The authors thank Ms. Makiko Kato for providing encouragement to complete this study. We also thank the Division of Experimental Animals and Medical Research Engineering, Nagoya University Graduate School of Medicine, for the housing of mice.

All experiments were carried out in accordance with protocols approved by the Animal Care and Use Committee of our institution. Sterilize all instruments prior to the procedure.
1. Preparation of a Mold for Creating the Custom-made Distractor
<ol>
	<li>Make two incomplete rings (outer diameter, 20 mm; inner diameter, 10 mm), which are a part of the distractor, with one sheet of paraffin wax (145 mm x 74 mm) using an Evans wax carver.
	<ol>
		<li>Make 4 of the same pieces. Use a wax spatula heated with a gas burner. Stack the four rings for a thickness of 5 mm. Provide space for the expansion screw and needle, 8 mm x 2 mm and 5 mm x 2 mm respectively with the Evans wax carver (Figures 1A, 1B).</li>
	</ol>
	</li>
	<li>Embed wax patterns in the silicone impression material and create a mold for the resin rings (Figure 1C).</li>
	<li>After curing the silicone impression material (approximately 5 min at room temperature), remove the wax patterns.</li>
</ol>
2. Production of the Custom-made Distractor
<ol>
	<li>Apply petroleum jelly thinly and evenly to the silicone mold. Mix 3 g of polymerization dental resin and immediately pour it into the silicone mold. Set for 5 min at room temperature (RT).</li>
	<li>Remove the polymerized resin ring from the mold and polish it with a carbide bar and a dental micromotor to remove the burr (about 1 min). Two resin rings are required for each custom-made distractor.
	<ol>
		<li>Repeat the procedure to create the required number of resin rings (Figure 1D).</li>
	</ol>
	</li>
	<li>Leave a gap of about 2 mm to fix the resin rings with utility wax and secure the outside of the expansion screw completely with transparent resin. Set the resin for 5 min at RT. When polymerization is complete, remove the expansion screw knob (Figure 1E).</li>
</ol>
3. Surgical Protocol
<ol>
	<li>Use 8 week-old male mice.</li>
	<li>Induce anesthesia with an intraperitoneal injection of medetomidine hydrochloride at 0.3 mg/g, midazolam at 4 mg/kg, and butorphanol tartrate at 5 mg/kg of body weight.
	<ol>
		<li>Carefully shave and disinfect the surgical area with 10% iodine solution, and then, administer 0.5% lidocaine hydrochloride at the right lower limb.</li>
	</ol>
	</li>
	<li>Make a longitudinal skin incision (approximately 15 mm in length) at the right lower leg with a No. 15 scalpel. Bluntly separate the underlying muscles, taking care not to remove all of the periosteum. Approach from the outside to easily reach the fibula. Cut the fibula with scissors.</li>
	<li>Grasp the ankle with narrow thin sterile forceps, and use a sterile 27 G needle to make a hole in the bone about 5 mm from the heel. The needle should penetrate the skin, bone, and skin in that order. When the needle penetrates, cut the tip and root with a nipper so that the needle is about 15 mm.
	<ol>
		<li>Make another hole in the same manner, about 2&#8211;3 mm proximal. Hold around the ankle and pass two 25 G needles under the knee in the same manner. After the needles penetrate, cut the tips and roots with a nipper (Figure 1F).</li>
	</ol>
	</li>
	<li>Place the custom-made distractor such that it is parallel to the extension direction. Fix the needles and device with enough polymerization dental resin to fill the grooves of the device. Wait for the polymerization to complete (approximately 5 min) (Figure 1G).</li>
	<li>Be careful not to damage the surrounding tissues. Cut the middle of the diaphysis of the tibia using a very thin cutting disc while applying a saline solution (1&#8211;2 mL).</li>
	<li>Close the wound with a 4-0 nylon suture.</li>
	<li>Give subcutaneous injections of buprenorphine (0.1 mg/kg) for analgesia immediately after operation. Continue buprenorphine every 12 h through postoperative day 7 and as needed thereafter.</li>
</ol>
4. Distraction Protocol
NOTE: There are various reports on the latency period and distraction rate, but here, representative protocols are shown.
<ol>
	<li>After a latency period of 5 days, start distraction at a rate of 0.2 mm/12 h. For extension, use the pin attached to the rapid expansion screw. Move the pin in the direction of the yellow arrow attached to the extension screw (Figure 1E).</li>
	<li>Anesthesia is not necessary during extension. Hold the tail with the little finger and palm, and fix the extension device with the forefinger and thumb.</li>
	<li>Perform extension (0.2 mm for 1/4 turn). Continue lengthening for 8 days, which will result in a total gap of 3.2 mm.</li>
</ol>
5. Analysis
<ol>
	<li>Radiography analysis: After completion of extension, evaluate bone regeneration with computed tomography under general anesthesia by using 2.0% isoflurane. 
	NOTE: If care has been taken with regard to the position of the extension device, it is possible to evaluate it with simple radiography even with the device attached.</li>
	<li>Histological analysis: Remove the apparatus carefully so as not to cause a fracture distraction site during sampling. Fix the sample in 10% neutral buffered formalin for 24 h at room temperature. Decalcify with Morse&#39;s solution (10% sodium citrate, 20% formic acid) overnight at 4 &#176;C.</li>
</ol>

<ol>
	<li>Watson, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distraction+osteogenesis.">Distraction osteogenesis.</a> The Journal of the American Academy of Orthopaedic Surgeons. 14, 168-174 (2006).</li><li>Ilizarov, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tension-stress+effect+on+the+genesis+and+growth+of+tissues:+Part+II.+The+influence+of+the+rate+and+frequency+of+distraction.">The tension-stress effect on the genesis and growth of tissues: Part II. The influence of the rate and frequency of distraction.</a> Clinical Orthopaedics and Related Research. 239, 263-285 (1989).</li><li>Wan, 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=Activation+of+the+hypoxia-inducible+factor-1+alpha+pathway+accelerates+bone+regeneration.">Activation of the hypoxia-inducible factor-1 alpha pathway accelerates bone regeneration.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (2), 686-691 (2008).</li><li>Fujio, 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=Stromal+cell-derived+factor-1+enhances+distraction+osteogenesis-mediated+skeletal+tissue+regeneration+through+the+recruitment+of+endothelial+precursors.">Stromal cell-derived factor-1 enhances distraction osteogenesis-mediated skeletal tissue regeneration through the recruitment of endothelial precursors.</a> Bone. 49 (4), 693-700 (2011).</li><li>Tong, L., 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=Focal+adhesion+kinase+expression+during+mandibular+distraction+osteogenesis:+evidence+for+mechanotransduction.">Focal adhesion kinase expression during mandibular distraction osteogenesis: evidence for mechanotransduction.</a> Plastic and reconstructive surgery. 111 (1), 211-222 (2003).</li><li>Rhee, S. T., El-Bassiony, L., Buchman, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+signal-+related+kinase+and+bone+morphogenetic+protein+expression+during+distraction+osteogenesis+of+the+mandible:+in+vivo+evidence+of+mechanotransduction+mechanism+for+differentiation+and+osteogenesis+by+mesenchymal+precursor+cells.">Extracellular signal- related kinase and bone morphogenetic protein expression during distraction osteogenesis of the mandible: in vivo evidence of mechanotransduction mechanism for differentiation and osteogenesis by mesenchymal precursor cells.</a> Plastic and reconstructive surgery. 117 (7), 2243-2249 (2006).</li><li>Isefuku, S., Joyner, C. J., Simpson, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+distraction+osteogenesis.">A murine model of distraction osteogenesis.</a> Bone. 27 (5), 661-665 (2000).</li><li>Tay, B. K., Le, A. X., Gould, S. E., Helms, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+and+molecular+analyses+of+distraction+osteogenesis+in+a+mouse+model.">Histochemical and molecular analyses of distraction osteogenesis in a mouse model.</a> Journal of Orthopaedic Research. 16 (5), 636-642 (1998).</li><li>Carvalho, 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=The+role+of+angiogenesis+in+a+murine+tibial+model+of+distraction+osteogenesis.">The role of angiogenesis in a murine tibial model of distraction osteogenesis.</a> Bone. 34 (5), 849-861 (2004).</li><li>Osawa, 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=Activated+FGFR3+promotes+bone+formation+via+accelerating+endochondral+ossification+in+mouse+model+of+distraction+osteogenesis.">Activated FGFR3 promotes bone formation via accelerating endochondral ossification in mouse model of distraction osteogenesis.</a> Bone. 105, 42-49 (2017).</li></ol>

The authors have nothing to disclose.

When a large animal is used as an experimental model, a ready-made extension device can be used, and it is easy to obtain good fixation and assess the extension operation itself and the extension amount. However, when a mouse is used as an experimental model, it is necessary to develop some or all of the equipment. Isefuku et al. and Tay et al. made the device and created a mouse model7,8. Carvahjo et al. adopted a method involving fixa...

Distraction osteogenesis (DO) is an established treatment method for a variety of skeletal disorders, such as limb length discrepancies, bone defects, and limb deformities1. This unique treatment strategy is based on the &#34;tension-stress principle&#34; proposed by Ilizarov. The method requires several days for latency, several weeks for active distraction, and several months for consolidation until the mature bone is formed2.
The local hypoxic conditions due to blockage of blood flow3,4 and mechanical stimulation5,6 are particularly important in the healing process of DO. Hypoxia-induced angiogenesis carries oxygen, nutrients, soluble factors and cells necessary for tissue repair locally through the blood flow. Mechanical stimulation by extension operation causes biological reactions such as differentiation of mesenchymal stem cells, bone formation, calcification and remodeling. Serial DO treatment allows the formation of not only hard tissues but also soft tissues, including nerves, muscles, blood vessels, and skin tissues, without the need for stem cell transplantation. Therefore, a DO model is considered to be an excellent model for analyzing the regeneration of various tissues.
Rabbits and dogs are the most widely used animals in basic research for DO; however, there are few analysis tools available for these animals. The use of a mouse DO model facilitates a more detailed analysis. It is particularly suitable for experiments using knockout mice. However, when using a mouse as an experimental animal, an extension device should be created. Here, we present a mouse tibial DO model developed using a custom-made distractor created using a dental laboratory tool and technique, which has been used in a previous study.

<table><tbody><tr><td>Paraffin wax</td><td>YAMAHACHI DENTAL MFG. CO.</td><td>-</td><td>For preparation a mold for resin rings</td></tr><tr><td>Labocone putty</td><td>GC Corporation</td><td>-</td><td>For preparation a mold for resin rings</td></tr><tr><td>Utility wax</td><td>GC Corporation</td><td>-</td><td>For preparation a mold for resin rings</td></tr><tr><td>Expansion screw</td><td>Ortho Dentaurum</td><td>600-301-30</td><td>Component of custom-made distractor</td></tr><tr><td>Unifast III</td><td>GC Corporation</td><td>-</td><td>Immediate polymerization resin Component of custom-made distractor</td></tr><tr><td>Ortho Crystal</td><td>NISSIN</td><td>-</td><td>Transparent resin Component of custom-made distractor</td></tr><tr><td>25 G needle</td><td>TERUMO</td><td>NN-2516R</td><td>For custom-made distractor</td></tr><tr><td>27 G needle</td><td>TERUMO</td><td>NN-2719S</td><td>For custom-made distractor</td></tr><tr><td>ICR mouse</td><td>Chubu Kagaku Shizai Corporation</td><td>-</td><td>Experimental animal</td></tr><tr><td>Somnopentyl</td><td>Kyoritsu Seiyaku</td><td>-</td><td>Pentobarbital sodium salt</td></tr><tr><td>Isoflurane</td><td>FUJIFILM Wako Pure Chemical Corporation</td><td>099-06571</td><td>Isoflurane inhalation solution</td></tr></tbody></table>

a mouse distraction osteogenesis model

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the following three phases: the latency phase after osteotomy and placement of the external distractor; the distraction phase, wherein the separated bone ends are gradually and continuously distracted; and the consolidation phase. This custom-made distractor used for DO is comprised of two incomplete acrylic resin rings and an expansion screw. The process was initiated by making a mold with silicone impression material and then creating the custom-made distractor. Dental resin was poured into the formwork made of silicone impression material, and it was allowed to polymerize to create the incomplete resin rings required for the custom-made distractor. These rings were fixed with an expansion screw using transparent resin. The custom-made distractor created via this approach was attached to the tibia of mice. The tibia was fixed to the device using one pair of 25 G needles proximally, one pair of 27 G needles distally, and acrylic resin. After a latency period of 5 days, distraction was initiated at a rate of 0.2 mm/12 h. The lengthening was continued for 8 days, resulting in a total gap of 3.2 mm. The mice were sacrificed 4 weeks after distraction. Bone formation in the distraction gap was confirmed using both radiography and histology.

Figures 1A and 1B present incomplete rings (outer diameter, 20 mm; inner diameter, 10 mm; thickness, 5 mm) with paraffin wax. Two wax patterns were embedded in silicone impression material, and a mold for the resin rings (Figure 1C) was formed. Polymerized resin was immediately poured into this mold, and resin rings were obtained (Figure 1D). A custom-made distra...

Watch this Scientific Journal Video about A Mouse Distraction Osteogenesis Model at JoVE.com

A Mouse Distraction Osteogenesis Model

We present a mouse tibial distraction osteogenesis model developed using a custom-made distractor. The use of a mouse as an analysis target is advantageous for advancing research.

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the ...

Research

JoVE Journal

Developmental Biology

6.2K Views.  Nagoya University Graduate School of Medicine. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in het harde en zachte weefsel regeneratie veld. Het belangrijkste voordeel van deze techniek is dat het gebruik van een muis zorgt voor een gedetailleerde analyse van botregeneratie. Begin met een volledig verdoofde volwassen muis.Scheer en ontsmet het chirurgische gebied zorgvuldig met een jodiumoplossing van 10%. Toedien vervolgens 0,5% lidocaïnehydrochloride aan de rechter onderste ledemaat. Dan, na het maken van ...

Video: A Mouse Distraction Osteogenesis Model

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Medicine

Direct Mouse Trauma/Burn Model of Heterotopic Ossification

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical procedures. The majority of animal models used to study HO rely on the application of exogenous substances, such as bone morphogenetic protein (BMP), exogenous cell constructs, or genetic mutations in BMP signaling. While these models are useful they do not accurately reproduce the inflammatory states that cause the majority of cases of HO. Here we describe a burn/tenotomy model in mice that reliably produces focused HO. This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin as well as division of the Achilles tendon at its midpoint. Relying solely on traumatic injury to induce HO at a predictable location allows for time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only. This method could prove instrumental in understanding the inflammatory and osteogenic pathways involved in trauma-induced HO. Furthermore, because HO develops in a predictable location and time-course in this model, it allows for research to improve early imaging strategies and treatment modalities to prevent HO formation.

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical ...

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

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 ...

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 ...

A Mouse Model of Ankle-Subtalar Complex Joint Instability

Ankle sprains are perhaps the most common sports injuries in daily life, often resulting in instability of the ankle-subtalar complex joint (ASCJ), and can eventually lead to post-traumatic osteoarthritis (PTOA) in the long term. However, due to the complexity of the injury mechanism and the clinical manifestations, such as ecchymosis, hematoma, or tenderness in the lateral foot, there is no clinical consensus on diagnosing and treating ASCJ instability. Since the musculoskeletal structure of the bones and ligaments of the mouse hindfoot is comparable to that of humans, an animal model of ASCJ instability in mice was established by the transection of ligaments around the ASCJ. The model was well-validated through a series of behavioral tests and histological analyses, including a balance beam test, a footprint analysis (an assessment of exercise level and balance ability in mice), a thermal nociception assessment (an assessment of foot sensory function in mice), micro-computed tomography (CT) scanning, and section staining of the articular cartilage (an assessment of articular cartilage damage and degeneration in mice). The successful establishment of a mouse model of ASCJ instability will provide a valuable reference for clinical research on the injury mechanism and result in better treatment options for ankle sprain.

The ankle-subtalar complex joint (ASCJ) is the core of the foot and plays a key role in balance control in daily activities. Sports injuries often lead to instability in this joint. Here, we describe a mouse model of ligament transection-induced instability of the ASCJ.

Ankle sprains are perhaps the most common sports injuries in daily life, often resulting in instability of the ankle-subtalar complex joint (ASCJ), and ...

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 ...

A Mouse Distraction Osteogenesis Model

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