Optimization of the Ligature-induced Periodontitis Mouse Model for Preclinical Research

Our lab investigates periodontal disease, examining how inflammation causes gingival and bone damage in different groups of individuals. We aim to hinder gingivitis and periodontitis by targeting novel players and novel pathways of disease. The simplified ligature-induced periodontitis mouse model utilizes a double-knotted single thread in between the teeth, utilizing 3D-printed tools, the mouse dental bed and the ligature holder, which happens to be less complex than traditional models.

This approach uses the technique of inhalation of anesthesia, which happens to result in a quicker recovery for mice, less than 10 minutes, compared to that of more traditional models that use an injectable anesthesia, where the recovery time can be around 30 minutes. To prepare the mouse dental bed assembly, lay out all dental bed parts and the glue on the working platform. Then, secure the head-stabilizing pieces in position using the glue.

Using a number three blade on a 15C scalpel blade handle, bevel the sharp edges on the exterior portion of the ligature holder lock at a 45-degree angle to reduce discomfort during locking or unlocking. Square off any internal rounded corners on the ligature holder lock. Remove the flash from the top and bottom edges of the 3D print, and bevel these edges at 45 degrees.

Take out a slice of each tip of the ligature holder at a 45-degree angle to prevent traumatic injury to the soft tissues. Ensure each tip on the ligature holder includes a V-shaped notch. If this V-shaped notch is visible, place a 15C scalpel blade gently into the base of the notch.

Now, carefully angle the blade towards the outer surface of the tip and refine the base of the V-shaped notch on the outer edge. If the V-shaped notch is not visible, place a 15C scalpel blade in the middle of the ligature holder tip, and in one deliberate motion, make a shallow cut to create a V-shaped notch. While threading the ligature into the grooves, ensure that two knots are centered and level when viewed from the front of the ligature holder.

Using a dental explorer, confirm that the ligature bounces back into place without slipping when gentle force is applied between the knots. For ligature placement, position the anesthetized mouse in the mouse dental bed with its back against the bed and the nose positioned in proximity to the nose cone. Using rubber bands, stabilize the mouse's head and open its mouth.

To place a ligature on the right side, hold the ligature holder in the right hand and the explorer in the left. Gently place the tip of the explorer below the interproximal contact of the first and second molars on the palatal side. Using the explorer tip, first touch the teeth, then, slide down and rest against the gingiva to avoid traumatizing the soft tissue.

Rest the explorer handle in a horizontal angulation once properly positioned. Ensure the correct usage of the explorer tip for ligature placement. Hook the explorer into the proximal contact area, and stabilize the head against reactionary forces.

Retract the cheek using the ligature holder, and position the ligature over the interproximal contact between the first and second molars. Next, rotate the handle of the ligature holder up to 45 degrees, and with a combination of gentle rocking and sliding, push the ligature through the contact. Using fine needle forceps, clasp the excess thread attached to the knot on the right side.

Pull the thread across the teeth towards the left, and slide the scissors down the thread until positioned approximately 1.5 millimeters from the knot before cutting. For micro CT analysis, open the software and click on File, then Import image. In the pop-up menu, click on Browse.

Go to the folder containing DICOM files for one scan and select the first file. Click on Examine to find and select any other files within the folder for the same scanned sample. Then, click OK.Once the images begin to load, click on Visualize and Isosurface.

Change the Image Threshold to 5, 000 and the Surface Quality Factor percent to 55. Check boxes marked Enable Image Smoothing and Clip Surface with current ROI, and click on Update. To adjust level properties, right-click on Window/Level at the bottom, and click on Edit Window/Level Properties.

Set the bone density value to 2, 000 and keep it constant for all measurements. To manipulate planes, use keyboard shortcuts IJK hide and show for the the three different planes. Right-click and move the cursor to zoom in and out.

Press Shift and left-click to pan. Then, click Slice to move and rotate the planes. Next, click on the center of the plane to move the plane forward or backward.

Alternatively, place the cursor in the center of the desired plane, and use the up and down arrow keys to move the plane one slice at a time. Click on the edge of the plane to rotate around a line parallel to this edge in the center of the plane. Click on the corner of a plane to rotate around the plane.

Next, orient the Z slice, seen in blue at the top of the screen, so that the surfaces of all three teeth become visible at the same time or as close as possible. Then, scroll through the same Z slice plane until the roots of the first and second molars become visible, ensuring the surrounding anatomy and canals are well-defined. Align the x-axis with the buccal roots of the first and second molar through the central part of each root canal.

Alternatively, align through the buccal root of the first molar and the distobuccal root of the second molar. Next, use the orientation of the X slice as a reference for the alignment of the Y slice. Align the Y slice vertically parallel through the root of the first molar in the X slice panel.

Rotate the plane to orient the root vertically, and observe that the blue lines cross through the middle of the root. In the Y slice panel, to measure the distance from the cemento-enamel junction, or CEJ, to the alveolar bone crest, or ABC, place the cursor in the deepest point of the groove at the CEJ and press 1. Then, place the cursor at the closer point of the ABC and press 2.

A blue arrow denotes the first point, and the second point is denoted by a green arrow. If the arrows indicating measurement points do not appear immediately, hover the arrow over the desired areas and press the number keys on the keyboard. If the CEJ is difficult to determine, use the up and down arrow keys to scroll through the adjacent slices to confirm its location.

When determining the closest point of the ABC is difficult, measure multiple points to ensure consistent measurement. Despite our success with our protocols paper, new researchers have still found difficulty in the assembly and utilization of the 3D-printed tools, the mouse dental bed, the ligature holder. In order to address this, we have created a step-by-step video which walks researchers through the assembly and the utilization of these 3D-printed tools so that they can be used in a laboratory setting.

Our research team has utilized clinical studies and the simplified ligature-induced periodontitis model to uncover how a group of proteins named inflammasome can be used to halt the development of inflammation and periodontitis.