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. 2023 Jun 25;52(3):285-295.
doi: 10.3724/zdxbyxb-2022-0702.

Preparation of silk fibroin/hyaluronic acid composite hydrogel based on thiol-ene click chemistry

[Article in English, Chinese]
Leidan Chen  1 , Mingqiang Zhong  2 , Jinyi Chen  3 , Zhenjie Liu  4 , Tairong Kuang  2 , Tong Liu  2 , Feng Chen  5
Affiliations

Preparation of silk fibroin/hyaluronic acid composite hydrogel based on thiol-ene click chemistry

[Article in English, Chinese]
Leidan Chen et al. Zhejiang Da Xue Xue Bao Yi Xue Ban. .

Abstract
in English, Chinese

Objectives: To design and prepare silk fibroin/hyaluronic acid composite hydrogel.

Methods: The thiol modified silk fibroin and the double-bond modified hyaluronic acid were rapidly cured into gels through thiol-ene click polymerization under ultraviolet light condition. The grafting rate of modified silk fibroin and hyaluronic acid was characterized by 1H NMR spectroscopy; the gel point and the internal microstructure of hydrogels were characterized by rheological test and scanning electron microscopy; the mechanical properties were characterized by compression test; the swelling rate and degradation rate were determined by mass method. The hydrogel was co-cultured with the cells, the cytotoxicity was measured by the lactate dehydrogenase method, the cell adhesion was measured by the float count method, and the cell growth and differentiation on the surface of the gel were observed by scanning electron microscope and fluorescence microscope.

Results: The functional group substitution degrees of modified silk fibroin and hyaluronic acid were 17.99% and 48.03%, respectively. The prepared silk fibroin/hyaluronic acid composite hydrogel had a gel point of 40-60 s and had a porous structure inside the gel. The compressive strength was as high as 450 kPa and it would not break after ten cycles. The water absorption capacity of the composite hydrogel was 4-10 times of its own weight. Degradation experiments showed that the hydrogel was biodegradable, and the degradation rate reached 28%-42% after 35 d. The cell biology experiments showed that the cytotoxicity of the composite gel was low, the cell adhesion was good, and the growth and differentiation of the cells on the surface of the gel were good.

Conclusions: The photocurable silk fibroin/hyaluronic acid composite hydrogel can form a gel quickly, and has excellent mechanical properties, adjustable swelling rate and degradation degree, good biocompatibility, so it has promising application prospects in biomedicine.

目的: 以丝素蛋白和透明质酸为原料设计并制备医用天然聚合物水凝胶。方法: 对丝素蛋白和透明质酸进行巯基化及双键化改性，在紫外光条件下，利用巯基-烯点击聚合快速固化成胶。通过氢-1核磁共振波谱法表征改性丝素蛋白及透明质酸的接枝率；流变学测试表征水凝胶的凝胶点；扫描电镜观察水凝胶的内部微观结构；压缩测试表征力学性能；质量法测定溶胀率及降解率；将水凝胶与细胞共培养，通过乳酸脱氢酶法测定细胞毒性，漂浮计数法测定细胞黏附性并用扫描电镜及荧光显微镜观察凝胶表面的细胞生长与分化状况。结果: 改性后的丝素蛋白和透明质酸的官能团取代度分别为17.99%和48.03%。制备所得的丝素蛋白/透明质酸复合水凝胶的凝胶点为40~60 s，凝胶内部具有孔状结构，压缩强度达450 kPa且循环十次不破裂。复合水凝胶可吸收自身质量4倍甚至10倍的水。水凝胶可生物降解，35 d后的降解率达28%~42%。复合水凝胶的细胞毒性较弱，细胞黏附性较好，细胞在凝胶表面生长和分化状况良好。结论: 光固化丝素蛋白/透明质酸复合水凝胶能快速成胶、力学性能优异、生物相容性好，具有可调的溶胀率及降解度，在生物医学上具有一定的应用潜力。.

Keywords: Biocompatibility; Hyaluronic acid; Hydrogel; Photo-initiated crosslinking; Silk fibroin; Thiol-ene click chemistry.

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Conflict of interest statement

所有作者均声明不存在利益冲突

The authors declare that there is no conflict of interests

Figures

图1
图1. 巯基化丝素蛋白的制备
图2
图2. 双键化透明质酸的制备
图3
图3. 丝素蛋白/透明质酸复合水凝胶制备的反应示意图
图4
图4. 巯基化丝素蛋白和双键化透明质酸的氢-1核磁共振波谱图
图5
图5. SH-gel的外观
图6
图6. SH-gel的流变学性能曲线
图7
图7. SH-gel的断面微孔形貌
图9
图9. SH-gel的溶胀性能
图10
图10. SH-gel的降解曲线
图11
图11. SH-gel的吸光度值比较（乳酸脱氢酶法）
图12
图12. SH-gel与HUVEC共培养72 h后材料表面扫描电子显微镜图
图13
图13. SH-gel表面培养人脐静脉内皮细胞的免疫荧光染色图
None
See this image and copyright information in PMC

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