This study was supported by R01DE023348 (RHK) from NIDCR/NIH and the Faculty Research Grant (RHK) from the Council on Research of the Academic Senate of the Los Angeles Division of the University of California.

Mice were purchased from Jackson Laboratory and kept in a pathogen-free vivarium in the UCLA Division of Laboratory Animal Medicine (DLAM). The experiments were performed according to the approved institutional guidelines from the Chancellor&#39;s Animal Research Committee&#160;(ARC#2016-037).
1. Mouse Anesthetization
<ol>
	<li>Use eight-week-old female C57/BL6 mice (n = 3).</li>
	<li>Anesthetize the mice using ketamine (80-120 mg/kg of mouse weight)/xylazine (5 mg/kg of mouse weight) solutions and administer in...

<ol>
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A pending public health crisis.</a> Am J Dent. 22 (1), 3-8 (2009).</li><li>Koliniotou-Koumpia, E., Tziafas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulpal+responses+following+direct+pulp+capping+of+healthy+dog+teeth+with+dentine+adhesive+systems.">Pulpal responses following direct pulp capping of healthy dog teeth with dentine adhesive systems.</a> J Dent. 33 (8), 639-647 (2005).</li><li>Tarim, B., Hafez, A. A., Cox, C. 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The authors have nothing to disclose.

Currently, there are several different experimental models available to validate the in vivo effects of dental materials, scaffolds, or growth factors on odontogenic differentiation of dental pulp stem cells (DPSCs)13. These models include ectopic autologous transplantation of DPSCs into an organ, such as the renal capsule, or subcutaneous transplantation of DPSCs into immunocompromised mice with scaffolds14,15. However, these methods are limited in that their odontogenic effect on DPSCs is...

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.

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	<colgroup>
		<col span="2" />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">BM-LED stereo microscope</td>
			<td style="width:164px;">MEIJI Techno</td>
			<td style="width:140px;"></td>
			<td style="width:173px;">Microscope&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optima MCX-LED&#160;</td>
			<td>Bien Air Dental</td>
			<td>1700588-001</td>
			<td>Electic motor engine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">isoflurane</td>
			<td>Henry schein animal health</td>
			<td>NDC 11695-0500-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1/4 round bur</td>
			<td>Brasseler</td>
			<td>001092T0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endodontic K-file</td>
			<td>Roydent</td>
			<td>98947</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProRoot MTA</td>
			<td>Dentsply</td>
			<td>PROROOT5W</td>
			<td>MTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paper point</td>
			<td>Henry schein</td>
			<td>100-3941</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra-Etch</td>
			<td>Ultradent product Inc.</td>
			<td></td>
			<td>Phosphoric acid etchant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OptiBond SoloPlus</td>
			<td>Kerr</td>
			<td>29669</td>
			<td>Adhesives</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coltolux LED</td>
			<td>Coltene/whaledent Inc.</td>
			<td>C7970100115</td>
			<td>Curing light unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Characterization tint</td>
			<td>Bisco</td>
			<td>T-14012</td>
			<td>Flowable composite</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Skyscan</td>
			<td>Breuker</td>
			<td>1275</td>
			<td>uCT scanner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microm</td>
			<td>Thermo</td>
			<td>HM355S</td>
			<td>Microtome</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematoxyline-1</td>
			<td>Thermo Scientific</td>
			<td>7221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eosin-Y</td>
			<td>Thermo Scientific</td>
			<td>7111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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.

Here, we showed the step-by-step procedures to perform pulp capping on mice teeth. One of the key aspects of pulp capping in mice is to have the appropriate apparatus. In this regard, having the microscope with a 10X power magnification is essential (Figure 1A). To create a Class-I-like preparation in the tooth, we used a &#188;-round burr in an electric high speed handpiece at 200,000 rpm (Figure 1B). Alternatively, any other engines, including those tha...

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

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

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 ...

The overall goal of this protocol is to present the direct pulp-capping procedure in mice. This method can help you answer key questions in pulp biology, such as, how pulp heals, or how reparative dentin forms, when you place trapped pulp-capping materials on the exposed pulp. The main advantage of this technique is that the actual direct pulp-capping procedure performed in humans can also be done in mice, in exactly the same manner.The implications of this technique extends towards improving material properties to save tooths that is otherwise doomed to be more invasive treatments, such as root canal therapy. After properly anesthetizing the mouse for the procedure, position the mouth holder in its mouth, and secure the other end to the table such that the head is facing upward. Using good lighting and a stereoscope, observe the first maxillary molar.Then, using the quarter-round bur in a high speed handpiece at 200, 000 rpm, remove the central region of the tooth's enamel until the pulp is visible through the transparent dentin. Be careful to not penetrate into the pulp. Next, using a no.15 endodontic k-file, perforate through the dentin and expose the pulp. Do not push any debris into the pulp;this can be avoided by rotating the k-file quarterly, and then pulling the k-file out. Now, prepare the MTA, and using the explorer, deliver the MTA, then pack it into the exposed region using the back side of the paper point and gentle tapping.Next, using a syringe loaded with 35%phosphoric acid etchant, cover the tooth with the etchant, avoiding the gingival tissues, and let the etchant work for 15 seconds. After 15 seconds, suck the etchant off the tooth, and use a water-moistened cotton applicator to wipe off any remaining etchant. Suck and wipe until all the etchant is removed.Follow by applying the dental adhesives using the backside of the paper point. Make the adhesive layer thin by blowing it with compressed air for a few seconds. Then cure the adhesive for 30 seconds with the appropriate light.Now, place small amounts of composite onto the tooth using the tip of the explorer to flow the composite into the tooth grooves. Once applied, cure the composite for 30 seconds. After euthanizing the mouse and removing the entire maxilla, place the jaw into 4%paraformaldehyde in PBS at pH 7.4.Keep the tissue at 4 degrees Celsius overnight, and on the following morning, transfer it to 70%ethanol for storage until it can be processed. To take a micro CT scan of the maxilla, first secure it in gauze soaked with 70%ethanol, then pack it into a 15 milliliter cell culture tube. Then mount the tube onto the micro CT scanning stage.Next, set the x-ray source to deliver a 145 microamp current with 55 peak kilovolts for 200 milliseconds. Using a 0.5 millimeter aluminum filter, acquire images with 20 micron resolution. After the scans are completed, start decalcifying the maxilla with 5%EDTA and 4%sucrose in PBS, at a pH of 7.4.Let this reaction go for two weeks at 4 degrees Celsius. After embedding the maxilla, make five micron thick slices. The pulp-capping areas usually coincide with the distal palatal root, which can be used as a landmark.Determine the precise area of interest by examining the histology under the light microscope, and comparing the results to the micro CT scans. For H&E staining, remove the paraffin, and rehydrate the slides with two baths in xylene for a few minutes each. Then pass them through a series of serially diluted ethanol.Each bath should be applied for just one minute. Follow the rehydration with a rinse. And then apply the hemotoxylin solution for two and a half minutes.Remove the dye with a rinse, and then put the slides in 95%ethanol for one minute. Next, stain the slides with eosin solution for one minute, and then rinse them off. Reverse the hydration steps to dehydrate the stained tissues, starting with ethanol.Then, treat them with xylene. The slides can then be mounted and imaged. Using the described procedures, pulp-capping was performed on 8-week-old mice.Six weeks later, their maxilla were harvested and examined. Curiously, H&E staining revealed dentin formation throughout the pulp chamber and the root canals. Comparatively, the uncapped molar did not show the same dentin formation.In the capped molar, there were characteristics of both dentin-like and bone-like formation, as both dentinal tubules, and osteocytes were found in the reparative dentin, suggesting that injured pulp may be mineralized by both residing odontoblasts and immigrated osteoblasts. Use of DMP1 immunohistochemical staining further confirmed increased activities of cells forming reparative dentin. Just like real dental clinical practices, having an assistant is extremely helpful.For example, with an assistant, this technique can be done in just ten minutes, once mastered. Importantly, when you're exposing the pulp, remember to use endofile, not the bur. And when you're handing the MTA, be careful.Don't forget that working with fixatives like paraformaldehyde can be extremely hazardous. Precautions such as wearing personal protective equipment should always be taken.

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

Research

JoVE Journal

Medicine

11.1K visualizações.  The UCLA School of Dentistry. The overall goal of this protocol is to present the direct pulp-capping procedure in mice. This method can help you answer key questions in pulp biology, such as, how pulp heals, or how reparative dentin forms, when you place trapped pulp-capping materials on the exposed pulp. The main advantage of this technique is that the actual direct pulp-capping procedure performed in humans can also be done in mice, in exactly the same manner.The implications of this technique extends towards im...