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.

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In this study, structure and biomechanical properties of freeze-dried decellularized porcine pulmonary heart valves were investigated. Heart valves were dissected from porcine hearts. The tissues were decellularized and separated in three groups: (1) without lyoprotectant, (2) with 5% sucrose, and (3) with a mixture of 2.5% sucrose and 2.5% hydroxyl ethylene starch (HES), and then underwent freeze-drying. Freeze-drying in the absence of lyoprotectants caused an overall more disintegrated appearance of the histological architecture of the porcine valves, especially between the fibrosa and the ventricularis layers. Freeze-dried tissues with lyoprotectants have a looser network of collagen and elastic fibers with bigger pore sizes. Tissue freeze-dried in the absence of lyoprotecants had the largest pore sizes, whereas the tissue freeze-dried in the presence of protectants showed pores of intermediate sizes between the decellularized tissue and the unprotected freeze-dried samples. Tissue freeze-dried with sucrose alone displayed less porosity than tissue freeze-dried with the sucrose/HES mixture, whereas no significant differences in biomechanical properties were observed. Decellularization decreased the elastic modulus of artery tissue. The elastic modulus of freeze-dried tissue without protectants resembled that of decellularized tissue. The elastic modulus values of freeze-dried tissue stabilized by lyoprotectants were greater than those of decellularized tissue, but similar to those of native tissue.

This new CPB mouse model will facilitate preclinical testing of therapeutic strategies in cardiovascular diseases and investigation of CPB in relation to different insults and pre-existing comorbidities. In combination with genetically modified mice, this model will be an important tool to dissect the molecular mechanisms involved in organ damage related to extracorporeal circulation.

Decellularized heart valves can be used as starter matrix implants for heart valve replacement therapies in terms of guided tissue regeneration. Decellularized matrices ideally need to be long-term storable to assure off-the-shelf availability. Freeze-drying is an attractive preservation method, allowing storage at room temperature in a dried state. However, the two inherent processing steps, freezing and drying, can cause severe damage to extracellular matrix (ECM) proteins and the overall tissue histoarchitecture and thus impair biomechanical characteristics of resulting matrices. Freeze-drying therefore requires a lyoprotective agent that stabilizes endogenous structural proteins during both substeps and that forms a protective glassy state at room temperature. To estimate incubation times needed to infiltrate decellularized heart valves with the lyoprotectant sucrose, temperature-dependent diffusion studies were done using Fourier transform infrared spectroscopy. Glycerol, a cryoprotective agent, was studied for comparison. Diffusion of both protectants was found to exhibit Arrhenius behavior. The activation energies of sucrose and glycerol diffusion were found to be 15.9 and 37.7 kJ·mol(-1), respectively. It was estimated that 4 h of incubation at 37°C is sufficient to infiltrate heart valves with sucrose before freeze-drying. Application of a 5% sucrose solution was shown to stabilize acellular valve scaffolds during freeze-drying. Such freeze-dried tissues, however, displayed pores, which were attributed to ice crystal damage, whereas vacuum-dried scaffolds in comparison revealed no pores after drying and rehydration. Exposure to a hygroscopic sucrose solution (80%) before freeze-drying was shown to be an effective method to diminish pore formation in freeze-dried ECMs: matrix structures closely resembled those of control samples that were not freeze-dried. Heart valve matrices were shown to be in a glassy state after drying, suggesting that they can be stored at room temperature.

OBJECTIVES Decellularized homograft valves (DHVs) have shown promising clinical results, particularly in the treatment of congenital heart disease. However, DHV appears to elicit an immune response in a subset of young patients, indicated by early valve degeneration. As the decellularization process is quality controlled for each DHV, we hypothesized that there may be residual immunogenicity within the extracellular matrix of DHV. METHODS A semi-quantitative dot blot analysis was established to screen for preformed recipient antibodies using secondary anti-human antibodies. Fifteen DHV samples (7 aortic, 8 pulmonary) were solubilized and exposed to serum from 20 healthy controls. RESULTS The sera from young controls (n = 10, 18–25 years) showed significantly stronger binding of preformed antibodies than sera from older individuals (n = 10, 48–73 years). The difference between the means of arbitrary units was 15.1 ± 6.5 (P = 0.0315). There was high intraindividual variance in the mean amounts of arbitrary units of antibody binding with some healthy controls showing >10 times higher antibody binding towards 2 different DHV. The amount of preformed antibodies bound to DHVs was higher in aortic than in pulmonary DHVs. The mean number of antibody binding (in arbitrary units) was 17.2 ± 4.5 in aortic and 14.5 ± 4.7 in pulmonary DHV (P = 0.27). The amount of preformed antibodies bound to pulmonary DHVs was statistically significantly higher in the sera of healthy males (n = 10) than in the sera of healthy females (n = 10). The mean number of arbitrary units was 17.2 ± 4.2 in male and 11.7 ± 5.3 in female sera (P = 0.036). Antibody binding to aortic DHV was also higher in males, but not significant (18.8 ± 5.0 vs 15.6 ± 4.0). Blood group (ABO) incompatibility between the serum from controls and DHV showed no impact on antibody binding, and there was no age-related impact among DHV donors. CONCLUSIONS Residual immunogenicity of decellularized homografts appears to exist despite almost complete cell removal. The established dot blot method allows a semi-quantitative assessment of the individual immune response towards extracellular DHV components and potentially the possibility of preoperative homograft matching.

Decellularization of ovine mitral valve results in a mitral valves scaffold with mechanical properties comparable with native tissue, and a graft surface, which can be repopulated by endothelial cells.

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