Biofabrication of a prevascularized functional trachea substitute

3D打印技术能够实现快速增材制造，确保生成部件的高空间分辨率和复杂性。应用于组织工程领域，3D打印技术显示出生成人造器官的高潜力，人造器官包括细胞和水凝胶，并在结构和组成上模仿天然组织的复杂性。例如，气管由几种不同的细胞类型和不同的功能组织类型（如肌肉、结缔组织和软骨）组成。作为TracheaPrint项目的延续，本文提出的研究项目基于以下假设：气管的管状结构可以在3D按需打印过程中进行模拟。打印包括两种不同类型的水凝胶：琼脂糖和I型胶原蛋白的细胞负载水凝胶混合物，以及类似天然气管软骨功能和形状的无细胞水凝胶。琼脂糖-胶原蛋白混合物已经在项目的第一阶段使用人类内皮细胞和成纤维细胞的共培养物证明了其可印刷性和高血管生成潜力。特别是，我们在项目的第二阶段专注于基于聚乙二醇（PEG）的软骨替代物的发展，该替代物形成具有可调机械性能的水凝胶。我们打算使用点击化学方法进一步缩短PEG基水凝胶的凝胶化时间，避免光交联剂的细胞毒性作用。本研究项目包括体外细胞诱导的组织重塑和组织成熟及其对血管生成和促血管生成标志物表达的影响。此外，将研究CAM模型中预血管化水凝胶样品的整合。此外，一种最适合特定应用的新型薄膜打印技术将被阐述并与现有的基于微阀的打印机相结合。最后，将载有细胞的气管替代物打印并在脉动生物反应器中以两步孵育进行培养。此外，将使用喷涂技术在内表面对结构进行上皮化。此外，我们研究了采用3D生物反应器进行逐层生物打印管状结构的组织工程的一般可行性。该项目的科学发现随后可用于开发个性化的气管替代品。

3D printing technologies enable a rapid additive manufacturing that ensures high spatial resolution and complexity of generated parts. Applied on the field of tissue engineering, 3D printing technologies show high potential for the generation of artificial organs which comprise cells and hydrogels and mimic the complexity of natural tissue in structure and composition. A trachea for example consists of several different cell types and different functional tissue types such as muscle, connective tissue, and cartilage.The research project proposed here as a continuation of the project TracheaPrint is based on the hypothesis that the tubular structure of a trachea can be resembled in a 3D drop-on-demand printing procedure. The printing includes two different types of hydrogels: a cell-laden hydrogel blend of agarose and type I collagen and further a cell-free hydrogel that resembles function and shape of native tracheal cartilage. The agarose-collagen blend already proved its printability and high angiogenic potential in the first phase of the project using a co-culture of human endothelial cells and fibroblasts. Particularly, we focus in the second phase of the project on the advancement of a cartilage substitute based on polyethylene glycol (PEG) which forms a hydrogel with tunable mechanical properties. We intend to further shorten the gelation time of a PEG-based hydrogel using a click-chemistry approach avoiding the cytotoxic effect of photo-crosslinkers. The research project includes studies on the cell induced remodeling and tissue maturation in vitro and its influence on angiogenesis and the expression of proangiogenic markers. Furthermore, the integration of pre-vascularized hydrogel samples in a CAM-model will be investigated. Moreover, a novel membrane printing technology best suitable for the specific application will be elaborated and combined with the existing micro-valve based printer. Finally, a cell-laden trachea substitute is printed and cultured in two-step incubation in a pulsatile bioreactor. Additionally, the construct will be epithelialized at the inner surface using a spraying technique. Furthermore, we investigate the general feasibility of employing 3D-bioreactors for tissue engineering of layer-by-layer bioprinted tubular structures. The scientific findings from this project could subsequently be used to develop individualized trachea substitutes.