Melanie Klingenberg

The effect of detergent-based decellularization procedures on cellular proteins and immunogenicity in equine carotid artery grafts

Decellularized equine carotid arteries (dEAC) may represent a reasonable alternative to alloplastic materials in vascular replacement therapy. Acellularity of the matrix is standardly evaluated by DNA quantification what however may not record sufficiently the degree of matrix immunogenicity. Thus, our aim was to analyze dEAC with a low DNA content for residual cellular proteins. A detergent-based decellularization protocol including endonuclease treatment resulted in dEAC with 0.6 ± 0.15 ng DNA/mg dry weight representing 0.33 ± 0.14% of native tissue DNA content. In contrast, when matrices were homogenized and extracted by high detergent concentrations westernblot analyses revealed cytosolic and cytosceleton proteins like GAPDH and smooth muscle actin which were depleted to 4.1 ± 1.9% and 13.8 ± 0.55%, resp. Also putative immunogenic MHC I complexes and the alpha-Gal epitop were reduced to only 14.8 ± 1.2% and 15.1 ± 2.05%. Mass spectrometry of matrix extracts identified 306 proteins belonging to cytosol, organelles, nucleus and cell membrane. Moreover, aqueous matrix extracts evoked a pronounced antibody formation when administered in mice and thus display high immunogenic potential. Our data indicate that an established decellularization protocol which results in acellular matrices evaluated by low DNA content reduces but not eliminates cellular components which may contribute to its immunogenic potential in vivo.

Immunogenicity of Intensively Decellularized Equine Carotid Arteries Is Conferred by the Extracellular Matrix Protein Collagen Type VI

The limited biocompatibility of decellularized scaffolds is an ongoing challenge in tissue engineering. Here, we demonstrate the residual immunogenicity of an extensively decellularized equine carotid artery (dEACintens) and identify the involved immunogenic components. EAC were submitted to an elaborated intensified decellularization protocol with SDS/sodium desoxycholate for 72 h using increased processing volumes (dEACintens), and compared to dEACord prepared by an ordinary protocol (40 h, normal volumes). Matrix integrity was checked via correlative volumetric visualization which revealed only minor structural changes in the arterial wall. In dEACintens, a substantial depletion of cellular components was obvious for smooth muscle actin (100%), MHC I complexes (97.8%), alphaGal epitops (98.4% and 91.3%) and for DNA (final concentration of 0.34±0.16 ng/mg tissue). However, dEACintens still evoked antibody formation in mice after immunization with dEACintens extracts, although to a lower extent than dEACord. Mouse plasma antibodies recognized a 140 kDa band which was revealed to contain collagen VI alpha1 and alpha2 chains via mass spectrometry of both 2D electrophoretically separated and immunoprecipitated proteins. Thus, even the complete removal of cellular proteins did not yield non-immunogenic dEAC as the extracellular matrix still conferred immunogenicity by collagen VI. However, as lower antibody levels were achieved by the intensified decellularization protocol, this seems to be a promising basis for further development.

The immune response to crosslinked tissue is reduced in decellularized xenogeneic and absent in decellularized allogeneic heart valves

Background The degeneration and failure of xenogeneic heart valves, such as the Matrix P Plus valve (MP-V) consisting of decellularized porcine valves (dec-pV) and equine glutaraldehyde-fixed conduits (ga-eC) have been linked to tissue immunogenicity accompanied by antibody formation. In contrast, decellularized allograft valves (dec-aV) are well-tolerated. Here, we determined tissue-specific antibody levels in patients after implantation of MP-V or dec-aV and related them to valve failure or time period after implantation. Methods and Results Specific antibodies toward whole tissue-homogenates or alphaGal were determined retrospectively by ELISA analyses from patients who received MP-V with an uneventful course of 56.1 ± 5.1 months (n = 15), or with valve failure after 25.3 ± 14.6 months (n = 3), dec-aV for various times from 4 to 46 months (n = 14, uneventful) and from healthy controls (n = 4). All explanted valves were assessed histopathologically. MP-V induced antibodies toward both tissue components with significantly higher levels toward ga-eC than toward dec-pV (68.7 and 26.65 μg/ml IgG). In patients with valve failure, levels were not significantly higher and were related to inflammatory tissue infiltration. Anti-Gal antibodies in MP-V patients were significantly increased in both, the uneventful and the failure group. In contrast, in dec-aV patients only a slight tissue-specific antibody formation was observed after 4 months (6.24 μg/ml) that normalized to control levels after 1 year. Conclusions The strong humoral immune response to glutaraldehyde-fixed tissues is reduced in decellularized xenogeneic valves and almost absent in decellularized allogeneic tissue up to 4.5 years after implantation.

Antibody formation towards porcine tissue in patients implanted with crosslinked heart valves is directed to antigenic tissue proteins and αGal epitopes and is reduced in healthy vegetarian subjects.

Glutaraldehyde‐fixed porcine heart valves (ga‐pV) are one of the most frequently used substitutes for insufficient aortic and pulmonary heart valves which, however, degenerate after 10‐15 years. Yet, xeno‐immunogenicity of ga‐pV in humans including identification of immunogens still needs to be investigated. We here determined the immunogenicity of ga‐pV in patients with respect to antibody formation, identity of immunogens and potential options to reduce antibody levels.

Evaluation of autologous tissue sources for the isolation of endothelial cells and adipose tissue-derived mesenchymal stem cells to pre-vascularize tissue-engineered vascular grafts

Abstract Currently used synthetic vascular grafts bear a high infection risk due to insufficient microvascularization of the graft wall disabling the infiltration of immune cells. Tissue-engineered grafts with a functional pre-vascularization thus would be desirable. However, autologous tissue sources for capillary forming cells need to be evaluated. Here, peripheral blood outgrowth endothelial cells (PB-OEC) from 17 healthy donors and pericyte-like mesenchymal stem cells derived from adipose tissue (ASC) of 17 patients scheduled for visceral surgery were characterized and investigated regarding their ability to form capillary-like networks in plasma-derived fibrin gels. To obtain proliferating PB-OEC with endothelial cell-specific properties (CD31-, VE-cadherin-expression, ac-LDL uptake and three-dimensional (3D)-tube formation in fibrin gels) both enrichment of CD34+ blood cells and young donor age was necessary (7/17, age≤24 years). In contrast, all isolated ASC revealed the expression of surface antigens expressed on pericytes [human neural/glial antigen-2 (hNG2), platelet-derived growth factor receptor β (PDGF-Rβ)] and showed mesodermal differentiation capacity. Moreover, co-culture of PB-OEC and ASC in fibrin gels resulted in highly branched capillary-like networks with significantly increased tube length (2.9-fold, p<0.0001) and number of junctions (8-fold, p<0.0001). In conclusion, successful cell isolation from autologous tissues for pre-vascularization of vascular grafts has been demonstrated although certain limitations for autologous EC require further strategies to enable the use of allogeneic cells.