Name: development of a direct pulp-capping model for the evaluation of pulpal wound healing and reparative dentin formation in mice
Uploaded: 2017-01-12T12:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice at JoVE.com

这项研究是由来自NIDCR / NIH R01DE023348（RHK）和理事会学院研究基金（RHK）在加州大学洛杉矶分部的学术评议的研究的支持。

小鼠从杰克逊实验室购买并保存在实验动物医学UCLA司（DLAM）无病原体动物饲养。实验是按照从校长的动物研究委员会（ARC＃2016-037）批准的机构准则进行的。 1.鼠标麻醉<ol><li>使用八周龄雌性C57 / BL6小鼠（n = 3）。 </li><li>麻醉用氯胺酮（80-120毫克/千克小鼠体重）/赛拉嗪（5mg / kg的小鼠体重的）解决方案的小鼠和在剂量为10毫升/千克腹膜内（IP）施用。 </li><li>制备氯胺酮（80 - 120毫克/千克）/赛拉嗪（5mg / kg的）解决方案，并在剂量为10毫升/千克腹膜内（IP）施用它们。 </li><li>确认小鼠完全通过执行脚趾捏麻醉。 </li></ol> 2.盖髓过程<ol><li>放置在鼠标的嘴的嘴保持器。 </li><li>固定口保持器上的表，使得他广告朝上。 </li><li>放置在口的顶部的显微镜（10X），使得第一磨牙完全可见。 </li><li>使用¼ - 圆钻在高速手机为20万转，除去中间的牙釉质一部分，直到纸浆是通过透明牙本质可见。不要暴露与钻纸浆。 </li><li>使用＃15牙髓K-文件（150微米直径），通过穿孔牙本质和揭露浆。 注:特别应注意，使牙本质碎片不会被压入纸浆。这可以通过每季度转动K-文件，然后拉K-鱼贯而出避免。 </li><li>根据制造商的说明用无菌H 2ö混合MTA。交付和MTA放置到露髓的探险家的一角。使用纸张点（精）的背面侧到MTA包入露髓通过轻轻敲击。纸点的较厚侧是平坦的，因此可以对MTA的正常凝结成露髓。 </li><li>通过将35％的磷酸刻蚀剂，其中它只是覆盖了牙齿蚀刻15秒的牙齿。要特别小心，以限制蚀刻剂的位置，因为它可能会刺激牙龈组织。 注:蚀刻剂是在一个注射器中，并用来粗糙化的牙齿表面，使牙科用粘合剂可以流动到调解微机械粘合到牙齿。因为它们是粘性的，它可以是自包含的直接涂布少量到牙齿。 </li><li>使用负压力抽吸去除腐蚀剂。使用所轻轻用H 2 O中浸泡以除去蚀刻液的残差一个棉球。重复此步骤，直至蚀刻液完全从牙齿去除。 </li><li>使用压缩空气除尘器，轻轻擦干牙齿。 </li><li>申请使用的纸张点的背面的牙科粘合剂。 </li><li>使用压缩空气3秒的粘合剂层薄。 </li><li> CURE使用固化光用单元20秒的牙科粘合剂。 </li><li>放入少量的流动树脂到这是与MTA皑皑的牙齿。使用浏览器的尖端复合流入齿槽。 </li><li>固化复合用于使用光固化单元聚合它30秒。确认复合材料完全固化并用坚硬的探险家。 </li></ol> 3.后运护理<ol><li>管理该盖髓过程后立即卡洛芬（5毫克/千克）皮下（SC）。 </li><li>放置在一个加热垫的老鼠在低功耗，以保持温暖的动物，他们醒过来了。 </li><li>返回小鼠动物饲养住房。 </li></ol> 4.组织采购<ol><li>经过5 - 6周，安乐死与异氟醚麻醉完备的条件下通过颈椎脱位的小鼠。 </li><li>小心取出颌骨出头骨的基地，放入50毫升管。修复entir包含纸浆封端齿与在PBS中的4％多聚甲醛，pH值7.4对侧未加帽的齿都在4℃下过夜，然后将其存储在70％的乙醇溶液E颌骨。 注:多聚甲醛是有毒和致癌性。作为标准操作程序（SOP）中列出的正确使用多聚甲醛进行监测。 </li><li>扫描使用扫描μCT鼠标上颌骨。在扫描期间固定上颌骨，用纱布用70％乙醇浸泡包裹样本，将它们放置在15 mL细胞培养管。 </li></ol> 5.μCT扫描<ol><li>准备μCT扫描样本。简言之，将包裹的试样用纱布用70％乙醇浸泡和在通用的15毫升细胞培养物的锥形管固定。安装在管上的μCT扫描阶段，在制造商的说明所述。 </li><li>的X射线源设定为145微安的电流，55 kVp的的一个电压，和2的曝光时间00毫秒。 </li><li>用μCT扫描器在20微米分辨率和0.5mm的铝过滤器执行图像采集。 </li><li>重建图像和可视化是11。 </li><li>一旦μCT扫描完成后，2周开始脱钙，用5％EDTA和PBS中4％蔗糖（pH 7.4）中。 </li></ol> 6.组织处理和染色<ol><li>嵌入在石蜡脱钙组织。之前嵌入，通过矢状精确剪裁上颌骨前壁立即向第一磨牙。而嵌入，向下定位该表面，使得第一磨牙的纵向截面是切割表面。 </li><li>使用切片机，准备5微米厚的幻灯片。纸浆封区域通常与distopalatal（DP）的根，它可被用作一个划时代重合。通过检查在光学显微镜下组织学和比较μCT图像确定感兴趣的精确区域。 </li><li>对于H＆E染色，deparaffinize并用二甲苯（2×）和连续稀释的乙醇（100％EtOH中2倍，95％的EtOH 2x，以及70％乙醇1×）再水合的幻灯片。 </li><li>冲洗用流动的自来水的幻灯片。 </li><li>与2.5分钟苏木素液染色，用自来水冲洗。 </li><li>浸在95％乙醇的幻灯片1分钟。 </li><li>用1分钟伊红染色解决方案，并与自来水冲洗。 </li><li>用连续稀释的乙醇（70％EtOH中1个，95％的EtOH 2x，以及100％乙醇3×）和二甲苯（3×）脱水。 </li><li>安装与安装解决方案的幻灯片。 </li></ol>

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The authors have nothing to disclose.

目前，有可用来验证牙科材料，支架，或生长因子对牙髓干细胞（牙髓干）13的牙源性分化的体内作用几个不同的实验模型。这些模型包括牙髓干细胞的异位自体移植到器官，如肾包膜，或牙髓干细胞的皮下移植入与支架14,15免疫功能低下的小鼠。然而，这些方法是有限的，不是在原位纸浆环境中执行对牙髓干细胞的牙源性作用。另一方面，原位移植到牙齿上的纸浆或纸浆封盖程序在较大的动物16,17被使用。虽然这些模型都是在原位环境评估牙源性潜在价值，使用这些模式在本质上是主要的观测，提供有关纸浆伤口的愈合和修复性牙本质形成机械有限见解。 </p&gt; 在本文中，我们呈现给小鼠进行盖髓的详细方法。这一步一步的过程包括麻醉的小鼠，制备类我状腔，放置盖髓材料，收获的上颌骨，用μCT分析扫描，并评估组织样品用于修复性象牙质的形成。我们的盖髓小鼠模型将在通过使利用转基因或基因敲除小鼠，这是在研究界广泛使用的调查体内牙髓创伤愈合修复性牙本质的上下文中的基本分子机制的工具。 最近的研究表明，其中观察到18,19象牙质形成几个小鼠模型。 Saito 等。创建了一个类，我一样不准备牙髓暴露，刺激反动的，而不是修复性牙本质的形成。无论反动牙本质和修复性牙本质被列为三级牙本质，其形成外部刺激之后到牙齿。然而，与反动牙本质，其通过现有牙本质形成，修复性象牙质是由成牙本质细胞样细胞，例如牙髓干细胞，当纸浆变成露出，成齿质细胞层被破坏20形成。因此，它并不代表在临床实际盖髓过程。在另一项研究中，玻璃离聚物被用来盖住牙髓暴露19。然而，临床研究表明，玻璃离子诱导的慢性炎症，但不修复牙本质21。在这方面，我们的盖髓小鼠模型更好地表示在患者的实际盖髓过程。 值得注意的是，当我们超过6周后收获小鼠，修复性象牙质的形成发生在整个纸浆室和根管（ 图4）。这种观察是相当意外，因为我们在君预期修复性牙本质的形成该盖髓材料和纸浆之间ction。然而，纸浆的效矿化在临床设置中也观察到，特别是在相对年轻的患者22。因为在这项研究中所用的8周龄的小鼠被认为是"年轻人"23，一个可能性是存在的，由此这些小鼠仍然存有显著牙源性潜力。因此，这将是值得研究修复性牙本质形成的小鼠老化效应。 我们的组织学检查显示，虽然修复性象牙质是在纸浆封端齿明确形成，有两个牙质和骨形成的特点，通过牙质小管中的修复存在（红色箭头）和骨细胞（黑色箭头）所证明牙本质（ 图5）。这些观察表明，修复性象牙质的形成可通过本地驻留牙本质状牙髓干细胞，以及infiltratin诱导克间质干细胞从周围的骨。 相比于牙本质形成细胞中，我们发现在纸浆内没有牙本质再吸收细胞，如通过抗酒石酸酸性磷酸酶（TRAP）染色测定（数据未显示）。事实上，牙髓或根尖周炎诱导牙齿周围的骨表面上的破骨细胞形成，但不能在牙质表面上由于的尚未未知的机制24。值得注意的是，有现有的牙本质和新成立的修复性牙本质（ 图4）之间有明显的分界。先前的研究证实了类似的现象;当齿在二膦酸或抗RANKL抗体，这两者的抑制破骨细胞的功能的存在下被提取，有现有的板层骨和新形成的编织骨25之间的明确分界。此概念还支持不存在在纸浆牙本质骨吸收的细胞。总的来说，我们建立了小鼠模型将provide检查纸浆伤口愈合和体内修复性牙本质形成的机制独特的机会。 有限制的盖髓小鼠模型。人类和小鼠之间的遗传妆显然是不同的。完整的基因组已经被测序在人类和小鼠中，并且在小鼠和人类26,27之间的蛋白编码区约85％的相似性。与此概念行，有人认为与纸浆动物封盖发现并不一定反映在人类28。然而，动物模型在研究界被广泛用来概括在体内的人类疾病，如类风湿关节炎29 ovariactomy诱导的骨损失为骨质疏松30，脂多糖（LPS）给药用于全身性休克31和结扎胶原诱导的关节炎安置牙周炎32。这样，盖髓谋本身模型将是至关重要的审查纸浆伤口愈合和体内修复象牙质形成的分子机制。然而，就像其他的动物模型，解释和从盖髓小鼠模型研究结果的确认应仔细评估。 综上所述，目前的研究表明成功的纸浆小鼠封盖。不像其它已知的型号，该纸浆封盖的小鼠模型将提供在纸浆再生和修复性象牙质的形成，因为它提供的领域中的宝贵的研究工具:1），以利用广泛可用遗传工程小鼠品系阐明在基本机制的机会分子级和2级）一个经济有效的方法通过增加样本量，以获得统计显著的效果。进一步的研究等候， 包括体内修复性牙本质形成的客观量化，修复性牙本质形成的年龄依赖性的影响，evalua临床上可用的盖髓材料的灰，和所需要的适当的牙髓创伤愈合和修复性象牙质再生的分子决定簇的验证。

Dental caries are one of the most prevalent oral diseases and the leading cause of surgical interventions to dentitions in almost all individuals1,2. The prognosis of surgical interventions and restorations of a tooth largely depends upon proper pulpal response and successful wound healing. Indeed, dental caries that penetrate deeply through the enamel and dentin frequently lead to the exposure of the underlying pulp tissue that is often "capped" with dental materials, such as calcium hydroxide (Ca(OH)2) or hydraulic calcium-silicate cements (HCSCs), including mineral trioxide aggregates (MTA). The ultimate goal of such a pulp-capping procedure is to expedite pulpal wound healing by regenerating reparative dentin, a physical barrier that functions as a "biological seal" to protect the underlying pulp tissue and to increase the life expectancy of the tooth and the overall oral health. However, the underlying mechanism of pulpal wound healing and reparative dentin formation is not fully understood.
To better understand the mechanisms of pulpal wound healing and reparative dentin formation in vivo, several animals were previously used, including monkeys, dogs, and pigs3-5. Among them, rats are frequently used because they are relatively smaller in sizes compared to the other animals, but their teeth are large enough to perform direct pulp capping without any technical difficulties6-10. These animal models are ideal alternatives to human studies for examining pulpal responses and reparative dentin formation. However, their utilization is limited to observational studies at the cellular level, and they scarcely provide mechanistic insights during reparative dentin formation at the molecular level.
Recent technical advances in genetic engineering provided invaluable and indispensable research tools-mice that harbor a gene that is either overexpressed or deleted-that are instrumental to studying molecular mechanisms of human diseases in vivo. The numbers of different strains of transgenic or knockout mice that are strategically inducible in a cell-specific manner are continually growing in the scientific community. Therefore, examining pulpal wound healing and reparative dentin regeneration in these mice would greatly help to expedite our understanding of these processes at the molecular level. However, the use of mice is significantly dampened, as performing a pulp-capping procedure on a mouse tooth is technically challenging due to its miniature size. Here, we present our reproducible method of performing direct pulp capping in mice for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

<table><tbody><tr><td>BM-LED stereo microscope</td><td>MEIJI Techno</td><td></td><td>Microscope&amp;nbsp;</td></tr><tr><td>Optima MCX-LED&amp;nbsp;</td><td>Bien Air Dental</td><td>1700588-001</td><td>Electic motor engine</td></tr><tr><td>isoflurane</td><td>Henry schein animal health</td><td>NDC 11695-0500-2</td><td></td></tr><tr><td>1/4 round bur</td><td>Brasseler</td><td>001092T0</td><td></td></tr><tr><td>Endodontic K-file</td><td>Roydent</td><td>98947</td><td></td></tr><tr><td>ProRoot MTA</td><td>Dentsply</td><td>PROROOT5W</td><td>MTA</td></tr><tr><td>Paper point</td><td>Henry schein</td><td>100-3941</td><td></td></tr><tr><td>Ultra-Etch</td><td>Ultradent product Inc.</td><td></td><td>Phosphoric acid etchant</td></tr><tr><td>OptiBond SoloPlus</td><td>Kerr</td><td>29669</td><td>Adhesives</td></tr><tr><td>Coltolux LED</td><td>Coltene/whaledent Inc.</td><td>C7970100115</td><td>Curing light unit</td></tr><tr><td>Characterization tint</td><td>Bisco</td><td>T-14012</td><td>Flowable composite</td></tr><tr><td>Skyscan</td><td>Breuker</td><td>1275</td><td>uCT scanner</td></tr><tr><td>Microm</td><td>Thermo</td><td>HM355S</td><td>Microtome</td></tr><tr><td>Hematoxyline-1</td><td>Thermo Scientific</td><td>7221</td><td></td></tr><tr><td>Eosin-Y</td><td>Thermo Scientific</td><td>7111</td><td></td></tr><tr><td>Cytoseal 60</td><td>Thermo Scientific</td><td>8310-16</td><td>Mounting solution</td></tr></tbody></table>

development of a direct pulp-capping model for the evaluation of pulpal wound healing and reparative dentin formation in mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

在这里，我们展示了一步一步的过程来执行盖髓对老鼠牙齿。其中纸浆小鼠封盖的关键方面是有相应的设备。在这方面，具有与10倍放大的显微镜是必要的（ 图1A）。要创建在齿类-I样的准备，我们为20万转（ 图1B）使用1/4轮毛刺在电动高速手机。备选地，任何其他的发动机，包括那些使用压缩空气，可用于制备牙齿。 在图2A-2E，用于进行盖髓代表步骤被示出。进行（ 图2B）的Class-Ⅰ类制剂。因为喷水可能在手术过程中淹没的小鼠，不推荐使用它。出于这个原因，它与温柔intermi制备牙齿是必不可少ttent招，以防止经济过热。还建议利用压缩空气以提供冷却效果。而纸浆与根管锉曝光，采取小心不要牙本质碎片推入纸浆，因为这可能在修复性象牙质的形成（ 图2C）中的数据解释干扰。这可以通过使用压缩空气来避免。同样地，在MTA应放置在暴露的纸浆没有推得太远进入纸浆。 MTA放置可以通过使用纸点的背面用轻轻拍打运动（ 图2D）来实现。以下的MTA放置，齿应使用的 H 2 O形浸透棉花球粒以除去任何剩余的MTA在槽中，这可能与在复合体上的齿的结合干扰进行清洗。复合恢复的常规方法使用，包括蚀刻牙齿表面，引发和粘合用粘合剂，以及放置和固化该可流动的复合材料（图2E）。 盖髓后六周，将小鼠收获，并在顶视图被拍到以确认该复合仍然完好无损（ 图3A）。 μCT扫描显示的纸浆封端基团（ 图3B）在牙髓空间显著衰退，这表明在纸浆形成修复性牙本质。脱钙组织样品进行H＆E染色组织学上，进一步研究修复性牙本质在体内的形成。在对照组中，成齿质细胞层（OB）均围绕牙质（ 图4A - 4C）的边缘突出地明显。与此相反，盖髓组显著量形成在牙髓空间（ 图4D - 4F）修复性象牙质（RD）的。有趣的是，仔细检查发现，修复性牙本质（RD）展出牙本质的典型特征（ 如 S<html>代表牙本质小管，红色箭头）triated线，以及作为骨（ 例如，骨细胞代表被困成骨细胞，黑色箭头）。当我们染色牙本质基质蛋白1（DMP1），对牙源性分化12的标记物，我们发现在纸浆封端的齿的纸浆DMP1表达一个显著增加相比，该未封端的齿的（ 图5A和5B）时，表示修复性象牙质是在纸浆内形成。 <img alt="图1" src="/files/ftp_upload/54973/54973fig1.jpg" /> 图1:设备安装了盖髓过程。 （A）的可视化鼠标牙齿显微镜（10X）。 （B）高速手机和制备I类制剂，露出牙髓电动机引擎。="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54973/54973fig2.jpg" /> 图2:在旋盖过程代表步骤。 （一）对鼠标的上颌第一磨牙牙毫无准备。 （B）使用四分之一圆钻初始釉质去除。用根管锉（C），牙髓暴露。 （D）MTA在露髓位置。 （五）对齿复合修复位置。该标尺代表500微米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54973/54973fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54973/54973fig3.jpg" /> 图3:临床表现和μCTScannin克小鼠纸浆封顶，无上限齿。 （一）对纸浆皑皑的齿（左）和不封顶的牙齿（右）鼠标上颌骨咬合视图。 （B）中的上颌骨的剖μCT图像。该标尺代表500微米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54973/54973fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54973/54973fig4.jpg" /> 图4:修复性牙本质形成体内的组织学证据。在100X，200X，400X和鼠标不封顶的上颌第一磨牙（AC）H＆E染色。在100X，200X，400X和鼠标纸浆皑皑的上颌第一磨牙（DE）H＆E染色。酒吧为100μm（OB =牙本质层; RD =修复性牙本质;黑色箭头=骨细胞;红色箭头=牙本质小管）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54973/54973fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54973/54973fig5.jpg" /> 图5:牙本质基质蛋白的免疫组织化学染色。在400X鼠标不封顶的上颌第一磨牙（A）DMP1染色。 （B）DMP1在400X鼠标纸浆皑皑的上颌第一磨牙的染色。所述条为100μm。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54973/54973fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice at JoVE.com

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

我们描述了执行直接纸浆对小鼠齿封盖为牙髓创伤愈合和体内修复象牙质形成的所述评估的步骤的分步方法。

伤口的牙髓评价一个直接盖髓模型的开发和治疗小鼠中修复性牙本质形成

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

development-direct-pulp-capping-model-for-evaluation-pulpal-wound

Universidad San Sebastian

Research

JoVE Journal

Medicine

11.2K 次观看.  The UCLA School of Dentistry. 我们描述了执行直接纸浆对小鼠齿封盖为牙髓创伤愈合和体内修复象牙质形成的所述评估的步骤的分步方法。 

视频: Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

伤口愈合的小鼠模型

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

科研

医学

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

研究对牙髓寄生虫信号反应的神经质生长的共同培养方法

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

发育生物学

Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, proliferation and remodeling. Murine wounds have a histologically well-defined and easily recognizable wound bed over which these different phases of the healing process are measurable. Within the field, it is common to use an arbitrarily defined &#8220;middle&#8221; of the wound for histological analyses. However, wounds are a three-dimensional entity and often not histologically symmetrical, supporting the need for a well-defined and robust method of quantification to detect morphometric defects with a small effect size. In this protocol, we describe the procedure for creating bilateral, full-thickness excisional wounds in mice as well as a detailed instruction on how to measure morphometric parameters using an image processing program on select serial sections. The two-dimension measurements of wound length, epidermal length, epidermal area, and wound area are used in combination with the known distance between sections to extrapolate the three-dimension epidermal area covering the wound, overall wound area, epidermal volume and wound volume. Although this detailed histological analysis is more time and resource consuming than conventional analyses, its rigor increases the likelihood of detecting novel phenotypes in an inherently complex wound healing process.

This protocol describes how to generate bilateral, full-thickness excisional wounds in mice and how to subsequently monitor, harvest, and prepare the wounds for morphometric analysis. Included is an in-depth description of how to use serial histological sections to define, precisely quantify and detect morphometric defects.

穆林切除伤口愈合模型与组织学形态性伤口分析

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, ...

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 ...

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.

使用多盐皮下聚乙烯醇海绵植入和外向尾皮肤伤口模型进行急性伤口愈合评估。

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine ...

免疫与感染

Digital Planimetry for Assessing Wound Closure Kinetics in a Mouse Model

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, thereby reducing the risk of complications and alleviating the economic burden on healthcare systems. Although numerous experimental studies have investigated wound healing, most rely on qualitative observations or quantitative direct measurements. The objective of this study was to standardize an indirect wound measurement method using digital planimetry, incorporating digital scaling and segmentation. This approach addresses the lack of detailed, step-by-step methodologies for accurate wound assessment. A photodocumentation booth was designed and constructed, and computer-assisted digital planimetry tools were employed to minimize variability in measurements of the wound area, perimeter, and the distance from the wound center to its edges. A circular traumatic wound (5 mm in diameter) was created on the dorsal midline at the shoulder blade level of male CD1 mice (n = 4, 10 weeks old, 30-35 g). Wound evolution was photodocumented for 14 days using the custom-designed photo booth, which controlled lighting conditions, focal distance, and subject positioning. Scaling and wound measurements were performed using segmentation in ImageJ software, and statistical analysis was conducted using statistical analysis software. The kinetics of wound closure showed a slight increase in wound size and perimeter between day 0 and day 2, followed by a gradual decrease until complete closure by day 14. The photodocumentation booth and computer-assisted digital planimetry enabled quantitative measurements with minimal variability. In conclusion, these tools provide a reliable and reproducible method for evaluating wound closure kinetics in pre-clinical models.

Wounds represent a global health challenge. This study developed a standardized photo booth utilizing digital planimetry to minimize wound measurement variability. Monitoring wounds in mice over 14 days revealed an initial increase in wound area and perimeter, followed by gradual closure. This methodology may aid in evaluating wound closure kinetics in pre-clinical models.

用于评估小鼠模型中伤口闭合动力学的数字平面测量法

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, ...

生物工程

使用新型 Mouth-Gag 创建牙髓炎症小鼠模型 

Establishment of a Murine Pulp Exposure Model with a Novel Mouth-Gag for Pulpitis Research

Pulpitis, a common cause of natural tooth loss, leads to necrosis and loss of bioactivity in the inflamed dental pulp. Unraveling the mechanisms underlying pulpitis and its efficient treatment is an ongoing focus of endodontic research. Therefore, understanding the inflammatory process within the dental pulp is vital for improving pulp preservation. Compared to other in vitro experiments, a murine pulpitis model offers a more authentic and genetically diverse context to observe the pathological progression of pulpitis. However, using mice, despite their cost-effectiveness and accessibility, poses difficulties due to their small size, poor coordination, and low tolerance, complicating intraoral and dental procedures. This protocol introduces a novel design and application of a mouth-gag to expose mouse pulp, facilitating more efficient intraoral procedures. The mouth-gag, comprised of a dental arch readily available to most dentists and can significantly expedite surgical preparation, even for first-time procedures. Micro-CT, hematoxylin-eosin (HE) staining, and immunofluorescence staining were used to identify changes in morphology and cell expression. The aim of this article is to help researchers establish a more reproducible and less demanding procedure for creating a pulp inflammation model using this novel mouth-gag.

作者聚焦：通过改进的小鼠模型增强牙髓研究

This article presents a streamlined protocol for establishing a pulpitis model in mice using an innovative mouth-gag, followed by subsequent histological analysis.

伤口的牙髓评价一个直接盖髓模型的开发和治疗小鼠中修复性牙本质形成

摘要

探索更多视频

此视频中的章节

伤口愈合的小鼠模型

研究对牙髓寄生虫信号反应的神经质生长的共同培养方法

穆林切除伤口愈合模型与组织学形态性伤口分析

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

使用多盐皮下聚乙烯醇海绵植入和外向尾皮肤伤口模型进行急性伤口愈合评估。

用于评估小鼠模型中伤口闭合动力学的数字平面测量法

基于新型口堵的鼠牙髓暴露模型的建立及其在牙髓炎研究中的应用

基于新型口堵的鼠牙髓暴露模型的建立及其在牙髓炎研究中的应用

伤口的牙髓评价一个直接盖髓模型的开发和治疗小鼠中修复性牙本质形成

摘要

探索更多视频

此视频中的章节

伤口愈合的小鼠模型

研究对牙髓寄生虫信号反应的神经质生长的共同培养方法

穆林切除伤口愈合模型与组织学形态性伤口分析

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 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 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

使用多盐皮下聚乙烯醇海绵植入和外向尾皮肤伤口模型进行急性伤口愈合评估。

用于评估小鼠模型中伤口闭合动力学的数字平面测量法

基于新型口堵的鼠牙髓暴露模型的建立及其在牙髓炎研究中的应用

基于新型口堵的鼠牙髓暴露模型的建立及其在牙髓炎研究中的应用

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