Magnesium Presence Prevents Removal of Antigenic Nuclear-Associated Proteins from Bovine Pericardium for Heart Valve Engineering

Abstract

Current heart valve prostheses are associated with significant complications, including aggressive immune response, limited valve life expectancy, and inability to grow in juvenile patients. Animal derived "tissue" valves undergo glutaraldehyde fixation to mask tissue antigenicity; however, chronic immunological responses and associated calcification still commonly occur. A heart valve formed from an unfixed bovine pericardium (BP) extracellular matrix (ECM) scaffold, in which antigenic burden has been eliminated or significantly reduced, has potential to overcome deficiencies of current bioprostheses. Decellularization and antigen removal methods frequently use sequential solutions extrapolated from analytical chemistry approaches to promote solubility and removal of tissue components from resultant ECM scaffolds. However, the extent to which such prefractionation strategies may inhibit removal of antigenic tissue components has not been explored. We hypothesize that presence of magnesium in prefractionation steps causes DNA precipitation and reduces removal of nuclear-associated antigenic proteins. Keeping all variables consistent bar the addition or absence of magnesium (2 mM magnesium chloride hexahydrate), residual BP ECM scaffold antigenicity and removed antigenicity were assessed, along with residual and removed DNA content, ECM morphology, scaffold composition, and recellularization potential. Furthermore, we used proteomic methods to determine the mechanism by which magnesium presence or absence affects scaffold residual antigenicity. This study demonstrates that absence of magnesium from antigen removal solutions enhances solubility and subsequent removal of antigenic nuclear-associated proteins from BP. We therefore conclude that the primary mechanism of action for magnesium removal during antigen removal processes is avoidance of DNA precipitation, facilitating solubilization and removal of nuclear-associated antigenic proteins. Future studies are necessary to further facilitate solubility and removal of nuclear-associated antigenic proteins from xenogeneic ECM scaffolds, in addition to an in vivo assessing of the material.

Graft-specific immune tolerance is determined by residual antigenicity of xenogeneic extracellular matrix scaffolds

“…Indeed, this finding is supported by previous work, which demonstrated that removal of hydrophilic antigens only resulted in increased cell-mediated and humoral immune response compared to removal of both hydrophilic and lipophilic antigens[13]. Importantly, the present study demonstrates that ASB-16 at an optimal concentration of 3%, in the absence of magnesium chloride, is essential to overcome both hydrophilic and lipophilic antigen mediated graft-specific humoral responses [20,21,38]. …”

“…Lipophilic antigen removal plateaued at 79% removed for ASB-14 at 5% w/v and 92% removed for ASB-16 at 3% w/v. Although neither detergent statistically increased hydrophilic antigen removal compared to no detergent use, the level of hydrophilic antigen removal achieved was greater than any previously reported method for both ASB-14 (95%) and ASB-16 (96%) [13,20,38]. The results of this study demonstrate that although sulfobetaines show great promise in solubilization and subsequent removal of lipophilic antigens from bovine pericardium, the ability of individual sulfobetaines to achieve antigen removal cannot be predicted from their solubilizing profile determined through analytical chemistry.…”

Immunogenicity of decellularized extracellular matrix scaffolds: a bottleneck in tissue engineering and regenerative medicine

“…Chelators such as ethylenediaminetetraacetic acid (EDTA) detach these cations from cell membrane binding sites and release cell components out of ECM [48,93]. Furthermore, chelators may facilitate removing nuclear immunogens via depleting free divalent cations, which precipitate DNA on ECM [94]. However, mere EDTA treatment is insufficient to achieve suitable acellular ECM scaffolds [95].…”

“…Salts containing magnesium, calcium, and other divalent cations may precipitate nuclear antigens and hinder the solubilization and elimination of nuclear antigens from ECM. They also may co-precipitate DNA-related proteins [94].…”