Wiet Matthew G, Dharmadhikari Sayali, White Audrey, Reynolds Susan D, Johnson Jed, Breuer Christopher K, Chiang Tendy
Department of Otolaryngology Head & Neck Surgery, Nationwide Children's Hospital; The Ohio State University College of Medicine.
Department of Otolaryngology Head & Neck Surgery, Nationwide Children's Hospital; Center for Regenerative Medicine, Research Institute at Nationwide Children's Hospital.
J Vis Exp. 2019 Apr 1(146). doi: 10.3791/59173.
Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.
由于无法替换功能性组织，先天性或继发性长节段气管缺损的治疗选择在历史上一直有限。组织工程作为一种潜在的解决方案具有巨大的前景，因为它能够将细胞和信号分子整合到三维支架中。最近使用组织工程气管移植物（TETG）的研究取得了一些成功，但其转化受到移植物狭窄、移植物塌陷和上皮化延迟的限制。为了研究导致这些问题的机制，我们开发了一种用于组织工程气管移植物植入的小鼠模型。TETG是使用电纺聚合物聚对苯二甲酸乙二酯（PET）和聚氨酯（PU）以PET和PU的混合物（重量比20:80）构建的。然后通过梯度离心从6-8周龄的C57BL/6小鼠中分离骨髓单个核细胞接种到支架上。每个移植物接种1000万个细胞到支架腔内，在植入第三至第七气管环之间之前孵育过夜。如组织学分析以及免疫荧光显示缺乏角蛋白5和角蛋白14基底上皮细胞所表明的，这些移植物能够重现狭窄和上皮化延迟的结果。该模型将作为研究宿主重塑中涉及的细胞和分子机制的工具。
