Endothelial cells support osteogenesis in an in vitro vascularized bone model developed by 3D bioprinting
Endothelial cells support osteogenesis in an in vitro vascularized bone model developed by 3D bioprinting
复制标题
DOI：
10.1088/1758-5090/ab6a1d
复制
发表时间：
2020-04-01
期刊：
影响因子：
9
通讯作者：
Gottardi, Riccardo
中科院分区：
文献类型：
--
作者：
Chiesa, Irene;De Maria, Carmelo;Gottardi, Riccardo
骨是一种高度血管化的组织，其中血管化和矿化是骨骼发育过程中同时发生的过程。事实上，这两种成分都应该包含在任何可靠且一致的体外模型平台中，用于研究骨生理学和骨骼疾病的发病机制。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（hMSC）接种在支架上，并进行两周的成骨分化。然后，我们将慢病毒-GFP 转染的人脐静脉内皮细胞 (HUVEC) 纳入 3D 生物打印支架大孔内，在培养 2 周后形成毛细血管样网络。我们测试了三种实验条件：条件1，在1:1成骨培养基（OM）：内皮培养基（EM）中培养HUVEC的骨构建体；条件2，在1:1 OM:EM中培养的不含HUVEC的骨结构；条件3：用在1:1生长培养基:EM中培养的HUVEC进行骨构建。所有样品均产生工程骨基质。在条件 1 和 3 中，HUVEC 在骨结构内形成管状结构，并在活体组织和组织学中通过荧光显微镜观察到复杂的毛细血管样网络的组装。 CD31免疫染色证实了显着的血管腔形成。使用定量实时PCR来量化成骨分化和内皮反应。碱性磷酸酶和 runt 相关转录因子 2 上调证实了 hMSC 的早期成骨作用。即使在条件 3 下去除 OM，我们也观察到明显的成骨作用，这明显伴随着骨桥蛋白、血管内皮生长因子和 I 型胶原的上调。这些发现表明，我们在短短 4 周的培养中成功地实现了具有强血管化的骨模型，并且我们强调了内皮细胞的包含如何更现实地支持成骨作用。这里报道的方法产生了受生物学启发的骨血管化体外模型，模拟组织发育过程中发生的毛细血管的从头形态发生。
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.