Osteogenic Differentiation of Three-Dimensional Bioprinted Constructs Consisting of Human Adipose-Derived Stem Cells In Vitro and In Vivo

Here, we aimed to investigate osteogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3D) bioprinted tissue constructs in vitro and in vivo. A 3D Bio-plotter dispensing system was used for building 3D constructs. Cell viability was determined using live/dead cell staining. After 7 and 14 days of culture, real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of osteogenesis-related genes (RUNX2, OSX, and OCN). Western blotting for RUNX2 and immunofluorescent staining for OCN and RUNX2 were also performed. At 8 weeks after surgery, osteoids secreted by osteogenically differentiated cells were assessed by hematoxylin-eosin (H&E) staining, Masson trichrome staining, and OCN immunohistochemical staining. Results from live/dead cell staining showed that most of the cells remained alive, with a cell viability of 89%, on day 1 after printing. In vitro osteogenic induction of the 3D construct showed that the expression levels of RUNX2, OSX, and OCN were significantly increased on days 7 and 14 after printing in cells cultured in osteogenic medium (OM) compared with that in normal proliferation medium (PM). Fluorescence microscopy and western blotting showed that the expression of osteogenesis-related proteins was significantly higher in cells cultured in OM than in cells cultured in PM. In vivo studies demonstrated obvious bone matrix formation in the 3D bioprinted constructs. These results indicated that 3D bioprinted constructs consisting of hASCs had the ability to promote mineralized matrix formation and that hASCs could be used in 3D bioprinted constructs for the repair of large bone tissue defects.

本研究旨在探究人脂肪间充质干细胞（human adipose-derived stem cells, hASCs）在三维（3D）生物打印组织构建体中的体外及体内成骨分化情况。本研究采用三维生物打印分配系统（3D Bio-plotter dispensing system）制备三维构建体：通过活死细胞染色检测细胞活力；于培养7天和14天后，采用实时荧光定量聚合酶链反应（PCR）分析成骨相关基因（RUNX2、OSX及OCN）的表达水平；此外还开展了RUNX2的蛋白质免疫印迹（Western blotting）检测，以及OCN与RUNX2的免疫荧光染色。术后8周，通过苏木精-伊红（H&E）染色、马松三色染色及OCN免疫组织化学染色，评估成骨分化细胞分泌的类骨质。活死细胞染色结果显示，打印后第1天，绝大多数细胞保持存活，细胞存活率达89%。体外成骨诱导实验表明，与正常增殖培养基（PM）培养的细胞相比，成骨培养基（OM）培养的细胞在打印后第7天和第14天，RUNX2、OSX及OCN的表达水平显著升高。荧光显微镜观察与蛋白质免疫印迹结果显示，成骨培养基培养的细胞中成骨相关蛋白的表达水平显著高于正常增殖培养基培养的细胞。体内实验证实，三维生物打印构建体内可形成明显的骨基质。综上，由人脂肪间充质干细胞构建的三维生物打印体具备促进矿化基质形成的能力，hASCs可应用于三维生物打印构建体以修复大型骨组织缺损。