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  • 1
    Keywords: CELLS ; TISSUE ; GRAFTS ; RE-ENDOTHELIALIZATION ; ENGINEERED HEART-VALVES
    Abstract: Cell-matrix interactions in a three-dimensional (3D) extracellular matrix (ECM) are of fundamental importance in living tissue, and their in vitro reconstruction in bioartificial structures represents a core target of contemporary tissue engineering concepts. For a detailed analysis of cell-matrix interaction under highly controlled conditions, we developed a novel ECM evaluation culture device (EECD) that allows for a precisely defined surface-seeding of 3D ECM scaffolds, irrespective of their natural geometry. The effectiveness of EECD was evaluated in the context of heart valve tissue engineering. Detergent decellularized pulmonary cusps were mounted in EECD and seeded with endothelial cells (ECs) to study EC adhesion, morphology and function on a 3D ECM after 3, 24, 48 and 96 h. Standard EC monolayers served as controls. Exclusive top-surface-seeding of 3D ECM by viable ECs was demonstrated by laser scanning microscopy (LSM), resulting in a confluent re-endothelialization of the ECM after 96 h. Cell viability and protein expression, as demonstrated by MTS assay and western blot analysis (endothelial nitric oxide synthase, von Willebrand factor), were preserved at maintained levels over time. In conclusion, EECD proves as a highly effective system for a controlled repopulation and in vitro analysis of cell-ECM interactions in 3D ECM.
    Type of Publication: Journal article published
    PubMed ID: 22761130
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  • 2
    Keywords: DIFFERENTIATION ; TISSUE ; SCAFFOLD
    Abstract: Decellularized cardiac extracellular matrix (ECM) has been introduced as a template for cardiac tissue engineering, providing the advantages of a prevascularized scaffold that mimics native micro- and macro-architecture to a degree difficult to achieve with synthetic materials. Nonetheless, the decellularization protocols used to create acellular myocardial scaffolds vary widely throughout the literature. In this study we performed a direct comparison of three previously described protocols while introducing and evaluating a novel, specifically developed fourth protocol, by decellularizing whole rat hearts through software-controlled automatic coronary perfusion. Although all protocols preserved the macroarchitecture of the hearts and all resulting scaffolds could successfully be reseeded with C2C12 myoblasts, assessing their biocompatibility for three-dimensional in vitro studies, we found striking differences concerning the microcomposition of the ECM scaffolds on a histological and biochemical level. While laminin could still be detected in all groups, other crucial ECM components, like elastin and collagen IV, were completely removed by at least one of the protocols. Further, only three protocols maintained a glycosaminoglycan content comparable to native tissue, whereas the remaining DNA content within the ECM varied highly throughout all four tested protocols. This study showed that the degree of acellularity and resulting ECM composition of decellularized myocardial scaffolds strongly differs depending on the decellularization protocol
    Type of Publication: Journal article published
    PubMed ID: 21548726
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  • 3
    Abstract: Whole-organ engineering is an innovative field of regenerative medicine with growing translational perspectives. Recent reports suggest the feasibility of decellularization and repopulation of entire human size hearts. However, little is known about the susceptibility of epicardial adipose tissue (EAT) to decellularization. Here, human size hearts of ovine donors were subjected to perfusion-based decellularization using detergent solutions. Upon basic histological evaluation and total DNA measurement myocardial regions prove largely decellularized while EAT demonstrated cellular remnants, further confirmed by transmission electron microscopy. Western blot analysis showed a significant reduction in lipid-associated and cardiac proteins. However, gas chromatography revealed unchanged proportional composition of fatty acids in EAT of decellularized whole-hearts. Finally, cell culture medium conditioned with EAT from decellularized whole hearts had a significant deleterious effect on cardiac fibroblasts. These data suggest that perfusion decellularization of human size whole-hearts provides inconsistent efficacy regarding donor material removal from myocardial regions as opposed to EAT.
    Type of Publication: Journal article published
    PubMed ID: 28895502
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  • 4
    Keywords: PROTEINS ; TISSUE ; MATRIX ; MECHANICAL-PROPERTIES ; ALLOGRAFTS ; HEART-VALVES ; RECELLULARIZATION ; DETERGENT ; LEAFLET ; FRESH
    Abstract: Hulsmann J, Grun K, El Amouri S, Barth M, Hornung K, Holzfuss C, Lichtenberg A, Akhyari P. Transplantation material bovine pericardium: biomechanical and immunogenic characteristics after decellularization vs. glutaraldehyde-fixing. Xenotransplantation 2012; 19: 286-297. (c) 2012 John Wiley & Sons A/S. Abstract: Background: Today, bovine pericardium (BP) is extensively investigated as a biomaterial for the generation of various bioimplants. But despite the commercial distribution, and the development of methods either to remove (decellularization) or to mask (chemical cross-linking, for example by glutaraldehyde [GA] treatment) the xenogeneic antigen epitopes, yet questions around the immunogenic reactivity of BP remain. The aim of this study is the comparison of crucial tissue characteristics, that is, biomechanical properties, the presence of alphaGal epitopes, and residual DNA in acellular vs. GA-fixed BP. Methods: Bovine pericardium was either cross-linked with 0.6% GA or decellularized according to two common protocols using either sodium dodecyl sulfate (SDS) and desoxycholic acid (DCA) or trypsin and ethylenediaminetetraacetic acid (EDTA). The resulting extracellular matrix was prone to one-dimensional tensile testing. The tissue content for alphaGal was evaluated by immunoblotting, and residual DNA was determined by a commercial assay. Untreated BP served as control. Results: In contrast to previous reports, we found a pronounced decrease in the elastic modulus (E-Modulus) for common GA treatment and overall smaller values for the elastic moduli after decellularization (P 〈 0.05). In parallel, we observed an overall increased ultimate elongation of acellular and cross-linked BP, although ultimate stress values did not significantly differ. SDS/DCA decellularized BP revealed a dramatic reduction in the DNA content and an almost complete removal of alphaGal epitopes, whereas the trypsin/EDTA protocol retained a residual DNA content of almost 50% and with a great trail of alphaGal signal. GA-treated tissue had a remarkable content of DNA and alphaGal. Conclusions: Although chemically fixated BP is clinically still in wide use, for example, for biological heart valve engineering, our results suggest that an improved biomaterial preparation may be provided by appropriate decellularization. SDS/DCA decellularized BP shows similar biomechanical characteristics as GA treatment, paired with reduced potential immunogenic reactivity. Furthermore, decellularized BP holds the potential of cellular repopulation in vivo or in vitro, to enable an endogenous regenerative capacity in contrast to the toxic effects of GA fixing.
    Type of Publication: Journal article published
    PubMed ID: 22978462
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