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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Generating cellularized 3D constructs with clinical relevant dimensions is challenged by nutrition supply. This is of utmost importance for cardiac tissue engineering, since cardiomyocytes are extremely sensitive to malnutrition and hypoxia in vitro and after implantation. To develop a perfusable myocardial patch, we have focused on seeding a decellularized biological vascularized matrix (BioVaM) with endothelial cells. BioVaM is produced by decellularization of porcine small intestinal segments with preserved arterial and venous pedicles, which can be connected to a perfusion system in vitro or the host vasculature in vivo. The BioVaM vessel bed was re-seeded with porcine primary endothelial cells (pCEC). Seeding efficiency was influenced by detergent composition used for decellularization (sodium dodecyl sulfate (SDS) and/or Triton X-100) and the medium composition used for re-seeding. After decellularization, residual SDS was detected in the matrix affecting the survival of pCEC which showed a low tolerance to SDS and Triton X-100. Sensitivity to detergents was attenuated by supplementation of the medium with bovine serum albumin (BSA) or fetal calf serum (FCS). Pre-conditioning of the BioVaM with 20% FCS was not sufficient to attain pCEC survival in the vascular bed. However, re-cellularization was achieved by prolonged FCS supplementation during cultivation, resulting in a perfusable, re-endothelialized matrix of 11 cm2 in size. This achievement represents a promising step towards engineering of perfusable, 3D cardiac constructs with clinically relevant dimensions.

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.