Multi-tissue type condensations for trachea tissue regeneration via individual cell bioprinting

摘要 由于长时间插管、气管切开术、外部创伤、穿透性碎片射弹、枪击 战斗中的伤口和简易爆炸装置，以及良性或恶性肿瘤，许多士兵和 美国军队中的退伍军人患有严重的气管狭窄或损伤， 由于没有成功的长期治疗长段气管狭窄或损伤， 已经探索了使用不同的生物材料组合来开发新气管的工程策略 细胞来源。然而，基于生物材料支架的方法经常干扰关键细胞-细胞间的相互作用。 相互作用，细胞增殖和新的细胞外基质的产生，这是重要的形成过程中， 功能性气管组织功能性替代气管必须保持（1）径向刚度以防止再狭窄， (2)与宿主脉管系统紧密结合，以充分为植入物提供营养，以及（3）含有呼吸道 上皮以提供保护性粘膜层。将三维（3D）生物打印技术与 无支架组织工程原理提供了一个强大的平台，用于工程化多组织功能 气管，并且将规避基于支架的方法的上述限制。这项建议旨在 利用我们最近开发的单个细胞3D生物打印技术的优势， 打印复杂和高分辨率的基于细胞凝聚的组织构建体， 气管我们计划使用多个离散的单个细胞打印无支架的多组织新气管 用于由组织的空间受控呈现驱动的组织类型的空间上不同的区分的生物墨水， 特定的生长因子。构建自组装将由来自于 软骨组织用人骨髓间充质干细胞和自体内皮祖细胞 和hMSC用于血管前组织，自体人支气管上皮细胞用于内衬血管腔。 新生气管具体而言，该提案旨在（1）检查微凝胶的物理性质的作用 支持浆料上的细胞只有生物墨水打印，冷凝形成/维护，和软骨形成的3D 生物打印结构，（2）具有软骨形成生物墨水和预血管化环的3D生物打印软骨环构建体 使用仅单个细胞的生物打印技术的具有血管生成生物墨水的构建体，以及（3）工程化 用软骨形成和血管形成生物墨水预血管化和上皮化的气管组织， 纯细胞生物打印技术作为一个探索性的目标，工程气管的能力，以恢复气道 将在动物缺损模型中评价功能性。这项工作最终寻求利用一个简单而灵活的 单个细胞生物墨水3D打印平台，用于设计患者特定的替代气管， 在那些受长节段影响的患者中进行置换所需的生理和机械性能 气管狭窄这种仅含单个细胞的3D打印平台具有固有的灵活性，可以创建复杂的 可以利用由多种空间上不同的组织类型组成的结构来开发其它多组织 机关

Abstract As a result of prolonged intubation, tracheostomy, external trauma, penetrating fragment projectiles, gunshot wounds and improvised explosive devices during combat, and benign or malignant tumors, many soldiers and veterans in the US military suffer from severe trachea stenosis or damage that can cause complete airway failure, Since there is no successful long-term treatment for long-segment tracheal stenosis or damage, tissue engineering strategies have been explored to develop neotracheas using different combinations of biomaterials and cell sources. However, biomaterial scaffold-based approaches often interfere with critical cell-cell interactions, cell proliferation and new extracellular matrix production that are important during the formation of functional trachea tissue. A functional replacement trachea must retain (1) radial rigidity to prevent restenosis, (2) anastomose with host vasculature to adequately provide nutrients to the implant, and (3) contain respiratory epithelium to provide a protective mucosal layer. Combining three-dimensional (3D) bioprinting technologies with scaffold-free tissue engineering principles presents a powerful platform for engineering a multi-tissue functional trachea, and would circumvent the aforementioned limitations of scaffold-based approaches. This proposal aims to leverage the benefits of our recently developed individual cell-only 3D bioprinting technology, which allows for printing of complex and high-resolution cell condensation-based tissue constructs to engineer functional tracheas. We plan to print scaffold-free, multi-tissue neotracheas using multiple discrete individual cell-only bioinks for spatially distinct differentiation of tissue types driven by spatially controlled presentation of tissue- specific growth factors. Construct self-assembly will be driven by the condensation of autologously sourced human mesenchymal stem cells (hMSCs) for cartilaginous tissue and autologous endothelial progenitor cells and hMSCs for prevascular tissue, with autologous human bronchial epithelial cells applied to line the lumen of the neotrachea. Specifically, this proposal aims to (1) examine the role of the physical properties of the microgel support slurry on cell-only bioink printing, condensation formation/maintenance, and chondrogenesis of the 3D bioprinted structures, (2) 3D bioprint cartilage ring constructs with chondrogenic bioink and prevascularized ring constructs with vasculogenic bioink using the individual cell-only bioprinting technology, and (3) engineer prevascularized and epithelized tracheal tissue with chondrogenic and vasculogenic bioinks using the individual cell-only bioprinting technology. As an exploratory aim, the capacity of the engineered tracheas to restore airway functionality will be evaluated in an animal defect model. This work ultimately seeks to utilize a facile and flexible individual cell-only bioink 3D printing platform to engineer a patient-specific replacement trachea that provides requisite physiologic and mechanical properties for replacement in those that are affected by long-segment tracheal stenosis. The inherent flexibility of this individual cell-only 3D printing platform to create complex structures composed of multiple spatially distinct tissue types can be leveraged to develop other multi-tissue organs.