Name: 3d imaging of pdl collagen fibers during orthodontic tooth movement in mandibular murine model
Uploaded: 2021-04-15T13:30:00
Description: Watch this Scientific Journal Video about 3D Imaging of PDL Collagen Fibers during Orthodontic Tooth Movement in Mandibular Murine Model at JoVE.com

This study was supported by the NIH (NIDCR R00- DE025053, PI:Naveh). We would like to thank Harvard Center for Biological Imaging for infrastructure and support. All figures are generated with biorender.com.

All animal experiments were performed in compliance with NIH&#39;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
<ol>
	<li>To generate a mouse bed, use a flat plastic platform with a wedge shaped, 45&#176; angled headrest. The headrest can be generated by cutting a plastic box.
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		<li>Elevate the head end of the platform to generate an approx...

<ol>
	<li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthodontic+tooth+movement:+The+biology+and+clinical+implications.">Orthodontic tooth movement: The biology and clinical implications.</a> The Kaohsiung Journal of Medical Sciences. 34 (4), 207-214 (2018).</li><li>Meikle, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tissue,+cellular,+and+molecular+regulation+of+orthodontic+tooth+movement:+100+years+after+Carl+Sandstedt.">The tissue, cellular, and molecular regulation of orthodontic tooth movement: 100 years after Carl Sandstedt.</a> European Journal of Orthodontics. 28, 221-240 (2006).</li><li>Krishnan, V., Davidovitch, Z., molecular, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular,+molecular,+and+tissue-level+reactions+to+orthodontic+force.">Cellular, molecular, and tissue-level reactions to orthodontic force.</a> American Journal of Orthodontics and Dentofacial Orthopedics. 129 (4), 1-32 (2006).</li><li>Shoji-Matsunaga, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteocyte+regulation+of+orthodontic+force-mediated+tooth+movement+via+RANKL+expression.">Osteocyte regulation of orthodontic force-mediated tooth movement via RANKL expression.</a> Scientific Reports. 7 (1), 8753 (2017).</li><li>Oppenheim, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+changes,+particularly+of+the+bone,+incident+to+tooth+movement.">Tissue changes, particularly of the bone, incident to tooth movement.</a> European Journal of Orthodontics. 29, 2-15 (2007).</li><li>Unnam, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+Orthodontics-An+overview.">Accelerated Orthodontics-An overview.</a> Journal of Archives of Oral Biologyogy and Craniofacial Research. 3 (1), 4 (2018).</li><li>von Bohl, M., Kuijpers-Jagtman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyalinization+during+orthodontic+tooth+movement+:+a+systematic+review+on+tissue+reactions.">Hyalinization during orthodontic tooth movement : a systematic review on tissue reactions.</a> European Journal of Orthodontics. 31 (1), 30-36 (2009).</li><li>Kirschneck, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+assessment+of+mouse+models+for+experimental+orthodontic+tooth+movement.">Comparative assessment of mouse models for experimental orthodontic tooth movement.</a> Scientific Reports. 10 (1), 1-12 (2020).</li><li>Naveh, G. R. S., Weiner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+orthodontic+tooth+movement+of+a+multirooted+tooth:+a+3D+study+of+a+rat+molar.">Initial orthodontic tooth movement of a multirooted tooth: a 3D study of a rat molar.</a> Orthodontics &amp; Craniofacial Research. 18 (3), 134-142 (2015).</li><li>Nakamura, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+observation+of+rat+periodontal+ligament+during+function+and+tooth+movement,+using+microcomputed+tomography.">Time-lapse observation of rat periodontal ligament during function and tooth movement, using microcomputed tomography.</a> European Journal of Orthodontics. 30 (3), 320-326 (2008).</li><li>Kawarizadeh, A., Bourauel, C., Jager, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+numerical+determination+of+initial+tooth+mobility+and+material+properties+of+the+periodontal+ligament+in+rat+molar+specimens.">Experimental and numerical determination of initial tooth mobility and material properties of the periodontal ligament in rat molar specimens.</a> European Journal of Orthodontics. 25 (6), 569-578 (2003).</li><li>J&#243;nsd&#243;ttir, S. H., Giesen, E. B. W., Maltha, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+behavior+of+the+periodontal+ligament+of+the+beagle+dog+during+the+first+5+hours+of+orthodontic+force+application.">Biomechanical behavior of the periodontal ligament of the beagle dog during the first 5 hours of orthodontic force application.</a> European Journal of Orthodontics. 28, 547 (2006).</li><li>Lindhe, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+breakdown+of+peri-implant+and+periodontal+tissues.+A+study+in+the+beagle+dog.">Experimental breakdown of peri-implant and periodontal tissues. A study in the beagle dog.</a> Clinical Oral Implants Research. 3 (1), 9-16 (1992).</li><li>Salamati, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+tooth+mobility+in+young+pigs.">Functional tooth mobility in young pigs.</a> Journal of Biomechanics. 104, 109716 (2020).</li><li>Maria, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+unusual+disordered+alveolar+bone+material+in+the+upper+furcation+region+of+minipig+mandibles:+A+3D+hierarchical+structural+study.">An unusual disordered alveolar bone material in the upper furcation region of minipig mandibles: A 3D hierarchical structural study.</a> Journal of Structural Biology. 206 (1), 128-137 (2019).</li><li>Wang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+miniature+pig:+a+useful+large+animal+model+for+dental+and+orofacial+research.">The miniature pig: a useful large animal model for dental and orofacial research.</a> Oral Diseases. 10, 1-7 (2007).</li><li>Melsen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+reaction+to+orthodontic+tooth+movement--a+new+paradigm.">Tissue reaction to orthodontic tooth movement--a new paradigm.</a> European Journal of Orthodontics. 23 (6), 671-681 (2001).</li><li>Naveh, G. R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+MicroCT+imaging+of+non-mineralized+connective+tissues+at+high+resolution.">Direct MicroCT imaging of non-mineralized connective tissues at high resolution.</a> Connective Tissue Research. 55 (1), 52-60 (2014).</li><li>Naveh, G. R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonuniformity+in+ligaments+is+a+structural+strategy+for+optimizing+functionality.">Nonuniformity in ligaments is a structural strategy for optimizing functionality.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (36), 9008 (2018).</li><li>Naveh, G. R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tooth+periodontal+ligament:+Direct+3D+microCT+visualization+of+the+collagen+network+and+how+the+network+changes+when+the+tooth+is+loaded.">Tooth periodontal ligament: Direct 3D microCT visualization of the collagen network and how the network changes when the tooth is loaded.</a> Journal of Structural Biology. 181 (2), 108-115 (2013).</li><li>Naveh, G. R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tooth+movements+are+guided+by+specific+contact+areas+between+the+tooth+root+and+the+jaw+bone+:+A+dynamic+3D+microCT+study+of+the+rat+molar.">Tooth movements are guided by specific contact areas between the tooth root and the jaw bone : A dynamic 3D microCT study of the rat molar.</a> Journal of Structural Biology. 17 (2), 477-483 (2012).</li><li>Naveh, G. R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tooth-PDL-bone+complex:+Response+to+compressive+loads+encountered+during+mastication+-A+review.">Tooth-PDL-bone complex: Response to compressive loads encountered during mastication -A review.</a> Archives of Oral Biology. 57 (12), 1575-1584 (2012).</li><li>Ben-Zvi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+the+tooth-periodontal+ligament-bone+complex+to+load:+A+microCT+study+of+the+minipig+molar.">Response of the tooth-periodontal ligament-bone complex to load: A microCT study of the minipig molar.</a> Journal of Structural Biology. 205 (2), 155-162 (2019).</li><li>Klingberg, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+Automated+Evaluation+of+Total+Glomerular+Number+and+Capillary+Tuft+Size+in+Nephritic+Kidneys+Using+Lightsheet+Microscopy.">Fully Automated Evaluation of Total Glomerular Number and Capillary Tuft Size in Nephritic Kidneys Using Lightsheet Microscopy.</a> Journal of the American Society of Nephrology. 28 (2), 452 (2017).</li><li>Richardson, D. 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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&#160;for movement of up to approximately 1 mm. Howe...

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 formation1,2,3. 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 elucidated4,5.
Despite many studies aimed at revealing structure-function relations of the PDL during OTM, a clear functional mechanism is yet to be defined6,7. 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 rats9,10,11, dogs12,13, pigs14,15,16 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&#160;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;&#160;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 described9,18,19,20,21,22,23. Fresh tissues are scanned immediately after the animal is sacrificed without any staining or fixation, which reduces tissue artefacts as well as&#160; 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 method24,25. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-mL BD Luer-Lok syringe</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>1X phosphate buffered saline</td>
			<td>VWR Life Sciences</td>
			<td>0780-10L</td>
			<td></td>
		</tr>
		<tr>
			<td>200 proof ethanol</td>
			<td>VWR Life Sciences</td>
			<td>V1016</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum alloy 5019 wire</td>
			<td>Sigma-aldrich</td>
			<td>GF15828813</td>
			<td>0.08 mm diameter wire, length 100th, temper hard. Used as wire ligature around molar.</td>
		</tr>
		<tr>
			<td>Avizo 9.7</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td>Used to analyze microCT scans</td>
		</tr>
		<tr>
			<td>Castroviejo Micro Needle Holders</td>
			<td>Fine Science Tools</td>
			<td>12060-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Clr Plan-Apochromat 20x/1.0,CorrVIS-IR M27 85mm</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td>Used for second harmonic generation imaging</td>
		</tr>
		<tr>
			<td>Cone socket handle, single ended, hand-form</td>
			<td>G.Hartzell and son</td>
			<td>126-CSH3</td>
			<td>Handle of the inspection mirror</td>
		</tr>
		<tr>
			<td>EC Plan-Neofluar 5x/0.16</td>
			<td>Zeiss</td>
			<td>440321-9902</td>
			<td>Used for light-sheet imaging</td>
		</tr>
		<tr>
			<td>Elipar DeepCure-S LED curing light</td>
			<td>3M ESPE</td>
			<td>76985</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf safe-lock tubes, 1.5mL</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl cinnamate, &#62;= 98%</td>
			<td>Sigma-aldrich</td>
			<td>W243000-1KG-K</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic Needle, 27G x 1/2&#39;&#39;</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketathesia 100mg/ml</td>
			<td>Henry Schein Animal Health</td>
			<td>NDC:11695-0702-1</td>
			<td></td>
		</tr>
		<tr>
			<td>KIMWIPES delicate task wipers</td>
			<td>Kimberly-Clark</td>
			<td>21905-026 (VWR Catalog number)</td>
			<td>Purchased from VWR</td>
		</tr>
		<tr>
			<td>LightSheet Z.1 dual illumination microscope system</td>
			<td>Zeiss</td>
			<td>LightSheet Z.1/LightSheet 7</td>
			<td>Used for lightsheet imaging</td>
		</tr>
		<tr>
			<td>LSM 880 NLO multi-photon microscope</td>
			<td>Zeiss</td>
			<td>LSM 880 NLO</td>
			<td>Used for two-photon imaging</td>
		</tr>
		<tr>
			<td>MEGAmicro, plane, 5mm dia, SS-Thread</td>
			<td>Hahnenkratt</td>
			<td>6220</td>
			<td>Front surface inspectrio mirror</td>
		</tr>
		<tr>
			<td>MicroCT machine, MicroXCT-200</td>
			<td>Xradia</td>
			<td>MICRO XCT-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Colibri</td>
			<td>Fine Science Tools</td>
			<td>17000-01</td>
			<td></td>
		</tr>
		<tr>
			<td>PermaFlo Flowable Composite</td>
			<td>Ultradent</td>
			<td>948</td>
			<td></td>
		</tr>
		<tr>
			<td>Procedure platform</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom-made from lab materials</td>
		</tr>
		<tr>
			<td>Routine stereo micscope M80</td>
			<td>Leica Micosystems</td>
			<td>M80</td>
			<td></td>
		</tr>
		<tr>
			<td>Sentalloy NiTi open coil spring</td>
			<td>TOMY Inc.</td>
			<td></td>
			<td>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.&#160;</td>
		</tr>
		<tr>
			<td>T-304 stainless steel ligature wire, 0.009&#39;&#39; diameter</td>
			<td>Orthodontics</td>
			<td>SBLW109</td>
			<td>0.009&#39;&#39;(.23mm) diameter, Soft temper</td>
		</tr>
		<tr>
			<td>X-Ject E (Xylazine) 100mg/ml</td>
			<td>Henry Schein Animal Health</td>
			<td>NDC:11695-7085-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Z100 Restorative, A2 shade</td>
			<td>3M ESPE</td>
			<td>5904A2</td>
			<td></td>
		</tr>
	</tbody>
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3d imaging of pdl collagen fibers during orthodontic tooth movement in mandibular murine model

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&#160;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.

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 experime...

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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 of altered soft and hard tissue remodeling as a result of external forces. In order to ...

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.

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