Biofabrication of a prevascularized functional trachea substitute

3D打印技术实现了快速的加法制造，确保了生成部件的高空间分辨率和复杂性。3D打印技术在组织工程领域的应用显示出极大的潜力，可以生成由细胞和水凝胶组成的人造器官，并在结构和组成上模拟自然组织的复杂性。例如，气管由几种不同的细胞类型和不同的功能组织类型组成，如肌肉、结缔组织和软骨。作为TracheaPrint项目的继续，这里提出的研究项目基于这样的假设，即气管的管状结构可以在3D按需打印过程中类似。打印包括两种不同类型的水凝胶：一种是琼脂糖和I型胶原的细胞水凝胶混合物，另一种是类似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印刷性和高血管生成潜力，使用的是人内皮细胞和成纤维细胞的共同培养。特别是，在该项目的第二阶段，我们将重点放在基于聚乙二醇基的软骨替代物的发展上，这种软骨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基于聚乙二醇基的水凝胶的凝胶时间，避免光交联剂的细胞毒性效应。该研究项目包括体外细胞诱导的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的研究。此外，还将研究预血管化水凝胶样品在CAM模型中的整合。此外，还将阐述一种最适合具体应用的新型薄膜打印技术，并与现有的基于微阀的打印机相结合。最后，将负载细胞的气管替代物打印出来，并在脉动生物反应器中进行两步培养。此外，该结构将使用喷雾技术在内表面上皮化。此外，我们还探讨了将3D生物反应器用于层层生物打印的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.