3D bioprinted in vitro bone models

在拟议的项目中，将生成3D生物打印的体外骨骼模型。使用基于挤压的生物打印，我们想要创建复杂的3D结构，模仿自然的骨骼结构。我们的目标是涉及原代人类细胞，而不是永生化细胞系，最终建立成骨细胞、破骨细胞、骨细胞和内皮细胞的3D共培养模型。这个项目的关键是1)开发一种支持骨细胞分化、成骨细胞附着和铺展以及破骨细胞分化的墨水，2)不同细胞种类的空间排列，3)内皮细胞的包含，以研究骨细胞对血管形成的影响，反之亦然。骨细胞作为骨重建的调节者，以前很少涉及生物打印的结构，因为这些细胞的可获得性和它们的有丝分裂后特性是有限的。我们希望应用生物打印的人类原代成骨细胞，并在打印的结构中触发它们向骨细胞的转变，因为这一主题的初步工作已经显示出令人振奋的结果。含有骨细胞的构建物将与成骨细胞和破骨细胞或破骨细胞前体细胞一起种植，以模拟自然的骨结构，成骨细胞和破骨细胞在表面，骨细胞深入骨基质中。我们还计划在墨水中加入骨细胞外基质的成分，如I型胶原和羟基磷灰石，以获得更接近骨骼的模型。最后，将使用核壳生物打印来包括内皮细胞。将遵循两种不同的方法，水凝胶核心中的内皮细胞被其他类型的细胞包围，或者种植到含有骨细胞的中空链中。3D打印模型旨在测试骨组织对生长因子、药物、生物活性离子以及生物材料提取物等生物活性物质的反应。这将有助于减少动物实验，至少在生物材料和药物开发的早期阶段是这样。此外，该模型将有助于进一步了解骨细胞、成骨细胞、破骨细胞和内皮细胞之间的复杂相互作用。

In the proposed project, 3D bioprinted in vitro bone models will be generated. Using extrusion-based bioprinting we want to create complex 3D structures mimicking the natural osteon structure. Our ambition is to involve primary human cells instead of immortalised cell lines to finally establish a 3D co-culture model of osteoblasts, osteoclasts, osteocytes and endothelial cells. Crucial points in this projects are 1) the development of an ink supporting both osteocyte differentiation, attachment and spreading of osteoblasts as well as osteoclast differentiation, 2) the spatial arrangement of the different cell species and 3) the inclusion of endothelial cells to study effects of bone cells on vascularization and vice versa. Osteocytes, as regulators of bone remodeling have rarely been involved before in bioprinted structures, due to the limited availability of these cells and their postmitotic character. We want to apply bioprinted human primary osteoblasts and trigger their transition to osteocytes in the printed constructs, since preliminary work on this topic already showed promising results. Osteocyte containing constructs will be seeded with osteoblasts and osteoclasts or osteoclast progenitors to mimic natural bone structure with osteoblasts and osteoclasts at the surface and osteocytes deeply embedded into the bone matrix. We furthermore plan to include components of bone ECM like collagen I and hydroxyapatite into the inks to get a more bone-like model. Finally, core-shell bioprinting will be used to include endothelial cells. Two different approaches will be followed, with endothelial cells in a hydrogel core surrounded by the other cell types or seeded into osteocyte-containing hollow strands. The 3D printed model is intended to test the response of bone tissue to bioactive agents like growth factors, drugs, bioactive ions as well as biomaterials extracts. This would help to reduce animal experiments at least at the early stages of biomaterials and drug development. Furthermore, the model will help to further understand the complex interaction between osteocytes, osteoblasts, osteoclasts and endothelial cells.