中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (2): 196-203.doi: 10.3969/j.issn.2095-4344.1507

• 药物控释材料 drug delivery materials • 上一篇    下一篇

可注射性乙二醇壳聚糖/双醛功能化聚乙二醇水凝胶的细胞相容性

荆晓光1,2,刘舒云2,郭维民2,李 旭2,吕 奥2,刘士臣1,孟昊业2,陈明学2,张学亮2,张增增1,2,刘雪剑1,2,高 超1,2,王泽浩2,张 彬2,沈 师2,陶 磊3,杨建华1,4,郭全义2   

  1. 1佳木斯大学,黑龙江省佳木斯市 154007;2解放军总医院骨科研究所,北京市 100853;3清华大学化学系,北京市 100084;4深圳市龙岗区人民医院,广东省深圳市 518172
  • 收稿日期:2018-08-10 出版日期:2019-01-18 发布日期:2019-01-18
  • 通讯作者: 杨建华,教授,佳木斯大学,黑龙江省佳木斯市 154007
  • 作者简介:荆晓光,男,1989年生,河南省周口市人,汉族,佳木斯大学在读硕士,主要从事关节软骨再生方向的研究。
  • 基金资助:

    国家重点研发计划课题(2017YFC1104102,2017YFC1103404);国家自然科学基金(21134004;81472092,项目负责人:郭全义;81772319,项目负责人:郭全义);北京市自然科学基金(7172203,项目负责人:郭全义);北京市科技专项(Z161100005016059)

Cytocompatibility of injectable glycol chitosan/dibenzaldehyde-terminated poly-ethyleneglycol hydrogel

Jing Xiaoguang1, 2, Liu Shuyun2, Guo Weimin2, Li Xu2, Lü Ao2, Liu Shichen1, Meng Haoye2, Chen Mingxue2, Zhang Xueliang2  Zhang Zengzeng1, 2, Liu Xuejian1, 2, Gao Chao1, 2, Wang Zehao2, Zhang Bin2, Shen Shi2, Tao Lei3, Yang Jianhua1, 4, Guo Quanyi2   

  1. 1Jiamusi University, Jiamusi 154007, Heilongjiang Province, China; 2Institute of Orthopedics, Chinese PLA General Hospital, Beijing 100853, China; 3Department of Chemistry, Tsinghua University, Beijing 100084, China; 4Longguang People’s Hospital, Shenzhen 518172, Guangdong Province, China
  • Received:2018-08-10 Online:2019-01-18 Published:2019-01-18
  • Contact: Yang Jianhua, Professor, Jiamusi University, Jiamusi 154007, Heilongjiang Province, China; Longguang People’s Hospital, Shenzhen 518172, Guangdong Province, China
  • About author:Jing Xiaoguang, Master candidate, Jiamusi University, Jiamusi 154007, Heilongjiang Province, China; Institute of Orthopedics, Chinese PLA General Hospital, Beijing 100853, China
  • Supported by:

    the National Key Research and Development Project of China, No. 2017YFC1104102 and 2017YFC1103404; the National Natural Science Foundation of China, No. 21134004, 81472092 (to GQY), and 81772319 (to GQY); the Beijing Natural Science Foundation, No. 7172203 (to GQY); the Beijing Science and Technology Special Project, No. Z161100005016059

摘要:

文章快速阅读:

 

文题释义:
双醛功能化聚乙二醇
:是将聚乙二醇2000(3.26 g,1.63 mmol)、4-甲酰苯甲酸(0.98 g,6.52 mmol)和4-二甲氨基吡啶(0.05 g)溶解在100 mL四氢呋喃中,再加入二环己基碳二亚胺(1.68 g),在20 ℃环境下搅拌18 h,将析出的白色固体过滤并在四氢呋喃中反复溶解,在乙醚中沉淀3次后而得到的白色固体。双醛功能化聚乙二醇能与乙二醇壳聚糖在水溶液中形成希夫碱与醛胺反应之间的动态平衡,进而得到可注射性水凝胶。
可注射性水凝胶:水凝胶是以水为分散介质的凝胶,具有网状交联结构的水溶性高分子中引入一部分疏水基团和亲水残基,亲水残基与水分子结合,将水分子连接在网状内部,而疏水残基遇水膨胀的交联聚合物,是一种高分子网络体系,性质柔软,能保持一定的形状。可注射性水凝胶即是成胶后能经注射器推出的一类水凝胶。


背景:课题组前期研究自主研发了可注射性乙二醇壳聚糖/双醛功能化聚乙二醇水凝胶(glycol chitosan/ dibenzaldehyde-terminated poly-ethyleneglycol,GCS/DF-PEG),研究显示其具有较好的可注射性和自愈性。
目的:进一步检测可注射性GCS/DF-PEG的物理学性质及细胞相容性。
方法:将质量分数1.5%的乙二醇壳聚糖溶液与质量分数分别为2%,4%,8%的双醛功能化聚乙二醇溶液等体积混合,制备3组可注射性GCS/DF-PEG水凝胶,检测其弹性模量。将3组可注射性水凝胶分别浸入PBS中4周,检测凝胶的体外降解。提取第2代SD乳鼠脂肪间充质干细胞,实验组加入可注射性GCS/DF-PEG水凝胶浸提液,对照组常规培养,MTT法检测细胞增殖。将质量分数3%的乙二醇壳聚糖溶液与第2代SD乳鼠脂肪间充质干细胞混合,再与质量分数为4%的双醛功能化聚乙二醇溶液混合,培养第1,5 天,采用死活染色法检测细胞在水凝胶内死活状态。将质量分数3%的乙二醇壳聚糖溶液与第2代SD乳鼠脂肪间充质干细胞混合,再与质量分数分别为2%,4%,8%的双醛功能化聚乙二醇溶液混合,MTT法检测细胞增殖。
结果与结论:①质量分数分别为2%,4%,8%双醛功能化聚乙二醇组水凝胶的弹性模量分别为13.48,22.21,33.19 kPa;②随时间的延长,3组水凝胶的降解率均逐渐增加,质量分数2%双醛功能化聚乙二醇组水凝胶的降解速率明显快于其他两组;③培养7 d内,实验组细胞增殖与对照组比较无差异;④死活染色显示,脂肪间充质干细胞在水凝胶内呈球形,存活率在90%以上,且随时间延长细胞数量明显增多;⑤随着时间的延长,脂肪间充质干细胞在含不同质量分数双醛功能化聚乙二醇GCS/DF-PEG水凝胶内的数量逐渐增多,且含质量分数2%双醛功能化聚乙二醇组水凝胶内的细胞增殖快于含质量分数4%,8%双醛功能化聚乙二醇组(P < 0.05);⑥结果表明,可注射性GCS/DF-PEG水凝胶的细胞相容性良好,力学强度及降解具有可调性,有望成为软骨组织工程中干细胞移植的良好载体。

ORCID: 0000-0002-6424-3242(荆晓光)

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: 可注射水凝胶, 脂肪间充质干细胞, 扫描电镜, 降解实验, 细胞增殖, 细胞死活染色, 国家重点研发计划课题, 生物材料

Abstract:

 BACKGROUND: Our research group independently developed an injectable glycol chitosan/dibenzaldehyde-terminated poly-ethyleneglycol (GCS/DF-PEG) hydrogel, which has good injectability and self-healing properties.

OBJECTIVE: To test the physical properties and cytocompatibility of the GCS/DF-PEG hydrogel.
METHODS: The injectable GCS/DF-PEG hydrogel was prepared by mixing GCS solution at a mass fraction of 1.5% with an equal volume of DF-PEG solution at a mass fraction of 2%, 4%, and 8%, respectively. Their moduli of elasticity were measured. Three groups of injectable hydrogels were immersed in PBS for 4 weeks to detect the in vitro degradation of the hydrogels. Passage 2 adipose-derived mesenchymal stem cells from Sprague-Dawley neonatal rats were cultured in injectable GCS/DF-PEG hydrogel leaching solution as experimental group or cultured routinely as control group. MTT assay was used to detect the cell proliferation. Passage 2 adipose-derived mesenchymal stem cells from Sprague-Dawley neonatal rats were mixed with 3% GCS solution, and then mixed with 4% DF-PEG solution. On the 1st and 5th days of culture, the cell survival and death in the hydrogel were tested by live/dead staining. Passage 2 adipose-derived mesenchymal stem cells from Sprague-Dawley neonatal rats were mixed with 3% GCS solution, and then mixed with 2%, 4% and 8% DF-PEG solution, respectively. MTT method was used for testing the cell proliferation.
RESULTS AND CONCLUSION: (1) The moduli of elasticity of GCS/DF-PEG hydrogel with 2%, 4%, 8% DF-PEG were 13.48, 22.21 and 33.19 kPa, respectively. (2) In vitro degradation experiments showed that GCS/DF-PEG hydrogels gradually degraded in PBS over time. And the degradation rate of the 2% DF-PEG hydrogel was significantly faster than the other two groups. (3) Within 7 days of culture, there was no difference in the cell proliferation between the experimental and control groups. (4) Live/dead staining results showed that adipose-derived mesenchymal stem cells were spherical in the hydrogel, the survival rate was over 90%, and the number of cells increased significantly with time. (5) Over time, the number of adipose-derived mesenchymal stem cells in the GCS/DF-PEG hydrogel with different mass fractions gradually increased. And the cell proliferation in the hydrogel containing 2% DF-PEG was faster than 4% and 8% groups (P < 0.05). In conclusion, the injectable GCS/DF-PEG hydrogel has good cytocompatibility, mechanical strength and degradation, and it is expected to be a good carrier for stem cell transplantation in cartilage tissue engineering.  

Key words: Hydrogel, Materials Testing, Cell Proliferation, Tissue Engineering

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