Name: a surgical procedure for resecting the mouse rib: a model for large-scale long bone repair
Uploaded: 2015-01-21T15:00:00.000+00:00
Duration: 8 min 42 s
Description: Watch this Scientific Journal Video about A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair at JoVE.com

We thank members of the Mariani lab for critical reading of the manuscript. Our funding sources were: the Baxter Medical Scholar Research Fellowship (to M.K.S.), USC undergraduate fellowships and the Provost, Dean Joan M. Schaeffer, and Rose Hills Fellowships, (to M.K.S.). We also acknowledge a CIRM BRIDGES fellowship through Pasadena City College (to T.T.T). and the James H. Zumberge Research and Innovation Fund, the USC Regenerative Medicine Initiative, and the NIAMS NIH under Award Number R21AR064462 (to F.V.M).

NOTE: All procedures are in accordance with an animal protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Southern California.
1. Preparation for Surgery
<ol>
	<li>Lay out the necessary surgical tools and supplies (sterilized, items 1 - 18 in Materials list) and arrange a dissecting microscope and a fiber optics trans-illuminator in a hood or on a downdraft table.</li>
	<li>Place a sexually mature mouse (20 - 30 g, 1 - 3 months old) in an induction chamber with 4% isoflurane to induce anesthesia.</li>
	<li>Confirm anesthesia with a paw and/or tail pinch test.</li>
	<li>Apply ointment to the mouse&#8217;s eyes to avoid eye dryness during the surgery.</li>
	<li>Move the mouse to the microscope stage. Place the mouse into the nose cone of the maintenance hose and position laterally. Provide a warming pouch adjacent to the animal to help maintain body temperature. 
	NOTE: Position of the mouse is dependent on personal preference and/or handedness of the surgeon., either left or right side is fine.</li>
	<li>Adjust isoflurane to 2 - 3% for maintenance, and watch breathing rate.</li>
	<li>Inject buprenorphine (0.05 mg/kg) subcutaneously (lateral flank of the leg) for post-surgery pain with a 25 G needle. The leg may twitch which is a normal response.</li>
</ol>
2. Opening Incision
<ol>
	<li>Detect the desired rib by palpation, and clear the area of hair with a shaver. 
	NOTE: Ribs 8-10 are recommended since ribs 1-7 are closer to the lungs and 11-13 are considerably shorter. Counting the ribs should begin with the most rostral rib as number 1.</li>
	<li>Prepare the area with alternating povidone-iodine and isopropanol (70%) swabbing (3x is often recommended by animal use protocols).</li>
	<li>Cut a 2 cm incision through the skin, directly above and parallel to the desired rib with medium sized microsurgery scissors. Incise through the underlying muscle and fat layers.</li>
	<li>Place all 3 layers (skin, muscle, fat) into a retractor to expose the surgical area, while minimizing the size of the incision.</li>
</ol>
3. Excising the Rib
<ol>
	<li>Cut through the intercostal muscles overlying the desired section of rib with a 5.0 mm scalpel. Position this incision around 5 mm proximal to the chondrocostal joint as this is where the rib is not as highly curved. Carefully separate the muscle from the bone with fine tip forceps.</li>
	<li>To create a resection while retaining the periosteum in the animal, cut through the periosteum, along the length of rib with a 5.0 mm scalpel. Carefully separate the periosteum from the underlying bone laterally with fine tip forceps. Proceed with caution, as the periosteum is very delicate and has a gelatinous consistency.</li>
	<li>Next make a cross-section through the bone at one end with fine micro-scissors. If needed, measure the resection with a reticle in the microscope or a ruler type gauge. Then carefully lift the bone out of the periosteum and cut the other end. 
	NOTE: Proceed with caution. This is the most delicate step, as pulling up without excising the rib below will tear the pleural membrane and result in a pneumothorax. If the pleural membrane is torn this will be readily evident as the interior of the thoracic cavity will become visible.</li>
	<li>If bleeding occurs as a result of cutting the bone, apply pressure at the cut end with a cotton-tipped swab for 4 - 5 sec to stop the bleeding. 
	NOTE: On the rare occasion that bleeding continues, it may be necessary to stop and euthanize the animal as significant blood loss will compromise recovery (without fluid replacement, the maximum blood volume which can be safely be lost is 10% of the total blood volume or 7.7 - 8 &#181;l/g. For a 25 g mouse, this is equivalent to around 180 - 200 &#181;l9).</li>
	<li>Immediately place the removed rib into 4% PFA for future analysis. 
	NOTE: If the resection of both the bone and the periosteum is to be carried out, omit the periosteum incision and separation (step 3.2). Since the periosteum is very tightly attached to the pleural membrane proceed cautiously, teasing the rib away from the pleural membrane with forceps to avoid tearing.</li>
</ol>
4. Closing the Incision
<ol>
	<li>Suture the intercostal muscles over the top of the remaining periosteal sleeve with 9-0 sutures (2 sutures are usually sufficient). Place the sutures directly above the cut ends of the rib, to act as indicators for surgery location.</li>
	<li>Remove the retractor. Suture the overlying muscle and fat with 9-0 sutures (3 - 4 sutures are usually sufficient). In mice with particularly thick layers of muscle and/or fat, suture each layer separately (e.g., 1 layer of sutures for the muscular layer and 1 layer of sutures for the fatty layer).</li>
	<li>Close the skin with 7-0 sutures (4 - 5 sutures are usually sufficient).</li>
	<li>Secure the incision with suture glue, pinching the edges together with large forceps.</li>
	<li>Slowly wean the mouse off isoflurane by first adjusting to 1% for few minutes and then turn off.</li>
	<li>Place the mouse under a heat lamp and leave until consciousness is regained, 5 - 10 min. After consciousness is regained and throughout the healing period, the mouse should move and ambulate normally and show no signs of distress.</li>
</ol>
5. Recovery and Analysis
<ol>
	<li>Provide post-operative pain management including the administration of buprenorphine in an oral gelatin form at 0.5 mg/kg every 12 hr for 48 hr. Oral administration does not require restraining the animal which might cause pain to the thoracic region.</li>
	<li>Maintain the mouse in a cage with free access to food and water during the healing period. After the incision is dry, female animals can be co-housed while males must remain isolated to prevent fighting.</li>
	<li>After the healing period, follow the approved procedure for euthanasia. Remove the rib cage for fixation and analysis.</li>
</ol>

<ol>
	<li>Philip, S. J., Kumar, R. J., Menon, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+study+of+rib+regeneration+following+costectomy+in+adolescent+idiopathic+scoliosis.">Morphological study of rib regeneration following costectomy in adolescent idiopathic scoliosis.</a> Eur Spine J. 14 (8), 772-776 (2005).</li><li>Munro, I. R., Guyuron, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split-rib+cranioplasty.">Split-rib cranioplasty.</a> Ann Plast Surg. 7 (5), 341-346 (1981).</li><li>Taggard, D. A., Menezes, 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=Successful+use+of+rib+grafts+for+cranioplasty+in+children.+Pediatric+neurosurgery.">Successful use of rib grafts for cranioplasty in children. Pediatric neurosurgery.</a> 34 (3), 149-155 (2001).</li><li>Head, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+Regeneration+fo+the+Ribs:+A+problem+in+thoracic+surgery.">Prevention of Regeneration fo the Ribs: A problem in thoracic surgery.</a> Archives of Surgery. 14 (6), 1215-1221 (1927).</li><li>Kawanabe, Y., Nagata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+of+costal+cartilage+harvest+for+total+auricular+reconstruction:+part+I.+Avoidance+and+prevention+of+intraoperative+and+postoperative+complications+and+problems.">A new method of costal cartilage harvest for total auricular reconstruction: part I. Avoidance and prevention of intraoperative and postoperative complications and problems.</a> Plastic and reconstructive surgery. 117 (6), 2011-2018 (2006).</li><li>Cheung, K. 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=An+externally+fixed+femoral+fracture+model+for+mice.">An externally fixed femoral fracture model for mice.</a> Journal of orthopaedic research : official publication of the Orthopaedic Research Society. 21 (4), 685-690 (2003).</li><li>Matthys, R., Perren, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+fixator+for+use+in+the+mouse.">Internal fixator for use in the mouse.</a> Injury. 40, S103-S109 (2009).</li><li>Cooper, G. 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=Testing+the+critical+size+in+calvarial+bone+defects:+revisiting+the+concept+of+a+critical-size+defect.">Testing the critical size in calvarial bone defects: revisiting the concept of a critical-size defect.</a> Plastic and reconstructive surgery. 125 (6), 1685-1692 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ask+the+Vet.">Ask the Vet.</a> JAX NOTES. 499, (2005).</li><li>Flecknell, P. A., Roughan, J. V., Stewart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+oral+buprenorphine+('buprenorphine+jello')+for+postoperative+analgesia+in+rats--a+clinical+trial.">Use of oral buprenorphine ('buprenorphine jello') for postoperative analgesia in rats--a clinical trial.</a> Laboratory animals. 33 (2), 169-174 (1999).</li><li>Rigueur, D., Lyons, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+skeletal+staining.">Whole-mount skeletal staining.</a> Methods in molecular biology. 1130, 113-121 (2014).</li><li>Evans, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+somitic+cells+to+the+avian+ribs.">Contribution of somitic cells to the avian ribs.</a> Developmental biology. 256 (1), 114-126 (2003).</li><li>Colnot, C., Thompson, Z., Miclau, T., Werb, Z., 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=Altered+fracture+repair+in+the+absence+of+MMP9.">Altered fracture repair in the absence of MMP9.</a> Development. 130 (17), 4123-4133 (2003).</li><li>Lu, 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=Cellular+basis+for+age-related+changes+in+fracture+repair.">Cellular basis for age-related changes in fracture repair.</a> Journal of orthopaedic research : official publication of the Orthopaedic Research Society. 23 (6), 1300-1307 (2005).</li><li>Zilberman, Y., Gafni, Y., Pelled, G., Gazit, Z., Gazit, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+imaging+in+bone.">Bioluminescent imaging in bone.</a> Methods in molecular biology. 455, 261-272 (2008).</li><li>Pelled, G., Gazit, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+using+osteocalcin-luciferase.">Imaging using osteocalcin-luciferase.</a> Journal of musculoskeletal. 4 (4), 362-363 (2004).</li><li>Elefteriou, F., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mouse+models+for+bone+studies--strengths+and+limitations.">Genetic mouse models for bone studies--strengths and limitations.</a> Bone. 49 (6), 1242-1254 (2011).</li><li>Srour, M. 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=Natural+large-scale+regeneration+of+rib+cartilage+in+a+mouse.">Natural large-scale regeneration of rib cartilage in a mouse.</a> J. Bone Miner. , (2014).</li></ol>

The authors have nothing to disclose

When first learning this protocol, determining where to locate the initial incision can be challenging. However, practice on euthanized mice helps the surgeon learn where to place the initial incision and expose the desired rib to be resected. Working on cadavers also improves the fine-motor skills required to remove the rib portion with or without the periosteum. In addition, someone new to this procedure might find manipulating the fine tools and thin sutures to be difficult. While tying off, excess pulling on the suture can tear the tissues. Thus, some practice suturing also on cadavers could be helpful. Finally if the surgeon is new to surgical procedures with rodents, it may be useful to have another person assist with monitoring and adjusting the anesthesia during the first few surgeries.
The most critical step of this protocol is excising the bone without inducing a pneumothorax (an abnormal collection of air in the pleural space), which is difficult to treat in the mouse since the pleural membrane is too thin to close surgically. The layer of muscle and parietal pleura inferior to the rib is very thin and delicate; hence, precise movements are required to prevent a puncture in the pleural membrane that lies beneath the resection area. This occurs most commonly in steps 3.1 and 3.3. In step 3.1 (separating the bone from the surrounding intercostal muscles), the sharp scalpel and forceps will easily pierce the pleural layer if the point is not kept superficial. In step 3.3 (excising the bone with fine micro-scissors), the placement of the scissors beneath the bone may overcome the tensile strength of the pleural membrane, or the action of breaking the bone may cause the end of the bone fragment to perforate the pleural membrane. We have found that using fine micro-scissors that have a curved tip is beneficial. In addition, as above, it can also be useful to first practice the protocol on euthanized mice. Ultimately, however we have found that this problem can be easily overcome with patience and practice.
Although the rib is an endochondral bone, there may be some potentially important differences between the rib skeleton and the long bones of the appendicular skeleton. From an embryological perspective, the rib skeleton is derived from a different mesodermal compartment (somite) than the appendicular skeleton (lateral plate mesoderm)12. Thus it is possible that there are properties that are unique to the rib skeletal progenitors that reflect this difference in developmental history. Therefore, in the long-run, it may be necessary to determine what these unique properties are and then use this information to encourage the progenitors in the limb skeleton to mediate repair with the same facility as seen in the rib.
Another aspect that comes into play concerns the location of the ribs in relation to the respiratory system. Although the flanking ribs provide sufficient stability around the resection such that no external fixator is needed, the repair zone is under the constant movement and strain of lung inflation/deflation. It has been recognized that during bone repair, too much movement can be inhibitory to healing while some movement appears to be important for generating a cartilage intermediate13,14. At this point, it is not clear, however it is possible that formation of a cartilage intermediate may be a key step for effective large-scale repair. Thus, movement of the ribs during respiration might facilitate repair (when the periosteum is left behind in the animal). Since movement may be particularly important in this context, developing a method to place a fixator could be useful for studying the biomechanical influences on repair in the future.
By carrying out a rib repair assay in the mouse, one can take advantage of powerful genetic tools that have been developed. For example, using transgenic marks, the origin of the repair cells can be assessed. Transgenic reporters for bone formation (interesting new tools in development from Gazit et al.15,16), the endochondral process, and the read-out of important signal transduction pathways can be utilized12,17. In addition, mice carrying loss-of-function alleles could be used to determine the signaling pathways required. Currently, no other vertebrate model organism allows such a wide variety of genetic techniques to probe the basic underlying biology of this process.
In addition, while other excellent models for skeletal repair have been developed and have been in use for some time, another context to test scaffolds, compounds, and cell therapies considering the features of rib repair may be beneficial. The establishment of a standard rib repair model complements existing models as the ribs are not required to support the body and repair occurs without stabilization. In addition, the contribution of the periosteum to repair can be easily assessed. Futhermore, because the rib has both cartilage and bone segments, comparisons between the healing of these different but related tissue types may identify a common feature of effective repair18.

Debilitating skeletal injury, chronic osteoarthritis, and the severe problems associated with reconstructive surgery impact economic productivity, family well-being, and quality of life. While small breaks and lesions can heal fairly well, humans are not capable of repairing large defects and therefore must rely on reconstructive procedures&#160;to restore structure and function. Reconstruction may involve allogeneic or heterogeneic grafts, morcellized bone, implanted scaffolds, or distraction osteogenesis. Unfortunately, not only are there persistant morbidity factors associated with these treatments but the original strength of the repaired bone is rarely attained. Thus, new clinical approaches are needed.
One-way to develop innovative methods to treat segmental defects is to study situations in which large-scale repair occurs naturally. Amphibians famously can regenerate skeletal elements, while mammals are considered limited in this ability. However, since the early part of the 20th century, a few reports of regeneration in the human rib have been published suggesting that humans may not be so limited1-4 . Currently this phenomenon is best known by plastic surgeons who use rib material for jaw, face and ear reconstruction, but it is not more broadly appreciated5. In order to study this repair in more detail, we have developed a surgical model using the mouse. Using this protocol, researchers can identify the innate factors involved and use this information to facilitate skeletal healing in other locations.
There are many advantages to using the ribs as a model for studying skeletal repair. First, the surrounding ribs provide a natural fixator (as compared to resection of the femur6,7). This decreases the morbidity risk of internal and external fixators and simplifies the surgical procedure. Secondly, the thin muscular layers of the chest wall provide for easy access and excellent visibility which make the assay comparable to the convenience of calvarial resections8. Thirdly, in contrast to the calvariae which form by intramembranous ossification, the ribs form by endochondral ossification and grow in length via extension at growth plates located at either end of a central diaphysis. Therefore, repair of the ribs may be more comparable to repair of the long bones of the appendicular skeleton. Furthermore, we have found that compared to the femur, the periosteum of the rib is thicker and can be more easily manipulated. Thus, investigators who wish to assay bone repair for the purpose of studying the periosteum or testing cell therapies, pharmacological agents, and/or tissue scaffolds may find this surgical model useful. In summary, this rib resection model provides a context within which to study natural large-scale bone repair in mammals as no such model in general use currently exists.

<table><tbody><tr><td>Medium sized micro-dissection scissors (Vannas-T&amp;uuml;bingen Spring Scissors 5 mm)</td><td>Fine Science Tools</td><td>15003-08</td><td></td></tr><tr><td>Fine micro-dissection scissors (Vannas Spring Scissors - 2mm Cutting Edge)</td><td>Fine Science Tools</td><td>15000-04</td><td>curved tip is beneficial</td></tr><tr><td>Micro-scalpel 5.0 mm</td><td>Fine Science Tools</td><td>10315-12</td><td>other fine scalpels can be substituted</td></tr><tr><td>Dumont 55 forceps</td><td>Fine Science Tools</td><td>11295-51</td><td></td></tr><tr><td>Retractor&amp;nbsp;</td><td>Fine Science Tools</td><td>17004-05</td><td>adjustability is convenient</td></tr><tr><td>Micro-needle holders</td><td>Fine Science Tools</td><td>12060-01</td><td></td></tr><tr><td>9.0 nylon sutures (Ethilon), taper point best</td><td>Ethicon</td><td>2819G or similar</td><td>taper point best but reverse cutting is also good</td></tr><tr><td>7.0 prolene sutures (Prolene)</td><td>Ethicon</td><td>8700H or similar</td><td>6-0 can be used too, needle point can vary</td></tr><tr><td>Large forceps (Adson Forceps)</td><td>Fine Science Tools</td><td>11006-12</td><td>other brands are fine</td></tr><tr><td>Lubricant Eye Ointment (Akwa Tears)</td><td>Akorn</td><td>17478-062-35</td><td></td></tr><tr><td>Suture glue (GLUture Topical Tissue Adhesive)</td><td>Abbot</td><td>32046-01</td><td>has excellent working time</td></tr><tr><td>Shaver</td><td>Wahl</td><td>9918-6171 or similar</td><td></td></tr><tr><td>Clamp lamp</td><td>Zoo Med</td><td>LF-5</td><td></td></tr><tr><td>Infrared Bulb, 75W</td><td>Zoo Med</td><td>RS-75</td><td></td></tr><tr><td>RC&lt;sup&gt;2&lt;/sup&gt; Rodent Anesthesia System</td><td>VetEquip&amp;nbsp;</td><td>922100</td><td></td></tr><tr><td>IsoFlo (Isoflurane)</td><td>Abbot</td><td>05260-05</td><td></td></tr><tr><td>Buprenorphine (Buprenex)</td><td>Reckitt Benckiser</td><td>12496-0757-1</td><td></td></tr><tr><td>Betadine</td><td>Purdue Frederick</td><td>67618015017</td><td></td></tr><tr><td>Flavored Gelatin, raspberry</td><td>Jell-O</td><td>B000E1FYL0</td><td>made up firm, to the consistency of &#039;jigglers&#039;</td></tr></tbody></table>

a surgical procedure for resecting the mouse rib: a model for large-scale long bone repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

Animals typically recover quickly from this procedure, the incision heals well, and the animals exhibit normal behavior. The procedure uses commercially available supplies and after practice, can be completed in under 1 hr. Before using this procedure to generate experimental data, it is important to analyze the resection at the day 0 time point to determine if any large bone fragments might remain within the resection zone. One way to assess proper procedure is to assess the animals by X-ray or microCT imaging postoperatively, although this will typically require special radiation safety approvals. Instead, another simple assessment can be completed by carrying out a skeletal staining preparation as illustrated in Figure 1A. This involves collecting the rib cage, fixing it in EtOH, and staining with alizarin red using a standard protocol11. It can be useful to omit the potassium hydroxide (used to digest away the soft tissues) so that the sample can still be used afterwards for histological analysis if so desired. The sample can still be visualized without the potassium hydroxide steps by clearing in 80% glycerol because the overlying body wall muscle layer is thin. Or alternatively, these muscles can be removed manually prior to fixation/staining as is shown here (Figure 1A).
The portion of bone removed can also be analyzed. If the goal was to retain the periosteum in the animal, the bone portion should have a smooth surface as visualized by light microscopy and histological analysis (Figure 1B, B&#8217;). A bone portion removed with the periosteum should have a distinctly ragged surface and upon histological examination have an intact overlying periosteal layer, which has the characteristics of a dense irregular connective tissue (Figure 1C, C&#8217;).
Depending on the goal of the experiment, healing can be assessed at different time points. Observations indicate that 3 mm resections in which the periosteum is retained in the animal typically heal completely within 1 - 2 months. Repair occurs via the formation of a soft and hard callus, and remodeling. An example of complete repair at 2 months is shown in Figure 2A, A&#8217;; repair can be assessed by skeletal preparation (Figure 2A) and histological analysis (Figure 2A&#8217;). If the periosteum is also removed, the resection gap is never filled in. Figure 2B shows an example in which some repair has occurred at one end, however, it is more typical to observe blunt ends. Upon histological analysis, the resection zone is filled with adipose, granulation tissue, and muscle (Figure 2B&#8217;).
<img alt="Figure 1" src="/files/ftp_upload/52375/52375fig1highres.jpg" width="600" /> 
Figure 1. Example images of resections at day 0. (A) Illustration of a rib resection (4.5 mm) collected immediately after surgery. No bone fragments are left behind. Sample was stained with alizarin red (using a standard protocol but omitting any steps with potassium hydroxide to preserve the sample for future histological analysis) and cleared in 80% glycerol. Arrowheads indicate the location of the cut ends. (B) Removed rib portion from rib cage depicted in (A) without the periosteum attached. (B&#8217;) Histological section showing minimal periosteum attached to the bone (yellow arrowheads, left) and no periosteum to the right (cross section, stained with hematoxylin and eosin). The yellow asterisk indicates the bone marrow cavity. Mouse ribs do not have extensive trabeculae. (C) Removed rib portion showing the periosteum still attached. (C&#8217;) Histological section showing the intact periosteum overlying the bone indicated by a yellow bracket (longitudinal section, stained with hematoxylin and eosin). A yellow asterisk indicates the bone marrow cavity. Scale bars: A, B, C = 1 mm; B&#8217;,C&#8217; = 100 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52375/52375fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52375/52375fig2highres.jpg" width="700" /> 
Figure 2. Rib repair post-healing in resections with and without the periosteum. (A) Skeletal preparation following ~2 months of healing after resection, retaining the intact periosteum inside the mouse (prepared as in Figure 1A). Complete repair of the region can be observed. (A&#8217;) Histological section of B showing a restoration of the lamellar bone walls and the bone marrow cavity (hematoxylin and eosin). (B) Skeletal preparation (alizarin red) following ~3 months of healing after removing both a rib portion and the surrounding periosteum. There was only minimal repair at the cut end shown at the left of the panel. A yellow arrowhead points to the chondrocostal joint. (B&#8217;) Histological section of B showing granulation tissue, adipose, and muscle cells filling in the site of resection (hematoxylin and eosin). Scale bars: A, B = 1 mm; B&#8217; = 500 &#956;m; C&#8217; = 200 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52375/52375fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair at JoVE.com

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

a-surgical-procedure-for-resecting-mouse-rib-model-for-large-scale

Nanyang Technological University

Research

JoVE Journal

Medicine

13.0K Views.  Keck School of Medicine, University of Southern California. The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

Video: A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

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.

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 ...

Developmental Biology

Mouse Pneumonectomy Model of Compensatory Lung Growth

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to disability or death. Injury-based animal models of lung repair and regeneration are limited by injury-specific responses making it difficult to differentiate changes related to the injury response and injury resolution from changes related to lung repair and lung regeneration. However, use of animal models to identify these repair and regeneration signaling pathways is critical to the development of new therapies aimed at improving pulmonary function following lung injury. The mouse pneumonectomy model utilizes compensatory lung growth to isolate those repair and regeneration signals in order to more clearly define mechanisms of alveolar re-septation. Here, we describe our technique for performing mouse pneumonectomy and sham pneumonectomy. This technique may be utilized in conjunction with lineage tracing or other transgenic mouse models to define molecular and cellular mechanism of lung repair and regeneration.

Mouse pneumonectomy is a commonly employed model of compensatory lung growth. This procedure can be used in conjunction with lineage tracing or transgenic mouse models to elucidate underlying mechanisms.

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to ...

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 ...

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 ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

This protocol describes a method to perform fractures on adult mice and monitor the healing process.

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test ...

Biology

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 ...

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 ...