具有促进成骨及抗菌双重作用的新型碳点的制备及在感染性骨缺损中的应用评价

doi:10.19438/j.cjoms.2023.01.002

中国口腔颌面外科杂志 ›› 2023, Vol. 21 ›› Issue (1): 11-18.doi: 10.19438/j.cjoms.2023.01.002

具有促进成骨及抗菌双重作用的新型碳点的制备及在感染性骨缺损中的应用评价

刘一戈^1,2, 王梓霖¹, 郭陟永^1,2, 吴昊^1,3, 汪文颖⁴, 韩婧¹, 刘剑楠¹, 张陈平¹

1.上海交通大学医学院附属第九人民医院口腔颌面-头颈肿瘤科,上海交通大学口腔医学院, 国家口腔医学中心,国家口腔疾病临床医学研究中心,上海市口腔医学重点实验室, 上海市口腔医学研究所,上海 200011;
2.上海市组织工程重点实验室,上海 200011;
3.上海理工健康科学与工程学院,上海 200093;
4.上海交通大学口腔医学院,上海 200125

收稿日期:2022-08-09 修回日期:2022-11-02 出版日期:2023-01-20 发布日期:2023-06-12
通讯作者: 张陈平,E-mail: zhang.chenping@hotmail.com
作者简介:刘一戈（1997-）,女,硕士,E-mail: saltylight@sina.com

Synthesis of carbon dots with dual roles of osteogenic and antibacterial effects for infected bone defects treatment

LIU Yi-ge, WANG Zi-lin, GUO Zhi-yong, WU Hao, WANG Wen-ying, HAN Jing, LIU Jian-nan, ZHANG Chen-ping

1. Department of Oral Maxillofacial Head and Neck Oncology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology; Shanghai Research Institute of Stomatology. Shanghai 200011;
2. Shanghai Key Laboratory of Tissue Engineering Research. Shanghai 200011;
3. School of Health Science and Engineering, University of Shanghai for Science and Technology. Shanghai 200093;
4. College of Stomatology, Shanghai Jiao Tong University. Shanghai 200125, China

Received:2022-08-09 Revised:2022-11-02 Online:2023-01-20 Published:2023-06-12

摘要/Abstract

摘要： 目的： 探讨以庆大霉素和叶酸作为原料的新型碳点（carbon dots,CDots）对大鼠骨髓间充质干细胞（bone marrow stromal cells,BMSCs）成骨分化、自噬作用的影响,以及对口腔常见致病菌的抑制作用。方法： 以庆大霉素和叶酸作为原料,水热法制备CDots,通过透射电镜、紫外分光光度计进行表征。利用CCK-8实验及细胞凋亡实验评价生物相容性,激光共聚焦显微镜观察被细胞摄取情况。以碱性磷酸酶染色、茜素红染色检测对BMSCs成骨分化的影响,实时定量PCR检测成骨、自噬相关基因的mRNA表达水平。扫描电镜、平板菌落实验观察CDots对牙龈卟啉单胞菌、具核梭杆菌抑菌能力。采用Graph Pad Prism 9软件包对数据进行统计学分析。结果： CDots溶液在紫外光照射下发出蓝色荧光,在330 nm处有紫外吸收峰;在405 nm激光激发下,CDots的PL发射峰集中在440 nm处。透射电镜观察到CDots平均直径约为12 nm。CCK-8和细胞凋亡检测结果显示CDots生物相容性良好。激光共聚焦显微镜结果显示CDots可被BMSCs摄取。碱性磷酸酶染色、茜素红染色结果显示CDots能使染色增加。CDots能使成骨相关基因OPN、OCN、BMP-2,以及自噬相关基因LC3、Beclin1、ATG-12的mRNA表达较对照组升高（P<0.05）。CDots能使细菌数量、菌落数量较对照组显著减少（P<0.05）。结论： 以庆大霉素和叶酸作为原料制备的CDots生物相容性好,具有成骨抗菌双重作用,能促进BMSCs成骨分化,促进自噬水平升高,并且具有抑制牙龈卟啉单胞菌、具核梭杆菌生长的作用。

关键词: 碳点, 骨生成, 抗菌, 自噬

Abstract: PURPOSE: To investigate the effects of a new kind of carbon dots (CDots) made from gentamicin and folic acid on osteogenic differentiation and autophagy of rat bone marrow stromal cells (BMSCs), as well as the inhibition of common oral pathogenic bacteria. METHODS: CDots were prepared by hydrothermal method using gentamicin and folic acid as raw materials, and characterized by transmission electron microscopy and UV spectrophotometer. Biocompatibility was evaluated by CCK-8 assay and apoptosis assay, and uptake by cells was observed by laser confocal microscopy. Alkaline phosphatase staining and alizarin red staining were used to detect the effect on osteogenic differentiation of BMSCs. Real-time quantitative PCR was performed to detect the mRNA expression levels of osteogenic and autophagy-related genes. Scanning electron microscopy and plate colony assay were performed to observe the ability of CDots to inhibit Porphyromonas gingivalis and Fusobacterium nucleatum. Graph Pad Prism 9 software was used for statistical analysis. RESULTS: The CDots solution emitted blue fluorescence under UV light irradiation with a UV absorption peak at 330 nm; the PL emission peak of CDots was concentrated at 440 nm under the excitation of 405 nm laser. Transmission electron microscopy showed that the average diameter of CDots was about 12 nm. CCK-8 assay and apoptosis assay showed that CDots were biocompatible. Laser confocal microscopy results showed that CDots could be taken up by BMSCs. The results of alkaline phosphatase staining and alizarin red staining showed that CDots increased staining. mRNA expression of the osteogenic genes OPN, OCN, BMP-2, and autophagy-related genes LC3, Beclin1, ATG-12 was increased by CDots compared to the control group(P<0.05). In vitro antibacterial assays indicated that CDots could inhibit the growth of Porphyromonas gingivalis and Fusobacterium nucleatum. CONCLUSIONS: CDots prepared with gentamicin and folic acid are biocompatible, have dual osteogenic and antibacterial effects, promote osteogenic differentiation of BMSCs, promote elevated autophagy levels, and have inhibitory effects on the growth of Porphyromonas gingivalis and Fusobacterium nucleatum.

Key words: Carbon Dots, Osteogenesis, Anti-Bacterial Agents, Autophagy

中图分类号:

R318.08

刘一戈, 王梓霖, 郭陟永, 吴昊, 汪文颖, 韩婧, 刘剑楠, 张陈平. 具有促进成骨及抗菌双重作用的新型碳点的制备及在感染性骨缺损中的应用评价[J]. 中国口腔颌面外科杂志, 2023, 21(1): 11-18.

LIU Yi-ge, WANG Zi-lin, GUO Zhi-yong, WU Hao, WANG Wen-ying, HAN Jing, LIU Jian-nan, ZHANG Chen-ping. Synthesis of carbon dots with dual roles of osteogenic and antibacterial effects for infected bone defects treatment[J]. China J Oral Maxillofac Surg, 2023, 21(1): 11-18.

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具有促进成骨及抗菌双重作用的新型碳点的制备及在感染性骨缺损中的应用评价

Synthesis of carbon dots with dual roles of osteogenic and antibacterial effects for infected bone defects treatment

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