Nayakawde Nikhil B, Methe Ketaki, Banerjee Debashish, Berg Malin, Premaratne Goditha U, Olausson Michael
Laboratory for Transplantation and Regenerative Medicine, Sahlgrenska Academy at Gothenburg University and the Sahlgrenska Transplant Institute at Sahlgrenska University Hospital, Gothenburg, Sweden.
Department of Otolaryngology, Head and Neck Surgery, and Sahlgrenska Academy at Gothenburg University and the Sahlgrenska Transplant Institute at Sahlgrenska University Hospital, Gothenburg, Sweden.
Biores Open Access. 2020 Feb 21;9(1):22-36. doi: 10.1089/biores.2019.0054. eCollection 2020.
Decellularization of esophagus was studied using three different protocols. The sodium deoxycholate/DNase-I (SDC/DNase-I) method was the most successful as evidenced by histology and DNA quantification of the acellular scaffolds. Acellular scaffolds were further analyzed and compared with native tissue by histology, quantitative analysis of DNA, and extracellular matrix (ECM) proteins. Histologically, the SDC/DNase-I protocol effectively produced scaffold with preserved structural architecture similar to native tissue architecture devoid of any cell nucleus. ECM proteins, such as collagen, elastin, and glycosaminoglycans were present even after detergent-enzymatic decellularization. Immunohistochemical analysis of acellular scaffold showed weak expression of Gal 1, 3 Gal epitope compared with native tissue. For performing recellularization, human amnion-derived mesenchymal stem cells (MSCs) and epithelial cells were seeded onto acellular esophagus in a perfusion-rotation bioreactor. In recellularized esophagus, immunohistochemistry showed infiltration of MSCs from adventitia into the muscularis externa and differentiation of MSCs into the smooth muscle actin and few endothelial cells (CD31). Our study demonstrates successful preparation and characterization of a decellularized esophagus with reduced load of Gal 1, 3 Gal epitope with preserved architecture and ECM proteins similar to native tissue. Upon subsequent recellularization, xenogeneic acellular esophagus also supported stem cell growth and partial differentiation of stem cells. Hence, the current study offers the hope for preparing a tissue-engineered esophagus which can be transplanted further into pigs for further evaluation.
使用三种不同的方案研究了食管去细胞化。脱氧胆酸钠/脱氧核糖核酸酶I（SDC/DNase-I）方法最为成功，这在脱细胞支架的组织学和DNA定量分析中得到了证实。通过组织学、DNA定量分析以及细胞外基质（ECM）蛋白分析，对脱细胞支架进行了进一步分析，并与天然组织进行了比较。从组织学角度来看，SDC/DNase-I方案有效地制备出了结构与天然组织结构相似且无任何细胞核的支架。即使经过去污剂-酶法去细胞化处理后，胶原、弹性蛋白和糖胺聚糖等ECM蛋白依然存在。与天然组织相比，脱细胞支架的免疫组织化学分析显示半乳糖1,3半乳糖表位的表达较弱。为了进行再细胞化，将人羊膜来源的间充质干细胞（MSCs）和上皮细胞接种到灌注旋转生物反应器中的脱细胞食管上。在再细胞化的食管中，免疫组织化学显示MSCs从外膜浸润到外肌层，并分化为平滑肌肌动蛋白和少量内皮细胞（CD31）。我们的研究表明成功制备并表征了一种去细胞化食管，其半乳糖1,3半乳糖表位负载降低，结构和ECM蛋白与天然组织相似且得以保留。在随后的再细胞化过程中，异种脱细胞食管也支持干细胞生长以及干细胞的部分分化。因此，本研究为制备组织工程食管带来了希望，这种食管可进一步移植到猪体内进行进一步评估。
