国际口腔医学杂志

国际口腔医学杂志 ›› 2024, Vol. 51 ›› Issue (5): 596-607.doi: 10.7518/gjkq.2024054

• 论著 • 上一篇    下一篇

载黄连素的同轴静电纺丝膜对牙周致病菌及生物膜的抑菌性研究

张睿1(),郝婷1,吕闻1,任双双2,刘玉3,吴文蕾3,孙卫斌1()   

  1. 1.南京大学医学院附属口腔医院 南京市口腔医院牙周病科 南京大学口腔医学研究所 南京 210008
    2.南京大学医学院附属口腔医院;南京市口腔医院牙体牙髓病科;南京大学口腔医学研究所 南京 210008
    3.南京大学医学院附属口腔医院;南京市口腔医院高级专家诊疗科;南京大学口腔医学研究所 南京 210008
  • 收稿日期:2024-01-25 修回日期:2024-06-13 出版日期:2024-09-01 发布日期:2024-09-14
  • 通讯作者: 孙卫斌
  • 作者简介:张睿，住院医师，硕士，Email：zzzrrr2008@163.com
  • 基金资助:
    国家自然科学基金面上项目(51972167)

Antibacterial property of berberine-loaded coaxial electrospun membranes against periodontal pathogens and biofilms

Rui Zhang1(),Ting Hao1,Lü Wen1,Shuangshuang Ren2,Yu Liu3,Wenlei Wu3,Weibin Sun1()   

  1. 1.Dept. of Periodontics, Nanjing Stomatological Hospital, Affiliated Hospital of Medical School, Research Institute of Stomatology, Nanjing University, Nanjing 210008, China
    2.Dept. of Cariology and Endodontics, Nanjing Stomatological Hospital, Affiliated Hospital of Medical School, Research Institute of Stomatology, Nanjing University, Nanjing 210008, China
    3.Dept. of Senior Specialist, Nanjing Stomatological Hospital, Affiliated Hospital of Medical School, Research Institute of Stomatology, Nanjing University, Nanjing 210008, China
  • Received:2024-01-25 Revised:2024-06-13 Online:2024-09-01 Published:2024-09-14
  • Contact: Weibin Sun
  • Supported by:
    National Natural Science Foundation of China(51972167)

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摘要：

目的 制备并表征负载黄连素的同轴静电纺丝膜，评价其体外抑制牙周致病菌和生物膜的作用。 方法 采用同轴静电纺丝法制备负载黄连素的聚己内酯/明胶膜（PCL/Gel-BBR）并对其进行表征检测，黄连素负载质量分数分别为0.25%、0.5%、1.0%。通过抑菌试验评价载药膜对牙龈卟啉单胞菌（P. gingivalis，ATCC 33277）及具核梭杆菌（F. nucleatum，ATCC 25586）增殖的抑制作用。采用结晶紫染色法、苯酚-硫酸法、菌落计数法、活/死细菌染色等综合评价载药膜对P. gingivalis和F. nucleatum双菌种生物膜的影响。采用CCK-8法和共聚焦显微镜检测载药膜的细胞毒性。 结果 同轴黄连素载药膜理化性能良好且能在体外稳定释放黄连素。1.0%黄连素载药膜可以有效抑制P. gingivalis和F. nucleatum的增殖，对双菌种生物膜的形成和活性也有抑制作用，且以上效应呈现浓度依赖性。黄连素载药膜体外生物相容性良好。 结论 负载黄连素的同轴静电纺丝膜具备体外抑制牙周致病菌进而抑制生物膜形成的作用，生物相容性良好，有望用于牙周药物递送以辅助牙周炎治疗或预防牙周术后感染。

关键词: 黄连素, 同轴静电纺丝, 牙龈卟啉单胞菌, 生物膜

Abstract:

Objective　This study aimed to prepare berberine-loaded coaxial electrospun membranes, evaluate their antibacterial effects against periodontal pathogens, and explore their inhibitory effect on biofilm formation in vitro. Me-thods　Coaxial polycaprolactone/gelatin electrospun membranes loaded with berberine (drug loading capacity of 0.25%, 0.5% and 1.0%, respectively) were fabrica-ted and characterized. The bacteriostatic zone test was used to evaluate the antibacterial effect against Porphyromonas gingivalis (ATCC 33277) and Fusobacterium nucleatum (ATCC 25586). Crystal violet staining, phenol-sulfuric acid method, colony counting method, and live/dead cell staining were used to comprehensively evaluate the inhibitory effect of drug-loaded electrospun membranes on biofilm formation. The cell counting kit-8 and confocal microscopy me-thods was used to evaluate the biocompatibility of electrospun membranes. Results　The berberine-loaded electrospun membranes had stable physicochemical properties, and they could release berberine in vitro. The membranes (drug loa-ding capacity of 1.0%) exhibited efficient antibacterial activity against Porphyromonas gingivalis and Fusobacterium nucleatum, and inhibited the formation of biofilms in a concentration-dependent manner. The berberine-loaded membranes had good biocompatibility in vitro. Conclusion　The berberine-loaded coaxial electrospun membranes were biocompatible, and they had an antibacterial potential against periodontal pathogens and biofilm formation in vitro. Therefore, it was promising to use berberine-loaded membranes as adjunctive therapy or to prevent postoperative infection.

Key words: berberine, coaxial electrospinning, Porphyromonas gingivalis, biofilm

中图分类号: 

  • R781.4

图1

PCL/Gel和PCL/Gel-BBR-H纤维膜的表征A：PCL/Gel纤维膜 SEM × 20 000；B：PCL/Gel纤维膜大体观察；C：PCL/Gel-BBR-H纤维膜 SEM × 20 000；D：PCL/Gel-BBR-H纤维膜大体观察；E：PCL/Gel-BBR-H 纤维膜同轴结构 TEM × 100 000；F：PCL/Gel的X射线能谱分析；G：水接触角；H：应力-应变曲线；I：XRD图谱；J：傅里叶红外光谱图谱。"

图2

载药膜的黄连素释放曲线"

图3

载药膜体外抑菌效果A：P. gingivalis平板的抑菌圈，左为PCL/Gel膜，右为PCL/Gel-BBR-H膜；B：P. gingivalis抑菌圈直径；C：不同质量分数载药膜与P. gingivalis共培养后的OD值； D：F. nucleatum平板的抑菌圈，左为PCL/Gel膜，右为PCL/Gel-BBR-H膜；E：F. nucleatum抑菌圈直径；F：不同质量分数载药膜与F. nucleatum共培养后的OD值。Control：对照（无纤维膜）组；ns：P>0.05；**：P<0.01；***：P<0.001。"

图4

载药膜与P. gingivalis和F. nucleatum共培养下的细菌生物膜生物量A：对照组；B：PCL/Gel；C：PCL/Gel-BBR-L；D：PCL/Gel-BBR-M；E：PCL/Gel-BBR-H；F：不同质量分数载药膜与P. gingivalis和F. nucleatum菌液共培养后的OD值。Control：对照（无纤维膜）组；ns：P>0.05；***：P<0.001。"

图5

载药膜与P. gingivalis和F. nucleatum共培养下细菌生物膜EPS含量A：不同质量分数载药膜与P. gingivalis和F. nucleatum共培养；B：共培养后生物膜的EPS含量测定结果。Control：对照（无纤维膜）组；ns：P>0.05；*：P<0.05；**：P<0.01；***：P<0.001。"

图 6

载药膜与P. gingivalis和F. nucleatum共培养下细菌生物膜的细菌密度及菌落形成能力A：PCL/Gel；B：PCL/Gel-BBR-L；C：PCL/Gel-BBR-M；D：PCL/Gel-BBR-H；E：不同质量分数载药膜与P. gingivalis和F. nucleatum菌液共培养后的OD值；F：不同质量分数载药膜与P. gingivalis和F. nucleatum菌液共培养后的菌落计数结果。**：P<0.01，***：P<0.001。"

图7

载药膜与P. gingivalis和F. nucleatum共培养条件下，生物膜的活/死细菌染色FITC：绿色荧光通道，标记活细菌；TRITC：红色荧光通道，标记死细菌；Merge：合并图层后同时显示活细菌和死细菌。"

图8

载药膜与P. gingivalis和F. nucleatum共培养下，生物膜细菌的表面形态 SEMA：PCL/Gel × 5 000；B：PCL/Gel-BBR-L × 5 000；C：PCL/Gel-BBR-M × 5 000；D：PCL/Gel-BBR-H × 5 000；E：PCL/Gel-BBR-H × 50 000；F：PCL/Gel-BBR-H × 100 000。箭头示核分裂象。"

图9

载药膜共培养条件下细胞的相对增殖活性A：RAW264.7的细胞活性；B：hPDLSC的细胞活性。Contnsl：对照（无纤维膜）组；ns：P>0.05；*：P<0.05；***：P<0.001。"

图10

细胞在载药膜表面的黏附形态 共聚焦显微镜 × 10 000上：hPDLSC；下：RAW264.7；左：罗丹明-鬼笔环肽标记的细胞骨架；中：DAPI标记的细胞核；右：图层合并（Merge）。"

《孕期口腔保健与治疗》出版发行"

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