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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 therapies^2,3. 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 recovery^6,7. 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 vitro^8,9. Though in vitro 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 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.

Protokół

The experimental procedures in this study were approved by the ethical committee of the West China School of Stomatology, Sichuan University (WCHSIRB-D-2021-125). Adult C57BL/6 mice were obtained from Gempharmatech Experimental Animals Company, Chengdu, China. The whole crown of the maxillary first molar erupts 21 days after birth. Mice for surgery should be older than 21 days with normal vitality¹¹. Here, 6 to 8-week-old mice were used for modeling. Figure 1 is a flow diagram showing the protocol used.

1. Preoperative preparation (Figure 2)

1. Obtain the following instruments: Stereoscopic microscope, fixing plate, medical tape, mouth-gag, minimally invasive dental burr with a diameter of 0.6 mm, dental high-speed dental handpiece, 8# C+ file, heating pad, 1 mL syringe, sterile cotton ball, eye forceps.
2. Obtain the following drugs: anesthesia mix, veterinary ointment.

2. Preparation of the mouth-gag

1. Weigh and anesthetize the mouse by intraperitoneal injection of anesthesia mix solution (10% ketamine hydrochloride + 5% xylazine + 85% sterile isotonic saline) at 0.007 mL/g of body weight and confirm proper anesthetization through the toe pinch method. Apply ophthalmic lubricating ointment on the eyes to prevent eyes injury because of desiccation during operation.
   NOTE: Medical hats, masks, gloves and overalls and other basic protection are necessary. Make sure both surgery environment and mouse chamber are clean and safe. A heat pad for thermal support throughout the procedure is necessary.
2. Prepare the mouth-gag as described below (Figure 3).
   1. Obtain the following materials: Orthodontic arch wire with diameter of 8 µm, young loop bending plier, heavy wire cutter, marker pen, rubber cap with a length of 3 mm and a cross section diameter of 1 mm.
   2. First, straighten the arch wire with left hand for fixation and thumb, index finger of the right-hand bend slightly against the arc of the wire. Repeat this action several times, it will facilitate the bending to the correct three-dimensional Angle.
   3. Use the Yong loop bending plier to bend the top edge (Figure 3G, a-i) of the trapezoid (Figure 3C, a-l-k-b) about 8 mm long at the midpoint of the bow wire. Make sure that point a (Figure 3) is on the edge of the plier beak.
   4. Hold the pliers with left hand, clamp the free end of the bow wire with right thumb and forefinger, and bend the bow wire from point a to make an angle of about 120°. Duplicate the previous action at point i (Figure 3G). Check whether the arch wire is on one plane by putting it on a horizontal table without prying.
   5. Leave about 9 mm of length on each side (Figure 3D, a-b, l-k) and bend the free-end to a 75° angle by using the same skill as step 2.2.4, while ensuring that each edge is on one plane. Bend this acute angle with the tip of the plier beak.
   6. Find point c about 5 mm from point b. Follow the same skill to bend a 105° angle on point c. Bend another 105° angle at point d 5mm from point c. Leave about 4.5 mm from point d and find point e. Bend the free end at point e to form an angle of about 100 -105° (Figure 3E).
      NOTE: The 6-8-week-old C57 mice we used were about 20 g. The spacing of 5 mm could not only jam the upper and lower jaws of the mice without moving, but also would not press the skin of the mice and cause discomfort. If other species or ages of mice are used, please adjust the length of c-d and i-h parts according to the actual situation (Figure 3E,G).
   7. Bend an extra tongue depressor for mandibular part (Figure 3G, j-i-h-g).
   8. Duplicate bending steps of a-b-c part on l-k-j part. Clamp i-k and k-j parts at the same time and bend the free end at point j to make it vertical to i-k-j plane. Clamp point i which is 5 mm from point j, bend the free end to make it parallel to both i-k-j plane and c-d part (Figure 3H).
   9. Leave 5 mm length from point i, at point h, bend the arch vertical to i-h part and parallel to j-i-h plane. Find point g at 5mm from point h. Clamp j-i-h-g plane and bend the free end symmetrical to k-j part. Then free end after point f should be symmetrical to point e-free end (Figure 3H).
   10. Put rubber caps on the free end (Figure 3F).

3. Immobilization

1. Fix the mouse supine on the fixation plate with limbs secured by skin tape. Compress the free ends of mouth-gag with thumb and index finger.
2. Fix the mouse front incisors in the trapezoidal groove of two arms. Ensure that the arm with tongue depressor is for mandibula. Adjust the mouth-gag to make sure mouse's tongue is immobilized but not ischemic.

4. Tooth assessment

1. Ensure the maxillary first molar for surgery should be free of dental caries, trauma and odontogenesis. Ensure there is no redness, swelling, or fistula on the surrounding gingiva. Ensure the opposite teeth are healthy and available to act as the healthy control group.

5. Pulp exposure

1. Use dental burr to drill at the occlusal side of maxillary first molar at a speed of 20,000 rpm. Ensure that the enamel is removed. Keep the operation with dental handpiece only in shallow layer of dentine to prevent excessive thermal stimulation on dental pulp tissue¹².
2. At the same time, prevent overheating by using a syringe to drop normal saline on the tooth every 3 min during the operation.
3. Put an 8# or 10# C+ file on the lowest position of the drilled pit and pierce through the last dentine to expose the pulp chamber. It will be an obvious feeling of falling when local dentine is thoroughly removed. Do not prob too deep, or dental pulp tissue might be brought out of pulp chamber.
4. Clean the fragments around the tooth. Take off the mouth-gag; the surgery is finished. Use the opposite maxillary first molar as the control without operation.

6. Post-operative care

1. After surgery, administer carprofen (5 mg/kg) subcutaneously and place the mouse on the chemothermal heating pad in a prone position until recovery from anesthesia. Feed the mice and provide drinking water. The recovery process should be supervised. No other animals should be in the same chamber until the mouse is fully recovered.

7. Sample collection and storage

1. Euthanize the mouse with cervical dislocation under deep anesthetic condition 24 h after operation or any other time point as per experiment⁹. Cut the skeleton muscles attached to the maxilla and zygoma with ophthalmic scissors. Remove skeleton, frontal bone and soft tissue and take out the lamina gnathostegite with maxillary molars.
   NOTE: According to He et al., it's commended that pulpitis sample should be collected less than 72 h after surgery to avoid extensive necrosis in dental pulp tissue¹³.
2. Sagittally divide the gnathostegite in half and store the tissue in 4% paraformaldehyde in PBS, pH 7.4, at 4 °C for 24h-fixation.

8. Histological analysis

1. Wash the tissue with phosphate buffered saline (PBS) and decalcify them in daily-changed decalcification solution of 5% EDTA and 4% sucrose in PBS, pH 7.4, at 4 °C for 2-4 weeks¹⁰.
2. Embed the 1/2 gnathostegite in paraffin and make sure the sagittal face without teeth is at the bottom of tissue cassettes.
3. Cut the paraffin block to 5 µm thick slices with a paraffin microtome. Adjust the angle of the paraffin block according to the proximal, distal, upper as well as lower relationship observed under the microscope to ensure the complete crown pulp and perforation of the first molar can be cut.

Wyniki

The procedure described above was performed on the right maxillary first molar of 3, 6-8 weeks old C57BL/6 mice, while the left maxillary first molars were preserved as control. Histology and immunofluorescence results from blank control, 12 h pulpitis and 24 h pulpitis samples were utilized for demonstration.

Following protocol of CT analysis from Goldman et al.¹⁵, pulp exposure was confirmed through micro-CT and reconstruction modeling in Figure 4...

Dyskusje

As the solitary soft tissue within teeth, dental pulp fulfills a crucial role in maintaining bioactivity of tooth but remains highly sensitive. The preservation of this vital pulp has become the preferred initial approach in recent endodontic treatments, necessitating a comprehensive understanding of the inflammatory mechanisms of dental pulp¹⁶. The spatiotemporal fluctuation of the inflammatory microenvironment and interactions between resident cell types in pulpitis complicates its investigation...

Materiały

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