4D-bioprinting of vascularized bone tissue and evaluation of blood vessel and bone formation in an orthotopic bone defect model

在第一个资助期，我们开发了血管化骨组织生物打印的重要基础。在这种情况下，我们确定了两种合适的水凝胶，它们能够分别支持从生物打印内皮细胞（HUVECs）和间充质干细胞（MSCs）的成骨分化开始的血管形成。此外，我们还开发了一种3D生物打印机，结合了两种生物打印技术，即随需应变（DoD）和挤压打印。使用这种生物打印机，我们打印出边缘长度为1厘米的稳定立方体。在这些立方体中，血管成分是通过在纤维蛋白水凝胶中DoD打印高密度HUVECs细胞悬浮液来实现的，而骨成分是通过在由纤维蛋白、明胶、透明质酸、甘油和羟基磷灰石（骨水凝胶）组成的水凝胶中挤压打印MSCs来实现的。我们已经能够在体外和体内皮下植入模型中证明，生物打印的HUVECs能够形成血管，生物打印的MSCs能够形成钙化的骨基质。生物打印立方体的弹性模量约为1kpa，与人体软组织的弹性模量相当。然而，人类天然骨的刚度约为1x105 kPA，因此比我们目前构造的实际e模量高约100,000倍。因此，这个持续项目的主要目标是开发一种组合打印程序，用于打印含有细胞的水凝胶和产生稳定性的热塑性塑料和/或磷酸钙水泥（CPC），以生产具有生理相关刚性的人造血管化骨组织，类似于人类天然骨骼。第二个目标是实施所谓的4d生物打印（“时间”作为第四个维度），其中构建的时间和空间成熟应该由生长因子、分化因子和/或其他细胞实体的空间分辨打印来控制。继续项目的第三个主要目标是在生理相关的原位骨缺损模型中评估4d组合结构在血管化和骨形成方面的作用。在这种情况下，我们还想研究是否可以通过调节打印结构的弹性模量来控制血管形成和骨形成的数量和/或质量。

In the first funding period, we developed important basics for the bioprinting of vascularized bone tissue. In this context, we identified two suitable hydrogels, which were able to support blood vessel formation starting from bioprinted endothelial cells (HUVECs) and osteogenic differentiation of mesenchymal stem cells (MSCs), respectively. Moreover, we developed a 3D bioprinter which combines two bioprinting technologies, namely Drop on Demand (DoD) and extrusion printing.Using this bioprinter, we printed stabile cubes with edge lengths of 1 cm. In these cubes, the vascular component was realized by DoD printing of high density cell suspensions of HUVECs in fibrin-hydrogels, whereas the bone component was realized by extrusion printing of MSCs in a hydrogel composed of fibrin, gelatin, hyaluronic acid, glycerol and hydroxyapatite (osteo-Hydrogel). We have been able to show in vitro, as well as in vivo in a subcutaneous implantation model, that the bioprinted HUVECs were able to form blood vessels and the bioprinted MSCs were able to form a calcified bone matrix.The elastic modulus of the bioprinted cubes was around 1 kPa, which corresponds to native human soft tissue. However, the rigidity of human native bone is around 1x105 kPA and therefore about 100.000 times higher than the actual E-modulus of our current constructs.Therefore, on major goal of this continued project is to develop a combined printing procedure for printing cell-containing hydrogels and stability-generating thermoplastics and/or calcium phosphate cements (CPC) in order to produce artificial vascularized bone tissue with a physiological relevant rigidity which should resemble human native bone. The second goal is the implementation of the so called 4D-bioprinting (“time” as the fourth dimension) in which the temporal and spatial maturation of the construct should be controlled by the spatially resolved printing of growth factors, differentiation factors and/or additional cell entities. The third major goal of the continued project is the evaluation of the 4D-combination constructs in a physiologically-relevant orthotopic bone defect model in terms of vascularization and bone formation. In this context, we also want to investigate whether the quantity and/or quality of blood vessel formation and bone formation can be steered via modulation of the elastic moduli of the printed constructs.