3D Imaging of PDL Collagen Fibers during Orthodontic Tooth Movement in Mandibular Murine Model

We present a protocol for generating orthodontic tooth movement in mice and methods for 3D visualization of the collagen fibers and blood vessels of periodontal ligament without sectioning.

Orthodontic tooth movement is a complex biological process that involves external forces and remodeling of both soft and hard tissues. In order to understand these processes, it is critical to study the tooth and the periodontal tissues within the 3D context. Murine model is a good candidate for such studies due to its size, high metabolic rate and the vast genetic information we have.

However, creating orthodontic tooth movement in mice is not easy due to their small size. Additionally, structural analysis methods usually involve sectioning, which disrupts the 3D structure and context of the tissue. For a non-uniform tissue, such as the PDL, preserving the 3D structure and context is of a critical importance.

To address these two obstacles, we provide a protocol to generate mesial orthodontic tooth movement of the first mandibular molar in mice and describe two methods that enable 3D visualization of the periodontal ligament without sectioning the tissue. To position the anesthetized mouse for orthodontic device insertion, hook the top incisor onto the paper clip loop. Immobilize the lower incisors using the power chain And open the mouth further with mini-colibri mouse retractor.

Apply a small drop of saline on each eye to prevent corneal dehydration. These are the tools used in device insertion. Cut a piece of aluminum wire to one centimeter in length.

Using microsurgical needle holder, slide the wire from the buccal side lingually in the interproximal area below the contact point between first and second molars. Pull the wire from the lingual side towards the mesial side of the molar. Twist the free ends of the wire two to three times to secure.

Cut a piece of nickel titanium or NiTi coil spring around seven to nine threads in length. Insert the coil between lower first molar and incisor, pass one free end of the wire through two threads of the coil, then twist both ends of the wire tightly to fix the coil to the molar. Remove the power chain using a pair of tweezers.

Loop two to three threads of the coil over the incisor to anchor the coil. Leave exactly three active threads between first molar and the incisor. Slide the threats down to the incisor free gingival margin.

Place a layer of flowable composite resin on the incisal border of the coil and cure it with dental curing light. Replace the power chain after curing the resin. Trim excess wire around the molar to avoid injuries.

Using the same curing light, heat up the NiTi coil for 20 seconds. This will tighten up the coil, finished placement should look like this. Soft tissue on the dissected hemi-mandible can be removed with a lint-free wipe.

With a sample in between two fingers, rub the exterior in circular motion until most of the soft tissue is removed. The rest can be cut off using a pair of scissors. Cleaned mandible is shown here on the right.

To mount the dissected hemi-mandible in the sample chamber from micro CT scan, shape a rice grain sized packable dental resin into a log. Then place it into the sample slot of the stage. Place the hemi-mandible onto the composite.

Adjust the position until the first molar is centered at the midline groove and the occlusal surface is horizontal. Cure the composite when satisfied with the positioning. This is an example of the mandible in proper position.

Take a small ball of packable dental resin. Its diameter should be around the width of the molar. Place the resin onto the occlusal surface of the first molar.

Moisten lint-free wipe with water. Place the dampened wipe inside the humidity pools in the sample stage. Close the sample chamber and affix it to the micro CT with screws.

Now take 2D images while lowering the anvil vertically until the tip of the anvil is surrounded by the composite. Safely turn off the x-ray source, open the micro CT chamber and cure the composite through the clear plexiglass window. Now the sample is properly mounted and can be imaged as needed.

To generate optically cleared hemi-mandible sample, prepare the following solutions in 1.5 mil centrifuge tubes. 4%paraformaldehyde, 50%and 70%ethanol in deionized water. Two tubes of 100%ethanol and ethyl cinnamate.

Place dissected hemi-mandible in 4%paraformaldehyde. Cover the tube with aluminum foil and place on a rocker on gentle setting for six hours at room temperature. After six hours, transfer the sample into 50%ethanol.

Place the sample back on the rocker for 16 hours covered from light. Repeat the previous step with a sample in 70%ethanol for 16 hours followed by 100%ethanol twice for 16 hours each time. Finally move the sample to ethyl cinnamate for a minimum of 12 hours.

Reliable orthodontic movement is generated by the NiTi coil device in murine mandibular first molar. In nine week old male mice, the average interproximal space is 40 microns after seven days. Phase enhancement micro CT allows visualization of the PDL.

After three days of mesial orthodontic movement, PDL density is reduced. The bone PDL interface is rougher due to the development of craters in the bone surface, which are indicative of osteoclastic activity and bone resorption. Rough surface can be seen at all levels of the roots.

This shows that the movement is translational as opposed to a tipping motion. At day seven, PDL space is narrower than at day three and the bone PDL border at the distal root surface is smoother which are signs of bone opposition as expected when orthodontic tooth movement occurs. Due to the long micro CT imaging time and the rotation of the stage, secure mounting of the sample is essential.

Unstable sample where results in blurry scans where a silhouette can be seen around the crown and the PDL fibers are not seen. Optical clearing using ethyl cinnamate provides another method to view the PDL without sectioning. The substrate can be seen through the ramus of an adequately cleared sample shown here on the right.

This method, preserves endogenous fluorescence and can be used without chemical fixation. Lightsheet microscopy can be used on cleared samples. With a transgenic mouse, the fluorescent blood vessel linings can be observed.

If the bone was not properly cleared, blood vessels in the PDL are not visible as shown in panel F.This protocol described how to overcome two major challenges in generating and analyzing orthodontic tooth movement in murine mandibular molar teeth. Once the orthodontic tooth movement technique is mastered, it can be completed in 15 minutes. This process is challenging due to the small dimensions of the mouse teeth and the presence of the tongue.

However, we provide all the necessary solutions to overcome these challenges. The micro CT imaging requires a phase enhancement capability. We demonstrated our technique using a benchtop micro CT.However, any setup that can generate phase enhancement will do, for example, a synchrotron.

The ECi clearing results depend on the dehydration process. If the sample is not transparent enough, our suggestion is to increase the time of the dehydration steps. Our clearing method can be used on unfixed samples as well.

The clear tissues can be scanned in the micro CT since the clearing process does not affect the mineral content of the tissue. Therefore our method provides a correlative approach for fluorescence and structural information to generate a complete 3D analysis.