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北京大学学报（医学版） ›› 2022, Vol. 54 ›› Issue (3): 557-564. doi: 10.19723/j.issn.1671-167X.2022.03.024

• 论著 • 上一篇    下一篇

不同交联剂处理对脱细胞小肠黏膜下层多孔支架的影响

邓艺1,张一2,李博文1,王梅1,唐琳1,刘玉华1,*()   

  1. 1. 北京大学口腔医学院·口腔医院修复科，国家口腔医学中心，国家口腔疾病临床医学研究中心，口腔生物材料和数字诊疗装备国家工程研究中心，口腔数字医学北京市重点实验室，国家卫生健康委员会口腔医学计算机应用工程技术研究中心，国家药品监督管理局口腔生物材料重点实验室，北京 100081
    2. 北京大学口腔医学院·口腔医院综合二科，国家口腔医学中心，国家口腔疾病临床医学研究中心，口腔生物材料和数字诊疗装备国家工程研究中心，口腔数字医学北京市重点实验室，国家卫生健康委员会口腔医学计算机应用工程技术研究中心，国家药品监督管理局口腔生物材料重点实验室，北京 100081
  • 收稿日期:2020-10-10 出版日期:2022-06-18 发布日期:2022-06-14
  • 通讯作者: 刘玉华 E-mail:liuyuhua@bjmu.edu.cn
  • 基金资助:
    国家自然科学基金(81801027)

Effects of different crosslinking treatments on the properties of decellularized small intestinal submucosa porous scaffolds

Yi DENG1,Yi ZHANG2,Bo-wen LI1,Mei WANG1,Lin TANG1,Yu-hua LIU1,*()   

  1. 1. Department of Prosthodontics, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices & Beijing Key Laboratory of Digital Stomatology & NHC Research Center of Engineering and Technology for Computerized Dentistry & NMPA Key Laboratory for Dental Materials, Beijing 100081, China
    2. Department of General Dentistry Ⅱ, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices & Beijing Key Laboratory of Digital Stomatology & NHC Research Center of Engineering and Technology for Computerized Dentistry & NMPA Key Laboratory for Dental Materials, Beijing 100081, China
  • Received:2020-10-10 Online:2022-06-18 Published:2022-06-14
  • Contact: Yu-hua LIU E-mail:liuyuhua@bjmu.edu.cn
  • Supported by:
    the National Natural Science Foundation of China(81801027)

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摘要：

目的: 探讨不同交联剂处理对脱细胞小肠黏膜下层(decellularized small intestinal submucosa, SIS)多孔支架理化性能及生物相容性的影响。方法: (1) 将新鲜SIS塑形为三维多孔支架并采用戊二醛(glutaraldehyde, GA)、碳二亚胺[1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, EDC]和原花青素(procyanidine, PA)3种交联剂进行交联, 获得3组交联后支架, 分别为GA组、EDC组和PA组。(2)理化性能评价: 对3组多孔支架进行宏观形貌观察, 使用场发射环境扫描电镜进行微观形貌观察并测定孔径及孔隙率, 使用茚三酮法测定交联度, 使用酶促降解法评价抗降解性能, 使用万能力学测试机测定应力应变曲线及压缩强度。(3)生物相容性评价: 使用细胞计数试剂盒(cell counting kit-8, CCK-8)法检测交联处理对人骨髓间充质干细胞(human bone marrow mesenchymal stem cells, hBMSCs)增殖的影响, 使用Calcein-AM/PI活死细胞染色法评价交联处理后支架的细胞毒性。结果: 宏观形貌观察可见交联修饰未破坏支架三维结构; 微观形貌观察可见各组多孔支架具有大小均匀、相互连通的孔隙结构。EDC组孔径最大, 与另两组差异具有统计学意义(P < 0.05);PA组孔隙率最低, 与另两组比较差异有统计学意义(P < 0.05)。PA组交联度最高而溶胀率最低, EDC组溶胀率最高。EDC组和GA组的体外降解速率均高于PA组, 其中GA组降解速度最快, PA组抗降解能力最佳, 降解至第15天时3组质量丧失率间的差异有统计学意义(P < 0.05)。EDC组和GA组压缩强度近似, PA组压缩强度最高, 与另两组比较差异有统计学意义(P < 0.05)。GA组支架的细胞毒性最大, hBMSCs在EDC组和PA组支架上具有更好的增殖能力, 在GA组增殖较慢。结论: 3种交联剂处理均可达到较高的交联度, 经EDC和PA交联处理的SIS多孔支架在拥有较好理化性能的同时具有良好的生物相容性, 相比于GA是更有应用前景的交联处理方法。

关键词: 脱细胞小肠黏膜下层, 多孔支架, 戊二醛, 碳二亚胺, 原花青素

Abstract:

Objective: To compare the effects of three different crosslinkers on the biocompatibility, physical and chemical properties of decellularized small intestinal submucosa (SIS) porous scaffolds. Methods: The SIS porous scaffolds were prepared by freeze-drying method and randomly divided into three groups, then crosslinked by glutaraldehyde (GA), 1-ethyl-3-(3-dimethylaminopropyl) carbodi-imide (EDC) and procyanidine (PA) respectively. To evaluate the physicochemical property of each sample in different groups, the following experiments were conducted. Macroscopic morphologies were observed and recorded. Microscopic morphologies of the scaffolds were observed using field emission scanning electron microscope (FESEM) and representative images were selected. Computer software (ImageJ) was used to calculate the pore size and porosity. The degree of crosslinking was determined by ninhydrin experiment. Collagenase degradation experiment was performed to assess the resistance of SIS scaffolds to enzyme degradation. To evaluate the mechanical properties, universal mechanical testing machine was used to determine the stress-strain curve and compression strength was calculated. Human bone marrow mesenchymal cells (hBMSCs) were cultured on the scaffolds after which cytotoxicity and cell proliferation were assessed. Results: All the scaffolds remained intact after different crosslinking treatments. The FESEM images showed uniformed interconnected micro structures of scaffolds in different groups. The pore size of EDC group[(161.90±13.44) μm] was significantly higher than GA group [(149.50±14.65) μm] and PA group[(140.10±12.06) μm] (P < 0.05). The porosity of PA group (79.62%±1.14%) was significantly lower than EDC group (85.11%±1.71%) and GA group (84.83%±1.89%) (P < 0.05). PA group showed the highest degree of crosslinking whereas the lowest swelling ratio. There was a significant difference in the swelling ratio of the three groups (P < 0.05). Regarding to the collagenase degradation experiment, the scaffolds in PA group showed a significantly lower weight loss rate than the other groups after 7 days degradation. The weight loss rates of GA group were significantly higher than those of the other groups on day 15, whereas the PA group had the lowest rate after 10 days and 15 days degradation. PA group showed better mechanical properties than the other two groups. More living cells could be seen in PA and EDC groups after live/dead cell staining. Additionally, the proliferation rate of hBMCSs was faster in PA and EDC groups than in GA group. Conclusion: The scaffolds gained satisfying degree of crosslinking after three different crosslinking treatments. The samples after PA and EDC treatment had better physicochemical properties and biocompatibility compared with GA treatment. Crosslinking can be used as a promising and applicable method in the modification of SIS scaffolds.

Key words: Decellularized small intestinal submucosa, Porous scaffolds, Glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, Procyanidine

中图分类号: 

  • R783.1

图1

交联后SIS多孔支架的宏观形貌"

图2

交联后SIS多孔支架表面微观及横截面微观形貌"

图3

交联后SIS多孔支架的孔径及孔隙率"

图4

交联后SIS多孔支架的交联度"

图5

交联后SIS多孔支架的溶胀率"

图6

不同交联处理后SIS多孔支架的质量丧失率"

图7

第7、10、15天时不同交联处理后SIS多孔支架在降解液中的大体观"

图8

不同交联处理后SIS多孔支架的压缩强度和应力应变曲线"

图9

在不同交联处理后SIS多孔支架上培养hBMSCs 24 h后活死细胞染色图像"

图10

hBMSCs在不同交联处理后的SIS多孔支架上的增殖曲线"

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