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

Summary

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.

Abstract

Orthodontic tooth movement is a complex biological process of altered soft and hard tissue remodeling as a result of external forces. In order to understand these complex remodeling processes, it is critical to study the tooth and periodontal tissues within their 3D context and therefore minimize any sectioning and tissue artefacts. Mouse models are often utilized in developmental and structural biology, as well as in biomechanics due to their small size, high metabolic rate, genetics and ease of handling. In principle this also makes them excellent models for dental related studies. However, a major impediment is their small tooth size, the molars in particular. This paper is aimed at providing a step by step protocol for generating orthodontic tooth movement and two methods for 3D imaging of the periodontal ligament fibrous component of a mouse mandibular molar. The first method presented is based on a micro-CT setup enabling phase enhancement imaging of fresh collagen tissues. The second method is a bone clearing method using ethyl cinnamate that enables imaging through the bone without sectioning and preserves endogenous fluorescence. Combining this clearing method with reporter mice like Flk1–Cre;TdTomato provided a first of its kind opportunity to image the 3D vasculature in the PDL and alveolar bone.

Introduction

The basic underlying biological process in orthodontic tooth movement (OTM) is bone remodeling. The trigger for this remodeling process is attributed to changes in the structure of the periodontal ligament (PDL) such as extracellular matrix (ECM) stress, necrosis as well as blood vessel destruction and formation^1,2,3. Other possible triggers for alveolar bone remodeling are related to force sensing by osteocytes in the bone, as well as mechanical deformation of the alveolar bone itself; however their role in OTM is still not fully elucidated^4,5.

Despite many studies aimed at revealing structure-function relations of the PDL during OTM, a clear functional mechanism is yet to be defined^6,7. The major reason for this is the challenge in retrieving data of a soft tissue (PDL) located between two hard tissues (cementum and alveolar bone). The accepted methods to collect structural information usually necessitate fixation and sectioning that disrupt and modify the PDL structure. Moreover, most of these methods yield 2D data that even if not distorted, give only partial and localized information. Since the PDL is not uniform in its structure and function, an approach that addresses the intact 3D structure of the entire tooth-PDL-bone complex is warranted.

This paper will describe a method for generating an OTM in mice and two methods that enable 3D visualization of the collagen fibers in the PDL without any sectioning of the sample.

Murine models are widely used for in-vivo experiments in medicine, developmental biology, drug delivery and structural studies. They can be genetically modified to eliminate or enhance specific proteins and function; they provide fast, repeatable and predictable developmental control; they are also easy to image due to their small size⁸. Despite their many advantages, mouse models in dental research are not used frequently, especially when clinical manipulations are warranted, mostly due to the small sized teeth. Animal models such as rats^9,10,11, dogs^12,13, pigs^14,15,16 and monkeys¹⁷ are used more often than mice. With the recent development of high-resolution imaging techniques, the advantages of utilizing a mouse model to decipher the convoluted processes in OTM are numerous. This paper presents a method to generate a mesial movement of the molar tooth in the mandible with constant force levels that trigger bone remodeling. Most of the OTM experiments in rodents are done in the maxilla, since the mobility of the mandible and the presence of the tongue add another complexity level. However, the mandible has many advantages when 3D structural integrity is desired. It can be easily dissected as a whole bone; in some species it can be separated into two hemi-mandibles through the fibrous symphysis; it is compact, flat and contains only the teeth without any sinus spaces. In contrast, the maxilla is a part of the skull and closely related to other organs and structures, thus extensive sectioning is needed in order to dissect the alveolar bone with the associated teeth.

Using an in house humidity chamber coupled to a loading system inside a high resolution micro-CT that enables phase enhancement, we developed a method to visualize fresh fibrous tissues in 3D as previously described^{9,18,19,20,21,22,23}. Fresh tissues are scanned immediately after the animal is sacrificed without any staining or fixation, which reduces tissue artefacts as well as  alterations of biomechanical properties. These 3D data can be utilized for distribution and direction analyses of the fibers as described elsewhere¹⁹.

The second 3D whole tissue imaging method presented here is based on optical clearing of the mandible which enables imaging of the PDL fibers through the bone without any sectioning. Interestingly it also enables visualization of the collagen fibers of the bone itself, however this will not be discussed here. In general, there are two methods for tissue clearing. The first is aqueous-based clearing where the sample is immersed in an aqueous solution with a refractive index greater than 1.4 either through a simple immersion, hyperhydration or hydrogel embedding. However, this method is limited in the level of transparency as well as the structural preservation of the tissue and therefore necessitates fixation of the tissue. The second method which yields highly transparent samples and does not require fixation is the solvent-based clearing method^24,25. We generated a modified solvent-based clearing method based on ethyl-3-phenylprop-2-enoate (ethyl cinnamate, ECi) for the mandibular samples. This method has the advantages of using non-toxic food-grade clearing agent, minimal tissue shrinkage, and preservation of fluorescent proteins.

Protocol

All animal experiments were performed in compliance with NIH's Guidelines for the Care and Use of Laboratory Animals and guidelines from the Harvard University Institutional Animal Care and Use Committee (Protocol no. 01840).

1. Orthodontic Tooth Movement

1. To generate a mouse bed, use a flat plastic platform with a wedge shaped, 45° angled headrest. The headrest can be generated by cutting a plastic box.
   1. Elevate the head end of the platform to generate an approximately 30° angle between the headrest and the table plane. Attach a bent thick paper clip (0.036" in diameter) to the head side end to hold the upper incisors.
   2. On the tail end, generate an elevated surface to which an orthodontic power chain can be attached to hold the lower incisors. See Figure 1 for an example platform.
2. Anesthetize mouse by intraperitoneal injection of xylazine at 10 mg/kg and ketamine 100mg/kg using 1 mL syringe and a 27 Gauge needle.
3. Place anesthetized mouse on custom-made platform and immobilize upper jaw by hooking the upper incisors on the paperclip loop. Open the mouse lower jaw with the orthodontic power chain hooked on the lower incisors. Keep the cheeks retracted with the mini-colibri mouth retractor.
4. Place the platform under a surgical microscope or any other stereoscope that can reach to 5-6x magnification.
5. Apply 50 µL of saline (roughly 1 drop) on the mouse eyes to prevent corneal dehydration. Replenish saline every 20 min.
6. Cut a piece of an aluminum wire (0.08 mm diameter) 1 cm in length. Slide the wire from the buccal side lingually in the interproximal area bellow the contact point between first and second molars using a microsurgical needle holder. Leave 2 mm free edge in front of the first molar in order to be threaded into the spring end.
7. Cut a piece of nickel titanium (NiTi) coil, around 7 to 9 threads in length.
   NOTE: The elastic properties of the coil will provide constant force for orthodontic movement. The total unstrained length of the coil should be shorter than the gap between the incisor and the molar. Keep in mind that an extra 2 threads are needed on each end to anchor coil to the tooth. Aluminum wire is selected in order to reduce scanning artefacts such as beam hardening during the micro-CT scan.
8. Insert NiTi coil spring (0.15 mm wire diameter, 0.9 mm inner coil diameter; delivers a force of 10 g) between lower first molar and lower incisor. Use the wire ligature inserted around the first molar in step 1.6, twist the wire tightly around 2 threads of the coil spring to fix the coil on the molar side.
9. To ensure uniform force level, use exactly 3 active threads between the first molar and the incisor. Temporarily remove the power chain from the incisor and loop 2 to 3 unstrained threads over the incisor to anchor the coil. Slide the threads down to the incisor free gingival margin.
10. 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.
11. Using the same curing light, heat up the NiTi coil for 20 s. This will tighten the NiTi coil. The finished placement is shown Figure 1C.
12. Either leave the contralateral side intact or insert a sham such as the wire between the first and second molars.
13. Place the anaesthetized mouse under a heated light to keep the mouse warm until recovery.
14. Place the mouse back into an individual cage and monitor daily. No diet change is necessary during orthodontic movement.
    NOTE: OTM device on one side causes some discomfort but does not impair feeding. However, inserting devices on both sides are not advised due to the added amount of discomfort. Pain medication is not necessary unless outward signs of pain are seen.

2. Micro-CT scan of PDL fibers in fresh hemi-mandibles

1. Mounting the hemi-mandible (Figure 2)
   1. After the desired duration of orthodontic movement, sacrifice the mouse via cervical dislocation. Remove the mandible and separate into hemi-mandibles.
      ​NOTE: Since the sample will not be fixed, it is critical to perform the dissection of the jaw and mounting as soon as possible, ideally within 30 min.
   2. Remove the surrounding soft tissue gently with a clean lint-free wipe.
   3. Remove the orthodontic device using microsurgical scissors and tweezers under a stereo microscope with at least 4x magnification.
   4. Keep sample moist in a 1.5 mL volume, micro-centrifuge tube along with a piece of lint-free wipe moistened with water.
   5. Place packable dental composite resin into the sample slot on the stage, then place the fresh hemi-mandible into the composite. Before mounting make sure that the bone surface in contact with the dental composite is free from any soft tissues and dry, otherwise dental composite will not cure properly.
   6. Adjust the position of the hemi-mandible until the first molar is centered at the midline groove of the stage. Make sure the occlusal surface is horizontal. Cure the composite when satisfied with the positioning.
      NOTE: Additional small amounts of dental composites can be placed on the sides of the hemi-mandible and/or across the incisor to aid in stabilizing the sample.
   7. Place dampened lint-free wipe inside the humidity pools in the sample stage. Place dental composite on the occlusal surface of the first molar. Before closing the chamber, make sure nothing blocks the x-ray path at the specimen level.
   8. Affix the chamber in the micro-CT. Screw the chamber into the micro-CT sample stage so that movement during imaging is minimized.
   9. Turn on the x-rays and take 2D images while lowering the anvil vertically, until the tip of the anvil is surrounded by the composite but no increase in the force is detected.
   10. Once anvil is embedded in the composite, close the x-ray source. Then, open the micro-CT chamber and cure the composite through the clear Plexiglass window.
2. Micro-CT settings
   1. Set the source voltage to 40 kV and current to 200 µA. Using a 10x magnification detector, position the sample within the frame of view. Use binning of 2 for the captured images.
      NOTE: Since the PDL is significantly less dense than the bone and tooth, visualizing the PDL requires higher power and exposure time. This protocol will provide settings for visualizing the PDL.
   2. Set single image exposure time to 25 s. Set rotation of the sample stage to a range of 183 degrees or more. Set the scan for 2500 projections. Do not use any x-ray source filter, the resulting scans have a voxel size of 0.76 µm on each side.
   3. Collect a reference scan for proper reconstruction according to the micro-CT guidelines. Use 1/3 number of reference images as the total projections. Reconstruct the volume without additional binning, using a back projection filtered algorithm.

3. Clearing method (Figure 3)

1. Prepare five 1.5 mL micro-centrifuge tubes.
2. Prepare 1.4 mL of the following solutions in 1.5 mL micro-centrifuge tubes: 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS), 50% ethanol (EtOH) in deionized (DI) water, 70% EtOH in DI water, and two tubes of 100% EtOH.
   NOTE: PFA is used for fixation of the sample. ECi clearing will also work on unfixed samples. To clear unfixed samples, simply skip the PFA step.
3. Place dissected hemi-mandible in 4% PFA. Cover with aluminum foil and place on the rocker on gentle setting at room temperature for 6 h.
4. Move the hemi-mandible to 50% EtOH. Place it on the rocker covered from light for 16 h.
5. Move the hemi-mandible to 70% EtOH. Place it on the rocker covered from light for 16 h.
6. Move the hemi-mandible to 100% EtOH. Place it on the rocker covered from light for 16 h.
7. Repeat 3.6. in the second 100% EtOH tube.
8. Prepare 5 mL of ECi in a glass or polypropylene tube.
   NOTE: ECi dissolves polystyrene, but not polypropylene. Also, if not using a tissue with fluorescent proteins the sample can be exposed to light during the clearing procedure.
9. Move the hemi-mandible to ECi tube. Cover the tube with aluminum foil and place on the rocker on gentle setting for a minimum of 12 h.
   NOTE: The protocol can be paused here. Dehydrated sample can be stored in ECi at room temperature. The freezing or melting point of ECi is 6.5 to 8.0 °C. Do not store at 4 °C.
10. The hemi-mandible is ready for imaging with fluorescence microscope.
    NOTE: During imaging, the sample must be immersed in the ECi to remain optically transparent.

Representative Results

This paper presents a method to produce OTM as well as two methods for 3D imaging of collagen fibers inside the PDL without any sectioning. For animal research purposes, when alignment of the teeth is not necessary, a tooth movement is considered orthodontic if it generates remodeling of the alveolar bone at all root levels. Constant force level applied on teeth is required in order to generate a reliable OTM. Here, an activated shape-memory NiTi coil is used to generate a consistent force of 10 g throughout the experimental time of 7 days and beyond if warranted. The coil activation described here (Figure 1) generates strain in the NiTi coil within the martensitic phase and brings the coil to the hysteresis state, which delivers constant stress onto the tooth. Warming the coil with the curing light after the coil insertion will also make sure the alloy will shift to its austenitic form and the shape memory effect will take place.

Here we show representative results from 9-week old, male mice. The averaged mesiodistal space between the crowns of the first and second molars after 7 days of OTM is 40 µm as measured between the interproximal surfaces of the molars in the micro-CT with 1X magnification (n=12, st.dev.= 15 µm) (Figure 1E). The averaged space of the PDL in the mesiodistal direction is 80 µm before and after 7 days of OTM (Figure 4B). This confirms that the first molar translated mesially and 7 days is an adequate time for generating OTM in a mouse model while generating processes of bone resorption and apposition in nature (Figure 4). Mice were fed a standard hard pallet diet. No diet change was made post device insertion.

The first method presented to visualize the changes in the tooth-PDL-bone complex during OTM is based on phase-enhanced micro-CT imaging of fresh tissues (Figure 4) which was described in detail previously^{9,18,19,20,22,23}. In short, provided a phase enhancement capability of either a micro-CT or a synchrotron, mechanical stabilization of the fibrous tissue and humidified environment, fresh collagenous fibers can be visualized without any fixation or contrasting agents. In the PDL the fibers that are seen are those that are connected to both the tooth and the bone, mainly type I collagen¹⁹. This unique opportunity to visualize in 3D an intact PDL enables analysis of 3D fiber density, fibers orientation as well as the 3D movement of the tooth as previously described^9,19. Specifically, here we present the visualization of the fibrous network in the PDL. At time 0, physiological remodeling in both the bone and the PDL can be observed. Remodeling also occurs in the cellular cementum; however, this is not directly related to the presented method and therefore will not be elaborated. The bone-PDL interface is mostly smooth both in the transverse (Figure 4A) and sagittal (Figure 4B) planes prior to any force application. In the coronal plane (Figure 4C), the bone-PDL interface is rougher especially towards the apical region which might be indicative of the remodeling balance tends towards resorption. At 3 days of OTM (Figure 4D-F), upon which the first molar is moved mesially (direction is represented by the dashed arrow), the fibers density in the PDL is reduced (white arrow heads). The bone-PDL interface is rougher than at 0 days due to development of craters in the bone surface which are indicative of osteoclastic activity and bone resorption processes associated with mainly compression forces in the PDL²⁶, however here seen in tension areas at 3 days. Tissue destruction in tension areas within the PDL was suggested^27,28 and can be clearly seen using this method. The rough border is seen at different levels of the roots (white arrows) and therefore suggests that the tooth movement is translational in nature and not just tipping of the crown. At 7 days of OTM (Figure 4G-I), bone resorption signs, such as craters within the bone, rough borders and expansion of the PDL space, are seen at all planes but the averaged PDL space is narrower than at 3 days of OTM (Figure 4D-F). In some areas however, the bone-PDL border has become smoother after 7days of OTM these areas are located at the distal surfaces of the roots, which is most likely an indication for bone apposition, as expected in OTM to the mesial direction. 

Due to the long micro-CT imaging time (~19 h) and the rotation of the stage, mounting of the sample is essential to keep the sample still. Unstable sample will result in blurry scans. Figure 5 presents how the micro-CT scan looks when the sample has moved during the scan. The tooth and bone are blurry. Neither PDL fibers nor osteocytes are observed. In such incidents there is a silhouette present around the margin of an object. In Figure 5, multiple outlines of the tooth crown can be observed (arrows).

Depending on the research goal, the resolution and visualization of the PDL fibers may be sacrificed in trade for shorter scan time when only information on the hard tissues is desired.

A complementary method for 3D visualization of the PDL fibers without any sectioning is via optical microscopy on optically cleared samples using ECi  (Figure 3). This method can be used on a specimen without fixation and preserves fluorescent signals that exist in the tissue prior to clearing. Hemi-mandibles before and after ECi clearing are shown in Figure 3B and 3C. Adequate sample clearing of the PDL can be confirmed when a grid paper can be seen through the ramus of the mandible. The amount of clearing can be adjusted by lengthening of dehydration process. Figure 6 shows the second harmonic generation (SHG) signal from collagen fibers in both alveolar bone and the PDL in a cleared mandible. Imaging the collagen fibers of the bone in 3D is a complicated process, which often utilizes electron microscopy methods such as FIB/SEM. However, utilizing the ECi-based clearing method and SHG, the alveolar bone fibers are clearly seen, especially in the horizontal direction. When translating through the sample deep into the PDL from the bone surface, the transition to the PDL fiber level is very clear as the fibers suddenly change their orientation to a vertical one.

Lightsheet microscopy can also be utilized for imaging fluorescent proteins through the bone. In this case of a cleared sample from a transgenic Flk1-cre;Tdtomato mouse^19,29,30, the fluorescent endothelial cells lining the blood vessels are clearly observed (Figure 7A, B, C, E). Proper clearing is key to generating intelligible images with lightsheet microscopy. When the bone is not completely cleared, blood vessels within the PDL were not observed (Figure 7D, F).

Figure 1: Orthodontic appliance insertion set up. A. Mouse bed made from lab-supply to support the animal and keep the mouth open. Plastic platform (PP) for the body is on a 30° incline and the headrest (HR) is on a 45° angle from the surface of PP. A 2-tiered tube stand (TS) is used to elevate the end head of PP. The paper clip loop (black arrow) anchors the top incisors, and the bottom orthodontic power chain (white arrow) hooks onto the lower incisors. 5 mm diameter inspection mirror was used for visual inspection of the molars. B. Side view of the mouse bed. Angles between surfaces are marked (green and magenta). C. Representative image of the properly placed device. D. Molars seen through the inspection mirror prior to device implantation. E. Representative image of molars after the orthodontic movement. Dashed lines trace the outline of the first and second molars. F. Diagram of the device and its placement. Red line represents the wire ligature around first molar. Orange line represents flowable composite resin used to anchor the coil. NiTi coil is shown in blue and labeled. G. Dissected hemi-mandible with the device attached after 7-day orthodontic movement. Note how the 3 coil threads are still open, indicating that the coil is still active after 7 days. Scale bar = 1 mm in E and G. Please click here to view a larger version of this figure.

Figure 2: Hemi-mandible mounted in a custom-made chamber for micro-CT imaging. A. Full set-up of the sample chamber within the micro-CT machine. The x-ray source is seen on the left and the detector on the right. Red rectangle outlines the hemi-mandible mounted in the chamber on the sample stage (SS). The example chamber shown here is part of a mechanical testing set-up, including motor (M), anvil (A, outlined by white dashed lines) and anvil shaft (AS) atop the chamber. The full set up is screwed onto the CT stage. Inset image shows close-up of the red outlined region, containing the humidity chamber with sample inside. B. Top view of sample mounted on the sample stage. Humidity pools (grey arrow) are built-in on the perimeter to maintain humidity during imaging. On the circular stage in the middle, hemi-mandible can be mounted in the slanted deep groove (black arrow). A thin groove (white arrow) marks the midline of the stage to aid in orienting the sample. C. Diagram of the circular stage with the sample groove. The slant of the groove supports the mandible and allows the molars to be mounted along the roots' vertical axis. D. Representative micro-CT 2D slice combined with a 3D volume image of the hemi-mandible sample. The interproximal gap here is 52 µm. The sample is mounted onto the sample stage below (not shown) and the anvil (A) on top by dental composite (DC). Scale bar = 500 µm. Please click here to view a larger version of this figure.

Figure 3. ECi-based clearing method for dissected mouse hemi-mandibles. A. The dissected hemi-mandible is immersed in 4% PFA, 50% EtOH, 70% EtOH, and 100% EtOH consecutively. After dehydration, the hemi-mandible is stored in ECi for a minimum of 12 h until imaging. B. Hemi-mandible immediately after dissection. C. Hemi-mandible after completion of clearing. Scale bars = 5 mm Please click here to view a larger version of this figure.

Figure 4: Representative in-situ micro-CT scans of the PDL of a fresh sample in the different stages of the orthodontic movement. A-C, No orthodontic movement. A. Micro-CT 2D image in the transverse plane of the hemi-mandible showing the mesial (M) and distal (D) roots inside the alveolar bone, B-Buccal, L-Lingual sides of the alveolar bone. In between the tooth roots and the alveolar bone, the PDL space and the fibers within it are clearly observed. B. 2D image in the sagittal plane. C. 2D image in the coronal plane. D-E, 2D images after 3 days of OTM, arrow heads point at areas in the PDL with reduction in collagen fibers density, white arrows point at areas of bone resorption. G-I, 2D images after 7 days of OTM, black arrows point at regions of bone apposition. Scale bars = 150 µm. Please click here to view a larger version of this figure.

Figure 5: 2D micro-CT image in the sagittal plane, showing blurry structures of both tooth and bone due to movement of the tooth during the scan. Arrows point at multiple board lines of the tooth, indicating its movement. Scale bar 150 µm. Please click here to view a larger version of this figure.

Figure 6: ECi cleared mandible showing the first molar imaged with second harmonic generation (SHG). White arrow points at a region where collagen fibers of the PDL are seen, note the vertical orientation, black arrows point at a region where both vertical fibers of the PDL as well as horizontal fibers of the alveolar bone are seen. T-tooth, F-furcation, AB-alveolar bone, MR-mesial root, DR-Distal root, scale bar 150 µm. Images were obtained using a 20X multi-immersion lens for solutions with RI of 1.33-1.56. Excitation laser was set at 860nm at 10% power. Pixel dwelling time: 0.51µs; Scan mode: frame; Averaging: 16; Detector Type: nondescanned photomultiplier tube detector; Detector Gain 800V. Please click here to view a larger version of this figure.

Figure 7. Lightsheet microscope images of ECi-cleared Flk1-Cre;tdTomato mouse. A. Optimally cleared control hemi-mandible. The network of blood vessels within the bone (arrow head) and PDL space (arrow) is visible. B. inset of the mesiolingual region of the first molar (red outlined in A) shows the blood vessels. C. optimally cleared 7-day OTM hemi-mandible and D. sub-optimally cleared hemi-mandible. E. 2D image of panel C in the sagittal plane, the image demonstrates well defined blood vessels in bone (grey arrow) and PDL space (white arrows). F. Two-dimensional slice image of panel D, same region as images in E, resulting in a blurry image. Scale bars A, C, D = 500 µm, B, E, F = 100 µm Images were taken with a 5X plan objective, using camera as detector. Excitation laser was 561 nm at 4% power. Please click here to view a larger version of this figure.

Discussion

Generating OTM in mice is highly desired due to the size, genetics and handling advantages. Using the mandible provides an easy handling both in terms of tissue dissection as well as sample preparation and imaging. Here we presented a method to generate OTM with translational movement of the tooth inside the bone within 7 days of OTM. Using this protocol, the overall duration of the tooth movement can be extended, since the activated coil delivers a constant force level for movement of up to approximately 1 mm. However, the mesial side of the coil is fixed to the incisor, which is constantly erupting. As a result, the force vectors will gradually alter and start generating extrusion forces. This can be avoided if adjustment of the attachment level on the mesial end is performed every 7 days.

The PDL is the initiator for OTM, therefore understanding its structure and function during the different stages of the OTM is of great importance. However, the PDL is not uniform in both its structure and function^19,22,31,32. As a result, in order to retrieve meaningful data, the PDL needs to be studied in 3D and any tissue sectioning and manipulation should be avoided as much as possible. Yet, investigating a soft tissue situated between two hard tissues makes such requirements challenging to fulfill. Traditional methods of studying the PDL often involve compromising the 3D structure and removing the tissue out of its physiological environment, which consequently alters PDL structural and biomechanical properties. Both structural and biomechanical properties undergo dynamic changes during OTM which justify preserving the tissue 3D context even further. In order to do so we described two methods that enable whole tissue imaging without sectioning, which can also be used on the same sample co-localizing fluorescent signals, morphological and mineralization data.

The provided methodological description directs readers to apply the methods in their fields of study. The micro-CT imaging allows 3D visualization of PDL fibrous network. Images can be analyzed to produce directionality and density analyses and to quantitatively investigate changes in the PDL during OTM. We also described a clearing method that enables visualization with readily available optical microscopic methods such as lightsheet microscopy and confocal imaging. Lightsheet microscopy has the advantage of producing a 3D image of large specimens with relatively fast imaging speed. Confocal microscopy enables high resolution 3D visualization utilizing SHG signal for collagen fibers imaging and fluorescent tags. These methods independently or combined open many possibilities to 3D structural studies with minimal tissue preparation.

Several challenging steps in this protocol require extra attention:

Firstly, during the coil placement, the ligature wire must be placed securely between first and second molars. This process is challenging due to the small dimensions of the mouse teeth. We recommend the use of benchtop stereomicroscope to guide the placement. However, small movements during the procedure may move the mouse and cause the region of interest to go out of the field of view. As an alternative, we suggest using 4-5x magnifying loupes that can be worn on the operator, which could help view the area more dynamically.

Secondly, the clearing results depend on the dehydration process. If the sample has not reached the desired transparency level, our suggestion is to increase the dehydration time. More specifically, longer immersion time in 100% EtOH has been shown to improve the transparency of the final product. However, it should be noted that increased level of dehydration can dramatically reduce the fluorescence levels^24,25. The presented ECi-based method was shown to preserve fluorescent signals for longer than 2 weeks²⁴.

Aspects of this protocol can be modified to study a multitude of other purposes. The chamber we designed inside the micro-CT is coupled with a load cell and a motor and has the ability to perform tension/compression tests on the hemi-mandible samples. Combined with the visualization of the micro-CT, this set up can show changes in the PDL in-situ with varying mechanical loads²¹. The described clearing method could also be implemented on unfixed samples, which provides an opportunity for an interesting combination of various imaging modalities. 

Materials

1-mL BD Luer-Lok syringe	BD	309628
1X phosphate buffered saline	VWR Life Sciences	0780-10L
200 proof ethanol	VWR Life Sciences	V1016
Aluminum alloy 5019 wire	Sigma-aldrich	GF15828813	0.08 mm diameter wire, length 100th, temper hard. Used as wire ligature around molar.
Avizo 9.7	Thermo Fisher Scientific	N/A	Used to analyze microCT scans
Castroviejo Micro Needle Holders	Fine Science Tools	12060-01
Clr Plan-Apochromat 20x/1.0,CorrVIS-IR M27 85mm	Zeiss	N/A	Used for second harmonic generation imaging
Cone socket handle, single ended, hand-form	G.Hartzell and son	126-CSH3	Handle of the inspection mirror
EC Plan-Neofluar 5x/0.16	Zeiss	440321-9902	Used for light-sheet imaging
Elipar DeepCure-S LED curing light	3M ESPE	76985
Eppendorf safe-lock tubes, 1.5mL	Eppendorf	22363204
Ethyl cinnamate, >= 98%	Sigma-aldrich	W243000-1KG-K
Hypodermic Needle, 27G x 1/2''	BD	305109
Ketathesia 100mg/ml	Henry Schein Animal Health	NDC:11695-0702-1
KIMWIPES delicate task wipers	Kimberly-Clark	21905-026 (VWR Catalog number)	Purchased from VWR
LightSheet Z.1 dual illumination microscope system	Zeiss	LightSheet Z.1/LightSheet 7	Used for lightsheet imaging
LSM 880 NLO multi-photon microscope	Zeiss	LSM 880 NLO	Used for two-photon imaging
MEGAmicro, plane, 5mm dia, SS-Thread	Hahnenkratt	6220	Front surface inspectrio mirror
MicroCT machine, MicroXCT-200	Xradia	MICRO XCT-200
Mini-Colibri	Fine Science Tools	17000-01
PermaFlo Flowable Composite	Ultradent	948
Procedure platform	N/A	N/A	Custom-made from lab materials
Routine stereo micscope M80	Leica Micosystems	M80
Sentalloy NiTi open coil spring	TOMY Inc.		A 0.15mm diameter closed NiTi coil with an inner coil diameter of 0.9mm delivers a force of 10g. Similar products can be purchased from Dentsply Sirona.
T-304 stainless steel ligature wire, 0.009'' diameter	Orthodontics	SBLW109	0.009''(.23mm) diameter, Soft temper
X-Ject E (Xylazine) 100mg/ml	Henry Schein Animal Health	NDC:11695-7085-1
Z100 Restorative, A2 shade	3M ESPE	5904A2