Peptide-N-glycosidase F (PNGaseF). 33 Owing to the important role played by carbohydrates in pig-tohuman transplantation, our research efforts have been focusing on a better understanding of carbohydrates as xenoantigens.…”