Name: the establishment of a murine maxillary orthodontic model
Uploaded: 2023-10-27T12:40:00.000+00:00
Duration: 4 min 11 s
Description: Watch this Scientific Journal Video about Author Spotlight: Insights into an Efficient Murine Maxillary Orthodontic Model Protocol at JoVE.com

This work was supported by the National Natural Science Foundation of China grant 82100982 to F.L.

The animal procedures in this study were reviewed and approved by the Ethical Committee of the West China School of Stomatology, Sichuan University (WCHSIRB-D-2017-041). Adult C57BL/6 mice were used in this study (see the Table of Materials). This protocol adds mechanical load to the right maxillary first molar (M1) for mesial movement where a highly coordinated bone remodeling process is induced by coupling of bone resorption and bone formation (Figure 1).
1. Preoperative preparation
<ol>
	<li>Surgical items
	<ol>
		<li>Prepare the following surgical items for the operation: surgical platform (Figure 2A), fastener (Figure 2B), surgical instruments (Figure 2C and Supplementary Figure S1), orthodontic supplies (Figure 2C), and dental restoration supplies (Figure 2D). 
		NOTE: The customized coil spring is custom-made and provides a force of 10cN when stretched to 10 mm.</li>
	</ol>
	</li>
	<li>Sterilization
	<ol>
		<li>Sterilize the surgical instruments by autoclaving and all the surgical items with ultraviolet irradiation for at least 30 min.</li>
	</ol>
	</li>
	<li>Anesthesia
	<ol>
		<li>Anesthetize the mouse by administering ketamine (100 mg/kg) and diazepam (5 mg/kg) by intraperitoneal injection.</li>
		<li>Apply vet ointment to the eyes of the murine with a cotton stick to avoid eye dryness.</li>
		<li>Proceed with the surgery only when the mouse does not respond when its toes are pinched with forceps.</li>
	</ol>
	</li>
</ol>
2. Surgical process
<ol>
	<li>Spread and tape the limbs of the anesthetized mouse in a supine position to the surgical platform using adhesive tape.</li>
	<li>Pin a 27 G needle on either side above the head and another 27 G needle on either side below the axilla.</li>
	<li>Wind a rubber band around the above two needles and the upper incisors and another one around another two&#160;needles and the lower incisors. Change the needle positions to control the degree of opening and the orientation of the mouth (Figure 3A). 
	NOTE: For the orthodontic tooth movement operation, keep the mouth open to the maximum extent before the buccinator becomes completely tight. The tongue should be pulled towards the non-operative side to expose the surgical field and prevent ischemia.</li>
	<li>Bend the 1.5 mm end of a 3 cm 304 stainless steel wire and push the bent end through the interproximate space between the M1 and the maxillary second molar (M2) from the buccal side with curved ophthalmic tweezers (Figure 3B). When the palatal end of the ligature wire is seen from the palatal side, pull it out up to about half of its length and pass it through one end of the customized coil spring.</li>
	<li>Tie a square knot with the two ends of the ligature wire in the mesial direction of the maxillary M1 until the spring is firmly fixed to the tooth (Figure 3C). Subtract the excess wire.</li>
	<li>Similarly, pierce a second 3 cm 304 stainless steel wire through the other end of the coil spring.</li>
	<li>Clean and dry the incisors&#39; surfaces with cotton balls. Apply adhesives on all those surfaces with cotton sticks and light-cure them.</li>
	<li>Push the second stainless steel wire through the interproximate space between the maxillary incisors and tie a slip knot in the labial direction (Figure 3D). Subtract the excess wire and make the rest of the wire lie close to the tooth surface.</li>
	<li>Inject light-cured resin to cover the knot and incisors; light-cure the resin (Figure 3E).</li>
</ol>
3. Postoperative management
<ol>
	<li>After surgery, inject the mice with 0.05 mg/kg buprenorphine intraperitoneally&#160;for postoperative analgesia.</li>
	<li>Place the anesthetized mouse on a 37 &#176;C thermostatic electric blanket. When the murine regains consciousness with ambulation, return it to a separate housing cage.</li>
	<li>Due to the limited functioning of the incisors after the surgery, replace regular hard fodder with only a soft diet.</li>
	<li>Check the orthodontic appliances every day. If any condition are observed during the inspection that affects the conduction of orthodontic force, such as spring deformation, spring loosening, and device falling off, the mouse should be excluded from the experiment.</li>
	<li>In order to maintain the comparability of experiments, assess the weight of the mice daily post-surgery. Any mice exhibiting a weight loss exceeding 30% of their preoperative weight must be excluded from the experiment.</li>
</ol>

A Murine Maxillary Orthodontic Model Guide to Bone Remodeling Analysis

<ol>
	<li>Kenkre, J. S., Bassett, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bone+remodeling+cycle.">The bone remodeling cycle.</a> Annals of Clinical Biochemistry. 55 (3), 308-327 (2018).</li><li>Feng, X., McDonald, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorders+of+bone+remodeling.">Disorders of bone remodeling.</a> Annual Review of Pathology. 6 (1), 121-145 (2011).</li><li>Alliston, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+regulation+of+bone+quality.">Biological regulation of bone quality.</a> Current Osteoporosis Reports. 12 (3), 366-375 (2014).</li><li>Duncan, R. L., Turner, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+and+the+functional+response+of+bone+to+mechanical+strain.">Mechanotransduction and the functional response of bone to mechanical strain.</a> Calcified Tissue International. 57 (5), 344-358 (1995).</li><li>Garc&#237;a-Aznar, J. M., Nasello, G., Hervas-Raluy, S., P&#233;rez, M. &. #. 1. 9. 3. ;., G&#243;mez-Benito, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+modeling+of+bone+tissue+mechanobiology.">Multiscale modeling of bone tissue mechanobiology.</a> Bone. 151 (10), 1-12 (2021).</li><li>Rolvien, T., Amling, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disuse+osteoporosis:+clinical+and+mechanistic+insights.">Disuse osteoporosis: clinical and mechanistic insights.</a> Calcified Tissue International. 110 (5), 592-604 (2022).</li><li>Vico, L., Hargens, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+changes+during+and+after+spaceflight.">Skeletal changes during and after spaceflight.</a> Nature Reviews Rheumatology. 14 (4), 229-245 (2018).</li><li>Iwaniec, U. T., Turner, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+body+weight+on+bone+mass,+architecture+and+turnover.">Influence of body weight on bone mass, architecture and turnover.</a> Journal of Endocrinology. 230 (3), R115-R130 (2016).</li><li>Governale, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Craniosynostosis.">Craniosynostosis.</a> Pediatric Neurology. 53 (5), 394-401 (2015).</li><li>Sahoo, N. K., Issar, Y., Thakral, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mandibular+Distraction+osteogenesis.">Mandibular Distraction osteogenesis.</a> Journal of Craniofacial Surgery. 30 (8), e743-e746 (2019).</li><li>Roberts-Harry, D., Sandy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthodontics.+Part+1:+Who+needs+orthodontics.">Orthodontics. Part 1: Who needs orthodontics.</a> British Dental Journal. 195 (8), 433-437 (2003).</li><li>Li, Y., Jacox, L. A., Little, S. H., Ko, C. C. <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> Kaohsiung Journal of Medical Sciences. 34 (4), 207-214 (2018).</li><li>Will, L. A. <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:+a+historic+prospective.">Orthodontic tooth movement: a historic prospective.</a> Frontiers of Oral Biology. 18, 46-55 (2016).</li><li>Xu, H., Lee, A., Sun, L., Naveh, G. R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Imaging+of+PDL+collagen+fibers+during+orthodontic+tooth+movement+in+mandibular+murine+model.">3D Imaging of PDL collagen fibers during orthodontic tooth movement in mandibular murine model.</a> Journal of Visualized Experiments. (170), e62149 (2021).</li><li>Taddei, S. 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=Experimental+model+of+tooth+movement+in+mice:+a+standardized+protocol+for+studying+bone+remodeling+under+compression+and+tensile+strains.">Experimental model of tooth movement in mice: a standardized protocol for studying bone remodeling under compression and tensile strains.</a> Journal of Biomechanics. 45 (16), 2729-2735 (2012).</li><li>Deguchi, T., Takeshita, N., Balam, T. A., Fujiyoshi, Y., Takano-Yamamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galanin-immunoreactive+nerve+fibers+in+the+periodontal+ligament+during+experimental+tooth+movement.">Galanin-immunoreactive nerve fibers in the periodontal ligament during experimental tooth movement.</a> Journal of Dental Research. 82 (9), 677-681 (2003).</li><li>Gudhimella, 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=A+rodent+model+using+skeletal+anchorage+and+low+forces+for+orthodontic+tooth+movement.">A rodent model using skeletal anchorage and low forces for orthodontic tooth movement.</a> American Journal of Orthodontics and Dentofacial Orthopedics. 155 (2), 254-263 (2019).</li><li>Lira Dos Santos, E. 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=Orthodontic+tooth+movement+alters+cementocyte+ultrastructure+and+cellular+cementum+proteome+signature.">Orthodontic tooth movement alters cementocyte ultrastructure and cellular cementum proteome signature.</a> Bone. 153 (12), 116-139 (2021).</li><li>Danz, J. C., Bibby, B. M., Katsaros, C., Stavropoulos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+facial+tooth+movement+on+the+periodontium+in+rats:+a+comparison+between+conventional+and+low+force.">Effects of facial tooth movement on the periodontium in rats: a comparison between conventional and low force.</a> Journal of Clinical Periodontology. 43 (3), 229-237 (2016).</li><li>Kohno, T., Matsumoto, Y., Kanno, Z., Warita, H., Soma, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+tooth+movement+under+light+orthodontic+forces:+rates+of+tooth+movement+and+changes+of+the+periodontium.">Experimental tooth movement under light orthodontic forces: rates of tooth movement and changes of the periodontium.</a> Journal of Orthodontics. 29 (2), 129-135 (2002).</li><li>Gad, A. M., Soliman, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+systemic+Omega-3+PUFAs+effect+on+orthodontic+tooth+movement+in+a+rabbit+model:+RCT.">Evaluation of systemic Omega-3 PUFAs effect on orthodontic tooth movement in a rabbit model: RCT.</a> Angle Orthodontist. 93 (4), 476-481 (2023).</li><li>Huang, C. 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=Comparison+of+tooth+movement+and+biological+response+resulting+from+different+force+magnitudes+combined+with+osteoperforation+in+rabbits.">Comparison of tooth movement and biological response resulting from different force magnitudes combined with osteoperforation in rabbits.</a> Journal of Applied Oral Science. 29 (2), 20200734 (2021).</li><li>Alhasyimi, A. A., Pudyani, P. P., Asmara, W., Ana, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+post-orthodontic+tooth+stability+by+carbonated+hydroxyapatite-incorporated+advanced+platelet-rich+fibrin+in+rabbits.">Enhancement of post-orthodontic tooth stability by carbonated hydroxyapatite-incorporated advanced platelet-rich fibrin in rabbits.</a> Orthodontics &amp; Craniofacial Research. 21 (2), 112-118 (2018).</li><li>Elkattan, A. E., 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=Effects+of+Different+Parameters+of+Diode+Laser+on+Acceleration+of+Orthodontic+Tooth+Movement+and+Its+Effect+on+Relapse:+An+Experimental+Animal+Study.">Effects of Different Parameters of Diode Laser on Acceleration of Orthodontic Tooth Movement and Its Effect on Relapse: An Experimental Animal Study.</a> Open Access Macedonian Journal of Medical Sciences. 7 (3), 412-420 (2019).</li><li>von B&#246;hl, M., Maltha, J. C., Von Den Hoff, J. W., 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=Focal+hyalinization+during+experimental+tooth+movement+in+beagle+dogs.">Focal hyalinization during experimental tooth movement in beagle dogs.</a> American Journal of Orthodontics and Dentofacial Orthopedics. 125 (5), 615-623 (2004).</li><li>Machibya, F. M., 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=Effects+of+bone+regeneration+materials+and+tooth+movement+timing+on+canine+experimental+orthodontic+treatment.">Effects of bone regeneration materials and tooth movement timing on canine experimental orthodontic treatment.</a> Angle Orthodontist. 88 (2), 171-178 (2018).</li><li>Deguchi, T., 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=Histomorphometric+evaluation+of+alveolar+bone+turnover+between+the+maxilla+and+the+mandible+during+experimental+tooth+movement+in+dogs.">Histomorphometric evaluation of alveolar bone turnover between the maxilla and the mandible during experimental tooth movement in dogs.</a> American Journal of Orthodontics and Dentofacial Orthopedics. 133 (6), 889-897 (2008).</li><li>Tanimoto, K., 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+tooth+movement+into+new+bone+area+regenerated+by+use+of+bone+marrow-derived+mesenchymal+stem+cells.">Experimental tooth movement into new bone area regenerated by use of bone marrow-derived mesenchymal stem cells.</a> Cleft Palate-craniofacial Journal. 52 (4), 386-394 (2015).</li><li>Oltramari, P. V., 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+movement+in+bone+defects+filled+with+xenogenic+graft:+an+experimental+study+in+minipigs.">Orthodontic movement in bone defects filled with xenogenic graft: an experimental study in minipigs.</a> American Journal of Orthodontics and Dentofacial Orthopedics. 131 (3), e10-e17 (2007).</li><li>Verna, C., Dalstra, M., Lee, T. C., 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=Microdamage+in+porcine+alveolar+bone+due+to+functional+and+orthodontic+loading.">Microdamage in porcine alveolar bone due to functional and orthodontic loading.</a> European Journal of Morphology. 42 (1-2), 3-11 (2005).</li><li>Steiner, G. G., Pearson, J. K., Ainamo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+of+the+marginal+periodontium+as+a+result+of+labial+tooth+movement+in+monkeys.">Changes of the marginal periodontium as a result of labial tooth movement in monkeys.</a> Journal of Periodontology. 52 (6), 314-320 (1981).</li><li>Celebi, A. A., Demirer, S., Catalbas, B., Arikan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ovarian+activity+on+orthodontic+tooth+movement+and+gingival+crevicular+fluid+levels+of+interleukin-1&#946;+and+prostaglandin+E(2)+in+cats.">Effect of ovarian activity on orthodontic tooth movement and gingival crevicular fluid levels of interleukin-1&#946; and prostaglandin E(2) in cats.</a> Angle Orthodontist. 83 (2), 70-75 (2013).</li><li>Holmes, H. D., Tennant, M., Goonewardene, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Augmentation+of+faciolingual+gingival+dimensions+with+free+connective+tissue+grafts+before+labial+orthodontic+tooth+movement:+an+experimental+study+with+a+canine+model.">Augmentation of faciolingual gingival dimensions with free connective tissue grafts before labial orthodontic tooth movement: an experimental study with a canine model.</a> American Journal of Orthodontics and Dentofacial Orthopedics. 127 (5), 562-572 (2005).</li><li>Wennstr&#246;m, J. L., Lindhe, J., Sinclair, F., Thilander, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+periodontal+tissue+reactions+to+orthodontic+tooth+movement+in+monkeys.">Some periodontal tissue reactions to orthodontic tooth movement in monkeys.</a> Journal of Clinical Periodontology. 14 (3), 121-129 (1987).</li><li>Ibrahim, A. Y., Gudhimella, S., Pandruvada, S. N., Huja, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+differences+between+animal+models+for+expedited+orthodontic+tooth+movement.">Resolving differences between animal models for expedited orthodontic tooth movement.</a> Orthodontics &amp; Craniofacial Research. 20, 72-76 (2017).</li><li>Kirschneck, C., Bauer, M., Gubernator, J., Proof, P., Schr&#246;der, A. <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>Ransom, R. 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=Mechanoresponsive+stem+cells+acquire+neural+crest+fate+in+jaw+regeneration.">Mechanoresponsive stem cells acquire neural crest fate in jaw regeneration.</a> Nature. 563 (7732), 514-521 (2018).</li><li>Mardas, N., 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+model+for+bone+regeneration+in+oral+and+cranio-maxillo-facial+surgery.">Experimental model for bone regeneration in oral and cranio-maxillo-facial surgery.</a> Journal of Investigative Surgery. 27 (1), 32-49 (2014).</li></ol>

The authors declare no conflicts of interest.

In this paper, we tried to describe the simplest orthodontic tooth movement protocol on murine maxillary model step by step to study the latent mechanisms of mechanical load-induced bone remodeling. Apart from research on bone remodeling, there are some other mainstream applications of this method: 1) methodological research on the acceleration of orthodontic tooth movement; 2) research on orthodontic root resorption; 3) biological mechanisms of orthodontic tooth movement and pain; 4) research on the transgenic model.</p...

Bone is a highly active reconstructed tissue that adapts its size, shape, and properties through the lifetime of the individual1,2. In addition to hormones, aging, nutrition, and other biological or biochemical factors3, the idea that mechanical load is the most determining factor has garnered general acceptance4,5. Under some circumstances with abnormal mechanical load, the imbalance between bone resorption and bone formation may lead to abnormal bone remodeling and bone disorders. Bone diseases such as disuse osteoporosis and bone loss during long-term bed rest or in the presence of microgravity at spaceflight have a close relationship with abnormal mechanical load6,7,8.
Mechanical load has also been used to treat bone-related diseases such as distraction treatment and orthodontic treatment. Distraction treatment has been used in developmental diseases such as craniosynostosis and mandibular hypoplasia9,10, while orthodontic treatment has been widely used to rectify abnormal teeth position and any malocclusion11. The core of orthodontic treatment is also the management of mechanical load. When the bone tissue is subjected to mechanical load, a highly coordinated bone remodeling process is induced by coupling of bone resorption followed by bone formation, which can move teeth to achieve the orthodontic purpose12,13.
Although orthodontic treatment has been widely applied for clinical practice, as our knowledge of the biological effects of mechanical load is limited, the results of orthodontic treatment are uncontrollable. To overcome these limitations, several animal models such as mouse, rat, rabbit, cat, dog, monkey, and pig have been established to investigate the underlying mechanism of mechanical load-induced bone remodeling (Table 1)14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32. Large animals such as dogs, monkeys, and pigs have some advantages over small animals in orthodontic operation-they have more human-like teeth and dentition so that the surgical procedure is easy to replicate in humans. Additionally, a wide view can reduce the operation difficulty and make it possible to apply a variety of orthodontic schemes33,34. However, large animals are difficult to obtain, leading to challenges related to sample size, and they are subject to ethical restrictions35. Furthermore, routine extraction procedures and complex instruments make the experiments difficult to perform due to which large animals are rarely used.
Under such circumstances, rodents are mainly used to establish orthodontic models. Among these models, rats and rabbits have lower operating difficulty and more tooth movement schemes compared with mice. However, the murine model has the unique advantage that there are a large number of genetically modified mice available, which is especially crucial for investigating the underlying mechanisms36. However, the murine model is the most difficult model to manipulate because of its small size. Reviewing the current methods, moving the first molar in the mesial direction is the only practical method for an orthodontic model. Two devices are mainly used to move the tooth-coil spring and elastic band. Using an elastic band is easier, but the orthodontic force varies greatly, which makes it difficult to obtain stable results.
Xu et al.15 have established a murine model with a coil spring on the mandible. However, due to the mobility of the mandible and the obstructive nature of the tongue, operation on the maxilla is always the first choice for both intraoperative and postoperative considerations. Taddei et al.16 described a more detailed protocol on the murine maxilla 10 years ago and more visual and pellucid details should be added. In summary, this protocol has systematically described a detailed orthodontic tooth movement protocol in a murine maxillary model to help researchers master the modeling method in a standardized way and enable the comparative evaluation between different studies.

<table><tbody><tr><td>&lt;strong&gt;Experimental Models: Mouse Lines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>C57/B6J&amp;nbsp;</td><td>Gempharmatech Experimental Animals Company&amp;nbsp;</td><td>C57/B6J</td><td></td></tr><tr><td>&lt;strong&gt;Critical Commercial Assays&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Hematoxylin and Eosin Stain Kit</td><td>Biosharp</td><td>BL700B</td><td></td></tr><tr><td>Masson&amp;rsquo;s Trichrome Stain Kit</td><td>Solarbio</td><td>G1340</td><td></td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>27 G needle</td><td>Chengdu Xinjin Shifeng Medical Apparatus &amp;amp; Instruments Co. LTD.</td><td>SB1-074(IV)</td><td></td></tr><tr><td>Adhesives</td><td>Minnesota Mining and Manufacturing Co., Ltd.</td><td>41282</td><td></td></tr><tr><td>Corkboard</td><td>DELI Group Co., Ltd.</td><td>8705</td><td></td></tr><tr><td>Cotton balls</td><td>Haishi Hainuo Group Co.,&amp;nbsp; Ltd.</td><td>20120047</td><td></td></tr><tr><td>Cotton sticks</td><td>Lakong Medical Devices Co., Ltd.</td><td>M6500R</td><td></td></tr><tr><td>Customized coil spring</td><td>Chengdu Mingxing Spring Co., Ltd.</td><td>1109-02</td><td></td></tr><tr><td>Forceps</td><td>Chengdu Shifeng Co., Ltd.</td><td>none</td><td></td></tr><tr><td>Light-cured fluid resin</td><td>Shofu Dental Trading (SHANGHAI) Co., Ltd.</td><td>518785</td><td></td></tr><tr><td>Light curer</td><td>Liang Ya Dental Equipment Co., Ltd.</td><td>LY-A180</td><td></td></tr><tr><td>Medical adhesive tapes&amp;nbsp;</td><td>Haishi Hainuo Group Co.,&amp;nbsp; Ltd.</td><td>0008-2014</td><td></td></tr><tr><td>Medical non-woven fabric</td><td>Henan Yadu Industrial Co., Ltd.</td><td>01011500018</td><td></td></tr><tr><td>Needle holders</td><td>Chengdu Shifeng Co., Ltd.</td><td>none</td><td></td></tr><tr><td>Rubber bands</td><td>Haishi Hainuo Group Co.,&amp;nbsp; Ltd.</td><td>32X1</td><td></td></tr><tr><td>Surgical scissors</td><td>Chengdu Shifeng Co., Ltd.</td><td>none</td><td></td></tr><tr><td>Tweezers</td><td>Chengdu Shifeng Co., Ltd.</td><td>none</td><td></td></tr></tbody></table>

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.

We have performed the OTM surgery on 11 adult male mice (C57/BL6, 3 months old). They were euthanized for results on days 3, 8, and 14 post surgery. In these experiments, the right maxillary side is the operation side, while the left maxillary side is the control side. The micro-CT showed that there was a temporal consecutive increase in the distance between M1 and M2: 30 &#181;m, 70 &#181;m, and 110 &#181;m at days 3, 8, and 14 post surgery, respectively (Figure 4). The low-density periodon...

Watch this Scientific Journal Video about Author Spotlight: Insights into an Efficient Murine Maxillary Orthodontic Model Protocol at JoVE.com

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.

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 ...

the-establishment-of-a-murine-maxillary-orthodontic-model

Research

JoVE Journal

Medicine

584 Views.  Sichuan University. 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.

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

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

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.

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 ...

Development of an Effective Mice Model for Studying Oronasal Fistulas

Establishment of an Oronasal Fistula Mice Model

This study presents a method utilizing heated ophthalmologic cautery to develop a viable model for investigating oronasal fistulas. C57BL/6 mice were used to establish the oronasal fistula (ONF) model. To create the ONF, the mice were anesthetized, immobilized, and their hard palates were exposed. During the surgical procedure, a 2.0 x 1.5 mm full-thickness mucosal injury was induced in the midline of the hard palate using ophthalmologic cautery. It was crucial to control the size of the ONF and minimize bleeding in order to ensure the success of the experiment. Verification of the ONF model's effectiveness was conducted on the 7th-day post-operation, encompassing both anatomical and functional assessments. The presence of the nasal septum within the oral cavity and the outflow of sterile water from the nostrils upon injection into the oral cavity confirmed the successful establishment of the ONF model. The model demonstrated a practical and successful oronasal fistula, characterized by a low mortality rate, significant weight changes, and minimal variation in ONF size. Future studies may consider adopting this methodology to elucidate the mechanisms of palate wound healing and explore novel treatments for oronasal fistulas.

Author Spotlight: Investigating Wound Healing in Mice Models of Oronasal Fistulas 

This article outlines a step-by-step procedure for establishing a mice model with an oronasal fistula. The oronasal fistula was created by employing heated ophthalmologic cautery to damage the midline portion of the hard palate, resulting in the formation of an opening between the oral and nasal cavities.

This study presents a method utilizing heated ophthalmologic cautery to develop a viable model for investigating oronasal fistulas. C57BL/6 mice were used ...

Creating a Pulp Inflammation Mouse Model Using a Novel Mouth-Gag 

Establishment of a Murine Pulp Exposure Model with a Novel Mouth-Gag for Pulpitis Research

Pulpitis, a common cause of natural tooth loss, leads to necrosis and loss of bioactivity in the inflamed dental pulp. Unraveling the mechanisms underlying pulpitis and its efficient treatment is an ongoing focus of endodontic research. Therefore, understanding the inflammatory process within the dental pulp is vital for improving pulp preservation. Compared to other in vitro experiments, a murine pulpitis model offers a more authentic and genetically diverse context to observe the pathological progression of pulpitis. However, using mice, despite their cost-effectiveness and accessibility, poses difficulties due to their small size, poor coordination, and low tolerance, complicating intraoral and dental procedures. This protocol introduces a novel design and application of a mouth-gag to expose mouse pulp, facilitating more efficient intraoral procedures. The mouth-gag, comprised of a dental arch readily available to most dentists and can significantly expedite surgical preparation, even for first-time procedures. Micro-CT, hematoxylin-eosin (HE) staining, and immunofluorescence staining were used to identify changes in morphology and cell expression. The aim of this article is to help researchers establish a more reproducible and less demanding procedure for creating a pulp inflammation model using this novel mouth-gag.

Author Spotlight: Enhancing Dental Pulp Research with Improved Mouse Models

This article presents a streamlined protocol for establishing a pulpitis model in mice using an innovative mouth-gag, followed by subsequent histological analysis.

Pulpitis, a common cause of natural tooth loss, leads to necrosis and loss of bioactivity in the inflamed dental pulp. Unraveling the mechanisms ...

Evaluation of Maxillary Posterior Tooth Movement Using Digital Superimposition

Measuring Maxillary Posterior Tooth Movement: A Model Assessment using Palatal and Dental Superimposition

Since the introduction of Invisalign by Align Technology, Inc. in 1999, questions and debates have persisted regarding the precision of Invisalign (clear aligner) therapy, particularly when compared to the use of traditional fixed appliances. This becomes particularly significant in cases involving anteroposterior, vertical, and transverse corrections, where precise comparisons are of paramount importance. To address these inquiries, this study introduces a meticulously devised protocol, placing a primary emphasis on digitally superimposing the movement of maxillary posterior teeth to facilitate accurate analysis. The sample included 25 patients who had completed their first series of Invisalign (clear) aligners. Four maxillary digital models (pre-treatment, post-treatment, ClinCheck-initial, and final models) were digitally superimposed using the palate rugae and dentitions as stable references. A software combination was used for model superimposition and tooth segmentation. Transformation matrices then expressed the differences between the achieved and predicted tooth positions. Thresholds for clinically relevant differences were at &plusmn;0.25 mm for linear displacement and &plusmn;2&#176; for rotation. Differences were assessed using Hotelling's T-squared tests with Bonferroni correction. The mean differences in rotation (2.036&#176; &plusmn; 4.217&#176;) and torque (-2.913&#176; &plusmn; 3.263&#176;) were significant statistically and clinically, with p-values of 0.023 and 0.0003 respectively. De-rotation of premolars and torque control for all posterior teeth were less predictable. All mean differences for the linear measurements were statistically and clinically insignificant, except that the first molars seemed slightly (0.256 mm) more intruded than their predicted position. The clear aligner system appears to meet its prediction for most translational tooth movements and mesial-distal tipping in maxillary posterior teeth for non-extraction cases with mild to moderate malocclusions.

Author Spotlight: 3D Movement Assessment of Maxillary Posterior Teeth in Clear Aligner Treatment

This manuscript presents a comprehensive protocol to evaluate the three-dimensional (3D) movement of maxillary posterior teeth with clear aligners using digital model superimposition, an invaluable tool in orthodontics and dentofacial orthopedics.

Since the introduction of Invisalign by Align Technology, Inc. in 1999, questions and debates have persisted regarding the precision of Invisalign (clear ...

The Establishment of a Murine Mandibular Molar Extraction Socket Healing Model

This study introduces the development of a molar extraction model in the murine mandible to provide a practicable model for studying alveolar bone regeneration and intramembranous ossification. C57/J6 mice were used to extract the mandibular first molar to establish this model. They were euthanized, and the bilateral mandibles harvested, at 1 week and 4 weeks post-surgery, respectively. Subsequent serial stereoscopic harvest, histological assessment, and immunofluorescence staining were performed to demonstrate successful surgery. Immediately after surgery, the stereoscopic images displayed an empty extraction socket. The hematoxylin and eosin (H&#38;E) at 1 week and Masson staining at 4 weeks post-surgery showed that the area of the original root was partially and fully filled with bone trabeculae, respectively. The immunofluorescence staining showed that, compared with the homeostasis side, the Sp7 expression increased at 1 week post-surgery, suggesting vigorous osteogenesis in the alveolar fossa. All these results demonstrated a practicable murine tooth extraction socket healing model. Upcoming studies revealing the mechanisms of jawbone defect healing or socket healing could adopt this method.

This protocol demonstrates step-by-step details of how to extract the mandibular first molar in the mouse. It provides an alternative method for researchers focusing on jawbone healing and regeneration.

This study introduces the development of a molar extraction model in the murine mandible to provide a practicable model for studying alveolar bone ...

Biology

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 ...