牙种植体不同材料的口腔生物膜形成
Summary
在这里, 我们提出了一个评估口腔生物膜形成的方法在钛和氧化锆材料的牙科修复台, 包括分析细菌细胞的生存能力和形态学特征。在口腔生物膜分析中采用了一种与功能强大的显微技术相关联的原位模型。
Abstract
牙科植入物及其修复成分容易发生细菌定植和生物膜形成。使用提供低微生物黏附力的材料可以减少围植病的患病率和进展。鉴于口腔环境的复杂性和口腔生物膜的异质性, 需要显微技术来对牙齿和牙科材料的表面进行生物膜分析。本文介绍了用于比较人工牙基钛和陶瓷材料的口腔生物膜形成的一系列协议, 以及在形态学和细胞层面分析口腔生物膜的方法。用原位模型评价钛和氧化锆材料在牙科修复体上的口腔生物膜形成这项研究为 48 h 生物膜提供了令人满意的保护, 从而证明了方法上的充分性。光子显微镜允许分析在测试材料上形成的生物膜的区域代表。此外, 利用多光子显微镜对显影和图像进行处理, 可以对非常异构的微生物种群中的细菌生存能力进行分析。电子显微镜生物标本的制备促进了生物膜的结构保存, 图像分辨率好, 无工件。
Introduction
细菌生物膜是复杂的, 功能上和结构上有组织的微生物群落, 其特点是多种微生物物种, 合成胞外, 生物活性高分子基质1,2。细菌粘附到生物或非的表面之前, 形成的获得的薄膜, 主要包括唾液糖蛋白1,3,4。微生物与薄膜之间的物理化学相互作用初步建立, 其次是获得的薄膜的细菌 adhesins 和糖蛋白受体之间的相互作用更强。微生物多样性逐渐增加, 通过 coaggregation 的次级殖民者的受体已经附着的细菌, 形成一个多种群社区1,3,4, 5。
口腔微生物群的稳态及其与宿主的共生关系对维持口腔健康具有重要意义。口腔生物膜内的 dysbiosis 可能会增加龋病和牙周病2、5的发展风险。临床研究表明, 在牙齿或牙种植体上的生物膜积累与牙龈炎或周围种植体粘膜炎的形成有因果关系6,7。炎症过程的进展导致围 implantitis 和随后的损失种植体8。
牙科植入物及其修复成分容易发生细菌定植和生物膜形成9。使用具有化学成分和表面形貌的材料, 提供低微生物黏附力, 可减少9、10围植体疾病的患病率和进展。钛是用于制造植入物的假肢基台的最常用材料;然而, 陶瓷材料最近被介绍了并且越来越受欢迎作为替代钛由于他们的审美特性和生物相容性11,12。同样重要的是, 陶瓷材料已经与一个假想的减少可能坚持微生物, 主要是由于其表面粗糙度, 润湿性, 和表面自由能源10,13。
体外研究有助于了解微生物黏附力对修复桥台表面9、14、15、16、17的重要进展。然而, 口腔的动态环境, 其特点是其变化的温度和 pH 值和养分的可用性, 以及由存在的剪切力, 是不可再生的体外实验协议18, 19。为了克服这一问题, 另一种选择是使用原位模型的生物膜形成, 有利地保留其三维结构的前体分析10,20,21,22,23,24。
对口腔基质形成的生物膜复杂结构的分析需要使用能够显示光学致密物质25的显微技术。光子激光扫描显微镜是生物膜结构分析的现代选择26。它的特点是利用非线性光学光源接近红外波长, 脉冲到飞秒27。这一方法是为图像获取的荧光材料或显影标记的材料, 除了所产生的图像由非线性光学信号所衍生的现象称为第二次谐波产生。光子显微镜的优点之一是, 由于激发光强度的影响, 获得的最大图像深度为27。
对于非生物表面生物膜的生物活性分析, 用光子显微镜观察, 在细菌细胞中使用不同光谱特征和渗透能力的荧光核酸染料需要28。显影 SYTO9 (绿荧光) 和碘碘 (红荧光) 可用于视觉分化的活菌和死细菌28,29,30。碘碘化物只穿透受损膜的细菌, 而 SYTO9 进入细菌细胞, 并有完整且受损的膜。当两种染料都存在于细胞内时, 碘碘对核酸有更强的亲和力, 并取代 SYTO9, 标志着红色28,30。
鉴于口腔环境的复杂性和口腔生物膜的异质性, 需要显微技术来对牙齿和牙科材料表面进行生物膜分析。本文介绍了用于比较人工牙基钛和陶瓷材料的口腔生物膜形成的一系列协议, 以及在形态学和细胞层面分析口腔生物膜的方法。
Protocol
这项研究获得了 Ribeirão 欧鲁普雷图牙科学院机构审查委员会的批准, 志愿者参加者签署了书面同意书 (程序 2011.1. 371.583)。 1.原位生物膜形成 参加者的选择 根据以下纳入标准选择患者: 一个健康的个体, 有完整的牙列, 没有口腔疾病的临床症状。 根据以下排除标准排除病人: 怀孕、哺乳期、龋齿、牙周疾病或抗生素治疗在过去3月里, ?…
Representative Results
本研究以 48 h 的原位生长后生物膜的定植密度为依据, 用光子显微镜观察了钛和氧化锆圆盘上的占地面积与试样总扫描面积的比例 (26.64 毫米2)。图 2表示3测试材料表面的细菌定植密度。在铸件表面和在机械加工的钛盘 (分别为0.0292 µm2和0.0213 µm2) 上观察到的生物膜密度高于氧化锆圆盘 (0.0099 µm2;p < 0.05;克?…
Discussion
本文所述的协议是为了评价人工牙基钛和氧化锆材料的生物膜形成, 包括细菌细胞活力和形态学特征的分析。为了达到这一目的, 设计了一种生物膜形成的原位模型, 由口内装置组成, 能够容纳试验材料样品, 并使其暴露在动态口腔环境中48小时。该设备被认为是舒适和容易插入, 删除和清洁的志愿者。然而, 它对语音学和美学的影响甚少, 制作简单、成本低廉, 并使标本易于回收, 不破坏生物膜…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢 Maulin 从显微镜多用户实验室 (Ribeirão 欧鲁普雷图医学院) 的慷慨协助与 EDS 和 SEM 分析和老兄特谢拉马查多为他在视频版的慷慨技术援助。
Materials
| Hydrogum 5 | Zhermack Dental | C302070 | |
| Durone IV | Dentsply | 17130500002 | |
| NiCr wire | Morelli | 55.01.070 | |
| JET auto polymerizing acrylic | Clássico | ||
| Dental wax | Clássico | ||
| Pressure pot | Essencedental | ||
| Sandpapers 600 grit | NORTON | T216 | |
| Sandpapers 1200 grit | NORTON | T401 | |
| Sandpapers 2000 grit | NORTON | T402 | |
| Metallographic Polishing Machine | Arotec | ||
| Isopropyl alcohol | SIGMA-ALDRICH | W292907 | |
| Hot melt adhesive | TECSIL | PAH M20017 | |
| Filmtracer LIVE/DEAD Biofilm Viability Kit | Invitrogen | L10316 | |
| Pipette Tips, 10 µL | KASVI | K8-10 | |
| Pipette Tips, 1,000 µL | KASVI | K8-1000B | |
| 24-well plate | KASVI | K12-024 | |
| Glass Bottom Dish | Thermo Scientific | 150680 | |
| AxioObserver inverted microscope | ZEISS | ||
| Chameleon vision ii laser | Coherent | ||
| Objective EC Plan-Neofluar 40x/1.30 Oil DIC | ZEISS | 440452-9903-000 | |
| SDD sensors – X-Max 20mm² | Oxford Instruments | ||
| Glutaraldehyde solution | SIGMA-ALDRICH | G5882 | |
| Sodium cacodylate Buffer | SIGMA-ALDRICH | 97068 | |
| Osmium tetroxide | SIGMA-ALDRICH | 201030 | |
| Na2HPO4 | SIGMA-ALDRICH | S9638 | Used for preparation of phosphate buffered saline |
| KH2PO4 | SIGMA-ALDRICH | P9791 | |
| NaCl | MERK | 1.06404 | |
| Kcl | SIGMA-ALDRICH | P9333 | |
| Ethanol absolute for analysis EMSURE | MERK | 1.00983 | |
| CPD 030 Critical Point Dryer | BAL-TEC | ||
| JSM-6610 Series Scanning Electron Microscope | JEOL | ||
| SCD 050 Sputter Coater | BAL-TEC |
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Citar este artigo
Silva, T. S. O., Freitas, A. R., Pinheiro, M. L. L., do Nascimento, C., Watanabe, E., Albuquerque, R. F. Oral Biofilm Formation on Different Materials for Dental Implants. J. Vis. Exp. (136), e57756, doi:10.3791/57756 (2018).