间充质干细胞与内皮细胞3D仿生分布设计促进组织工程支架血管化的效果

3D biomimetic design of mesenchymal stem cells and endothelial cells promotes the vascularization of tissue engineering scaffold

  • 摘要: 目的 通过同轴生物3D打印技术构建间充质干细胞(MSCs)与人脐静脉内皮细胞(HUVECs)的3D仿生分布设计支架(Shell-Core支架),并对该种支架的仿生血管化功能进行实验验证。方法 通过同轴生物3D打印技术构建Shell-Core支架,并通过光镜、染色实验等方式对其仿生结构进行检测。通过分组观察比对、细胞划痕实验检测其体外促血管化效果。采用实时定量聚合酶链反应(qRT-PCR)检测仿生支架细胞的RNA表达水平。结果 成功构建了Shell-Core支架,其结构具有高度保真性,该结构可以保持到构建后的第7日。细胞增殖实验显示在第7日可以观察到支架中细胞增殖速度超过2D平面下混合培养的细胞增殖速度。细胞划痕实验显示Shell-Core条件培养基处理组的HUVECs划痕缩短距离大于细胞随机散在分布的3D-Mix条件培养基处理组和无血清DMEM处理组(431.6±=-33.6)μm vs.(378.7±22.5)μm vs.(302.3±20.1)μm,均P < 0.01。将仿生分布设计的组织工程体外培养7 d后在荧光显微镜下可以观察到表达绿色荧光蛋白的HUVECs仍维持在设计的核通道内,并向四周自组装出内皮细胞芽。qRT-PCR的结果显示Shell-Core支架中细胞的MMP-9基因表达高于3D-Mix支架(1.55±0.06)倍,P < 0.01. 结论 Shell-Core支架在确保了仿生设计结构高度保真性的同时具有促进血管化的作用,为解决组织缺损的修复、制造血管化的工程器官提供了新思路,也可为药物测试、研究血管发生等提供新的组织模型。

     

    Abstract: Objective To fabricate 3D biomimetic design of tissue engineering scaffolds(Shell-Core scaffolds)incorporating mesenchymal stem cells(MSCs)and human umbilical vein endothelial cells(HUVECs)by using advanced coaxial 3D bioprinting technology,and to validate the biomimetic vascularization capacity of the scaffolds by experiments. Methods The Shell-Core scaffolds were successfully fabricated via coaxial 3D bioprinting technology. The biomimetic structural features were characterized using optical microscopy and histological staining assay. The in vitro pro-angiogenic capacity was evaluated through inter-group comparative observations and cell scratch assays. Furthermore,qRT-PCR was employed to quantify RNA expression levels of angiogenesis-related markers in cells cultured on the Shell-Core scaffolds. Results Shell-Core scaffolds were successfully fabricated. The scaffolds possessed high structural fidelity,which could be maintained at 7 d after scaffold fabrication. Cell proliferation assay showed that the cell proliferation rate in the scaffolds at 7 d was higher than that in the mixed culture on the 2D plane. Cell scratch assay showed that the shortening scratch distance of HUVECs treated by Shell-Core-CM was significantly greater than those treated by 3D-Mix-CM and blank groups (431.6±33.6)μm vs.(378.7±22.5)μm vs.(302.3±20.1)μm,both P < 0.01. At 7 d after in vitro cultured of engineered biomimetic tissues,under fluorescence microscope,HUVECs expressing green fluorescent protein remained in the designed core channel,and self-assembled endothelial buds in all directions. Furthermore,the results of qRT-PCR show that quantified RNA expression levels of angiogenesis-related markers(MMP-9)in cells cultured on the Shell-Core scaffolds was significantly higher(1.55±0.06,P < 0.01)than that in 3D-Mix scaffolds. Conclusions The Shell-Core scaffolds integrating MSCs and HUVECs demonstrates high structural fidelity and pro-angiogenic capacity,offering a novel strategy for addressing tissue defect repair and fabricating vascularized engineered organs. This platform further provides a physiologically relevant tissue model for drug testing and mechanistic investigation of angiogenesis.

     

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