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

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。

摘要

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.

引言

Dental caries are one of the most prevalent oral diseases and the leading cause of surgical interventions to dentitions in almost all individuals^1,2. 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 pigs^3-5. 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 difficulties^6-10. 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.

研究方案

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

1. 使用八周龄雌性C57 / BL6小鼠（n = 3）。
2. 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。
3. 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。
4. 确认小鼠完全通过执行脚趾捏麻醉。

2.盖髓过程

1. 放置在鼠标的嘴的嘴保持器。
2. 固定口保持器上的表，使得他广告朝上。
3. 放置在口的顶部的显微镜（10X），使得第一磨牙完全可见。
4. 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。
5. 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。
6. 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。
7. 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。
8. 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。
9. 使用压缩空气除尘器，轻轻擦干牙齿。
10. 申请使用的纸张点的背面的牙科粘合剂。
11. 使用压缩空气3秒的粘合剂层薄。
12. CURE使用固化光用单元20秒的牙科粘合剂。
13. 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。
14. 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。

3.后运护理

1. 管理该盖髓过程后立即卡洛芬（5毫克/千克）皮下（SC）。
2. 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。
3. 返回小鼠动物饲养住房。

4.组织采购

1. 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。
2. 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。
3. 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。

5.μCT扫描

1. 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。
2. 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。
3. 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。
4. 重建图像和可视化是^11。
5. 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。

6.组织处理和染色

1. 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。
2. 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。
3. 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。
4. 冲洗用流动的自来水的幻灯片。
5. 与2.5分钟苏木素液染色，用自来水冲洗。
6. 浸在95％乙醇的幻灯片1分钟。
7. 用1分钟伊红染色解决方案，并与自来水冲洗。
8. 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。
9. 安装与安装解决方案的幻灯片。

结果

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。

讨论

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?...

材料

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