Saremco Dental Switzerland and Olympus Microsystems France Companies are gratefully acknowledged for their financial support. Saremco Dental is also acknowledged for the generous donation of the dental composites ELS extra low shrinkage. The authors also wish to express their appreciation to Dr Simon Banks from the department of Chemistry (University College London) for his assistance with English revision.

该协议包括三个主要步骤:第一步包括在培养基中复合提取物的制备方法;第二个关注的小学的人牙龈成纤维细胞（HGF）细胞培养，细胞与复合提取物和细胞染色的接触;第三步骤包括共焦成像和荧光信号的分析。对于牙龈组织采取，该协议被批准，符合法国法律，知情同意和当地伦理委员会。 1，复合提取物的制备用Dulbecco改进的Eagle培养基（DMEM）作为洗脱介质。保证样品的表面积和提取介质的体积之间的比率是在所述的ISO一十二分之一万〇九百九十三要求的最低水平。这个程序是最近描述的11和下面简要概述: <ol><li>形成的未固化牙科复合使用一个定制的保持球形样品呃。支架的大小为2毫米的半径，这是根据在口腔临床治疗与一般宜固化深度。 </li><li>制备板的孔中，每个含有1ml培养基（DMEM）中，具有青霉素/链霉素的5％的混合物和两性霉素的1％，但无胎牛血清（FBS）中。 </li><li>使用LED灯灯带1070毫瓦cm -2的强度（λ= 465-475纳米）为40秒的很好的DMEM培养基中的固化样品。确保固化尖端和样品之间的距离是在0.5毫米的范围内。使用该固化模式来模拟牙齿和未固化的牙科用复合之间的临时接触。在临床情况下，当恢复性复合材料被放置在口腔中发生接触。 </li><li> 5％CO 2的空气中的潮湿气氛下孵育样品（复合材料在DMEM培养基样品）在37℃下。孵育的DMEM培养基（无混合样品）为对照组细胞在相同的条件。 </li></ol> 2.肝细胞生长因子细胞培养，复合萃取物测试和细胞染色<ol><li>生长和维持细胞培养物的 隔离期间常规正畸拔牙的患者采取健康的牙龈组织活检人牙龈成纤维细胞。 <ol><li>培养在DMEM中的人牙龈成纤维细胞在10％FBS的存在下，5％青霉素/链霉素和1％两性霉素B.保持在培养箱中的细胞在37℃，5％CO 2的空气的潮湿气氛下。 </li><li>改变的培养基，每3天，并传代细胞，每5天。后经胰蛋白酶达到融合，收获细胞。 </li><li>离心细胞，在90×g离心5分钟，并在培养基中再悬浮其中。细胞计数与手持权杖自动细胞计数器。 </li><li>种细胞悬液，以2.5×10 4个细胞/ ml，在腔室载玻片系统中的细胞密度。允许细胞在5％的气氛中温育过夜，在37℃。 </li></ol></li><li>除了复合提取物中的细胞培养<ol><li> （24小时后培养）以1毫升试验的复合提取物的替代两个孔腔室载玻片中的介质。 </li><li>维持剩下的两个孔中（对照细胞）中的相同条件。 </li><li>孵育含有四个井在37℃下在恒温箱腔室载玻片，5％CO 2的空气中的潮湿气氛下。 </li></ol></li><li>荧光染色 根据制造商的说明使用活/死细胞毒性染色为真核细胞。 注:该试剂盒提供了一种快速的双色荧光测定法来确定在基于质膜完整性和酯酶活性的细胞群的生存能力。色斑区分活从受损细胞通过与绿色荧光钙黄绿素-AM同时标记，以指示MEMBRAN的胞内酯酶活性和红色荧光乙锭同型二聚体-1（EthD-1），以指示丢失Ë完整性。 <ol><li>制备含有2μM的钙黄绿素AM和4微米EthD-1（报道为适合于成纤维细胞的染色最终浓度）的2染色试剂的工作溶液。污渍添加到培养的贴壁细胞在选定的复合提取物的存在或不存在。 </li><li>染色后，并通过延时成像观察直接和快速地被染色的细胞群的至少15分钟。不要离心，不还是传统的染色细胞。 </li></ol></li></ol> 3.激光共聚焦时间流逝成像和信号分析所使用的系统配备有一个4二极管激光器单元和一个多染色图像为多达四个荧光染料为每个试样。该系统可以执行的映射图像（10X放大倍率），使该测试样品上的哪一个可以移动到观察的兴趣（600X放大倍率）的区域中进行了概述。 <ol><li>时间推移成像<ol><li>请将Specimens（腔室载玻片含有染色的样品）在黑暗的房间中的共焦集成培养箱（37 ℃，CO 2的湿润气氛中的5％）。 </li><li>选择相应的激光器。使用两个激光光源473纳米（15毫瓦）和559纳米（18毫瓦），以激发钙黄绿素和EthD-1。 </li><li>用两个连续的方式获取通过线序列图像的串扰，而不在成像与两个常用的荧光染料。 </li><li>使用了新开发的光谱检测器来自动设置的条件按照在扫描单元中的荧光染料。 </li><li>优化了检测到的荧光的带宽为每个通道的最大值。记录所有的收购顺序，以避免潜在的交叉谈话。 </li><li>选择基于使用采集软件的荧光染料的选择最合适的成像条件。 </li><li>取得时自动从中心以螺旋状图案创建的地图图像，与感兴趣的区域容易地选择用于进一步检查。 </li><li>选择观察模式。选择多达5种观察模式包括时间推移，Z堆叠，并根据需要进行多区。用在本研究中Z堆叠和时间推移模式的组合，并完成快速图像捕获， </li><li>改变和切换荧光染料。可替换地，覆盖荧光和光对比度的图像彼此。利用左下地图画面上所选择的点观察实时图像。 </li><li>通过使用帧和变焦功能确定的成像区域。任选地，对于每种类型的荧光染料的显示之间进行切换。 注:在本协议中所使用的显微镜​​具有相同的功能在一个紧凑的设计，较高的传统共焦。该显微镜配备有被最佳地适合于活细胞的简化内置的保育箱的稳定时间推移成像倒浸水目标。 </l我&gt; </ol></li><li>信号分析和定量<ol><li>使用图像处理软件来定量荧光信号。获得5的地图图像，10倍的目标为测试样本和细胞的控制。 </li><li>获得1024×1024像素的普通图像，用0.231微米×0.231微米像​​素尺寸。将图像存储为奥林巴斯映像格式（OIF）进行信号分析。这种格式可以存储各种参数设置和图像组合在一起。使用不同的分析工具（的强度分布的测量，这两个使用的信道间的强度比）。 </li><li>存储的最大投影图像为12位/像素的TIFF文件。 </li><li>瓶坯使用R指挥官软件统计分析。分析采用单因素方差分析（ANOVA）分析了重复试验的差异。报告结果的平均值±标准差（±SD）和P &lt;0.05有统计学意义。 </li></ol></li></ol>

The authors declare that they have no competing financial interests, they have nothing to disclose.

生物相容性材料的行为是最先决条件参数的医疗用途之前进行评估。国际标准涵盖医疗器械的ISO 10993 33和专门的牙科材料的ISO 7405 34。缺乏的毒性作用于周围的细胞是在这样的材料的生物相容性评价的关键标准。这就是为什么细胞毒性试验在牙科材料生物相容性35的评价如此重要- 37。了ISO建议的测试方法，可以基于代谢活性或膜完整性。选择的端点应遵循的排名，以减少时间和评估测定的成本由试验材料引起的不利影响的特定条件。调查当前方法（3D共焦时间推移成像）是一种基于膜的完整性。 通过这个端点的细胞毒性评价似乎是细胞biocompa的一个有价值的指标tibility与供试材料，也是ISO标准端点。 体外试验的目的是确定一种材料样品影响特定细胞类型。比色MTT法最初是由莫斯曼（1983）被描述为用于体外细胞毒性和细胞增殖的测量的有用方法。的MTT试验是基于四唑鎓盐转化成甲臜结晶活性的细胞，它决定线粒体活性。这可以通过存活率38进行翻译。 Gupta和合作者报道，MTT检测是最广泛使用的方法，用于测量复合树脂的细胞毒性。琥珀酸脱氢酶和碱性磷酸酶的反应也被用于相同的目的39。然而，命运的细胞不能跟进，由于MTT法要求在每个测量时间点杀死细胞。为了更密切地监测染色的细胞的行为在当前的研究中，二进行矩形观察活细胞，由于时间的推移激光共聚焦显微镜成像结合短协议染色，自动图像分析和荧光信号的量化。 牙科复合材料都与口腔组织，特别是牙龈和牙髓细胞紧密接触。成纤维细胞是从这些修复剂组合物40释放组分的靶- 41。此外，一些研究已经报道，永生化细胞和原代细胞可以具有与生物材料接触的不同的细胞反应。原代细胞被认为是评估生物材料的影响42更合适。这解释了使用初级人牙龈成纤维细胞作为细胞模型在本研究中所测试的牙科用复合材料的细胞毒性的评估。从牙科复合释放物质的毒性潜力已被广泛研究43 - 44。为了评估吨他的细胞毒性的材料的潜力，两个接触模型细胞可以被执行。在直接接触的模型系统的材料直接置于与靶细胞，而在间接接触系统中，靶细胞是在与来自生物介质洗脱物接触。在临床上的应用程序和用于恢复过程，牙科用复合被放置在与牙龈组织间接接触。出于这个原因，目前的实验方案集中在间接接触系统的人牙龈成纤维细胞上（细胞在测试的复合提取物的存在下）。如先前所报道的达尔和合作者，细胞毒性应答列为基础的活性相对于对重度（&lt;30％），中度（30-60％），轻微（60-90％）或无细胞毒性的（&gt; 90％）获得控件45的值。因此，根据该排名，并在所得结果的光（ 表1），复合的ELS额外低收缩可列为版本Ÿ略有毒性，并显示相媲美的行为在整个时间间隔期来控制细胞。使用精确相同的调查方法，这项研究的结果可能比那些最近发表的研究报告。相比于用于相同的临床应用，它是直接恢复11两个先前测试的复合材料在ELS额外低收缩复合证实优越的生物相容性。进一步的研究还需要建立更全面的比较。 当决定批准用于特定端点评估其细胞毒性测定，理解的优点，每个测定法的限制是重要的。共焦成像的主要目的是实现细胞和器官的3-D结构。该技术是能够揭示不仅检体的表面上，而且它的表面下。本文所描述的协议强调了利用三维CLSM时间推移共焦成像，作为森西略去方法来评估牙科复合材料的体外生物相容性行为。然而，为了避免在这项工作中所遇到的限制，抗振动的工作站可以以允许更稳定和更长的时间推移成像使用;桌面可以分离使用共聚焦系统的不利影响，在实验室中的振动可能会导致。此外，要使用在口腔中的材料应理想地是无害的牙龈组织，并应含有不物质，可能引起全身毒性。根据所使用的方法，并从当前的研究中获得的结果，可以得出结论，测试复合物（ELS的额外低收缩率）不是在本实验条件下的细胞毒性。作为CLSM方法能够灵敏地给活细胞的初始速率，其他的可能性可以预见，以评估多个端点。特别是，因为它已被最近报道，复合单体影响细胞苏氨酸ough活性氧（ROS）的46- 48，进一步的研究是必要的，以评估的相关性（如果有）的细胞毒性之间的信令网络和ROS产生。未来的协议可以被设计成同时评估ROS的产生（H 2 O 2和/或O 2 -染色），并使用时间的推移激光共聚焦显微镜成像的细胞毒性比（活/死染色）。

其中引用了ISO建议定义生物相容性的主要标准是缺乏物质毒性的细胞。基于树脂的牙科复合材料可释放从树脂基质组分，它可以是最初由于部分聚合，和/或购买由于降解过程随着时间的推移1-4。这些释放的部件接触到口腔组织接触，并且可以具有各种缺点像荨麻疹和粘膜反应5中，在患者6发展过敏和过敏性反应，牙龈成纤维细胞形态和还原上式改型I型胶原蛋白7，免疫抑制或免疫刺激对有丝分裂原驱动的扩散纯化的T淋巴细胞和脾细胞8和初级人牙龈成纤维细胞的DNA损伤，从而强调其潜在遗传毒性9。许多参数会影响牙科复合毒性，如C的荫omposite，光固化时，树脂单体的化学组成中，填料和转换10- 13度。自体外毒性的材料潜在可预测在体内的情况，并且可以相对于由相关的一些端点的研究的临床情况。牙科用复合材料和它们的组件的细胞毒性已使用细胞培养系统14-16广泛评价。 这些体外系统已被开发，以评估的术语牙科复合材料的生物相容性:细胞生长，并使用THP-1monocytes样细胞17细胞凋亡，细胞毒性中的人牙龈成纤维细胞18，抗炎潜力的HaCaT角质形成细胞样细胞2，细胞功能odontoblast-像MDPC-23细胞19，减少总RNA水平，以及在细胞凋亡的诱导对人牙龈的显著增加角化细胞20和对牙龈和牙髓成纤维细胞21的DNA损伤。 不同的方法被提出来探讨牙科复合材料的生物相容性。比色测 ​​定法，其中涉及特定的着色剂和光吸收测量，仍然是最常用的，由于它们的相对简单性和成本22- 26低。通过光显微镜分析经常会消耗更多的时间，且需要更高的成本。然而，这些显微技术具有一些重要的优点。细胞结构的观察提供了关于经历的毒性作用的扩展信息，从而提供更相关和敏感数据。特别是，引入共聚焦显微镜27的已经允许用于生物结构的观测与改善轴向分辨率相比于传统的宽视场成像的荧光显微镜。因此，激光共聚焦成像已经成为不同领域的功能强大的调查工具医学和科学的研究。而相比之下，电子显微镜技术，共聚焦显微镜，提供通过特定的荧光标记物掺入到可视化细胞的不同的部件的能力。大多数这些具体示踪剂是稳定在含水环境中，明智地衡量，廉价和无毒性的28，从而允许其在临床上使用，以及实验室研究报告。此外，所需的常规的电子显微镜分析的样品干燥和固着没有必要为时间的经过共焦成像，从而依赖于时间的获取，也可以对样品。相比于二维成像，共焦成像原理使样品表面下的可视化和三维结构重建从不同得到的深度图像;这导致，更精确，更详细结构29，30，但本视频研究是利用三维时间流逝的C的示例onfocal激光扫描显微镜（3D-CLSM）结合活/死荧光标记和信号量化为一个创新的方法。在本文所提出的技术具有使活细胞的敏感性评价和延时成像而不改变评估的单元结构的优点。共聚焦分析之前不需要任何准备步骤。先前，相同的染色，以评估培养的松质骨芯31的存活力，但没有共焦时间推移以下。类似地，相同的时间推移共焦步骤，用于根据使用的特定的细菌活/死染色的革兰式32，以评估活性污泥的细菌红霉素时间-杀活性。这些方法近来已经适于和用于比较基于甲基丙烯酸酯单体11的牙科复合材料的毒性。

<table><tbody><tr><td>Olympus Fluoview FV10i&amp;nbsp;</td><td>Olympus</td><td></td><td>Confocal microscope</td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt;/CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Pnasonic</td><td>MCO 5M</td><td>Cell incubation</td></tr><tr><td>Live/Dead Kit</td><td>Invitrogen&amp;nbsp;</td><td>MP 03224</td><td>Stain&amp;nbsp;</td></tr><tr><td>ELS extra low shrinkage &amp;nbsp;</td><td>Saremco&amp;nbsp;</td><td></td><td>Dental composite</td></tr><tr><td>DMEM medium</td><td>Sigma</td><td>D 6046</td><td>Culture medium</td></tr><tr><td>Trypsin</td><td>Sigma</td><td>T1426</td><td>harvesting cells</td></tr><tr><td>chamber slide system&amp;nbsp;&amp;nbsp;</td><td>PAA</td><td>14220040X</td><td>Culture chamber</td></tr><tr><td>penicillin/streptomycin&amp;nbsp;</td><td>PAA</td><td>P11-010</td><td>Cell culture</td></tr><tr><td>Amphotericin B</td><td>PAA</td><td>P11-001</td><td>Cell culture</td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>PAA</td><td>A11-101</td><td>Cell culture</td></tr><tr><td>Cell Counter</td><td>Merck Millipore</td><td>00110230TP1</td><td>Cell counting&amp;nbsp;</td></tr></tbody></table>

confocal time lapse imaging as an efficient method for the cytocompatibility evaluation of dental composites

人们普遍认为在体外的细胞物质的相互作用是在牙科材料的生物相容性评价一个有用的标准。本研究的目的是利用3D激光共聚焦显微镜时间推移共焦成像，以评估在体外生物相容性牙科复合材料。此方法提供了存活细胞率与牙科用复合提取物接触的精确和灵敏的指示。在ELS额外收缩率低，用于直接修复的牙科用复合材料，已被作为例子， 在体外评估用活/死细胞染色的原代培养的人牙龈成纤维细胞中进行。图像得到的FV10i共聚焦生物倒置制度，并与FV10-ASW 3.1软件进行分析。图像分析显示，在所测试的复合物的存在下，很轻微的细胞毒性5小时时间流逝之后。细胞活力略有下降与测试复合提取物对比例接触被证明ED控制单元。研究结果强调了利用3D激光共聚焦显微镜时间推移成像作为一个敏感的方法，定性和定量评价的牙科复合材料的生物相容性行为。

图1显示了活/死染色的细胞获得的映射图像（10X物镜），用于控制和细胞暴露于测试的复合提取物（ELS的额外低收缩率）中进行了概述。细胞的三维结构，可以在放大600X进行可视化。收集在收集在测试的复合提取物的存在下，腔室单元的控制腔室和细胞可视化示于图2的最大投影（目标60X）显示了选定的细胞群的命运，在时间经过的开始（在15分钟），并在时间流逝期结束时（在5个小时）。在此期间获得了57个筹码的图像（XYZT MOD扫描，每堆39的图像，像素深度1.00微米）。在暴露的细胞中得到的绿色荧光信号略有下降，非常轻微的变化，在红色荧光。研究结果显示，控制无显著变化细胞。复合提取物暴露细胞假设类似的主轴形态来控制细胞;形态没有在这些实验条件下改变（尽管绿信号降低，并在检测复合物的存在下，红色信号增加）。相比于阴性对照细胞为绿色和红色信号在相应于从所获得的供试合成图像不同细胞群体选择的五个不同图像堆栈进行图像分析。染细胞的强度分布示于图3;将细胞与测试复合接触的绿色和红色信号进化比得上那些控制单元。钙黄绿素和EthD-1荧光发射强度进行测量的对照细胞和复合暴露的细胞。测定是进行每隔一小时长达5小时。绿/红荧光比（基于所述绿色495-515纳米的积分强度和红色620-650 nm的信号）期间的时间推移周期进行计算。生存能力比率控制和暴露的细胞为ELS额外低收缩萃取示在 T 能够1.在测试复合物的存在下，活细胞的百分数为100％，1小时后稍有下降到93.9％接触。 5小时所指示的时间推移的周期后的测试复合材料和阴性对照（细胞无任何复合提取物）之间观察显著差异。 <img alt="图1" src="/files/ftp_upload/51949/51949fig1highres.jpg" /> 图1:映射图像概述表现为染色复合提取物（ELS超低收缩率）的时间间隔周期（T = 15分钟）的开头存在细胞目标10X，（A）控制单元和（B）细胞。绿色区域是活细胞和红色区域的受损细胞。在这实验条件下，没有变化（用于绿色和红色荧光）观察到暴露的复合材料的细胞相比阴性对照细胞。 <img alt="图2" src="/files/ftp_upload/51949/51949fig2highres.jpg" /> 图2:CLSM从（A）的控制腔和（B）的复合材料的萃取腔室中的开始和曝光的时间推移期末图像在一个细胞群体的目标60X（Ⅰ:15分钟和II:5小时后，分别）。绿色区域是活细胞和红色区域的受损细胞。图像的面板显示出在强度信号微小变化（在绿色信号略有下降，并在红1非常轻微的增加）。无细胞形态的改变，观察在复合提取物的存在;暴露维持其梭形和成纤维细胞形态作为对照的细胞的细胞。 图3:细胞的时间推移显微镜观察到的线轮廓 ，在15分钟和（II）以5小时（A）的控制单元（B）的复合材料的萃取暴露的细胞，（Ⅰ）。没有可见的变化，在检测复合物的存在下的绿色信号进化不管时间接触。然而，观察到复合物的存在下的红色信号的轻微增加5小时的接触后。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td rowspan="2"> 接触时间（小时） </td><td colspan="5"> 细胞存活率（％） </td></tr><tr><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td>五</td></tr><tr><td> 对照细胞 </td><td> 100 </td><td> 100 </td><td> 100 &lt;/ TD&gt; <td> 100 </td><td> 100 </td></tr><tr><td> ELS超低收缩® </td><td> 93.9±7 </td><td> 91.3±5 * </td><td> 89.5±3 * </td><td> 87.6±2 * </td><td> 87.7±3 * </td></tr></tbody></table> 表1:速率的活肝细胞生长因子的细胞进化后的1，2，3，4和5小时，用复合材料的萃取细胞活性接触的测定通过使用活/死细胞毒性试剂盒染色。数据显示平均值±5幅图像的SD栈分析。值是显著不同于对照细胞当p &lt;0.05。

Watch this Scientific Journal Video about Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites at JoVE.com

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

牙科用复合材料的生物相容性研究最多的方面是其细胞毒性三维CLSM时间推移成像结合的荧光信号的量化允许细胞命运的敏感性评价随时间。利用这种方法可能是一种有效的工具，以评估牙科复合材料的相容性。

焦时间推移成像为牙科复合材料的细胞相容性评价的有效方法

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

confocal-time-lapse-imaging-as-an-efficient-method-for

Research

JoVE Journal

Medicine

13.5K 次观看.  UMR CNRS 5615, Université Lyon1. 牙科用复合材料的生物相容性研究最多的方面是其细胞毒性三维CLSM时间推移成像结合的荧光信号的量化允许细胞命运的敏感性评价随时间。利用这种方法可能是一种有效的工具，以评估牙科复合材料的相容性。 

视频: Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

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 ...

科研

医学

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e.&#160;geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

原位压极限和骨牙周韧带齿纤维联合的相关性无创成像

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

生物工程

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 ...

免疫与感染

Shrinkage of Dental Composite in Simulated Cavity Measured with Digital Image Correlation

Polymerization shrinkage of dental resin composites can lead to restoration debonding or cracked tooth tissues in composite-restored teeth. In order to understand where and how shrinkage strain and stress develop in such restored teeth, Digital Image Correlation (DIC) was used to provide a comprehensive view of the displacement and strain distributions within model restorations that had undergone polymerization shrinkage.
Specimens with model cavities were made of cylindrical glass rods with both diameter and length being 10 mm. The dimensions of the mesial-occlusal-distal (MOD) cavity prepared in each specimen measured 3 mm and 2 mm in width and depth, respectively. After filling the cavity with resin composite, the surface under observation was sprayed with first a thin layer of white paint and then fine black charcoal powder to create high-contrast speckles. Pictures of that surface were then taken before curing and 5 min after. Finally, the two pictures were correlated using DIC software to calculate the displacement and strain distributions.
The resin composite shrunk vertically towards the bottom of the cavity, with the top center portion of the restoration having the largest downward displacement. At the same time, it shrunk horizontally towards its vertical midline. Shrinkage of the composite stretched the material in the vicinity of the &#8220;tooth-restoration&#8221; interface, resulting in cuspal deflections and high tensile strains around the restoration. Material close to the cavity walls or floor had direct strains mostly in the directions perpendicular to the interfaces. Summation of the two direct strain components showed a relatively uniform distribution around the restoration and its magnitude equaled approximately to the volumetric shrinkage strain of the material.

In order to understand the spatial development of polymerization shrinkage stress in dental resin-composite restorations, Digital Image Correlation was used to provide full-field displacement/strain measurement of restored model glass cavities by correlating images of the restoration taken before and after polymerization.

牙科复合材料在模拟腔收缩测量与数字图像相关

Polymerization shrinkage of dental resin composites can lead to restoration debonding or cracked tooth tissues in composite-restored teeth. In order to ...

通过双光子显微镜实时跟踪牙齿细胞

Using Ex Vivo Live Imaging to Investigate Cell Divisions and Movements During Mouse Dental Renewal

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal stem cells and tooth regeneration. These progenitor populations actively divide, move, and differentiate to maintain tissue homeostasis and regenerate lost cells in a responsive manner. However, traditional analyses using fixed tissue sections could not capture the dynamic processes of cellular movements and interactions, limiting our ability to study their regulations. This paper describes a protocol to maintain whole mouse incisors in an explant culture system and live-track dental epithelial cells using multiphoton timelapse microscopy. This technique adds to our existing toolbox for dental research and allows investigators to acquire spatiotemporal information on cell behaviors and organizations in a living tissue. We anticipate that this methodology will help researchers further explore mechanisms that control the dynamic cellular processes taking place during both dental renewal and regeneration.

作者聚焦:了解成年小鼠牙齿组织更新和修复中的动态细胞行为 

Ex vivo live imaging is a powerful technique for studying the dynamic processes of cellular movements and interactions in living tissues. Here, we present a protocol that implements two-photon microscopy to live track dental epithelial cells in cultured whole adult mouse incisors.

使用 离体 实时成像研究小鼠牙齿更新过程中的细胞分裂和运动

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal ...

发育生物学

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

透过玻璃看:时间推移显微镜和单细胞的纵向跟踪研究抗癌治疗

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空 体内 成像

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

焦时间推移成像为牙科复合材料的细胞相容性评价的有效方法

摘要

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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

原位压极限和骨牙周韧带齿纤维联合的相关性无创成像

细胞和生物膜细胞外成分的定量并发

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使用 离体 实时成像研究小鼠牙齿更新过程中的细胞分裂和运动

透过玻璃看:时间推移显微镜和单细胞的纵向跟踪研究抗癌治疗

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使用离体实时成像研究小鼠牙齿更新过程中的细胞分裂和运动

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空体内成像