中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (27): 4308-4313.doi: 10.12307/2022.860

• 复合支架材料 composite scaffold materials • 上一篇    下一篇

可注射葡萄糖酸内酯-海藻酸钠/β-磷酸三钙-聚乙二醇复合水凝胶支架的性能评价

刘  印1，2，3，刘  琴1，2，3，陈  娇1，2，3，古先扬1，2，3，陈佳文1，3，4，马敏先1，3，4，5   

  1. 1贵州医科大学，贵州省贵阳市  550004；2贵州医科大学附属口腔医院，贵州省贵阳市  550004；3贵州医科大学组织工程与干细胞实验中心，贵州省贵阳市  550004；4贵阳市口腔医院口腔种植科，贵州省贵阳市  550004；5贵州医科大学细胞工程生物医药技术国家地方联合工程实验室，贵州省贵阳市  550004
  • 收稿日期:2021-04-09 接受日期:2021-05-20 出版日期:2022-09-28 发布日期:2022-03-11
  • 通讯作者: 马敏先，主任医师，贵州医科大学，贵州省贵阳市 550004；贵州医科大学组织工程与干细胞实验中心，贵州省贵阳市 550004；贵阳市口腔医院口腔种植科，贵州省贵阳市 550004；贵州医科大学细胞工程生物医药技术国家地方联合工程实验室，贵州省贵阳市 550004
  • 作者简介:刘印，女，1995 年生，贵州省遵义市人，土家族，贵州医科大学在读硕士，医师，主要从事骨组织工程生物材料的研究。
  • 基金资助:
    贵阳市科技计划项目(筑科合同[2018]1-81号)，项目负责人：马敏先

Properties of injectable gluconolactone-sodium alginate/beta-tricalcium phosphate/polyethylene glycol composite hydrogel scaffold

Liu Yin1, 2, 3, Liu Qin1, 2, 3, Chen Jiao1, 2, 3, Gu Xianyang1, 2, 3, Chen Jiawen1, 3, 4, Ma Minxian1, 3, 4, 5   

  1. 1Guizhou Medical University, Guiyang 550004; 2Stomatological Hospital of Guizhou Medical University, Guiyang 550004; 3Research Center of Tissue Engineering and Stem Cell Technique, Guizhou Medical University, Guiyang 550004; 4Department of Oral Implantology, Guiyang Hospital of Stomatology, Guiyang 550004; 5National-Guizhou Joint Engineering Laboratory of Cell Engineering and Biomedicine Technique, Guizhou Medical University, Guiyang 550004
  • Received:2021-04-09 Accepted:2021-05-20 Online:2022-09-28 Published:2022-03-11
  • Contact: Ma Minxian, Chief physician, Guizhou Medical University, Guiyang 550004, Guizhou Province, China; Research Center of Tissue Engineering and Stem Cell Technique, Guizhou Medical University, Guiyang 550004, Guizhou Province, China; Department of Oral Implantology, Guiyang Hospital of Stomatology, Guiyang 550004, Guizhou Province, China; National-Guizhou Joint Engineering Laboratory of Cell Engineering and Biomedicine Technique, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • About author:Liu Yin, Master candidate, Physician, Guizhou Medical University, Guiyang 550004, Guizhou Province, China; Stomatological Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China; Research Center of Tissue Engineering and Stem Cell Technique, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • Supported by:
    the Guiyang Science and Technology Planning Project, No. [2018]1-81 (to MMX)

PDF

62

摘要：

文题释义：
葡萄糖酸内酯：一般是白色晶体或粉末，水解呈酸性，是一种用途十分广泛的食品添加剂，无毒，具有良好的生物相容性。
可注射水凝胶：是一种高含水率的三维结构，与细胞外基质相似，因具有可注射性可填充不规则的骨缺损，并可通过针头注射的方式实现微创治疗，可将细胞、药物、生长因子、基因、纳米粒子等包覆在其中，以满足不同组织的再生需求，其多孔结构和溶胀性能允许营养和氧气有效扩散到组织结构中，同时可促进代谢产物的排出。

背景：可注射海藻酸钠水凝胶可以通过非侵入性或微创方式修复骨缺损，但力学性能欠佳，聚乙二醇基水凝胶具有弹性，将材料复合有望提高水凝胶支架的机械性能及细胞生物相容性。
目的：探索葡萄糖酸内酯-海藻酸钠/β-磷酸三钙-聚乙二醇复合水凝胶的理化性能，以及对鼠骨髓间充质干细胞增殖和分化的影响。
方法：以葡萄糖酸内酯为交联剂，分别制备葡萄糖酸内酯-海藻酸钠/β-磷酸三钙-聚乙二醇水凝胶(交联剂的浓度分别为5，10，20 g/L，依次记为A、B、C组)与葡萄糖酸内酯-海藻酸钠/β-磷酸三钙水凝胶(交联剂的浓度为10 g/L，记为D组)，表征4组水凝胶的形态、机械性能与凝胶时间。将SD大鼠骨髓间充质干细胞分别与4组水凝胶共培养，利用CCK-8法检测细胞增殖，Live/Dead 荧光染色观察细胞存活情况，Ⅰ型胶原免疫细胞化学染色检测成骨分化情况。
结果与结论：①扫描电镜下，A、B、C组可见类似黏结性的丝状结构，A组孔径分布不均匀，B组孔径分布均匀且孔隙率高，C组孔隙率高但孔径大小不一，D组孔径分布不均且孔隙率低。②理化性质：B组的压缩应力高于D组(P < 0.05)，并且随着交联剂浓度的增加，A、B、C组支架的压缩应力增强(P < 0.05)；压缩50%后水凝胶的大体观显示，压缩后D组有较大裂纹，B组仅有较小裂纹；A、B、C组凝胶时间随加入交联剂浓度的增加而缩短，B组凝胶时间较D组稍延长(P < 0.05)。③CCK-8实验检测显示，A、B组的细胞增殖速率高于C组(P < 0.05)，B组高于D组(P < 0.05)；Live/Dead 染色显示，各组细胞存活率均较高，但C组死细胞较其他3组多。④免疫细胞化学染色显示，A、B、D组细胞呈多角形，C组细胞部分呈圆形，B组绿色荧光强度较其他3组高，且细胞骨架和微丝的形态及细胞数量也优于其他3组。⑤结果表明，葡萄糖酸内酯-海藻酸钠/β-磷酸三钙-聚乙二醇水凝胶具有良好的机械性能和稳定性，其中以10 g/L葡萄糖酸内酯制备的水凝胶能有效促进鼠骨髓间充质干细胞的增殖和分化。
缩略词：葡萄糖酸内酯-海藻酸钠/β-磷酸三钙-聚乙二醇：D-gluconic acid δ-lactone-sodium alginate/beta-tricalcium phosphate-polyethylene glycol，GDL-SA/β-TCP-PEG

https://orcid.org/0000-0001-9655-9527 (刘印) 

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料;口腔生物材料;纳米材料;缓释材料;材料相容性；组织工程

关键词: 组织工程支架, 可注射水凝胶, 海藻酸钠, β-磷酸三钙, 聚乙二醇, 葡萄糖酸内酯

Abstract: BACKGROUND: Injectable sodium alginate hydrogel can repair bone defects by non-invasive or minimally invasive methods, but the mechanical properties are poor and polyethylene glycol-based hydrogel has elasticity. The composite of materials is expected to improve the mechanical properties and cellular biocompatibility of hydrogel scaffolds.
OBJECTIVE: To investigate the physical and chemical properties of glucolactone-sodium alginate/β-tricalcium phosphate-polyethylene glycol hydrogel and its effect on the proliferation and differentiation of murine bone marrow mesenchymal stem cells. 
METHODS: Gluconolactone as cross-linking agent was set to prepare glucolactone-sodium alginate/β-tricalcium phosphate-polyethylene glycol hydrogel (concentrations of cross-linking agent were 5, 10, and 20 g/L, respectively, which were marked as groups A, B, and C) and glucolactone-sodium alginate/β-tricalcium phosphate hydrogel (concentration of cross-linking agent was 10 g/L, which was marked as group D). The morphology, mechanical properties, and gelation time of hydrogels in the four groups were characterized. Rat bone marrow mesenchymal stem cells were co-cultured with four groups of hydrogels. The proliferation of cells was detected by CCK-8 assay. Cell survival was observed by Live/Dead fluorescence staining. Osteogenic differentiation was detected by immunocytochemical staining of type I collagen. 
RESULTS AND CONCLUSION: (1) The filamentous structure similar to adhesion was observed by scanning electron microscope in the groups A, B and C. The pore size distribution of group A was uneven. The pore size of group B was uniform and the porosity was high. The group C had high porosity but different pore sizes. The group D had uneven pore size distribution and low porosity. (2) Physicochemical properties: The compressive stress in group B was higher than that in group D (P < 0.05). With the increase of gluconolactone concentration, the compressive stress of hydrogel in groups A, B and C increased (P < 0.05). The macroscopic performance of hydrogel after 50% compression showed that there were larger cracks in group D and only smaller cracks in group B after compression. The gelation time of groups A, B and C shortened with the increase of gluconolactone concentration, and the gelation time of group B was slightly longer than that of group D (P < 0.05). (3) CCK-8 assay showed that the cell proliferation rate of groups A and B was higher than that of group C (P < 0.05), and that of group B was higher than that of group D (P < 0.05). Live/Dead staining showed that the cell survival rate of each group was higher, but the number of dead cells was more in group C than that in the other three groups. (4) Immunocytochemical staining showed that the cells in groups A, B, and D were polygonal, and the cells in group C were partially round. The green fluorescence intensity in group B was higher than that in the other three groups, and the morphology of the cytoskeleton and microfilaments and the number of cells were also better than those in the other three groups. (5) The results show that the glucolactone-sodium alginate/β-tricalcium phosphate-polyethylene glycol hydrogel has perfect mechanical properties and stability; hydrogel prepared with 10 g/L gluconolactone can effectively promote the proliferation and differentiation of murine bone marrow mesenchymal stem cells.

Key words: tissue-engineered scaffold, injectable hydrogel, sodium alginate, β-tricalcium phosphate, polyethylene glycol, gluconolactone

中图分类号: