Mechanical Compliance and Immunological Compatibility of Fixative-Free Decellularized/Cryopreserved Human Pericardium

Abstract

BackgroundThe pericardial tissue is commonly used to produce bio-prosthetic cardiac valves and patches in cardiac surgery. The procedures adopted to prepare this tissue consist in treatment with aldehydes, which do not prevent post-graft tissue calcification due to incomplete xeno-antigens removal. The adoption of fixative-free decellularization protocols has been therefore suggested to overcome this limitation. Although promising, the decellularized pericardium has not yet used in clinics, due to the absence of proofs indicating that the decellularization and cryopreservation procedures can effectively preserve the mechanical properties and the immunologic compatibility of the tissue.Principal FindingsThe aim of the present work was to validate a procedure to prepare decellularized/cryopreserved human pericardium which may be implemented into cardiovascular homograft tissue Banks. The method employed to decellularize the tissue completely removed the cells without affecting ECM structure; furthermore, uniaxial tensile loading tests revealed an equivalent resistance of the decellularized tissue to strain, before and after the cryopreservation, in comparison with the fresh tissue. Finally, immunological compatibility, showed a minimized host immune cells invasion and low levels of systemic inflammation, as assessed by tissue transplantation into immune-competent mice.ConclusionsOur results indicate, for the first time, that fixative-free decellularized pericardium from cadaveric tissue donors can be banked according to Tissue Repository-approved procedures without compromising its mechanical properties and immunological tolerance. This tissue can be therefore treated as a safe homograft for cardiac surgery.

Feasibility of pig and human‐derived aortic valve interstitial cells seeding on fixative‐free decellularized animal pericardium

“…Moreover, the impact of these components on cell‐mediated immune reaction would have to be carefully evaluated by in vitro and in vivo testing before considering immunologically safe the decellularized tissue for a perspective in human transplantation. On the other hand, the evidence (Figure ) that the decellularization procedure adopted for the porcine pericardium reduced below the detection limit the content of the 1,3 α‐galactose, confirms the potential advantage of osmotic/detergent decellularization procedures to strongly reduce the level of xenoantigens and enhance immunological tolerance …”

“…This was performed by histological sectioning and observing under the light/fluorescence microscope after histochemistry and staining with nuclear dye DAPI. As already shown in the human pericardium, the structure and the composition of the tissue was not grossly affected by the osmotic treatment [Figure (A)]. In addition, both the Masson's trichrome [Figure (A)] and the fluorescent nuclear staining with DAPI [Figure (B), left] confirmed the absence of cells and a complete removal of DNA.…”

“…Pig hearts (6–9 months of age) were obtained at the local slaughterhouse. Parietal pericardial tissue was dissected from the left ventricle and then treated as described in our previous work . This procedure is a modification of the protocol originally used by others and consists in a sequential incubation of the pericardial tissue into (i) a hypotonic buffer (10 m M Tris‐HCl; pH 8.0), (ii) a detergent hypotonic solution containing 0.1% (w/v) sodium dodecylsulfate (SDS), and (iii) a nucleic acid removal solution containing 50 U mL −1 deoxyribonuclease‐I and 1 U mL −1 ribonuclease‐A.…”

“…These methods, originally used to decellularize porcine valves, are not based on GA fixation but on osmotic lysis of the cells populating the tissue, followed by extraction of cell remnants with ionic/nonionic detergents, and treatment with enzymes to remove the genetic material (DNA/RNA). Although fixation‐free cell removal procedures have been proven successful for the lower inflammatory potential of the decellularized grafts in experimental models, and have been employed to perform decellularization of human‐derived pericardium for potential employment as an homograft tissue in surgery, or the production of scaffolds for the derivation of tissue engineered heart valve (TEHV) leaflets, they are not free of contraindications. In fact, cases of decellularized valve implant rejection have been reported in children due to strong immune and foreign body reactions …”

Evaluation of transcatheter heart valve biomaterials: Biomechanical characterization of bovine and porcine pericardium

“…However, cryopreservation has been shown to retain the native tissue’s structural integrity and mechanical properties [20, 57]. Vinci et al (2013) [58] investigated the mechanical properties of decellularized cryopreserved human pericardium. Uniaxial tensile loading tests revealed equivalent elastic modulus, UTS and UTE of the decellularized tissues, before and after the cryopreservation, in comparison with the fresh tissue.…”