Tissue engineering technology provides effective alternative treatments for tracheal reconstruction. The formation of a functional microvascular network is essential to support cell metabolism and ensure the long-term survival of grafts. However, given the lack of an identifiable vascular pedicle of the trachea that could be anastomosed to the blood vessels directly in the recipient's neck, successful tracheal transplantation faces significant challenges in rebuilding the adequate blood supply of the graft. Herein, we describe a one-step method to construct microvascularization of tissue-engineered trachea in orthotopic transplantation. Forty rabbit tracheae were decellularized using a vacuum-assisted decellularization (VAD) method. Histological appearance and immunohistochemical (IHC) analysis demonstrated efficient removal of cellular components and nuclear material from natural tissue, which was also confirmed by 4′-6-diamidino-2-phenylindole(DAPI) staining and DNA quantitative analysis, thus significantly reducing the antigenicity. Scanning electron microscopy (SEM), immunofluorescence (IF) analysis, GAG and collagen quantitative analysis showed that the hierarchical structures, composition and integrity of the extracellular matrix (ECM) were protected. IF analysis also demonstrated that basic fibroblast growth factor (b-FGF) was preserved during the decellularization process, and also exerted biocompatibility and proangiogenic properties by the chick chorioallantoic membrane(CAM) assay. Xenotransplantation assays indicated that the VAD tracheal matrix would no longer induced inflammatory reactions implanted in the body for 4 weeks after treated by VAD more than 16 h. Furthermore, we seeded the matrix with bone marrow-derived endothelial cells (BMECs) in vitro and performed in vivo tracheal patch repair assays to prove the biocompatibility and neovascularization of VAD-treated tracheal matrix, and the formation of a vascular network around the patch promoted the crawling of surrounding ciliated epithelial cells to the surface of the graft. We conclude that this natural VAD tracheal matrix is non-immunogenic and no inflammatory reactions in vivo transplantation. Seeding with BMECs on the grafts and then performing orthotopic transplantation can effectively promote the microvascularization and accelerate the native epithelium cells crawling to the lumen of the tracheal graft.

组织工程技术为气管重建提供了有效的替代治疗方法。功能性微血管网络的形成对于支持细胞代谢和确保移植物的长期存活至关重要。然而，由于缺乏可直接与受者颈部血管吻合的可识别气管血管蒂，成功的气管移植在重建移植物充足的血液供应方面面临重大挑战。在此，我们描述了一种在原位移植中构建组织工程气管微血管化的一步方法。使用真空辅助脱细胞 (VAD) 方法对 40 只兔气管进行脱细胞。组织学外观和免疫组化 (IHC) 分析表明，可有效去除天然组织中的细胞成分和核物质，4'-6-二脒基-2-苯基吲哚 (DAPI) 染色和 DNA 定量分析也证实了这一点，从而显着降低了抗原性. 扫描电子显微镜 (SEM)、免疫荧光 (IF) 分析、GAG 和胶原蛋白定量分析表明，细胞外基质 (ECM) 的分层结构、组成和完整性受到保护。IF 分析还表明，碱性成纤维细胞生长因子 (b-FGF) 在脱细胞过程中得以保留，并且通过鸡绒毛尿囊膜 (CAM) 测定也发挥了生物相容性和促血管生成特性。异种移植试验表明，VAD 气管基质植入体内 4 周后，经 VAD 治疗 16 小时以上，不再诱发炎症反应。此外，我们在体外用骨髓源性内皮细胞 (BMECs) 接种基质并进行体内气管补片修复试验，以证明 VAD 处理的气管基质的生物相容性和新血管化，并促进了补片周围血管网络的形成周围的纤毛上皮细胞爬到移植物表面。我们得出结论，这种天然 VAD 气管基质在体内移植中是非免疫原性的，也没有炎症反应。