Corlife (Germany)

Decellularized equine carotid arteries (dEAC) are potential alternatives to alloplastic vascular grafts although there are certain limitations in biocompatibility and immunogenicity. Here, dEAC were coated with the matricellular protein CCN1 and evaluated in vitro for its cytotoxic and angiogenic effects and in vivo for cellular repopulation, local biocompatibility, neovascularization, and immunogenicity in a sheep model. CCN1 coating resulted in nontoxic matrices not compromising viability of L929 fibroblasts and endothelial cells (ECs) assessed by WST-8 assay. Functionality of CCN1 was maintained as it induced typical changes in fibroblast morphology and MMP3 secretion. For in vivo testing, dEAC±CCN1 (n=3 each) and polytetrafluoroethylene (PTFE) protheses serving as controls (n=6) were implanted as cervical arteriovenous shunts. After 14 weeks, grafts were harvested and evaluated immunohistologically. PTFE grafts showed a patency rate of only 33% and lacked cellular repopulation. Both groups of bioartificial grafts were completely patent and repopulated with ECs and smooth muscle cells (SMCs). However, whereas dEAC contained only patch-like aggregates of SMCs and a partial luminal lining with ECs, CCN1-coated grafts showed multiple layers of SMCs and a complete endothelialization. Likewise, CCN1 coating reduced leukocyte infiltration and fibrosis and supported neovascularization. In addition, in a three-dimensional assay, CCN1 coating increased vascular tube formation in apposition to the matrix 1.6-fold. Graft-specific serum antibodies were increased by CCN1 up to 6 weeks after implantation (0.89±0.03 vs. 1.08±0.04), but were significantly reduced after 14 weeks (0.85±0.04 vs. 0.69±0.02). Likewise, restimulated lymphocyte proliferation was significantly lower after 14 weeks (1.78±0.09 vs. 1.32±0.09-fold of unstimulated). Thus, CCN1 coating of biological scaffolds improves local biocompatibility and accelerates scaffold remodeling by enhancing cellular repopulation and immunologic tolerance, making it a promising tool for generation of bioartificial vascular prostheses.

Disinfection of biological implants is indispensable for clinical safety. Here, decellularized equine carotid arteries (dECAs) were disinfected by polyhexanide (PHX), an effective, well-tolerated and nontoxic wound disinfectant and evaluated as vascular grafts for their repopulation and local biocompatibility in vivo. dECAs were terminally disinfected by a combination of 0.1% PHX and 70% ethanol (dECA_PHX-ET) or exclusively ethanol (dECA-ET) and subsequently implanted as arteriovenous shunts in sheep for 14 weeks. Repopulation was determined by immunohistochemistry for endothelial- (ECs) or smooth muscle cells (SMCs) using antibodies against CD31 and smooth muscle actin. Histological evaluation was performed on HE-stained sections. Cytotoxicity of dECAs was measured directly by seeding the scaffolds with L-929 fibroblasts, which were visualized by calcein staining. Indirect cytotoxicity was determined by WST-8 viability assay by incubation of L-929 with dECA extracts. dECA_PHX-ET completely lacked repopulation with ECs and SMCs, showed leukocyte infiltration, strong calcification and poor neovascularization indicating insufficient biocompatibility and inflammatory graft degeneration. PHX-treatment reduced cell viability to 33.2 ± 12.6% and disturbed cell growth at direct contact. In contrast, dECA_ET had no direct cytotoxic effect and only slightly influenced cell viability (82.9 ± 12.5%), showed a substantial repopulation by ECs and SMCs including neovascularization, and were only slightly calcified. The disinfectant polyhexanide seems to exert severe cytotoxic effects when used for the processing of decellularized matrices and may result in degenerative graft deterioration. In contrast, dECAs exclusively disinfected with ethanol were well integrated. Thus, ethanol seems to be a more suitable tool for graft processing than polyhexanide.