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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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's Animal Research Committee (ARC#2016-037).

1. Mouse Anesthetization

1. Use eight-week-old female C57/BL6 mice (n = 3).
2. Anesthetize the mice using ketamine (80-120 mg/kg of mouse weight)/xylazine (5 mg/kg of mouse weight) solutions and administer intraperitoneally (ip) at a dose of 10 mL/kg.
3. Prepare ketamine (80 - 120 mg/kg)/xylazine (5 mg/kg) solutions and administer them intraperitoneally (ip) at a dose of 10 mL/kg.
4. Confirm that the mice are fully anesthetized by performing a toe pinch.

2. Pulp-capping Procedure

1. Place the mouth holder in the mouse's mouth.
2. Secure the mouth holder on the table such that the head is facing upward.
3. Place the microscope (10X) on top of the mouth so that the first maxillary molar is fully visible.
4. Using the ¼-round bur in a high-speed handpiece at 200,000 rpm, remove the enamel part of the tooth in the middle until the pulp is visible through the transparent dentin. Do not expose the pulp with the bur.
5. Using a #15 endodontic K-file (diameter of 150 µm), perforate through the dentin and expose the pulp.
   NOTE: Special care should be taken so that the dentin debris does not get pushed into the pulp. This can be avoided by rotating the K-file quarterly and then pulling the K-file out.
6. Mix MTA with sterile H₂O according to the manufacturer's instructions. Deliver and place the MTA onto the exposed pulp with the tip of the explorer. Use the back side of the paper point (fine) to pack the MTA into the exposed pulp by gentle tapping. The thicker side of the paper point is flat and therefore allows for the proper condensation of the MTA into the exposed pulp.
7. Etch the tooth for 15 s by placing the 35% phosphoric acid etchant where it just covers the tooth. Take special care to limit the placement of the etchant, as it may irritate gingival tissues.
   NOTE: The etchant comes in a syringe and is used to roughen the tooth surfaces so that dental adhesives can flow in to mediate micromechanical bonding onto the tooth. Because they are viscous, it can be self-contained by applying small amounts directly onto the tooth.
8. Use negative-pressured suction to remove the etchant. Use a cotton pellet that is lightly soaked with H₂O to remove the residuals of the etchant. Repeat this step until the etchant is completely removed from the tooth.
9. Using a compressed air duster, gently dry the tooth.
10. Apply the dental adhesives using the backside of the paper point.
11. Make the adhesive layer thin using compressed air for 3 s.
12. Cure the dental adhesives for 20 s using the curing-light unit.
13. Place the flowable composite in small amounts onto the tooth that was capped with MTA. Use the tip of the explorer to flow the composite into the tooth grooves.
14. Cure the composite for 30 s using a light-curing unit to polymerize it. Confirm that the composite is fully cured and hard using the explorer.

3. Post-op Care

1. Administer carprofen (5 mg/kg) subcutaneously (sc) immediately after the pulp-capping procedure.
2. Place the mice on a heating pad at low power to keep the animals warm before they wake up.
3. Return the mice to the vivarium for housing.

4. Tissue Procurement

1. After 5 - 6 weeks, euthanize the mice by cervical dislocation under a complete anesthetic condition with isoflurane.
2. Carefully remove the maxilla out of the base of the skull and put it into a 50-mL tube. Fix the entire maxilla that contains both the pulp-capped tooth and the contralateral uncapped tooth in 4% paraformaldehyde in PBS, pH 7.4, at 4 °C overnight, and then store it in a 70% ethanol solution.
   NOTE: Paraformaldehyde is toxic and carcinogenic. The proper use paraformaldehyde should be monitored as outlined in the standard operating procedures (SOP).
3. Scan the mouse maxillae using the µCT scan. To secure the maxillae during scanning, wrap the samples with gauze soaked with 70% ethanol and place them in the 15-mL cell culture tube.

5. µCT Scanning

1. Prepare the samples for µCT scanning. Briefly, wrap the samples with gauze soaked with 70% ethanol and secure them in a generic 15-mL cell culture conical tube. Mount the tube onto the µCT scanning stage, as outlined in the manufacturer's instructions.
2. Set the X-ray source to a current of 145 µA, a voltage of 55 kVp, and an exposure time of 200 ms.
3. Perform image acquisition with the µCT scanner at a 20-µm resolution and with a 0.5 mm Al filter.
4. Reconstruct the image and visualize it¹¹.
5. Once the µCT scan is complete, start decalcification with 5% EDTA and 4% sucrose in PBS (pH 7.4) for 2 weeks.

6. Tissue Processing and Staining

1. Embed the decalcified tissues in paraffin. Before embedding, trim the maxilla by making a sagittal cut immediately anterior to the first molar. While embedding, position this surface downward, such that the longitudinal section of the first molar is the cutting surface.
2. Using the microtome, prepare 5 µm-thick slides. The pulp-capping areas usually coincide with the distopalatal (DP) 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 µCT images.
3. For H&E staining, deparaffinize and rehydrate the slides with xylene (2x) and serially diluted ethanol (100% EtOH 2x, 95% EtOH 2x, and 70% EtOH 1x).
4. Rinse the slides with running tap water.
5. Stain with Hematoxylin solution for 2.5 min and rinse with tap water.
6. Dip the slides in 95% ethanol for 1 min.
7. Stain with Eosin solution for 1 min and rinse with tap water.
8. Dehydrate with serially diluted ethanol (70% EtOH 1x, 95% EtOH 2x, and 100% EtOH 3x) and xylene (3x).
9. Mount the slides with mounting solution.

Results

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 ¼-round burr in an electric high speed handpiece at 200,000 rpm (Figure 1B). Alternatively, any other engines, including those that us...

Discussion

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 scaffolds^14,15. However, these methods are limited in that their odontogenic effect on DPSCs is...

Materials

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