Bone is a highly vascularized tissue, in which vascularization and mineralization are concurrent processes during skeletal development. Indeed, both components should be included in any reliable and adherent in vitro model platform for the study of bone physiology and pathogenesis of skeletal disorders. To this end, we developed an in vitro vascularized bone model, using a gelatin-nanohydroxyapatite (gel-nHA) three-dimensional (3D) bioprinted scaffold. First, we seeded human mesenchymal stem cells (hMSCs) on the scaffold, which underwent osteogenic differentiation for 2 weeks. Then, we included lentiviral-GFP transfected human umbilical vein endothelial cells (HUVECs) within the 3D bioprinted scaffold macropores to form a capillary-like network during 2 more weeks of culture. We tested three experimental conditions: condition 1, bone constructs with HUVECs cultured in 1:1 osteogenic medium (OM): endothelial medium (EM); condition 2, bone constructs without HUVECs cultured in 1:1 OM:EM; condition 3: bone construct with HUVECs cultured in 1:1 growth medium:EM. All samples resulted in engineered bone matrix. In conditions 1 and 3, HUVECs formed tubular structures within the bone constructs, with the assembly of a complex capillary-like network visible by fluorescence microscopy in the live tissue and histology. CD31 immunostaining confirmed significant vascular lumen formation. Quantitative real-time PCR was used to quantify osteogenic differentiation and endothelial response. Alkaline phosphatase and runt-related transcription factor 2 upregulation confirmed early osteogenic commitment of hMSCs. Even when OM was removed under condition 3, we observed clear osteogenesis, which was notably accompanied by upregulation of osteopontin, vascular endothelial growth factor, and collagen type I. These findings indicate that we have successfully realized a bone model with robust vascularization in just 4 weeks of culture and we highlighted how the inclusion of endothelial cells more realistically supports osteogenesis. The approach reported here resulted in a biologically inspired in vitro model of bone vascularization, simulating de novo morphogenesis of capillary vessels occurring during tissue development.

中文翻译：

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内皮细胞支持通过3D生物打印开发的体外血管化骨模型中的成骨作用。

骨骼是高度血管化的组织，在骨骼发育过程中，血管化和矿化是同时发生的过程。确实，这两个组件都应包括在任何可靠的，附着的体外模型平台中，以研究骨骼生理学和骨骼疾病的发病机理。为此，我们使用明胶-纳米羟基磷灰石（gel-nHA）三维（3D）生物打印支架开发了体外血管化骨模型。首先，我们将人间充质干细胞（hMSCs）播种在支架上，进行了2周的成骨分化。然后，我们将慢病毒-GFP转染的人脐静脉内皮细胞（HUVEC）纳入了3D生物打印的支架大孔中，在培养的另外2周内形成了毛细管样网络。我们测试了三个实验条件：条件1 HUVEC的骨构建物在1：1成骨培养基（OM）中培养：内皮培养基（EM）；条件2，无HUVEC的骨骼构建物以1：1 OM：EM培养；条件3：具有在1：1生长培养基：EM中培养的HUVEC的骨构建体。所有样品均产生工程骨基质。在条件1和3中，HUVEC在骨骼结构中形成管状结构，并通过活体组织和组织学中的荧光显微镜观察到了复杂的毛细管状网络的组装。CD31免疫染色证实明显的血管腔形成。实时定量PCR用于量化成骨分化和内皮反应。碱性磷酸酶和矮子相关的转录因子2上调证实了hMSCs的早期成骨作用。即使在条件3下移除OM，我们也观察到明显的成骨作用，这些发现表明，仅在培养的4周内我们就成功实现了具有强大血管生成的骨模型，并且我们着重指出了如何更现实地包含内皮细胞支持成骨。本文报道的方法产生了生物学上受启发的骨血管化体外模型，模拟了组织发育过程中毛细血管的新生形态发生。这些发现表明，我们在短短4周的培养中就成功实现了具有强大血管生成的骨模型，并且我们着重指出了内皮细胞的包容如何更现实地支持成骨作用。本文报道的方法产生了生物学上受启发的骨血管化体外模型，模拟了组织发育过程中毛细血管的新生形态发生。这些发现表明，我们在短短4周的培养中就成功实现了具有强大血管生成的骨模型，并且我们着重指出了内皮细胞的包容如何更现实地支持成骨作用。本文报道的方法产生了生物学上受启发的骨血管化体外模型，模拟了组织发育过程中毛细血管的新生形态发生。