Hofmann Alejandro D, Hilfiker Andres, Haverich Axel, Andree Birgit, Kuebler Joachim, Ure Benno
Department of Pediatric Surgery, Hannover Medical School, Hannover, Germany.
Leibniz Research Laboratories for Biotechnology and Artificial Organs, Hannover Medical School, Hannover, Germany.
Eur J Pediatr Surg. 2015 Apr;25(2):181-8. doi: 10.1055/s-0034-1370778. Epub 2014 May 2.
A major obstacle in tissue engineering is to create a surgically implantable tissue with long-term viability. Several promising techniques have focused on biological vascularized matrices (BioVaM) with preserved vascular pedicles in the porcine model. However, the handling of this model is time-consuming and expensive. Therefore, our aim was to establish a BioVaM in the rat.
Small bowel segments of Sprague-Dawley rats were isolated and perfused via cannulation of the superior mesenteric artery and the portal vein. All cellular matrix components were removed by sequential treatment with sodium dodecyl sulfate, sodium deoxycholate, and DNase. Quality of decellularization was investigated by histology and potential residual DNA by spectrophotometry. Primary endothelial cells (ECs) isolated from the major vessels of Sprague-Dawley rats. Cells were labeled with fluorescent cell tracker and injected into the vascular pedicles of the matrix. Attachment of ECs was assessed using fluorescence microscopy of the whole mount.
Decellularized matrix demonstrated the absence of cellular components but conserved matrix architecture as determined by immune fluorescent, pentachrome, and hematoxylin and eosin stains. DNA content was reduced by more than 99%. ECs were characterized by specific staining against endothelial nitric oxide synthase and von Willebrand factor; when injected, ECs attached along the vessel walls including the capillaries of the intestinal wall.
Rat small bowel segments harvested with intact vascular pedicles and associated vascular network can be successfully decellularized and re-endothelialized ex vivo. This model is an inexpensive and easy to handle alternative and appears to be a promising approach for establishing vascularized tissue constructs.
组织工程中的一个主要障碍是创建具有长期生存能力的可手术植入组织。在猪模型中，几种有前景的技术聚焦于带有保留血管蒂的生物血管化基质（BioVaM）。然而，该模型的操作既耗时又昂贵。因此，我们的目标是在大鼠中建立BioVaM。
分离Sprague-Dawley大鼠的小肠段，通过肠系膜上动脉和门静脉插管进行灌注。通过用十二烷基硫酸钠、脱氧胆酸钠和脱氧核糖核酸酶依次处理去除所有细胞基质成分。通过组织学研究脱细胞质量，通过分光光度法检测潜在的残留DNA。从Sprague-Dawley大鼠的主要血管中分离原代内皮细胞（ECs）。用荧光细胞追踪器标记细胞并注入基质的血管蒂。使用整装荧光显微镜评估ECs的附着情况。
免疫荧光、五色染色以及苏木精和伊红染色显示，脱细胞基质显示无细胞成分，但保留了基质结构。DNA含量降低了99%以上。ECs通过针对内皮型一氧化氮合酶和血管性血友病因子进行特异性染色来表征；注射后，ECs沿着血管壁附着，包括肠壁的毛细血管。
完整血管蒂及相关血管网络的大鼠小肠段可在体外成功脱细胞并重新内皮化。该模型是一种廉价且易于操作的替代方法，似乎是建立血管化组织构建体的一种有前景的方法。
