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的内皮细胞（HUVEC）开始的血管形成和间充质干细胞（MSC）的成骨分化。此外，我们还开发了一种3D生物打印机，它结合了两种生物打印技术，即按需喷墨（DoD）和挤出打印。使用这种生物打印机，我们打印了边长为1 cm的稳定立方体。在这些立方体中，血管组分通过DoD打印HUVEC在纤维蛋白-水凝胶中的高密度细胞悬浮液来实现，而骨组分通过在由纤维蛋白、明胶、透明质酸、甘油和羟基磷灰石组成的水凝胶（骨-水凝胶）中挤出打印MSC来实现。我们已经能够在体外以及在皮下植入模型中的体内显示，生物打印的HUVEC能够形成血管，生物打印的MSC能够形成钙化的骨基质。生物打印的立方体的弹性模量约为1 kPa，这对应于天然的人体软组织。然而，人类天然骨的刚度约为1 × 105 kPA，因此比我们目前构建体的实际E模量高约100.000倍。该持续项目的主要目标是开发一种用于打印含细胞水凝胶和稳定性生成热塑性塑料和/或磷酸钙水泥（CPC）的组合打印程序以便产生具有生理相关硬度的人造血管化骨组织，该硬度应类似于人的天然骨。第二个目标是实现所谓的4D生物打印（“时间”作为第四维），其中构建体的时间和空间成熟应该通过生长因子、分化因子和/或另外的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价4D组合结构的血管化和骨形成。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.