Medizinische Hochschule Hannover

[Alpha-Gal in therapeutics : More relevant than thought?].

Development and Characterization of a Porcine Mitral Valve Scaffold for Tissue Engineering.

Decellularized scaffolds represent a promising alternative for mitral valve (MV) replacement. This work developed and characterized a protocol for the decellularization of whole MVs. Porcine MVs were decellularized with 0.5% (w/v) SDS and 0.5% (w/v) SD and sterilized with 0.1% (v/v) PAA. Decellularized samples were seeded with human foreskin fibroblasts and human adipose-derived stem cells to investigate cellular repopulation and infiltration, and with human colony-forming endothelial cells to investigate collagen IV formation. Histology revealed an acellular scaffold with a generally conserved histoarchitecture, but collagen IV loss. Following decellularization, no significant changes were observed in the hydroxyproline content, but there was a significant reduction in the glycosaminoglycan content. SEM/TEM analysis confirmed cellular removal and loss of some extracellular matrix components. Collagen and elastin were generally preserved. The endothelial cells produced newly formed collagen IV on the non-cytotoxic scaffold. The protocol produced acellular scaffolds with generally preserved histoarchitecture, biochemistry, and biomechanics.

Tissue-engineered mitral valve: morphology and biomechanics †.

The present study aimed at developing tissue-engineered mitral valves based on cell-free ovine mitral allografts. The ovine mitral valves (OMVs) (n = 46) were harvested in the local slaughter house. They were decellularized using detergent solutions and DNase. The effectiveness of decellularization was assessed by histological (haematoxylin-eosin, Movat's pentachrome) and immunofluorescent staining (for DNA and α-Gal), and DNA-quantification. To reveal the receptiveness of decellularized tissue to endothelial cells (ECs), the valve leaflets were reseeded with ovine ECs, derived from endothelial progenitor cells in vitro. For assessment of biomechanical properties, uniaxial tensile tests were carried out. Histology and immunofluorescent staining revealed absence of cell nuclei in decellularized leaflets, chordae and papillary muscles. According to the software for immunofluorescence analysis, reduction in DNA and α-Gal was 99.9 and 99.6%, respectively. DNA-quantification showed 71.2% reduction in DNA content without DNase and 96.4% reduction after DNase treatment. Decellularized leaflets were comparable with native in ultimate tensile strain (native, 0.34 ± 0.09 mm/mm, vs decellularized, 0.44 ± 0.1 mm/mm; P = 0.09), and elastin modulus (native, 0.39 ± 0.27, vs decellularized, 0.57 ± 0.55, P = 0.46), had increased ultimate tensile stress (native, 1.23 ± 0.35 MPa, vs decellularized 2.12 ± 0.43 MPa; P = 0.001) and collagen modulus (native, 5.5 ± 1.26, vs decellularized, 8.29 ± 2.9; P = 0.04). After EC seeding, immunofluorescent staining revealed a monolayer of CD31-, eNOS- and vWF-positive cells on the surface of the leaflet, as well as a typical cobble-stone morphology of those cells. Decellularization of ovine mitral valve results in a mitral valves scaffold with mechanical properties comparable with native tissue, and a graft surface, which can be repopulated by endothelial cells.

Development and characterization of acellular porcine pulmonary valve scaffolds for tissue engineering.

Currently available replacement heart valves all have limitations. This study aimed to produce and characterize an acellular, biocompatible porcine pulmonary root conduit for reconstruction of the right ventricular outflow tract e.g., during Ross procedure. A process for the decellularization of porcine pulmonary roots was developed incorporating trypsin treatment of the adventitial surface of the scraped pulmonary artery and sequential treatment with hypotonic Tris buffer (HTB; 10 mM Tris pH 8.0, 0.1% (w/v) EDTA, and 10 KIU aprotinin), 0.1% (w/v) sodium dodecyl sulfate in HTB, two cycles of DNase and RNase, and sterilization with 0.1% (v/v) peracetic acid. Histology confirmed an absence of cells and retention of the gross histoarchitecture. Immunohistochemistry further confirmed cell removal and partial retention of the extracellular matrix, but a loss of collagen type IV. DNA levels were reduced by more than 96% throughout all regions of the acellular tissue and no functional genes were detected using polymerase chain reaction. Total collagen levels were retained but there was a significant loss of glycosaminoglycans following decellularization. The biomechanical, hydrodynamic, and leaflet kinematics properties were minimally affected by the process. Both immunohistochemical labeling and antibody absorption assay confirmed a lack of α-gal epitopes in the acellular porcine pulmonary roots and in vitro biocompatibility studies indicated that acellular leaflets and pulmonary arteries were not cytotoxic. Overall the acellular porcine pulmonary roots have excellent potential for development of a tissue substitute for right ventricular outflow tract reconstruction e.g., during the Ross procedure.

[Urticaria. Sometimes IgE-mediated?].

There are specific diagnostic recommendations for approaching chronic spontaneous urticaria, physical urticaria and special forms of urticaria. They include the detection of subclinical viral and bacterial infections, particularly with Helicobacter pylori. Frequently an autoimmune urticaria is diagnosed by using the autologous serum test. Less specific findings, such as leukocytosis and elevated CRP can sometimes be helpful in suggesting subclinical infections as the cause of acute urticaria; usually no further procedures are recommended. However, in cases with a long history of intermittent attacks, particularly with severe and generalized symptoms, it is recommended to look for IgE-mediated reactions (e.g. alpha-Gal, Omega-5-Gliadin). In our hands at the Department of Dermatology and Allergy at the Hannover Medical School a standardized set of diagnostic procedures was successfully established, and is now individually completed with selected single allergens and allergen components.

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.

Detection of elicited antiporcine antibodies by FACS and Galalpha1-3Gal-specific ELISA in cynomolgus recipients after porcine kidney transplantation.