Ⅰ型胶原/海藻酸钠/透明质酸复合水凝胶用于血管组织工程细胞负载与3D培养

doi:10.11868/j.issn.1001-4381.2021.001166

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摘要

胶原、海藻酸钠和透明质酸是天然来源的高分子材料，具有良好的细胞相容性与生物安全性，在细胞培养、组织工程、药物负载等方面具有广泛应用。单纯的胶原力学性能较差，将胶原与海藻酸钠制备成复合水凝胶材料后，可以通过调节海藻酸钠与Ca²⁺交联程度来改变水凝胶支架的力学性能和孔隙率，模拟细胞培养的力学环境和细胞微环境。本研究通过PIUMA纳米压痕仪和DHR流变仪表征Ⅰ型胶原/海藻酸钠/透明质酸水凝胶的杨氏模量和溶胶-凝胶转变温度。并将内皮细胞与间充质干细胞在水凝胶微环境内进行3D培养，倒置荧光显微镜观察细胞培养0，3，5，7 d时细胞的活力情况，表征Ⅰ型胶原/海藻酸钠/透明质酸水凝胶的细胞相容性，并在内皮细胞与间充质干细胞培养0，1，4，6 d时，观察内皮细胞的迁移、成血管情况，在培养1，6，9 d时，观察内皮细胞的生长扩散情况。结果表明：水凝胶杨氏模量为（600±81）Pa，水凝胶的溶胶-凝胶转变温度为23.2℃。细胞培养0，3，5，7 d时，活力持续增强，培养4，6 d时，观测到共培养下内皮细胞的迁移，培养1，6，9 d时，水凝胶内的内皮细胞球体持续生长扩散。本工作表明，Ⅰ型胶原/海藻酸钠/透明质酸水凝胶对内皮细胞与间充质干细胞具有良好的细胞相容性，可用于细胞3D培养的理想支架材料。水凝胶的杨氏模量和溶胶-凝胶转变温度对细胞活力无损害，可作为研究血管新生的相关体外模型，在血管组织工程研究中具有重要的应用前景。

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	谢航
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	王志伟

关键词 ：细胞3D培养, Ⅰ型胶原, 海藻酸钠, 水凝胶微环境

Abstract：

Collagen, sodium alginate and hyaluronic acid are natural-derived polymer materials with good cell compatibility and bio-safety, which are widely used in cell culture, tissue engineering and drug delivery, and so on. Pure collagen has poor mechanical properties. When preparing collagen and sodium alginate to form a composite hydrogel material, the mechanical properties and porosity of the hydrogel scaffold can be improved by adjusting the degree of cross-linking of sodium alginate and Ca²⁺ mimicking extracellular matrix. The Young's modulus and the sol-gel transition temperature of the hydrogel were characterized by PIUMA nanoindenter and DHR rheometer in this study. Microscopic images of endothelial cells expressing red fluorescent proteins and mesenchymal stem cell expressing green fluorescent proteins were captured with Olympus fluorescence microscope after cell cultured for 0 day, 3 days, 5 days and 7 days in hybrid hydrogel microenvironment, and the images of endothelial cell spheroid growth diffusion after cell cultured for 1 day, 6 days and 9 days. The results show that the hybrid hydrogel is cytocompatible. The Young's modulus of the hydrogel is (600±81) Pa and its sol-gel transition temperature is 23.2℃. In conclusion, type Ⅰ collagen/sodium alginate/hyaluronic acid hydrogel has good cytocompatibility for endothelial cells and mesenchymal stem cells, and can be used as an ideal scaffold material for cell 3D culture. The Young's modulus and sol-gel transition temperature of the hydrogel have no damage to cell viability, which can be used as an in vitro model for studying angiogenesis and has important application prospects in vascular tissue engin-eering.

Key words： 3D cell culture type Ⅰ collagen sodium alginate hydrogel microenvironment

收稿日期: 2021-11-30 出版日期: 2022-11-17

中图分类号:

R318

基金资助:广东省基础与应用基础研究基金委员会区域联合基金青年基金项目(2019A1515110005);中国博士后科学基金(2021M703710)

通讯作者: 王志伟 E-mail: jnuwzhw@sina.com

作者简介: 王志伟(1965—)，男，副教授，博士，研究方向为口腔修复与仿生矿化，联系地址：广东省广州市天河区黄埔大道西601号暨南大学口腔医学院(510632)，E-mail：jnuwzhw@sina.com

引用本文:

谢航, 刘纯, 胡灏, 王志伟. Ⅰ型胶原/海藻酸钠/透明质酸复合水凝胶用于血管组织工程细胞负载与3D培养[J]. 材料工程, 2022, 50(11): 26-33.
Hang XIE, Chun LIU, Hao HU, Zhiwei WANG. Type Ⅰ collagen/sodium alginate/hyaluronic acid composite hydrogel for cell loading and 3D culture in vascular tissue engineering. Journal of Materials Engineering, 2022, 50(11): 26-33.

链接本文:

http://jme.biam.ac.cn/CN/10.11868/j.issn.1001-4381.2021.001166 或 http://jme.biam.ac.cn/CN/Y2022/V50/I11/26

Table 1 Ⅰ型胶原/海藻酸钠/透明质酸复合水凝胶的成分添加顺序

Fig.1 Ⅰ型胶原/海藻酸钠/透明质酸复合水凝胶合成示意图

Fig.2 Ⅰ型胶原/海藻酸钠/透明质酸复合水凝胶的杨氏模量表征

Fig.3 Ⅰ型胶原/海藻酸钠/透明质酸水凝胶的温度依赖性流变行为

Fig.4 载细胞水凝胶中细胞培养0 d(1), 3 d(2), 5 d(3), 7 d(4)表达荧光信号强度
(a)红色：bEnd.3-RFP细胞; (b)绿色：MSC-GFP细胞

Fig.5 水凝胶内3D培养0, 3, 5, 7 d后细胞数定量

Fig.6 水凝胶内3D培养0, 3, 5, 7 d后细胞荧光强度定量

Fig.7 培养0 d(1), 1 d(2), 4 d(3), 6 d(4)时内皮细胞球状体迁移情况
(a)MSC-GFP球状体; (b)bEnd.3-RFP球状体; (c)MSC-GFP球状体和bEnd.3-RFP球体

Fig.8 载bEnd.3-RFP水凝胶培养0 d(1), 1 d(2), 4 d(3), 6 d(4)时bEnd.3-RFP未见小管形成
(a)载bEnd.3-RFP水凝胶；(b) 载bEnd.3-RFP水凝胶内bEnd.3-RFP球体；(c) 载bEnd.3-RFP水凝胶内MSC-GFP球体

Fig.9 bEnd.3-RFP球状体在空载水凝胶和载MSC-GFP水凝胶中培养1 d(1), 6 d(2), 9 d(3)时的生长扩散情况
(a)空载水凝胶; (b)载MSC-GFP的水凝胶

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