Fixative-free Decellularization Of Human Pericardium Ensures Maintenance Of Biomechanical Properties And Maximizes Graft Immunotolerance After Cryopreservation

Background - The pericardium is a natural tissue used for derivation of bio-prosthetic cardiac valves. Common decellularization procedures are not optimized providing risks of post- implantation inflammatory reaction. Specific aims and Results - The aim of this work was to devise a procedure to decellularize the pericardium maintaining mechanical compliance with reduced risks for rejection into an immunocompetent environment, also after cryopreservation. Pericardium sheets from tissue donors were dissected and incubated into a hypotonic buffer containing DNAse I, removing all cellular remnants. The decellularized tissue, with or without 60 days cryopreservation in N2 vapor was used for mechanical testing and implanted into immunocompetent mice. Mechanical response was assessed by uniaxial stress/strain tests. These showed minimal changes in decellularized (DE) vs. decellularized/cryopreserved (DE/CR) tissues. In particular, the elastic modulus at low (0.3±0.16 vs. 0.5±0.3) and high (26.5±16.6 vs. 31.0±13.7 MPa) strains were similar, while stress at failure (0.4±0.2 vs. 0.47±0.2 MPa) and at transition (3.7±1.8 vs. 5.1±2.4 MPa) were only slightly higher (P<.05, t-test) in DE/CR samples. Fresh, DE and DE/CR tissues were implanted into immunocompetent mice for 60 days. Post-graft rejection was evaluated by determining the relative CD4+/CD8+ circulating lymphocytes ratio. Results a persistently reduced (3.6±0.2 in controls vs. 2.2±0.4, 2.2±0.2, 2.5±0.5 and 2.5±0.4 at 15, 30, 45 and 60 days) CD4/CD8 ratio in mice receiving fresh pericardium, indicating rejection. By contrast, mice implanted with DE or DE/CR tissues had CD4/CD8 ratios in the controls range. Histology on pericardium fragments recovered at 60 days showed higher invading cell number (P<.05, ANOVA) in fresh pericardial implants (553.4±39.6 nuclei/0.15mm2) compared with DE (265.1±65.7 nuclei/0.15mm2) or DE/CR tissues (318.6±44.5 nuclei/0.15mm2), confirming improved tolerance after decellularization. Conclusions - Decellularization by hypotonic lysis preserves mechanical compliance and increases immunologic tolerance of pericardial tissue. This method is proposed as an alternative to prepare pericardial sheets for bio-prosthetic cardiac valves fabrication.