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.
	<ol>
		<li>Elevate the head end of the platform to generate an approximately 30&#176; angle between the headrest and the table plane. Attach a bent thick paper clip (0.036&#34; in diameter) to the head side end to hold the upper incisors.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Anesthetize mouse by&#160;intraperitoneal injection of xylazine at 10 mg/kg and ketamine 100mg/kg using 1 mL syringe and a 27 Gauge needle.</li>
	<li>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.</li>
	<li>Place the platform under a surgical microscope or any other stereoscope that can reach to 5-6x magnification.</li>
	<li>Apply 50 &#181;L of saline (roughly 1 drop) on the mouse eyes to prevent corneal dehydration. Replenish saline every 20 min.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Either leave the contralateral side intact or insert a sham such as the wire between the first and&#160;second molars.</li>
	<li>Place the anaesthetized mouse under a heated light to keep the mouse warm until recovery.</li>
	<li>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.</li>
</ol>
2. Micro-CT scan of PDL fibers in fresh hemi-mandibles
<ol>
	<li>Mounting the hemi-mandible (Figure 2)
	<ol>
		<li>After the desired duration of orthodontic movement, sacrifice the mouse via cervical dislocation. Remove the mandible and separate into hemi-mandibles. 
		&#8203;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.</li>
		<li>Remove the surrounding soft tissue gently with a clean lint-free wipe.</li>
		<li>Remove the orthodontic device using microsurgical scissors and tweezers under a stereo microscope with at least 4x magnification.</li>
		<li>Keep sample moist in a 1.5 mL volume, micro-centrifuge tube along with a piece of lint-free wipe moistened with water.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Affix the chamber in the micro-CT. Screw the chamber into the micro-CT sample stage so that movement during imaging is minimized.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Micro-CT settings
	<ol>
		<li>Set the source voltage to 40 kV and current to 200 &#181;A. 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.</li>
		<li>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 &#181;m on each side.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Clearing method (Figure 3)
<ol>
	<li>Prepare five 1.5 mL micro-centrifuge tubes.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Move the hemi-mandible to 50% EtOH. Place it on the rocker covered from light for 16 h.</li>
	<li>Move the hemi-mandible to 70% EtOH. Place it on the rocker covered from light for 16 h.</li>
	<li>Move the hemi-mandible to 100% EtOH. Place it on the rocker covered from light for 16 h.</li>
	<li>Repeat 3.6. in the second 100% EtOH tube.</li>
	<li>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.</li>
	<li>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 &#176;C. Do not store at 4 &#176;C.</li>
	<li>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.</li>
</ol>

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</ol>

The authors have nothing to disclose.

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><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, &gt;= 98%</td><td>Sigma-aldrich</td><td>W243000-1KG-K</td><td></td></tr><tr><td>Hypodermic Needle, 27G x 1/2&#039;&#039;</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.&amp;nbsp;</td></tr><tr><td>T-304 stainless steel ligature wire, 0.009&#039;&#039; diameter</td><td>Orthodontics</td><td>SBLW109</td><td>0.009&#039;&#039;(.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></table>

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...

Watch this Scientific Journal Video about 3D Imaging of PDL Collagen Fibers during Orthodontic Tooth Movement in Mandibular Murine Model at JoVE.com

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 ...

3d-imaging-pdl-collagen-fibers-during-orthodontic-tooth-movement

Research

JoVE Journal

Medicine

4.9K Views.  School of Dental Medicine, Harvard University. 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. 

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

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e.&#160;geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

Bioengineering

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. At present, it is common practice to collect 3 mm skin biopsies from the distal leg (DL) and the proximal thigh (PT) for the evaluation of length-dependent polyneuropathies 3. However, due to the multidirectional nature of IENFs, it is challenging to examine overlapping nerve structures through the analysis of two-dimensional (2D) imaging. Alternatively, three-dimensional (3D) imaging could provide a better solution for this dilemma.
In the current report, we present methods for applying 3D imaging to study painful neuropathy (PN). In order to identify IENFs, skin samples are processed for immunofluorescent analysis of protein gene product 9.5 (PGP), a pan neuronal marker. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP immunohistochemistry using brightfield microscopy 4. In the current study, we applied double immunofluorescent analysis to identify total IENFD, using PGP, and nociceptive IENF, through the use of antibodies that recognize tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for nerve growth factor 5. The advantages of co-staining IENF with PGP and Trk A antibodies benefits the study of PN by clearly staining PGP-positive, nociceptive fibers. These fluorescent signals can be quantified to determine nociceptive IENFD and morphological changes of IENF associated with PN. The fluorescent images are acquired by confocal microscopy and processed for 3D analysis. 3D-imaging provides rotational abilities to further analyze morphological changes associated with PN. Taken together, fluorescent co-staining, confocal imaging, and 3D analysis clearly benefit the study of PN.

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...

Immunology and Infection

Experimental Model of Ligature-Induced Peri-Implantitis in Mice

Dental implants have a high success and survival rate. However, complications such as peri-implantitis (PI) are highly challenging to treat. PI is characterized by inflammation in the tissues around dental implants with progressive loss of supporting bone. To optimize dental implants' longevity in terms of health and functionality, it is crucial to understand the peri-implantitis pathophysiology. In this regard, using mouse models in research has proven clear benefits in recreating clinical circumstances. This study aimed to describe an experimental model of ligature-induced peri-implantitis in mice and determine whether there is effectiveness in inducing this disease, given the observed bone and tissue changes. The experimental peri-implantitis induction comprehends the following steps: teeth extraction, implant placement, and ligature-inducted PI. A sample of eighteen 3-week-old C57BL/6J male mice was divided into two groups, ligature (N=9) and control non-ligature (N=9). The evaluation of clinical, radiographical, and histological factors was performed. The ligature group showed significantly higher bone loss, increased soft tissue edema, and apical epithelial migration than the non-ligature group. It was concluded that this pre-clinical model can successfully induce peri-implantitis in mice.

This is a report on an experimental model of ligature-induced peri-implantitis in mice. We describe all surgical steps, from pre- and post-operative management of the animals, extractions, implant placement, and ligature-induced peri-implantitis.

Dental implants have a high success and survival rate. However, complications such as peri-implantitis (PI) are highly challenging to treat. PI is ...

A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the structural and cellular components of the mandibular condylar cartilage (MCC). This manuscript describes methods for analyzing these changes and a method for altering the loading of the TMJ in mice (i.e., compressive static TMJ loading). The structural evaluation illustrated here is a simple morphometric approach that uses the Digimizer software and is performed in radiographs of small bones. In addition, the analysis of cellular changes leading to alterations in collagen expression, bone remodeling, cell division, and proteoglycan distribution in the MCC is described. The quantification of these changes in histological sections - by counting the positive fluorescent pixels using image software and measuring the distance mapping and stained area with Digimizer - is also demonstrated. The methods shown here are not limited to the murine TMJ, but could be used on additional bones of small experimental animals and in other regions of endochondral ossification.

This manuscript presents methods for analyzing morphometric and cellular changes within the mandibular condyle of rodents.

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the ...

Biology

Analyzing Alveolar Bone Remodeling to Explore Skeletal Mechanical Biology

Using Inducible Osteoblastic Lineage-Specific Stat3 Knockout Mice to Study Alveolar Bone Remodeling During Orthodontic Tooth Movement

The alveolar bone, with a high turnover rate, is the most actively-remodeling bone in the body. Orthodontic tooth movement (OTM) is a common artificial process of alveolar bone remodeling in response to mechanical force, but the underlying mechanism remains elusive. Previous studies have been unable to reveal the precise mechanism of bone remodeling in any time and space due to animal model-related restrictions. The signal transducer and activator of transcription 3 (STAT3) is important in bone metabolism, but its role in osteoblasts during OTM is unclear. To provide in vivo evidence that STAT3 participates in OTM at specific time points and in particular cells during OTM, we generated a tamoxifen-inducible osteoblast lineage-specific Stat3 knockout mouse model, applied orthodontic force, and analyzed the alveolar bone phenotype. 
Micro-computed tomography (Micro-CT) and stereo microscopy were used to access OTM distance. Histological analysis selected the area located within three roots of the first molar (M1) in the cross-section of the maxillary bone as the region of interest (ROI) to evaluate the metabolic activity of osteoblasts and osteoclasts, indicating the effect of orthodontic force on alveolar bone. In short, we provide a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describe methods for analyzing alveolar bone remodeling during OTM, thus shedding new light on skeletal mechanical biology.

Author Spotlight: Comparing Alveolar and Long Bone Remodeling to Explore OTM Model Potential 

This study provides a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describes methods for analyzing alveolar bone remodeling during orthodontic tooth movement, thus shedding light on skeletal mechanical biology.

The alveolar bone, with a high turnover rate, is the most actively-remodeling bone in the body. Orthodontic tooth movement (OTM) is a common artificial ...

A Murine Maxillary Orthodontic Model Guide to Bone Remodeling Analysis

The Establishment of a Murine Maxillary Orthodontic Model

Due to the lack of reproducible protocols for establishing a murine maxillary orthodontic model, we present a reliable and reproducible protocol to provide researchers with a feasible tool to analyze mechanical loading-associated bone remodeling. This study presents a detailed flowchart in addition to different types of schematic diagrams, operation photos, and videos. We performed this protocol on 11 adult wide-type C57/B6J mice and harvested samples on postoperative days 3, 8, and 14. The micro-CT and histopathological data have proven the success of tooth movements coupled with bone remodeling using this protocol. Furthermore, according to the micro-CT results on days 3, 8, and 14, we have divided bone modeling into three stages: preparation stage, bone resorption stage, and bone formation stage. These stages are expected to help researchers concerned with different stages to set sample collection time reasonably. This protocol can equip researchers with a tool to carry out regenerative analysis of bone remodeling.

Author Spotlight: Insights into an Efficient Murine Maxillary Orthodontic Model Protocol

Here we demonstrate step-by-step a manageable, orthodontic tooth movement protocol operated on a murine maxillary model. With the explicit explanation of each step and visual demonstration, researchers can master this model and apply it to their experimental needs with a few modifications.

Due to the lack of reproducible protocols for establishing a murine maxillary orthodontic model, we present a reliable and reproducible protocol to ...

Investigating Orthodontic Tooth Movement and Bone Adaptation Mechanisms in Mice

Studying Orthodontic Tooth Movement in Mice

Orthodontic tooth movement (OTM) represents a dynamic process in which the alveolar bone undergoes resorption at compression sites and deposition at tension sites, orchestrated by osteoclasts and osteoblasts, respectively. This mechanism serves as a valuable model for studying various aspects of bone adaptation, including root resorption and the cellular response to mechanical force stimuli. The protocol outlined here offers a straightforward approach to investigate OTM, establishing 0.35 N as the optimal force in a mouse model employing a nickel-titanium (NiTi) coil spring. Utilizing micro-computed tomography analysis, we quantified OTM by assessing the discrepancy in the linear distance at the cement-enamel junction. The evaluation also included an analysis of orthodontic-induced inflammatory root resorption, assessing parameters such as root mineral density and the percentage of root volume per total volume. This comprehensive protocol contributes to advancing our understanding of bone remodeling processes and enhancing the ability to develop effective orthodontic treatment strategies.

Here, we present a protocol for studying orthodontic tooth movement (OTM), serving as a suitable model for investigating the mechanisms of bone adaptation, root resorption, and the response of bone cells to mechanical stimuli. This comprehensive guide provides detailed information on the OTM model, micro-computed tomography acquisition, and subsequent analysis.

Orthodontic tooth movement (OTM) represents a dynamic process in which the alveolar bone undergoes resorption at compression sites and deposition at ...

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

Podsumowanie

Przeglądaj więcej filmów

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

Experimental Model of Ligature-Induced Peri-Implantitis in Mice

A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

Using Inducible Osteoblastic Lineage-Specific Stat3 Knockout Mice to Study Alveolar Bone Remodeling During Orthodontic Tooth Movement

The Establishment of a Murine Maxillary Orthodontic Model

Studying Orthodontic Tooth Movement in Mice