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。

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
<ol><li>
参加者的选择
	<ol>
<li>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。</li>
		<li>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, 吸烟者, 或任何可能影响牙周状态的系统性疾病。</li>
	</ol>
</li>
	<li>
口腔内设备的研制
	<ol>
<li>用海藻酸盐的印象记录上颌弓。</li>
		<li>将19毫升的水添加到100克石粉中, 制备 IV. 型石材。将石头倒入海藻酸盐霉菌中, 使其成为上颌拱的模型。</li>
		<li>设计和制造一个丙烯酸口内设备, 以支持测试标本 (钛和陶瓷圆盘)。<ol>
<li>用正畸钳制作 NiCr 线 (直径0.7 毫米) 的固定扣 #139 并将其放置在模型上。要在上前磨牙之间定位夹钳, 请弯曲每根导线的一端, 使其形成循环。<ol>
<li>适应循环在牙缝的区域。在咬合中, 插入柔和的曲率, 以免干扰接触点和遮挡。在夹钳的末端做一个90°折叠, 以保持丙烯酸。</li>
				<li>为了适合在上第二磨牙钳, 调整在颈部第三 (前庭) 线的曲率, 以轮廓的牙齿 (远端), 并作出一个90°折叠在钳位的一端保持在丙烯酸。 注: 为了提供足够的保留/稳定的口内设备, 固定扣被放置在上前磨牙和远端的第二磨牙两侧的牙弓。</li>
			</ol>
</li>
			<li>根据制造商的指示操作自固化丙烯酸树脂, 在塑料阶段将丙烯酸树脂压在2玻璃板 (3 毫米厚的垫片中间), 使3毫米厚的板材。</li>
			<li>在模型的腭区上放置丙烯酸片, 根据装置的设计, 在聚合前用雕刻机修剪多余的丙烯酸树脂。</li>
			<li>用金属基体 (直径为10毫米, 厚度2毫米 (表面面积78.5 毫米2)) 制作蜡盘, 在基体中放置熔融蜡, 等待蜡凝固。手动将4个蜡盘嵌入丙烯酸树脂中, 其中2是前磨牙和第二磨牙旁边的2个。</li>
			<li>让丙烯酸树脂在 20 psi 压缩空气的压力罐中聚合20分钟。</li>
			<li>用电牙科实验室手机和切割机完成口腔内设备, 用磨料橡胶抛光。</li>
			<li>使用上面描述的设备, 调整设备的适当配合。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
钛及氧化锆试样的制备 注: 试样 (n = 14) 直径为10毫米, 厚度为2毫米, 表面面积为78.5 毫米2。<ol>
<li>用水冷 sandpapers 减少磨损 (600、1200和2000粒) 来抛光试样表面, 大约20分钟。 注: 对试样进行抛光, 以规范0.2 µm 表面粗糙度。</li>
		<li>用液体洗涤剂和自来水清洁和消毒标本和口内设备, 然后使用异丙醇超声波浴15分钟. 用吸水纸巾擦干。</li>
		<li>用0.1 毫升的无毒热熔胶 (图 1) 固定口腔内口服装置中的标本。</li>
		<li>安装包含口腔标本的装置。 注: 含标本的口内装置应佩戴48小时。该设备被删除, 并储存在磷酸盐缓冲盐水 (PBS), 而病人正在吃和执行口腔清洁。</li>
	</ol>
</li>
</ol>2. 对细菌生存能力的评估
注: 样品大小n = 10。
<ol><li>通过添加3µL 的 SYTO9 (绿色荧光核酸染料) 和3µL 碘碘化 (红色荧光核酸染料) 到1毫升的无菌蒸馏水中制备染色溶液。 注意: 准备防光保护的解决方案。</li>
	<li>将标本转移到24井板上, 用 PBS 彻底清洗, 去除任何非附着细胞。</li>
	<li>添加适当的荧光染料溶液体积 (1 毫升), 以覆盖含生物膜的试样。非常小心地添加染料, 以免瓦解生物膜。</li>
	<li>在室温下孵化标本 20-30 分钟, 免受光照。</li>
	<li>用无菌蒸馏水轻轻冲洗生物膜样品, 除去多余的染料。</li>
	<li>将试样放置在玻璃底碟中, 并执行微光子激光扫描显微镜进行生物膜分析。 注: 利用光子显微系统对细菌细胞生存能力进行分析。用 546/680 nm 和 477/600 nm 的激发/发射波长的过滤器检测了碘碘化物的荧光, SYTO9。获得的图像的大小为 5.16188 x 5.16188 毫米, 对应于每个标本 (78.5 毫米2) 或总面积33.94% 的总面积的26.64 毫米2 。这些图像是从样本的最中心部分制作的, 分辨率为 1024 x 1024 像素。用斐济软件31分别分析了红色通道和绿色通道图像。每个单元格都使用选择工具进行选择, 并通过像素的积分密度测量荧光的强度, 减去图像背景。</li>
</ol>3. 用能量色散谱法分析试样的化学成分 (EDS)
注: 样品大小n = 3。
<ol><li>选择每种无生物膜试验材料的3个标本, 并使用扫描电子显微镜与色散能量光谱仪相耦合, 评估每个试样2个不同区域的化学成分, 电子束电压为10伏32。</li>
</ol>4. 细菌生物膜的扫描电镜形态学分析
注: 样品大小n = 1。
<ol><li>将生物膜浸泡在2.5% 戊二醛稀释0.1 米常使用钠缓冲液中, pH 7.0-7.3, 为 24 h。</li>
	<li>在 PBS 缓冲器中清洗样品 (pH 值 = 7.6)。</li>
	<li>用1% 锇毒气对生物膜进行1小时的后缀。</li>
	<li>在 PBS 缓冲器中清洗样品 (pH 值 = 7.6)。</li>
	<li>仔细脱水生物膜样品, 使它们浸入乙醇溶液浓度的增加 (50%、70%、90%、95% 和 100%)。 注: 在100% 浓度下进行3的乙醇交换。总脱水步骤大约需要2小时。</li>
	<li>将试样转移到临界点干燥机上, 用二氧化碳 (CO2) 进行多次置换, 直到试样干燥。</li>
	<li>从仪器中取出干标本, 将其装入扫描电子显微镜支架上。</li>
	<li>溅射涂层20纳米的黄金层到标本的表面为120s。</li>
	<li>在扫描电镜室插入镀金圆盘。从每个标本的5个不同区域获得图像, 在可变压力下 20-30 伏, 在650X 放大12。</li>
</ol>

作者没有什么可透露的。

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...

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。

<table><tbody><tr><td>Hydrogum 5</td><td>Zhermack Dental</td><td>C302070</td><td></td></tr><tr><td>Durone IV</td><td>Dentsply</td><td>17130500002</td><td></td></tr><tr><td>NiCr wire&amp;nbsp;</td><td>Morelli</td><td>55.01.070</td><td></td></tr><tr><td>JET auto polymerizing acrylic</td><td>Cl&amp;aacute;ssico</td><td></td><td></td></tr><tr><td>Dental wax&amp;nbsp;</td><td>Cl&amp;aacute;ssico</td><td></td><td></td></tr><tr><td>Pressure pot&amp;nbsp;</td><td>Essencedental</td><td></td><td></td></tr><tr><td>Sandpapers 600 grit</td><td>NORTON</td><td>T216</td><td></td></tr><tr><td>Sandpapers 1200 grit</td><td>NORTON</td><td>T401</td><td></td></tr><tr><td>Sandpapers 2000 grit</td><td>NORTON</td><td>T402</td><td></td></tr><tr><td>Metallographic Polishing Machine</td><td>Arotec</td><td></td><td></td></tr><tr><td>Isopropyl alcohol</td><td>SIGMA-ALDRICH</td><td>W292907</td><td></td></tr><tr><td>Hot melt adhesive</td><td>TECSIL</td><td>PAH M20017</td><td></td></tr><tr><td>Filmtracer LIVE/DEAD Biofilm Viability Kit</td><td>Invitrogen</td><td>L10316</td><td></td></tr><tr><td>Pipette Tips, 10 &amp;micro;L</td><td>KASVI</td><td>K8-10&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Pipette Tips, 1,000 &amp;micro;L</td><td>KASVI</td><td>K8-1000B&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>24-well plate&amp;nbsp;</td><td>KASVI</td><td>K12-024</td><td></td></tr><tr><td>Glass Bottom Dish</td><td>Thermo Scientific</td><td>150680</td><td></td></tr><tr><td>AxioObserver inverted microscope&amp;nbsp;</td><td>ZEISS</td><td></td><td></td></tr><tr><td>Chameleon vision ii laser</td><td>Coherent</td><td></td><td></td></tr><tr><td>Objective EC Plan-Neofluar 40x/1.30 Oil DIC</td><td>ZEISS</td><td>440452-9903-000</td><td></td></tr><tr><td>SDD sensors - X-Max 20mm&amp;sup2;</td><td>Oxford Instruments</td><td></td><td></td></tr><tr><td>Glutaraldehyde solution</td><td>SIGMA-ALDRICH</td><td>G5882</td><td></td></tr><tr><td>Sodium cacodylate Buffer&amp;nbsp;</td><td>SIGMA-ALDRICH</td><td>97068&amp;nbsp;</td><td></td></tr><tr><td>Osmium tetroxide</td><td>SIGMA-ALDRICH</td><td>201030</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>SIGMA-ALDRICH</td><td>S9638</td><td>Used for preparation of phosphate buffered saline</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>SIGMA-ALDRICH</td><td>P9791&amp;nbsp;</td><td></td></tr><tr><td>NaCl</td><td>MERK</td><td>1.06404</td><td></td></tr><tr><td>Kcl</td><td>SIGMA-ALDRICH</td><td>P9333&amp;nbsp;</td><td></td></tr><tr><td>Ethanol absolute for analysis EMSURE</td><td>MERK</td><td>1.00983</td><td></td></tr><tr><td>CPD 030 Critical Point Dryer</td><td>BAL-TEC</td><td></td><td></td></tr><tr><td>JSM-6610 Series Scanning Electron Microscope</td><td>JEOL</td><td></td><td></td></tr><tr><td>SCD 050 Sputter Coater</td><td>BAL-TEC</td><td></td><td></td></tr></tbody></table>

oral biofilm formation on different materials for dental implants

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。

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?...

Watch this Scientific Journal Video about Oral Biofilm Formation on Different Materials for Dental Implants at JoVE.com

Oral Biofilm Formation on Different Materials for Dental Implants

牙种植体不同材料的口腔生物膜形成

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。

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

oral-biofilm-formation-on-different-materials-for-dental-implants

Research

JoVE Journal

Medicine

11.4K 次观看.  University of São Paulo. 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。

视频: Oral Biofilm Formation on Different Materials for Dental Implants

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

口腔生物膜的生物化学，结构和转录综合评价分析工具盒介导变形链球菌

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

科研

免疫与感染

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

腭扩展口腔生物膜分析，荧光原位杂交和激光共聚焦显微镜

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

医学

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

细胞和生物膜细胞外成分的定量并发

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

使用高通量的<em&gt;在体外</em&gt;微流控系统开发口服多物种生物膜

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

生物工程

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

在口腔生物膜细胞外pH值成比例成像

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition leads to a better understanding of cariogenic and periopathogenic mechanisms. Microbial changes taking place in the oral cavity during childhood are of interest for several reasons. The evolution of the child oral microbiota and shifts in its composition need to be analyzed further to understand and possibly prevent the onset of disease. At the same time, advanced knowledge of the natural composition of oral biofilm is needed. Early stages of caries-free permanent dentition with healthy gums provide a widely unaffected subgingival habitat that can serve as an in situ baseline for studying features of oral health and disease. Analysis of children's oral biofilm during different stages in life is thus an important theme in the field. Modern molecular analysis methods can provide comprehensive information about the bacterial diversity of such biofilms. To enable microbiota data comparison, it is important to standardize each step in the procedure for molecular data generation. This procedure spans from clinical sampling, Next Generation Sequencing (NGS), bioinformatic data processing, to taxonomic interpretation. One of the most critical factors here is biofilm sampling. Sampling in children is even more challenging in particular due to limited space in subgingival areas. We thus focus on the use of paper points for subgingival sampling. This article provides a detailed protocol for oral biofilm sampling of the subgingival sulcus, the mucosa, and saliva in children.

Changes in the oral microbiome throughout childhood are of growing interest. Comparison of different microbiome studies reveals a lack of standardized sampling protocols. Limited space makes sampling the sound subgingival sulcus of children challenging. Paper point sampling is presented here in detail as the method of choice for this area.

口腔生物膜取样在健康儿童中的微生物分析

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition ...

Direct and Indirect Culture Methods for Studying Biodegradable Implant Materials In Vitro

Over the past several decades, biodegradable materials have been extensively explored for biomedical applications such as orthopedic, dental, and craniomaxillofacial implants. To screen biodegradable materials for biomedical applications, it is necessary to evaluate these materials in terms of in vitro cell responses, cytocompatibility, and cytotoxicity. International Organization for Standardization (ISO) standards have been widely utilized in the evaluation of biomaterials. However, most ISO standards were originally established to assess the cytotoxicity of nondegradable materials, thus providing limited value for screening biodegradable materials. 
This article introduces and discusses three different culture methods, namely, direct culture method, direct exposure culture method, and exposure culture method for evaluating the in vitro cytocompatibility of biodegradable implant materials, including biodegradable polymers, ceramics, metals, and their composites, with different cell types. Research has shown that culture methods influence cell responses to biodegradable materials because their dynamic degradation induces spatiotemporal differences at the interface and in the local environment. Specifically, the direct culture method reveals the responses of cells seeded directly on the implants; the direct exposure culture method elucidates the responses of established host cells coming in contact with the implants; and the exposure culture method evaluates the established host cells that are not in direct contact with the implants but are influenced by the changes in the local environment due to implant degradation. 
This article provides examples of these three culture methods for studying the in vitro cytocompatibility of biodegradable implant materials and their interactions with bone marrow-derived mesenchymal stem cells (BMSCs). It also describes how to harvest, passage, culture, seed, fix, stain, characterize the cells, and analyze postculture media and materials. The in vitro methods described in this article mimic different scenarios of the in vivo environment, broadening the applicability and relevance of in vitro cytocompatibility testing of different biomaterials for various biomedical applications.

Direct and Indirect Culture Methods for Studying Biodegradable Implant Materials In Vitro

We introduce three methods of direct culture, direct exposure culture, and exposure culture for evaluating the in vitro cytocompatibility of biodegradable implant materials. These in vitro methods mimic different in vivo cell-implant interactions and can be applied to study various biodegradable materials.

体外研究可生物降解种植体材料的直接和间接培养方法

Over the past several decades, biodegradable materials have been extensively explored for biomedical applications such as orthopedic, dental, and ...

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active compounds from plants are challenging because of the complex plant matrix and time-consuming isolation and identification procedures. Therefore, a stepwise approach for screening natural compounds from plants, including the isolation and identification of potentially active molecules, is presented. It includes the collection of the plant material; preparation and fractionation of crude extracts; chromatography and spectrometry (UHPLC-DAD-HRMS and NMR) approaches for analysis and compounds identification; bioassays (antimicrobial and antibiofilm activities; bacterial &#34;adhesion strength&#34; to the salivary pellicle and initial glucan matrix treated with selected treatments); and data analysis. The model is simple, reproducible, and allows high-throughput screening of multiple compounds, concentrations, and treatment steps can be consistently controlled. The data obtained provide the foundation for future studies, including formulations with the most active extracts and/or fractions, isolation of molecules, modeling molecules to specific targets in microbial cells and biofilms. For example, one target to control cariogenic biofilm is to inhibit the activity of Streptococcus mutans glucosyltransferases that synthesize the extracellular matrix&#8217; glucans. The inhibition of those enzymes prevents the biofilm build-up, decreasing its virulence.

Natural products represent promising starting points for the development of new drugs and therapeutic agents. However, due to the high chemical diversity, finding new therapeutic compounds from plants is a challenging and time-consuming task. We describe a simplified approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions.

从植物提取物和分数中识别新型抗菌和抗生物膜分子的系统方法，以防止牙科蛀牙

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active ...

生物学

Effects of Mechanical Methods Used in Peri-implantitis Treatment on Implant Surface Decontamination and Roughness

Various mechanical methods have been proposed for decontaminating dental implant surfaces with varying success. This in vitro study evaluated the decontamination efficiency of an air abrasion (AA) system with erythritol powder, a polyether-ether-ketone (PEEK) ultrasonic tip, and titanium curettes (TIT) and their effects on implant surface topography using scanning electron microscopy (SEM). A total of 60 implants were stained with permanent red ink and placed in 3D-printed Class 1A and Class 1B peri-implantitis defects, forming six groups (n=10 per group) based on defect type and treatment protocol. Additionally, one positive and one negative control implant was used. Erythritol powder, PEEK ultrasonic tips, and titanium curettes were applied for 2 min in Class 1A defects and 3 minutes in Class 1B defects. Residual red ink areas were quantified with digital software, and implant surface changes were analyzed using SEM and EDS. None of the methods achieved complete decontamination. However, erythritol powder was significantly the most effective, leaving a residual ink rate of 24% &#177; 6% (p &#60; 0.001). PEEK ultrasonic tips resulted in 41% &#177; 4% residual ink, while titanium curettes left 55% &#177; 3%. Significant differences were observed among all methods. No significant difference in decontamination efficacy was found between Class 1A and Class 1B defects. SEM analysis showed minimal surface damage with erythritol powder and PEEK tips, whereas titanium curettes caused moderate to severe damage. Based on both decontamination efficiency and surface preservation, erythritol powder and PEEK tips are safe and effective options for peri-implantitis treatment, while titanium curettes are less effective and cause considerable surface damage. These findings may assist clinicians in peri-implantitis treatment planning.

The present protocol describes an experimental model based on ink-staining which can be used for in vitro implant surface decontamination and roughness research to contribute to clinical decision-making.

种植体周围炎治疗中使用的机械方法对种植体表面去污和粗糙度的影响

Various mechanical methods have been proposed for decontaminating dental implant surfaces with varying success. This in vitro study evaluated the ...

不动杆菌生物膜形成的评估和生物膜的表征

Quantification, Viability Assessment, and Visualization Strategies for Acinetobacter Biofilms

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, biofilm quantification and visualization are important methods to assess the potential of Acinetobacter strains to cause nosocomial infections. The biofilms forming on the surface of the microplate can be quantified in terms of volume and cell numbers. Biofilm volumes can be quantified by staining using crystal violet, washing, destaining using ethanol, then measuring the solubilized dye using a microplate reader. To quantify the number of cells embedded in the biofilms, the biofilms are scrapped off using cell scrapers, harvested in the saline, vigorously agitated in the presence of glass beads, and spread on Acinetobacter agar. Then, the plates are incubated at 30 &#176;C for 24-42 h. After incubation, the red colonies are enumerated to estimate the number of cells in biofilms. This viable count method can also be useful for counting Acinetobacter cells in mixed-species biofilms. Acinetobacter biofilms can be visualized using fluorescent dyes. A commercially available microplate designed for microscopic analysis is employed to form biofilms. Then, the bottom-surface attached biofilms are stained with SYTO9 and propidium iodide dyes, washed, then visualized with confocal laser scanning microscopy.

作者聚焦：不动杆菌生物膜定量、评估和可视化的综合方案 

This protocol describes the preparation of the inoculum, the biofilm quantification on microtiter plates using crystal violet dye, the viable count in biofilms, and the visualization of biofilms of Acinetobacter.

不动杆菌生物膜的定量、活力评估和可视化策略

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, ...