中国组织工程研究

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (34): 7333-7343.doi: 10.12307/2025.886

• 水凝胶材料Hydrogel materials • 上一篇    下一篇

聚多巴胺修饰还原石墨烯负载纳米银复合Gap19多肽水凝胶的制备及抗菌评价

李若彤1，2，3，吴悦宁1，2，3，邓云艺2，3，4，陈世超2，3，4，兰小蓉2，3，4，李适廷1，2，3，李广文2，3，4   

  1. 西南医科大学附属口腔医院，1牙体牙髓病科，4种植科，四川省泸州市   646000；2口颌面修复重建和再生泸州市重点实验室，四川省泸州市   646000；3西南医科大学口腔医学研究所，四川省泸州市 646000
  • 收稿日期:2024-09-18 接受日期:2024-10-11 出版日期:2025-12-08 发布日期:2025-01-17
  • 通讯作者: 李广文，博士在读，主治医师，西南医科大学附属口腔医院种植科，四川省泸州市 646000；口颌面修复重建和再生泸州市重点实验室，四川省泸州市 646000；西南医科大学口腔医学研究所，四川省泸州市 646000 李适廷，博士，副教授，西南医科大学附属口腔医院牙体牙髓病科，四川省泸州市 646000；口颌面修复重建和再生泸州市重点实验室，四川省泸州市 646000；西南医科大学口腔医学研究所，四川省泸州市 646000
  • 作者简介:李若彤，女，1998年生，山西省太原市人，汉族，硕士，主要从事水凝胶用作新型盖髓材料的研究。
  • 基金资助:
    四川省自然科学基金面上项目(2024NSFSC0680)，项目负责人：李广文；四川省科技厅重点研发项目(22YFS0634)，项目负责人：兰小蓉；泸州市科技局重点研发计划(面上)项目(2022-SYF-33)，项目负责人：李广文；西南医科大学自科重点项目(2022ZD015)，项目负责人：李广文；西南医科大学附属口腔医院重点项目(2022Z01)，项目负责人：李广文；四川省卫生健康委员会科技项目资助(24QNMP018)，项目负责人：李广文

Preparation and antibacterial evaluation of nanosilver-reduced graphene oxide/polydopamine/methacrylated gelatin@Gap19 hydrogel

Li Ruotong1, 2, 3, Wu Yuening1, 2, 3, Deng Yunyi2, 3, 4, Chen Shichao2, 3, 4, Lan Xiaorong2, 3, 4, Li Shiting1, 2, 3, Li Guangwen2, 3, 4   

  1. 1Department of Endodontics, 4Department of Implantology, Affiliated Stomatological Hospital, Southwest Medical University, Luzhou 646000, Sichuan Province, China; 2Luzhou Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Luzhou 646000, Sichuan Province, China; 3Institute of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Received:2024-09-18 Accepted:2024-10-11 Online:2025-12-08 Published:2025-01-17
  • Contact: Li Guangwen, Doctoral candidate, Attending physician, Luzhou Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Luzhou 646000, Sichuan Province, China; Institute of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China; Department of Implantology, Affiliated Stomatological Hospital, Southwest Medical University, Luzhou 646000, Sichuan Province, China Li Shiting, MD, Associate professor, Department of Endodontics, Affiliated Stomatological Hospital, Southwest Medical University, Luzhou 646000, Sichuan Province, China; Luzhou Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Luzhou 646000, Sichuan Province, China; Institute of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • About author:Li Ruotong, MS, Department of Endodontics, Affiliated Stomatological Hospital, Southwest Medical University, Luzhou 646000, Sichuan Province, China; Luzhou Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Luzhou 646000, Sichuan Province, China; Institute of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Supported by:
    Sichuan Provincial Natural Science Foundation (General Project), No. 2024NSFSC0680 (to LGW); Key Research and Development Project of Sichuan Provincial Science and Technology Department, No. 22YFS0634 (to LXR); Key Research and Development Plan of Luzhou Science and Technology Bureau (surface), No. 2022-SYF-33 (to LGW); Key Natural Science Project of Southwest Medical University, No. 2022ZD015 (to LGW); Hospital Level Key Project of Stomatology Hospital Affiliated to Southwest Medical University, No. 2022Z01 (to LGW); Science and Technology Project of Health Commission of Sichuan Province, No. 24QNMP018 (to LGW)

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摘要：


文题释义：

纳米银-还原氧化石墨烯：纳米银具有强效的抑菌和广谱杀菌能力，被广泛应用于生物医学领域，并被誉为“21世纪的先进材料”。还原氧化石墨烯是部分氧化还原的石墨烯产物，保留了石墨烯的强力学性能，并具有丰富的官能团，因此常用于制备抗菌水凝胶。将纳米银与还原氧化石墨烯按一定比例混合，超声2 h后形成均匀混悬液纳米银-还原氧化石墨烯，可有效提高材料的抗菌性。
Gap19：是由连接蛋白43胞质环衍生的9个氨基酸组成的多肽，通过诱导连接蛋白43的降解来阻断细胞间的交换，是连接蛋白43半通道的特异性抑制剂。生理状况下，连接蛋白43半通道处于闭合状态以防止ATP等丢失及钙离子内流等，而当机体处于炎症等病理状态时，连接蛋白43半通道被激活，可能促进疾病发展。课题组研究显示，Gap19能够抑制人牙髓细胞的炎症反应并促进其成牙本质分化和矿化。


背景：活髓保存疗法是治疗深龋等口腔常见疾病的主要疗法之一，盖髓材料的抗菌性能对疗效至关重要。目前常用的盖髓材料抗菌性较弱并会激起牙髓组织一定的炎症反应，从而导致治疗失败。

目的：探讨纳米银-还原氧化石墨烯/聚多巴胺/甲基丙烯酰化明胶@Gap19水凝胶材料的抗菌作用。
方法：制备纳米银-还原氧化石墨烯。使用含不同质量浓度纳米银-还原氧化石墨烯的完全培养基培养人牙髓干细胞，采用CCK-8实验检测细胞增殖。采用抑菌圈实验检测不同质量浓度纳米银-还原氧化石墨烯对金黄色葡萄球菌的抑制作用。通过CCK-8与抑菌圈实验结果筛选促细胞增殖与抑菌效果最明显的纳米银-还原氧化石墨烯质量浓度，负载于水凝胶中。使用含不同浓度Gap19(一种针对连接蛋白43半通道的特异性抑制剂)的完全培养基培养人牙髓干细胞，采用CCK-8实验检测细胞增殖，筛选促细胞增殖效果最明显的Gap19浓度，负载于水凝胶中。制备纳米银-还原氧化石墨烯/聚多巴胺/甲基丙烯酰化明胶@Gap19(AgNPs-rGO/PDA/GelMA@Gap19)水凝胶，表征该水凝胶材料的理化性质。将金黄色葡萄球菌(或大肠杆菌、变异链球菌、乳酸杆菌)悬液分别与三氧化矿物凝聚体、聚多巴胺/甲基丙烯酰化明胶水凝胶、纳米银-还原氧化石墨烯/聚多巴胺/甲基丙烯酰化明胶水凝胶与AgNPs-rGO/PDA/GelMA@Gap19水凝胶共培养，以单独培养的细菌悬液为空白对照组，检测各组水凝胶的抗菌率。

结果与结论：①通过CCK-8与抑菌圈实验结果确定纳米银-还原氧化石墨烯的最佳质量浓度为12.5 μg/mL，通过CCK-8实验确定Gap19的最佳浓度为20 μmol/L；②扫描电镜下可见AgNPs-rGO/PDA/GelMA@Gap19水凝胶呈褶皱多孔状，纳米银在水凝胶表面及内部均有负载；能谱分析结果显示纳米银-还原氧化石墨烯与Gap19成功负载至水凝胶上；③4种水凝胶对金黄色葡萄球菌、大肠杆菌、变异链球菌、乳酸杆菌均具有明显的抑制作用，并且以纳米银-还原氧化石墨烯/聚多巴胺/甲基丙烯酰化明胶水凝胶与AgNPs-rGO/PDA/GelMA@Gap19水凝胶的抗菌作用最显著，说明AgNPs-rGO/PDA/GelMA@Gap19水凝胶作为一种新型盖髓材料具备明显的抗菌作用。

https://orcid.org/0009-0003-3823-307X (李若彤) 

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: 纳米银, 还原氧化石墨烯, Gap19, 甲基丙烯酰化明胶, 水凝胶, 抗菌性, 工程化口腔材料

Abstract: BACKGROUND: Vital pulp therapy is one of the main treatments for common oral diseases such as deep caries. The antibacterial properties of pulp-capping materials are crucial to the efficacy of the treatment. Currently, commonly used pulp-capping material has weak antibacterial properties and may induce a certain degree of inflammatory response in the pulp tissue, leading to treatment failure. 
OBJECTIVE: To investigate the antibacterial effects of nanosilver-reduced graphene oxide/polydopamine/methacrylated gelatin@Gap19 (AgNPs-rGO/PDA/GelMA@Gap19) hydrogel material.
METHODS: Nanosilver-reduced graphene oxide was prepared. Human dental pulp stem cells were cultured in complete medium containing different mass concentrations of nanosilver-reduced graphene oxide. Cell proliferation was detected by CCK-8 assay. The inhibition zone assay was used to detect the inhibitory effect of different mass concentrations of nanosilver-reduced graphene oxide on Staphylococcus aureus. The nanosilver-reduced graphene oxide mass concentration with the most obvious cell proliferation and antibacterial effects was screened by the results of CCK-8 and inhibition zone assays, and loaded into hydrogels. Human dental pulp stem cells were cultured in complete medium containing different concentrations of Gap19 (a specific inhibitor of connexin 43 hemichannels), and cell proliferation was detected by CCK-8 assay. The Gap19 concentration with the most obvious cell proliferation effect was screened and loaded into hydrogels. AgNPs-rGO/PDA/GelMA@Gap19 hydrogel was prepared, and the physicochemical properties of the hydrogel material were characterized. The suspension of Staphylococcus aureus (or Escherichia coli, Streptococcus mutans, Lactobacillus) was co-cultured with mineral trioxide aggregates, polydopamine/methacrylated gelatin hydrogel, nanosilver-reduced graphene oxide/polydopamine/methacrylated gelatin hydrogel and AgNPs-rGO/PDA/GelMA@Gap19 hydrogel. The bacterial suspension cultured alone was used as the blank control group to detect the antibacterial rate of each group of hydrogels. 
RESULTS AND CONCLUSION: (1) The optimal mass concentration of nanosilver-reduced graphene oxide was determined to be 12.5 μg/mL by CCK-8 assay and inhibition zone test results, and the optimal concentration of Gap19 was determined to be 20 μmol/L by CCK-8 assay. (2) Scanning electron microscopy showed that AgNPs-rGO/PDA/GelMA@Gap19 hydrogel was wrinkled and porous, and nanosilver was loaded on the surface and inside of the hydrogel. Energy spectrum analysis results showed that nanosilver-reduced graphene oxide and Gap19 were successfully loaded on the hydrogel. (3) The four hydrogels had obvious inhibitory effects on Staphylococcus aureus, Escherichia coli, Streptococcus mutans, and Lactobacillus, and the antibacterial effects of nanosilver-reduced graphene oxide/polydopamine/methacryloyl gelatin hydrogel and AgNPs-rGO/PDA/GelMA@Gap19 hydrogel were the most significant, indicating that AgNPs-rGO/PDA/GelMA@Gap19 hydrogel, as a new type of pulp capping material, has an obvious antibacterial effect.

Key words: silver nanoparticle, reduced graphene oxide, Gap19, methacryloyl gelatin, hydrogel, antibacterial property, engineered dental material

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