Name: a mouse distraction osteogenesis model
Uploaded: 2018-11-14T12:45:00.000+00:00
Duration: 4 min 24 s
Description: Watch this Scientific Journal Video about A Mouse Distraction Osteogenesis Model at JoVE.com

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. 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.

Video: A Mouse Distraction Osteogenesis Model

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens4. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice 5-7, after application of bioactive molecules 8,9, after altering physiological parameters of healing 10, after modifying the amount or time of stabilization 11, after distraction osteogenesis 12, after creation of a non-union 13, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.

This article describes a method for stabilizing long bone fractures that is based on the application of modified Ilizarov external fixators 1-3. After application of the fixators and creation of the bone injury, healing can be assessed, distraction osteogenesis can be performed, or non-union or critical sized defect can be created and used to study therapeutic interventions.

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role ...

Medicine

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 ...

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

Bioengineering

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

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 ...

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

Here, we present a protocol to iatrogenically fracture the shaft of the femur of Wistar albino rats and follow up on the development of the callus. This femur osteotomy model can help researchers evaluate the process of fracture healing and to study how a drug could influence fracture healing.

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. ...

HE Staining and Micro-CT to Evaluate Murine Trabecular Bone Microarchitecture

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton is crucial for gaining insight into the pathophysiology of osteoporosis and improving its treatment. However, handling bone samples can be complex due to their hard and dense properties. Secondly, specialized software makes image processing and analysis difficult. In this protocol, we present a cost-effective and easy-to-use solution for trabecular bone microstructure analysis. Detailed steps and precautions are provided. Micro-CT is a non-destructive three-dimensional (3D) imaging technique that provides high-resolution images of trabecular bone structure. It allows for the objective and quantitative evaluation of bone quality, which is why it is widely regarded as the gold standard method for bone quality assessment. However, histomorphometry remains indispensable as it offers crucial cellular-level parameters, bridging the gap between two-dimensional (2D) and 3D assessments of bone specimens. As for the histologic techniques, we chose to decalcify the bone tissue and then perform traditional paraffin embedding. In summary, combining these two methods can provide more comprehensive and accurate information on bone microstructure.

Author Spotlight: An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model 

This protocol presents an economical and efficient method for quantitative evaluation of bone microarchitecture in a mouse model of osteoporosis by combining Hematoxylin-Eosin (HE) staining and micro-computed tomography (Micro-CT) techniques.

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton ...

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...

Biology

3D Visualization of Bone Remodeling Under Tensile Load in Suture Expansion Mice

A 3-D Visualization Technique for Bone Remodeling in a Suture Expansion Mouse Model

Craniofacial sutures play a crucial role beyond being fibrous joints connecting craniofacial bones; they also serve as the primary niche for calvarial and facial bone growth, housing mesenchymal stem cells and osteoprogenitors. As most craniofacial bones develop through intramembranous ossification, the sutures' marginal regions act as initiation points. Due to this significance, these sutures have become intriguing targets in orthopedic therapies like spring-assisted cranial vault expansion, rapid maxillary expansion, and maxillary protraction. Under orthopedic tracing force, suture stem cells are rapidly activated, becoming a dynamic source for bone remodeling during expansion. Despite their importance, the physiological changes during bone remodeling periods remain poorly understood. Traditional sectioning methods, primarily in the sagittal direction, do not capture the comprehensive changes occurring throughout the entire suture. This study established a standard mouse model for sagittal suture expansion. To fully visualize bone remodeling changes post-suture expansion, the PEGASOS tissue clearing method was combined with whole-mount EdU staining and calcium chelating double labeling. This allowed the visualization of highly proliferating cells and new bone formation across the entire calvarial bones following expansion. This protocol offers a standardized suture expansion mouse model and a 3-D visualization method, shedding light on the mechanobiological changes in sutures and bone remodeling under tensile force loading.

Author Spotlight: PEGASOS Tissue Clearing Technique to Visualize Bone Remodeling 

This protocol presents a standardized suture expansion mouse model and a 3-D visualization method to study the mechanobiological changes of the suture and bone remodeling under tensile force loading.

Craniofacial sutures play a crucial role beyond being fibrous joints connecting craniofacial bones; they also serve as the primary niche for calvarial and ...