Biofabrication of a prevascularized functional trachea substitute

3D打印技术实现了快速增材制造，确保了生成零件的高空间分辨率和复杂性。应用于组织工程领域，3D打印技术在生成由细胞和水凝胶组成的人造器官方面显示出很高的潜力，并在结构和组成上模仿自然组织的复杂性。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。作为气管打印项目的延续，这里提出的研究项目是基于一个假设，即气管的管状结构可以在3D按需打印过程中类似。打印的水凝胶包括两种不同类型的水凝胶：一种是琼脂糖和I型胶原蛋白混合的细胞水凝胶，另一种是类似于天然气管软骨功能和形状的无细胞水凝胶。在该项目的第一阶段，琼脂糖-胶原蛋白混合物已经证明了其可打印性和高血管生成潜力，该项目使用人类内皮细胞和成纤维细胞共同培养。特别是，我们在项目的第二阶段专注于基于聚乙二醇（PEG）的软骨替代品的进步，它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。本课题主要研究细胞诱导的体外血管重构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，将研究预血管化水凝胶样品在cam模型中的集成。此外，将阐述一种最适合特定应用的新型膜打印技术，并与现有的基于微阀的打印机相结合。最后，打印一个充满细胞的气管替代物，并在脉动生物反应器中进行两步培养。此外，结构将在内表面上皮化使用喷涂技术。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组织工程的总体可行性。这个项目的科学发现可以随后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.