Effect of Atmospheric-Pressure Cold Plasma on Pathogenic Oral Biofilms and In Vitro Reconstituted Oral Epithelium.
Effect of Atmospheric-Pressure Cold Plasma on Pathogenic Oral Biofilms and In Vitro Reconstituted Oral Epithelium.
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DOI：
10.1371/journal.pone.0155427
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Vergani CE
中科院分区：
文献类型：
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作者：
Delben JA;Zago CE;Tyhovych N;Duarte S;Vergani CE
考虑到常压冷等离子体(ACP)破坏生物膜基质和破坏细胞结构的能力，它可以作为一种有效的工具来对抗有毒的口腔生物膜。然而，ACP不会对口腔组织造成损害这一点是基本的。因此，本研究评价了(1)ACP对白色念珠菌和金黄色葡萄球菌单种和双种生物膜的抑菌作用，以及(2)ACP对体外重组口腔上皮的生物安全性。每种微生物的标准化细胞悬液在37°C的丙烯酸树脂盘上培养48h用于生物膜培养。生物膜在60秒内以10 mm的等离子体尖端到样品距离进行ACP处理。金黄色葡萄球菌和白色念珠菌的阳性对照分别为青霉素G和氟康唑。通过活菌落计数、激光共聚焦扫描显微镜、扫描电子显微镜和荧光显微镜检测活性氧等方法对生物膜进行分析。体外重组口腔上皮细胞经类似ACP处理后，通过组织学、细胞毒试验(LDH释放)、活性试验(四甲基偶氮唑盐比色法)和免疫组织化学(Ki67表达)进行分析。与阴性对照和阳性对照相比，所有经等离子体处理的生物膜均表现出显著的log10cfu/m L减少，微生物/生物膜形态发生改变，活性降低。此外，荧光显微镜显示，所有等离子体处理的生物膜中都存在活性氧物种。阴性对照组和血浆组口腔上皮细胞毒性低，细胞存活率高。组织学检查显示，血浆处理的上皮细胞既没有坏死的迹象，也没有明显的变化。Ki67阳性细胞显示经血浆处理的上皮细胞保持增殖。常压冷等离子体是一种很有希望的方法，可以消除口腔上皮中白色念珠菌和金黄色葡萄球菌的单物种和双物种生物膜，而不会产生毒性影响。
Considering the ability of atmospheric-pressure cold plasma (ACP) to disrupt the biofilm matrix and rupture cell structure, it can be an efficient tool against virulent oral biofilms. However, it is fundamental that ACP does not cause damage to oral tissue. So, this study evaluated (1) the antimicrobial effect of ACP on single- and dual-species biofilms of Candida albicans and Staphylococcus aureus as well as (2) the biological safety of ACP on in vitro reconstituted oral epithelium. Standardized cell suspensions of each microorganism were prepared for biofilm culture on acrylic resin discs at 37°C for 48 hours. The biofilms were submitted to ACP treatment at 10 mm of plasma tip-to-sample distance during 60 seconds. Positive controls were penicillin G and fluconazole for S. aureus and C. albicans, respectively. The biofilms were analyzed through counting of viable colonies, confocal laser scanning microscopy, scanning electron microscopy and fluorescence microscopy for detection of reactive oxygen species. The in vitro reconstituted oral epithelium was submitted to similar ACP treatment and analyzed through histology, cytotoxocity test (LDH release), viability test (MTT assay) and imunnohistochemistry (Ki67 expression). All plasma-treated biofilms presented significant log10 CFU/mL reduction, alteration in microorganism/biofilm morphology, and reduced viability in comparison to negative and positive controls. In addition, fluorescence microscopy revealed presence of reactive oxygen species in all plasma-treated biofilms. Low cytotoxicity and high viability were observed in oral epithelium of negative control and plasma group. Histology showed neither sign of necrosis nor significant alteration in plasma-treated epithelium. Ki67-positive cells revealed maintenance of cell proliferation in plasma-treated epithelium. Atmospheric-pressure cold plasma is a promissing approach to eliminate single- and dual-species biofilms of C. albicans and S. aureus without having toxic effects in oral epithelium.