PLG-g-TA/RGD酶催化交联水凝胶用于透明软骨细胞黏附和三维细胞培养

doi:10.7503/cjcu20190148

高等学校化学学报 ›› 2019, Vol. 40 ›› Issue (9): 2020.doi: 10.7503/cjcu20190148

PLG-g-TA/RGD酶催化交联水凝胶用于透明软骨细胞黏附和三维细胞培养

文静^1,²,徐志民¹,齐德胜^2,³,王佳玉^2,³,于双江^2,^*(),贺超良²,韩冰¹()

^1. 吉林大学口腔医院, 长春 130021
^2. 中国科学院长春应用化学研究所, 生态环境高分子材料重点实验室, 长春 130022
^3. 长春理工大学化学与环境工程学院, 长春 130022

收稿日期:2019-03-11 出版日期:2019-09-10 发布日期:2019-07-16
通讯作者: 于双江 E-mail:yushuangjiang@ciac.ac.cn;hbing@jlu.edu.cn
基金资助:
国家自然科学基金(81771041);吉林省发展和改革委员会项目(2018C052-12);吉林省科技发展计划项目(批准号: )资助.(20180201053YY)

PLG-g-TA/RGD Enzyme-catalyzed Crosslinked Hydrogel for Adhesion and Three-dimensional Culture of Hyaline Chondrocytes ^†

WEN Jing^1,²,XU Zhimin¹,QI Desheng^2,³,WANG Jiayu^2,³,YU Shuangjiang^2,^*(),HE Chaoliang²,HAN Bing¹()

^1. Stomatology Hospital, Jilin University, Changchun 130021, China
^2. Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry,Chinese Academy of Sciences, Changchun 130022, China
^3. School of Chemistry and Environmental Engineering, Changchun University of Science and Technology, Changchun 130022, China

Received:2019-03-11 Online:2019-09-10 Published:2019-07-16
Contact: YU Shuangjiang E-mail:yushuangjiang@ciac.ac.cn;hbing@jlu.edu.cn
Supported by:
? Supported by the National Natural Science Foundation of China(81771041);the Jilin Provincial Development and Reform Commission Project, China(2018C052-12);the Jilin Province Science and Technology Development Plan Project, China(20180201053YY)

摘要/Abstract

摘要：

制备了一种基于聚谷氨酸-g-酪胺/cRGDfk(PLG-g-TA/RGD)的新型酶催化交联水凝胶, 用于兔透明软骨细胞黏附和三维细胞的培养. PLG-g-TA/RGD聚合物材料在辣根过氧化物酶(HRP)和过氧化氢(H₂O₂)存在下, 能够通过酪氨基团的自交联快速形成水凝胶. 环状多肽(cRGDfk)的引入能够显著提高材料的溶液-凝胶转变速率和凝胶强度. 透明软骨细胞在水凝胶表面黏附3 d后, 在PLG-g-TA/RGD水凝胶表面有更多的细胞黏附; 将透明软骨细胞包裹在水凝胶内培养1, 4, 7 d后, 细胞在PLG-g-TA/RGD水凝胶内增殖效率明显高于对照组PLG-g-TA水凝胶. 细胞实验结果表明, 该水凝胶材料具有良好的生物相容性. cRGDfk的引入, 促进了透明软骨细胞的黏附和增殖, 显示了PLG-g-TA/RGD水凝胶材料在三维细胞培养方面的应用潜力.

关键词: 环状多肽, 酶催化交联, 细胞黏附, 三维细胞培养, 聚谷氨酸-g-酪胺

Abstract:

A novel enzyme-catalyzed cross-linking hydrogel based on poly(L-glutamic acid)-g-tyramine/cRGDfk cyclic peptide(PLG-g-TA/RGD) was prepared for the investigation of adhesion and the three-dimensional culture of hyaline chondrocytes. PLG-g-TA/RGD polymers could form a hydrogel by self-crosslinking of the tyrosine group rapidly in the presence of horseradish peroxidase(HRP) and hydrogen peroxide(H₂O₂). The further research results showed that both the gelation time and gel mechanical strength were significantly enhance with the presence of cRGDfk. More hyaline chondrocytes adhered to the surface of the cRGDfk containing hydrogel compared to the control group after incubation of 3 d. The proliferation efficiency of hyaline chondrocyte in PLG-g-TA/RGD hydrogel was significantly higher than PLG-g-TA formed hydrogel after culture with 1, 4 and 7 d, respectively. The results of the cells indicated that the obtained materials exhibited a good cell compatibility. Above all, the introduction of cRGDfk promotes the adhesion and proliferation of hyaline chondrocytes, and showed the potential application of the original PLG-g-TA/RGD hydrogel in the field of three-dimensional cell culture.

Key words: Cyclicpeptide, Enzyme-catalyzed cross-linking, Cell adhesion, Three-dimensional cell culture, Poly(L-glutamic acid)-g-tyramine

中图分类号:

O631

TrendMD:

文静, 徐志民, 齐德胜, 王佳玉, 于双江, 贺超良, 韩冰. PLG-g-TA/RGD酶催化交联水凝胶用于透明软骨细胞黏附和三维细胞培养. 高等学校化学学报, 2019, 40(9): 2020.

WEN Jing, XU Zhimin, QI Desheng, WANG Jiayu, YU Shuangjiang, HE Chaoliang, HAN Bing. PLG-g-TA/RGD Enzyme-catalyzed Crosslinked Hydrogel for Adhesion and Three-dimensional Culture of Hyaline Chondrocytes ^†. Chem. J. Chinese Universities, 2019, 40(9): 2020.

图/表 10

Scheme 1 Schematic diagram of enzyme-catalyzed crosslinking network of PLG-g-TA/RGD hydrogels

Scheme 2 Synthetic routes of the PLG-g-TA and PLG-g-TA/RGD

Table 1 Summary of various parameters of the polymer materials

Sample	PLG degree of polymerization	TA content (%)	RGD grafting rate(%)
PLG-g-TA	90	9.4	0
PLG-g-TA/RGD	90	9.4	0.8

Fig.1 1H NMR spectra of the polymer a. BLG-NCA, CDCl3-d; b. PBLG, TFA-d; c. PLG, D2O; d. PLG-g-TA, D2O; e. PLG-g-TA/RGD, D2O. a—k are the peaks corresponding to the positions in Scheme 2.

Fig.2 Gel-forming photographs of PLG-g-TA(A) and PLG-g-TA/RGD(B) polymer solution after enzyme-catalyzed reaction

Fig.3 Gelation time of PLG-g-TA/RGD polymers solution(HRP concentration was fixed at 3.2 Unit/mL as the polymer concentration changes, and the molar ratio of H2O2 to TA was fixed 0.4)(A), PLG-g-TA polymers solution(as the polymer concentration changes, the HRP concentration was fixed at 3.2 Unit/mL, and the molar ratio of H2O2 to TA was fixed at 0.4)(B) and rheological diagram of PLG-g-TA/RGD and PLG-g-TA hydrogels(the final concentration of the polymer was 40.0 g/L, the HRP concentration was fixed at 3.2 Unit/mL, and the molar ratio of H2O2 to TA was fixed at 0.4)(C)

Fig.4 P1 generation of rabbit hyaline chondrocytes

Fig.5 Cell compatibility of the co-culture of the hydrogel leaching solution and L929 cells for 48 h(n=3)(A), proliferation of hyaline chondrocytes cells in hydrogel(n=3)(B) * P<0.05; ** P<0.01; *** P<0.001.

Fig.6 Adhesion behavior of cells on the surface of PLG-g-TA hydrogel(A) and adhesion behavior of cells on the PLG-g-TA/RGD hydrogel(B)

Fig.7 Staining of chondrocytes cultured in hydrogels for 7 d Live cells were stained green by Calcein-AM, dead cells were dyed red by PI. (A) Chondrocytes survived inside the PLG-g-TA/RGD hydrogel; (B) chondrocytes survived within the PLG-g-TA hydrogel.

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PLG-g-TA/RGD酶催化交联水凝胶用于透明软骨细胞黏附和三维细胞培养

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PLG-g-TA/RGD酶催化交联水凝胶用于透明软骨细胞黏附和三维细胞培养

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PLG-g-TA/RGD Enzyme-catalyzed Crosslinked Hydrogel for Adhesion and Three-dimensional Culture of Hyaline Chondrocytes ^†