本研究由中国国家自然科学基金U21A20368（L.Y.）、82101000（H.W.）和82100982（F.L.）以及四川省科技计划2023NSFSC1499（H.W.）资助。所有原始数据和图像均包含在本文中。

本研究的实验程序经四川大学华西口腔医学院伦理委员会（WCHSIRB-D-2021-125）批准。成年C57BL / 6小鼠购自中国成都的Gempharmatech实验动物公司。上颌第一磨牙的整个牙冠在出生后 21 天萌出。手术小鼠的年龄应大于21天，具有正常活力11。在这里，使用 6 至 8 周龄的小鼠进行建模。 图 1 是一个流程图，显示了所使用的协议。
1.术前准备（图2）
<ol><li>获取以下仪器：立体显微镜、固定板、医用胶带、口腔堵嘴、直径0.6mm的微创牙科毛刺、牙科高速牙科手机、8# C+锉、加热垫、1 mL注射器、无菌棉球、眼钳。</li>
	<li>获得以下药物：麻醉混合物，兽药膏。</li>
</ol>2.口塞的准备
<ol><li>通过腹膜内注射麻醉混合溶液（10%盐酸氯胺酮+ 5%甲苯噻嗪+ 85%无菌等渗盐水）以0.007mL / g体重称重并麻醉小鼠，并通过脚趾捏法确认适当的麻醉。将眼科润滑膏涂抹在眼睛上，以防止手术时因干燥而造成眼睛受伤。 注意：医用帽子、口罩、手套和工作服以及其他基本保护措施是必要的。确保手术环境和小鼠室清洁安全。在整个过程中，用于热支撑的加热垫是必要的。</li>
	<li>如下所述准备嘴巴堵嘴（图3）。<ol><li>获得以下材料：直径为 8 μm 的正畸弓丝、年轻的环弯曲钳、重型钢丝钳、记号笔、长度为 3 mm、横截面直径为 1 mm 的橡胶帽。</li>
		<li>首先，用左手拉直弓形线进行固定，右手的拇指、食指稍微弯曲以抵挡线的弧度。重复此动作几次，将有助于弯曲到正确的三维角度。</li>
		<li>使用Yong环弯曲钳将梯形（图3C，a-l-k-b）的顶部边缘（图3G，a-i）弯曲在弓形丝的中点长约8 mm。确保点 a（图 3）位于钳嘴的边缘。</li>
		<li>用左手握住钳子，用右手拇指和食指夹住弓线的自由端，从a点弯曲弓线，形成约120°的角度。在点i处复制上一个操作（图3G）。将弓形线放在水平台上而不撬动，检查弓形线是否在一个平面上。</li>
		<li>每侧留出约 9 毫米的长度（图 3D、a-b、l-k），并使用与步骤 2.2.4 相同的技能将自由端弯曲到 75° 角，同时确保每条边都在一个平面上。用钳嘴的尖端弯曲这个锐角。</li>
		<li>找到距离 b 点约 5 毫米的 c 点。按照相同的技巧在点 c 上弯曲 105° 角。在距离 c 点 5 毫米的 d 点再弯曲 105° 角。距 d 点约 4.5 mm，找到 e 点。在点 e 处弯曲自由端以形成约 100 -105° 的角度（图 3E）。 注意：我们使用的 6-8 周龄的 C57 小鼠约为 20 g。5毫米的间距不仅可以卡住小鼠的上下颚而不动弹，而且不会压迫小鼠的皮肤而引起不适。如果使用其他种类或年龄的小鼠，请根据实际情况调整c-d和i-h部分的长度（图3E，G）。</li>
		<li>为下颌部分弯曲一个额外的压舌板（图3G，j-i-h-g）。</li>
		<li>a-b-c零件在l-k-j零件上的重复弯曲步骤。同时夹紧 i-k 和 k-j 零件，并在 j 点弯曲自由端，使其垂直于 i-k-j 平面。夹紧点 i 距点 j 5 mm，弯曲自由端以使其平行于 i-k-j 平面和 c-d 部分（图 3H）。</li>
		<li>从 i 点起留 5 mm 长度，在 h 点处，将拱形弯曲垂直于 i-h 部分并平行于 j-i-h 平面。在距点 h 5 毫米处找到点 g。夹紧 j-i-h-g 平面并弯曲对称于 k-j 部分的自由端。然后，点f之后的自由端应与点e的自由端对称（图3H）。</li>
		<li>将橡胶帽放在自由端（图3F）。</li>
	</ol></li>
</ol>3. 固定
<ol><li>将小鼠仰卧固定在固定板上，四肢用皮胶带固定。用拇指和食指压缩嘴巴的自由端。</li>
	<li>将鼠标前门牙固定在两条手臂的梯形凹槽中。确保带有压舌板的手臂用于下颌骨。调整嘴巴堵嘴，确保老鼠的舌头被固定但不会缺血。</li>
</ol>4. 牙齿评估
<ol><li>确保手术时上颌第一磨牙应没有龋齿、外伤和牙龈生成。确保周围的牙龈没有发红、肿胀或瘘管。确保对面的牙齿健康，并可以作为健康的对照组。</li>
</ol>5. 纸浆暴露
<ol><li>使用牙齿毛刺以 20,000 rpm 的速度在上颌第一磨牙的咬合侧钻孔。确保去除牙釉质。仅在牙本质的浅层中保持牙科手机的操作，以防止对牙髓组织进行过度热刺激12.</li>
	<li>同时，在手术过程中每 3 分钟使用注射器将生理盐水滴在牙齿上，以防止过热。</li>
	<li>将 8# 或 10# C+ 锉刀放在钻坑的最低位置，然后刺穿最后一个牙本质，露出牙髓室。当局部牙本质被彻底去除时，会有一种明显的坠落感。不要探得太深，否则牙髓组织可能会从牙髓室中带出。</li>
	<li>清洁牙齿周围的碎片。取下嘴巴堵嘴;手术已经完成。使用对面的上颌第一磨牙作为对照，无需手术。</li>
</ol>6. 术后护理
<ol><li>手术后，皮下给予卡洛芬（5mg / kg），并将小鼠置于化学热加热垫上，以俯卧姿势，直到从麻醉中恢复。喂老鼠并提供饮用水。恢复过程应该受到监督。在小鼠完全恢复之前，其他动物不应在同一腔室中。</li>
</ol>7. 样本采集和储存
<ol><li>在手术后 24 小时或根据实验9 的任何其他时间点，在深度麻醉条件下对颈椎脱位的小鼠实施安乐死。用眼科剪刀剪开附着在上颌骨和颧骨上的骨骼肌。去除骨骼、额骨和软组织，取出带有上颌磨牙的蚋臼片。 注意：根据 He 等人的说法，建议在手术后不到 72 小时收集牙髓炎样本，以避免牙髓组织广泛坏死13。</li>
	<li>矢状地将蚊蚋石分成两半，并将组织储存在PBS（pH 7.4）中的4%多聚甲醛中，在4°C下进行24小时固定。</li>
</ol>8. 组织学分析
<ol><li>用磷酸盐缓冲盐水（PBS）洗涤组织，并在每日更换的5%EDTA和4%蔗糖的PBS（pH 7.4）脱钙溶液中脱钙，在4°C下2-4周10。</li>
	<li>将 1/2 蚊蚋石嵌入石蜡中，并确保没有牙齿的矢状面位于组织盒的底部。</li>
	<li>用石蜡切片机将石蜡块切成5μm厚的切片。根据显微镜下观察到的近端、远端、上部和下部关系调整石蜡块的角度，以确保可以切割完整的冠髓和第一磨牙的穿孔。</li>
</ol>

使用新型 Mouth-Gag 创建牙髓炎症小鼠模型 

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作者声明没有利益冲突。

作为牙齿内孤立的软组织，牙髓在维持牙齿的生物活性方面发挥着至关重要的作用，但仍然高度敏感。保留这种重要的牙髓已成为最近牙髓治疗中首选的初始方法，因此需要全面了解牙髓的炎症机制16。炎症微环境的时空波动和牙髓炎中驻留细胞类型之间的相互作用使其通过 体外 研究的研究变得复杂11.相反， 体内 研究通过复制在人类中发现的生?...

牙髓是牙齿的一个组成部分，负责多种基本功能，如营养供应、牙本质形成、感觉功能和防御反应1。然而，被硬组织包围的牙髓容易受到深龋、牙髓炎、创伤或后续治疗的伤害和损伤 2,3。功能性牙髓的缺失会增加牙齿脆弱的风险4.此外，年轻恒牙牙髓活力的丧失会对牙齿成熟产生不利影响，而目前的假牙技术无法恢复健康牙髓提供的神经反馈4.这种情况促使研究人员探索管理发炎牙髓的替代解决方案，而不仅仅是去除。
2007 年，Murray 等人开始将组织工程应用于再生牙髓学，从而引发了人们对牙髓保存和再生的兴趣5。然而，发炎的牙髓组织带来了挑战，因为细胞会释放 IL-6 等炎症因子，IL-6 会招募炎症细胞并导致细胞坏死、牙髓活力丧失和功能恢复并发症 6,7。因此，了解炎症和相关的细胞死亡对于保护重要牙髓的进展至关重要。已经进行了许多实验来探索炎症牙髓在体内或体外的分子生物学 8,9。尽管像 2D 或 3D 细胞培养这样的体外实验已经发展了多年，并且已经变得成熟并广泛用于测试牙髓细胞对炎症因子的反应，但这些实验无法反映牙髓组织与全身免疫系统之间的相互作用10。如果正在研究的现象来源于其他组织来源的细胞，如免疫、血管和神经系统，那么纯牙髓细胞培养将导致死胡同。因此，体内实验是非常必要和具有参考意义的。
小鼠因其成本效益、高生育率和活力而日益成为 体内炎症研究中的常见选择。然而，目前尚无针对小鼠牙髓炎模型的综合方案，可作为参考。小鼠的小尺寸和它们对刺激的敏感性在实验过程中构成了重大挑战。观察隐藏在小鼠口腔深处的微小牙齿通常需要使用悬臂显微镜，尽管桌面显微镜在实验室中更为普遍。没有张口器需要其他人的帮助。为了解决这个问题，该小组使用现成的材料设计了一种口腔堵嘴器，旨在为构建小鼠牙髓炎模型提供标准化和可重复的协议。本文详细介绍了该过程，涵盖 C57 小鼠的术前准备、固定、牙髓暴露手术和样本采集。该协议建议使用口腔堵嘴，提供有关其结构、生产和应用的信息，以方便其他研究人员复制该程序。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Animal</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57/B6J mice</td>
			<td>Gempharmatech Experimental Animals Company</td>
			<td>C57/B6J</td>
			<td>For the establishment of pulp exposure</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>Chengdu Xinjin Shifeng Medical Apparatus &#38; Instruments Co. LTD.</td>
			<td>SB1-074(IV)</td>
			<td>Apply in drug injection.</td>
		</tr>
		<tr>
			<td>8# C+ file</td>
			<td>Readysteel</td>
			<td>0010047</td>
			<td>Apply in exposing the roof of pulp chamber.</td>
		</tr>
		<tr>
			<td>Anesthesia Mix solution</td>
			<td></td>
			<td></td>
			<td>10% ketamine hydrochloride+ 5% xylazine + 85% sterile isotonic saline.&#160;</td>
		</tr>
		<tr>
			<td>DAPI Staining Solution</td>
			<td>Beyotime</td>
			<td>C1005</td>
			<td>Apply in immunofluorescence staining for counter-staining of nucleus.</td>
		</tr>
		<tr>
			<td>Dental high-speed dental handpiece</td>
			<td>Jing yuan electronic commerce technology</td>
			<td>WJ-422</td>
			<td>Apply in pulp exposure.</td>
		</tr>
		<tr>
			<td>Heavy wire cutter</td>
			<td>Jirui Medical Instrument Co., Ltd.</td>
			<td>none</td>
			<td>Apply inarc cutting.</td>
		</tr>
		<tr>
			<td>Hematoxylin and Eosin Stain kit</td>
			<td>Biosharp</td>
			<td>BL700B</td>
			<td>For the histological analysis of the slides.</td>
		</tr>
		<tr>
			<td>IL-6 antibody</td>
			<td>Novus</td>
			<td>NBP2-89149</td>
			<td>Apply in immunofluorescence staining to detect the inflammation of the dental pulp.</td>
		</tr>
		<tr>
			<td>Ketamine(Ketamine hydrochloride)</td>
			<td>Vet One, Boise, Idaho, USA</td>
			<td>C3N VT1</td>
			<td>100mg/kg, IP. Apply in nesthetization.</td>
		</tr>
		<tr>
			<td>Medical tap</td>
			<td>3M</td>
			<td>1530</td>
			<td>Apply in mice immobilization.</td>
		</tr>
		<tr>
			<td>Orthodontic arch wire&#160;</td>
			<td>Shanghai Wei Rong Medical Apparatus Co. LTD.</td>
			<td>K417</td>
			<td>Diameter of 8&#181;m</td>
		</tr>
		<tr>
			<td>Round dental burr (0.6 mm)</td>
			<td>Shofu global</td>
			<td>072208</td>
			<td>Apply in removing enamel and shallow layer of dentin.</td>
		</tr>
		<tr>
			<td>Young loop bending plier</td>
			<td>Jirui Medical Instrument Co., Ltd.</td>
			<td>none</td>
			<td>Apply in arc bending.</td>
		</tr>
	</tbody>
</table>

establishment of a murine pulp exposure model with a novel mouth-gag for pulpitis research

牙髓炎是自然牙齿脱落的常见原因，会导致发炎牙髓中的坏死和生物活性丧失。揭示牙髓炎的机制及其有效治疗是牙髓病学研究的持续重点。因此，了解牙髓内的炎症过程对于改善牙髓保存至关重要。与其他体 外 实验相比，鼠牙髓炎模型为观察牙髓炎的病理进展提供了更真实和遗传多样化的背景。然而，尽管小鼠具有成本效益和可及性，但由于它们的体积小、协调性差和耐受性低，使用小鼠会带来困难，使口腔内和牙科手术复杂化。该协议引入了一种新颖的设计和应用，即口腔堵嘴以暴露小鼠牙髓，从而促进更有效的口内手术。口腔堵嘴由大多数牙医都可以轻松使用的牙弓组成，可以显着加快手术准备，即使是第一次手术也是如此。Micro-CT、苏木精-伊红（hematoxylin-eosin，HE）染色和免疫荧光染色（immunofluescing sing）检测形态和细胞表达的变化。本文的目的是帮助研究人员建立一种更具可重复性且要求更低的程序，以使用这种新型的口腔堵嘴创建牙髓炎症模型。

The dental pulp, an integral part of the tooth, is responsible for multiple essential functions such as nutrient supply, dentin formation, sensory function, and defense reactions1. Nevertheless, the dental pulp, surrounded by hard tissue, is susceptible to injuries and damages from deep caries, pulpitis, trauma, or subsequent therapies2,3. The absence of functional dental pulp increases the risk of tooth fragility4. Moreover, the loss of pulp vitality in young permanent teeth can adversely affect tooth maturation, and current denture techniques fail to restore the neural feedback offered by healthy pulp4. This situation has led researchers to explore alternative solutions for managing inflamed pulp beyond mere removal.
In 2007, Murray et al. initiated the application of tissue engineering in regenerative endodontics, thereby sparking increased interest in pulp preservation and regeneration5. However, inflamed pulp tissue poses a challenge as cells release inflammatory factors such as IL-6, which recruit inflammatory cells and results in cell necrosis, loss of pulp vitality, and complications in functional recovery6,7. Understanding inflammation and the associated cell death is, therefore, crucial for advancements in the preservation of vital pulp. There are a number of experiments that have been conducted to explore the molecular biology of the inflame pulp in vivo or in vitro8,9. Though in vitro&#160;experiments like 2D or 3D cell cultures have been developed for years and are becoming mature and widely used to test reactions of pulp cells to inflammatory factors, these experiments cannot reflect the interaction between pulp tissue and the systemic immune system10. If the phenomenon being studied is derived from cells of other tissue origin like immune, vascular and nervous system, then pure pulp cell culture will lead to a dead end. Therefore, in vivo experiments are very necessary and referential.
Mice have increasingly become a common choice in inflammation research in vivo&#160;due to their cost-effectiveness, high fertility, and vitality. However, a comprehensive protocol for mice pulpitis model is currently absent, which can serve as a reference. The small size of mice and their sensitivity to stimulation pose significant challenges during experimental procedures. Observing the minuscule teeth concealed deep within the mouse mouth often necessitates the use of a cantilever microscope, notwithstanding the more common presence of desktop microscopes in laboratories. The absence of a mouth opener requires assistance from others. To address this, the group has devised a mouth-gag using readily available materials which aims to provide a standardized and reproducible protocol for constructing the mice pulpitis model. This article details the procedure, covering preoperative preparation, immobilization, pulp exposure surgery, and sample collection on C57 mice. This protocol recommends the use of the mouth-gag, providing information on its structure, production, and application to facilitate other researchers in replicating the procedure.

作者聚焦：通过改进的小鼠模型增强牙髓研究

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

Individual experiments like 2D or 3D cell cultures have been widely used to test the reactions of dental pulp cells. These experiments cannot reflect the interaction between pulp tissue and the systemic immune system. Therefore, in vivo experiments are becoming increasingly popular in endodontic research.C57 mice have increasingly become a common choice in inflammation research on dental pulp due to their cost effectiveness, high fertility and vitality. However, the small size of mice and lack of coordination posed significant challenges during experimental procedures. The fixation process is an important factor that higher the technical threshold.We devised a novel mouth gag using readily available materials and simple assemblies to assist researchers in pulp exposure surgery. According to our research, this mouth gag can significantly shorten the time required for beginners to immobilize the mouth and expose the maxillary first molar of mice.

在3,6-8周龄C57BL / 6小鼠的右上颌第一磨牙上进行上述程序，同时保留左上颌第一磨牙作为对照。空白对照、12 h牙髓炎和24 h牙髓炎样品的组织学和免疫荧光结果用于示范。
根据 Goldman 等人 15 的 CT 分析方案，通过 图 4 A-C 中的显微 CT 和重建建模确认了牙髓暴露。来自对照侧和手术侧的上颌第一磨牙的矢状面切片都进行了 HE ?...

Watch this Scientific Journal Video about Establishment of a Murine Pulp Exposure Model with a Novel Mouth-Gag for Pulpitis Research at JoVE.com

本文提出了一种简化的方案，用于使用创新的口堵在小鼠中建立牙髓炎模型，然后进行随后的组织学分析。

Pulpitis, a common cause of natural tooth loss, leads to necrosis and loss of bioactivity in the inflamed dental pulp. Unraveling the mechanisms ...

2D或3D细胞培养等单个实验已被广泛用于测试牙髓细胞的反应。这些实验不能反映牙髓组织与全身免疫系统之间的相互作用。因此，体内实验在牙髓研究中越来越受欢迎。C57小鼠因其成本效益、高生育率和活力而日益成为牙髓炎症研究中的常见选择。然而，小鼠体积小和缺乏协调性在实验过程中带来了重大挑战。固定过程是提高技术门槛的重要因素。我们使用现成的材料和简单的组装设计了一种新颖的口腔堵嘴器，以帮助研究人员进行牙髓暴露手术。根据我们的研究，这种口腔堵嘴可以大大缩短初学者固定嘴巴和暴露小鼠上颌第一磨牙所需的时间。

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Research

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Medicine

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

621 次观看.  Sichuan University. 2D或3D细胞培养等单个实验已被广泛用于测试牙髓细胞的反应。这些实验不能反映牙髓组织与全身免疫系统之间的相互作用。因此，体内实验在牙髓研究中越来越受欢迎。C57小鼠因其成本效益、高生育率和活力而日益成为牙髓炎症研究中的常见选择。然而，小鼠体积小和缺乏协调性在实验过程中带来了重大挑战。固定过程是提高技术门槛的重要因素。我们使用现成?...