The authors would like to thank Sabine Stumpp for excellent technical support and Lidia Gr&#246;sser for assistance in the surgeries. This research was funded by the Austrian Science Fund [Hertha Firnberg Fellowship number T-1219], ERC [Advanced Grant, 742046 RegGeneMems], DFG [CRC 1444].

The following procedure was performed with approval from the Magistrate of Vienna (GZ: MA 58-65248-2021-26). 5-8 years old, &#8805; 20 cm snout-to-tail tip (snout to the tip of the tail) long axolotls (Ambystoma mexicanum) were used for fracture surgery and amputations. Both males and females were used for the surgeries. Axolotls were bred in the Research Institute of Molecular Pathology facility. Pain and risk of infections were managed with proper analgesics and antibiotics to ensure a successful outcome. The reagents and equipment used for the study are listed in the Table of Materials.
1. Animal preparation
<ol>
	<li>Bath the animal in 0.03% benzocaine solution for about 15-20 min until full sedation is reached and there is no reflexive movement upon limb touching with tweezers.</li>
	<li>Place the animal with the ventral side down on wet paper towels soaked in 0.03% benzocaine solution and cover it with benzocaine-soaked paper towels. The skin of aquatic animals, such as axolotls, is sensitive to drying, and thus, it is essential to cover the body surface to prevent dehydration and ensure cutaneous (skin) respiration.</li>
	<li>Stretch out the hindlimb to be operated using ring forceps. Do not apply disinfection reagents, such as ethanol, since axolotl skin is sensitive to chemicals and easily irritated. Instead, use 0.7x PBS (A-PBS) with 50 U/mL penicillin and 20 &#181;g/mL streptomycin for cleaning the limb and later on for bone irrigation upon sawing. 
	NOTE: The infection is generally not a concern for the surgeries performed on axolotls. However, due to the aquatic nature of these animals and the sutures placed on the skin surface, we recommend the use of antibiotics to prevent any contamination of the surgical site.</li>
</ol>
2. Surgery
NOTE: Sterilize all surgical tools. Common sterilization methods such as heat sterilization, autoclaving, and washing in 70% ethanol, followed by thorough removal of the alcohol remnants, are suitable for this purpose. If operating on multiple animals, sterilize tools in between using a hot bead sterilizer or 70% ethanol.
<ol>
	<li>Make a lateral longitudinal incision (1.5-2 cm) with a scalpel above the femur bone spanning the whole thigh in the upper hind limb. In order to do this, palpate the bone before cutting the skin.</li>
	<li>Carefully displace the muscles and nerves from the surgery site without cutting. Use bowed forceps to do it efficiently.</li>
	<li>Gently put bowed forceps under the femur to expose it for the surgery.</li>
	<li>Place a rigid 7.75 mm 4-hole fixator plate along with the femur diaphysis, avoiding touching the joints, and secure it in the aligned position with forceps.</li>
	<li>Use four 2 mm titanium screws to attach the bone to the plate. 
	NOTE: The screws used in this protocol have a complex design and consist of 4 parts: the main part (will be screwed into the bone), screw head (allows removal of the screws and plate using the square box wrench), narrower neck (used as a breaking point once the screw is tightened in the bone) and a screw handle (used for attaching to the screwdriver and the saw-guiding device).</li>
	<li>The order of screw attachment is important. Start with the inner screws first, and then the two outer screws to ensure the plate is aligned with the bone axis. Use a manual drill to create the first hole in the bone for easy insertion of the screw, followed by 1st screw placing. Drill in the middle of the bone circumference to avoid thinner bone on one side, which may result in spontaneous bone fracture. Use irrigation with 0.7x PBS + 1% Pen/Strep during drilling. Do not break off the handle of the 1st (optional: 1st and 2nd) screw(s).</li>
	<li>Apply the saw-guiding device onto the 1st (optional: 1st and 2nd ) screw(s) and align it with the bone and plate. 
	NOTE: In this protocol, a plate, screws, a saw-guiding device, and a saw are provided by the same manufacturer and optimized to fit each other. The saw-guiding device can come in different sizes to be compatible with different plates and saw sizes.</li>
	<li>Use the saw-guiding device to drill and insert the rest of the screws. Ensure alignment of the plate with the bone. Break off the handles of the screws.</li>
	<li>Place a piece of plastic film (6-7 mm by 4-5 cm), sterilized by wiping with 70% ethanol and then autoclaved or heat sterilized (140 &#176;C for 4 hours), under the femur to prevent soft tissue damage during the osteotomy process. 
	NOTE: For this purpose, a piece of plastic film, cut off a bag for heat sterilization can be used.</li>
	<li>Place Gigly wire saw between bone and protection film.</li>
	<li>Cut bone using the 0.66 mm Gigly wire saw, creating a single 0.7 mm cut in the femur. Use constant irrigation with 0.7x PBS + 1% Pen/Strep during sawing to minimize tissue damage and friction.</li>
	<li>Remove the saw, and saw guide and use a screwdriver to break off the screw handles from the screws.</li>
	<li>Remove the protection film, and irrigate the surgery site with 0.7x PBS + 1% Pen/Strep.</li>
	<li>Cover the top of the plate and screws with sterile bone bee wax to protect skin and muscles from irritation by the edges of the screws.</li>
	<li>Place muscles and skin on top of the bone bee wax.</li>
	<li>Close the incision site with a 7.0 synthetic (polypropylene/polyethylene) suture using simple interrupted stitches. Synthetic suture is used to minimize contamination with water-born bacteria and fungi.</li>
</ol>
3. Postoperative management
<ol>
	<li>To reawaken the animal, place it in a tank with fresh artificial pond water, supplemented with 50 U/mL penicillin, 20 &#181;g/mL streptomycin, and analgetic Butorphanol (0.5 mg/L water).</li>
	<li>Observe the animal to start moving gills, making steps, and swimming - usually within 1 h post-surgery.</li>
	<li>Keep the animal for 3 days in artificial pond water with 50 U/mL penicillin and 20 &#181;g/mL streptomycin before returning it to the holding tank. To ensure analgesia, add Butorphanol (0.5 mg/L water).</li>
	<li>Ensure that the sutures remain in place and proper wound healing occurs.</li>
</ol>

Using Plate Fixation to Study Bone Healing and Limb Regeneration in Axolotls

<ol><li>Amamoto, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+axolotls+can+regenerate+original+neuronal+diversity+in+response+to+brain+injury%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Adult axolotls can regenerate original neuronal diversity in response to brain injury.</a> Elife. 5, 13998(2016).</li>
<li>Butler, E. G., Ward, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reconstitution+of+the+spinal+cord+following+ablation+in+urodele+larvae%5BTitle%5D)+AND+%22J+Exp+Zool%22%5BJournal%5D)">Reconstitution of the spinal cord following ablation in urodele larvae.</a> J Exp Zool. 160 (1), 47-65 (1965).</li>
<li>Echeverri, K., Tanaka, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ectoderm+to+mesoderm+lineage+switching+during+axolotl+tail+regeneration%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Ectoderm to mesoderm lineage switching during axolotl tail regeneration.</a> Science. 298 (5600), 1993-1996 (2002).</li>
<li>Vargas-Gonzalez, A., Prado-Zayago, E., Leon-Olea, M., Guarner-Lans, V., Cano-Martinez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myocardial+regeneration+in+Ambystoma+mexicanum+after+surgical+injury%5BTitle%5D)+AND+%22Arch+Cardiol+Mex%22%5BJournal%5D)">Myocardial regeneration in Ambystoma mexicanum after surgical injury.</a> Arch Cardiol Mex. 75 (3), S321-S329 (2005).</li>
<li>Vieira, W. A., Wells, K. M., McCusker, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advancements+to+the+axolotl+model+for+regeneration+and+aging%5BTitle%5D)+AND+%22Gerontology%22%5BJournal%5D)">Advancements to the axolotl model for regeneration and aging.</a> Gerontology. 66 (3), 212-222 (2020).</li>
<li>Song, F., Li, B., Stocum, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amphibians+as+research+models+for+regenerative+medicine%5BTitle%5D)+AND+%22Organogenesis%22%5BJournal%5D)">Amphibians as research models for regenerative medicine.</a> Organogenesis. 6 (3), 141-150 (2010).</li>
<li>McCusker, C., Bryant, S. V., Gardiner, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+axolotl+limb+blastema%3A+Cellular+and+molecular+mechanisms+driving+blastema+formation+and+limb+regeneration+in+tetrapods%5BTitle%5D)+AND+%22Regeneration+%28Oxf%29%22%5BJournal%5D)">The axolotl limb blastema: Cellular and molecular mechanisms driving blastema formation and limb regeneration in tetrapods.</a> Regeneration (Oxf). 2 (2), 54-71 (2015).</li>
<li>Einhorn, T. A., Gerstenfeld, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fracture+healing%3A+Mechanisms+and+interventions%29%5BTitle%5D)+AND+%22Nat+Rev+Rheumatol%22%5BJournal%5D)">Fracture healing: Mechanisms and interventions).</a> Nat Rev Rheumatol. 11 (1), 45-54 (2015).</li>
<li>Polikarpova, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+specialist+in+regeneration-the+Axolotl-a+suitable+model+to+study+bone+healing%5BTitle%5D)+AND+%22NPJ+Regen+Med%22%5BJournal%5D)">The specialist in regeneration-the Axolotl-a suitable model to study bone healing.</a> NPJ Regen Med. 7 (1), 35(2022).</li>
<li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+axolotl+fibula+as+a+model+for+the+induction+of+regeneration+across+large+segment+defects+in+long+bones+of+the+extremities%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">The axolotl fibula as a model for the induction of regeneration across large segment defects in long bones of the extremities.</a> PLoS One. 10 (6), e0130819(2015).</li>
<li>Cosden-Decker, R. S., Bickett, M. M., Lattermann, C., MacLeod, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+and+functional+analysis+of+intra-articular+interzone+tissue+in+axolotl+salamanders%5BTitle%5D)+AND+%22Osteoarthritis+Cartilage%22%5BJournal%5D)">Structural and functional analysis of intra-articular interzone tissue in axolotl salamanders.</a> Osteoarthritis Cartilage. 20 (11), 1347-1356 (2012).</li>
<li>Williams, J. N., Li, Y., Valiya Kambrath, A., Sankar, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+generation+of+closed+femoral+fractures+in+mice%3A+A+model+to+study+bone+healing%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">The generation of closed femoral fractures in mice: A model to study bone healing.</a> J Vis Exp. (138), e58122(2018).</li>
<li>Cheung, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+externally+fixed+femoral+fracture+model+for+mice%5BTitle%5D)+AND+%22J+Orthop+Res%22%5BJournal%5D)">An externally fixed femoral fracture model for mice.</a> J Orthop Res. 21 (4), 685-690 (2003).</li>
<li>Jiang, S., Knapstein, P., Donat, A., Tsitsilonis, S., Keller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+optimized+protocol+for+a+standardized%2C+femoral+osteotomy+model+to+study+fracture+healing+in+mice%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">An optimized protocol for a standardized, femoral osteotomy model to study fracture healing in mice.</a> STAR Protoc. 2 (3), 100798(2021).</li>
<li>Matthys, R., Perren, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Internal+fixator+for+use+in+the+mouse%5BTitle%5D)+AND+%22Injury%22%5BJournal%5D)">Internal fixator for use in the mouse.</a> Injury. 40, Suppl 4 S103-S109 (2009).</li>
<li>Manassero, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+of+a+segmental+femoral+critical-size+defect+model+in+mice+stabilized+by+plate+osteosynthesis%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Establishment of a segmental femoral critical-size defect model in mice stabilized by plate osteosynthesis.</a> J Vis Exp. (116), e52940(2016).</li>
<li>Gunderson, Z. J., Campbell, Z. R., McKinley, T. O., Natoli, R. M., Kacena, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comprehensive+review+of+mouse+diaphyseal+femur+fracture+models%5BTitle%5D)+AND+%22Injury%22%5BJournal%5D)">A comprehensive review of mouse diaphyseal femur fracture models.</a> Injury. 51 (7), 1439-1447 (2020).</li>
<li>Riquelme-Guzman, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Postembryonic+development+and+aging+of+the+appendicular+skeleton+in+Ambystoma+mexicanum%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">Postembryonic development and aging of the appendicular skeleton in Ambystoma mexicanum.</a> Dev Dyn. 251 (6), 1015-1034 (2022).</li>
<li>Gentz, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Medicine+and+surgery+of+amphibians%5BTitle%5D)+AND+%22ILAR+J%22%5BJournal%5D)">Medicine and surgery of amphibians.</a> ILAR J. 48 (3), 255-259 (2007).</li>
<li>Lang, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Collagen+I-based+scaffolds+negatively+impact+fracture+healing+in+a+mouse-osteotomy-model+although+used+routinely+in+research+and+clinical+application%5BTitle%5D)+AND+%22Acta+Biomater%22%5BJournal%5D)">Collagen I-based scaffolds negatively impact fracture healing in a mouse-osteotomy-model although used routinely in research and clinical application.</a> Acta Biomater. 86, 171-184 (2019).</li>
</ol>

The authors declare no competing interests.

The currently described method of femur plate fixation and osteotomy allows for its application in aquatic animals, such as Ambystoma mexicanum (axolotl). This surgical method was recently used to compare fracture healing and limb regeneration in axolotls to fracture healing in mice9. As in mice, a 4-hole fixator plate can be attached to the bone with self-breaking screws, and a Gigly saw can be used to create a fracture of uniform size15. Plate fixation facilitate...

The axolotl (Ambystoma mexicanum) is an important model for organ regeneration, including the tail, spinal cord, brain, heart, gills, and limbs1,2,3,4,5. Detailed studies of axolotl limb regeneration uncovered mechanisms of cell dedifferentiation and the formation of a stem cell pool, blastema, at the amputation site. Due to the ability of the blastema cells to reconstruct all missing limb parts, including a patterned skeleton6,7, the axolotl appears to be an attractive model organism for bone healing studies. Recently, several studies focused more on bone biology in axolotls, describing skeletal morphology, cellular composition, and ossification dynamics.
It was found in mammals that the bone healing process in long bones occurs via endochondral ossification and consists of several stages: hematoma, granulation tissue, and soft callus formation, callus ossification into hard callus and woven bone, and bone remodeling8. A recent study has shown that similar stages can be observed in axolotl bone healing9.
Until now, axolotl fractures were studied in a non-stabilized system, whereby bone is simply cut with iridectomy scissors. The large fractures were created in the zeugopod, where osteotomy is performed on one of the bones, whereas the other serves as support10,11. In contrast, fractures are routinely studied in mammals, including rats and mice, using reliable fixation systems, such as intramedullary pin and bone-aligning plates, to control fracture size and ensure bone alignment.
Thus, the method aims to ensure stabilized and uniform fixation of the axolotl femur prior to osteotomy. In order to make axolotl studies more comparable to mammals, including mice and humans, intramedullary pin12, external plate fixator13,14, and internal bone aligning plate15,16,17&#160;fixation were considered. The latter was shown to ensure proper bone fixation and allow for creating a gap of a certain size by using one or two cuts with a Gigly saw of a specific diameter. As axolotls represent the aquatic larvae of Ambystoma mexicanum, the external fixator might have caused post-surgical complications due to the open wound and contact with water. As axolotls do not develop secondary ossification centers even until very late in their development (20 years old18), and thus the standard intramedullary nail used in mice might not be prevented from puncturing the epiphyses, a decision was made to apply an internal plate fixation method to large axolotls. In large axolotls, the femur size and degree of ossification resemble that of an adult mouse, thus allowing for mid-diaphyseal osteotomy with titanium plate fixation1.
The fracture gap size largely determines the healing dynamics and outcome. For example, in a mouse, a 0.25 mm stabilized fractures heal mostly through intramembranous&#160;ossification due to their small size and rigid stabilization; a 0.7 mm fracture heals by endochondral ossification, with the formation of a cartilaginous callus around the fracture; large defects, such as 3.5 mm critical-sized defects do not heal completely and thus are used to model bone fracture non-union16. In this study, the plate fixation protocol of the axolotl femur prior to the osteotomy using the example of a 0.7 mm fracture gap was established with the ultimate goal of comparing axolotl bone healing to that of the mouse9.
After osteotomy, the fractures underwent the process of endochondral ossification, albeit slower than in mice, possibly due to the aquatic lifestyle of axolotls and slower cell division rates. In the method presented here, the 0.7 mm gap osteotomy with rigid plate fixation is shown; however, other gap sizes and semi-flexible fixators, as well as plates of different materials, are potentially possible. Overall, the method presented here can be used for standardized bone fixation and will be helpful for studies comparing axolotl limb regeneration to bone healing or studying bone healing in axolotls under different conditions to ensure standardized fracture fixation.

<table><tbody><tr><td>0.66 mm Gigly wire saw&amp;nbsp;</td><td>RISystem</td><td>RIS.590.120</td><td></td></tr><tr><td>7.0 Optilene suture&amp;nbsp;</td><td>Braun</td><td>C3090538</td><td></td></tr><tr><td>Benzocaine&amp;nbsp;</td><td>Sigma-Aldrich</td><td>E1501</td><td>dilute to 0.03%&amp;nbsp; prior to using</td></tr><tr><td>Butorphanol (Butomidor 10 mg/mL)</td><td>Richter Pharma AG</td><td>-</td><td>dilute to 0.5 mg/L&amp;nbsp; prior to using</td></tr><tr><td>Drill bit 0.30 mm</td><td>RISystem</td><td>RIS.590.200</td><td></td></tr><tr><td>Dumont #5 Forceps - Standard/Inox</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Hand drill</td><td>RISystem</td><td>RIS.390.130</td><td>better to have at least 3 pieces</td></tr><tr><td>Micro CT data analyzer</td><td>Bruker, Billerica, MA, USA</td><td>SkyScan NRecon software</td><td></td></tr><tr><td>Micro CT specimen scanner</td><td>Bruker, Billerica, MA, USA</td><td>SkyScan 1172&amp;nbsp;</td><td></td></tr><tr><td>Moria MC31b Iris forceps - smooth, curved, 10 cm</td><td>Fine Science Tools</td><td>11373-12FST</td><td>2 pieces</td></tr><tr><td>MouseFix Drill-&amp;amp;Saw guide 1.75 mm, rigid</td><td>RISystem</td><td>RIS.301.102</td><td></td></tr><tr><td>MouseFix plate 4 hole, rigid</td><td>RISystem</td><td>RIS.401.110</td><td></td></tr><tr><td>MouseFix screw, L =2.00 mm</td><td>RISystem</td><td>RIS.401.100</td><td>need 4 per bone</td></tr><tr><td>Narrow Pattern Forceps</td><td>VWR</td><td>FSCI11002-12</td><td></td></tr><tr><td>penicillin/streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Ring forceps</td><td>Fine Science Tools</td><td>11103-09</td><td></td></tr><tr><td>scalpel #15</td><td>B Braun, Thermo Fischer Scientific</td><td>5518032</td><td></td></tr><tr><td>Square box wrench 0.50 mm</td><td>RISystem</td><td>RIS.590.111</td><td></td></tr><tr><td>Sterile bone wax, 2.5 g</td><td>Ethicon, Johnson &amp;amp; Johnson</td><td>W810</td><td></td></tr><tr><td>Student Fine Scissors - Straight/11.5cm</td><td>Fine Science Tools</td><td>91460-11</td><td></td></tr></tbody></table>

stabilizing a femur osteotomy with a plate fixation in ambystoma mexicanum

The axolotl (Ambystoma mexicanum) is a promising model organism for regenerative medicine due to its remarkable ability to regenerate lost or damaged organs, including limbs, brain, heart, tail, and others. Studies on axolotl shed light on cellular and molecular pathways ruling progenitor activation and tissue restoration after injury. This knowledge can be applied to facilitate the healing of regeneration-incompetent injuries, such as bone non-union. In the current protocol, the femur osteotomy stabilization using an internal plate fixation system is described. The procedure was adapted for use in aquatic animals (axolotl, Ambystoma mexicanum). &#8805;20 cm snout-to-tail tip axolotls with fully ossified, mouse-size comparable femurs were used, and special attention was paid to the plate positioning and fixation, as well as to the postoperative care. This surgical technique allows for standardized and stabilized bone fixation and could be useful for direct comparison to axolotl limb regeneration and analogous studies of bone healing across amphibians and mammals.

The surgical procedure described here (Figure 1) lasts between 20 min and 30 min and requires a surgeon and an assistant. Optionally, use a binocular dissection microscope or magnifying glass system.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66648/66648fig01.jpg" /> 
Figure 1: Schematics of the surgical procedure and the experimental setup. (A</s...

Read this Scientific Article Protocol about Author Spotlight: Exploring Regeneration in Axolotls Through Insights in Cellular and Molecular Mechanisms, Bone Healing, and Implications for Human Therapi

Author Spotlight: Exploring Regeneration in Axolotls Through Insights in Cellular and Molecular Mechanisms, Bone Healing, and Implications for Human Therapies

A protocol for femoral osteotomy surgery with the use of internal plate fixation in mature axolotls is presented. The procedure can be used to perform comparative studies on limb regeneration and fracture healing in aquatic amphibians.

Stabilizing a Femur Osteotomy with a Plate Fixation in Ambystoma mexicanum

The axolotl (Ambystoma mexicanum) is a promising model organism for regenerative medicine due to its remarkable ability to regenerate lost or damaged ...

stabilizing-femur-osteotomy-with-plate-fixation-ambystoma

Research

JoVE Journal

Biology

546 Views.  Research Institute of Molecular Pathology. A protocol for femoral osteotomy surgery with the use of internal plate fixation in mature axolotls is presented. The procedure can be used to perform comparative studies on limb regeneration and fracture healing in aquatic amphibians.

Video: Author Spotlight: Exploring Regeneration in Axolotls Through Insights in Cellular and Molecular Mechanisms, Bone Healing, and Implications for Human Therapies

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 Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to restrict knee joint mobility is a widely accepted model to generate experimental contracture. However, implanting application will inevitably cause surgical trauma to the animals. Aiming to develop a less invasive approach, we combined a muscle-gap separation modus with a previously reported mini-incision skill during the surgical procedure: Two mini skin incisions were made on the lateral thigh and leg, followed by performing muscle-gap separation to expose the bone surface. The rat knee joint was gradually immobilized by a preconstructed internal fixation at approximately 135&#176; knee flexion without interfering essential nerves or blood vessels. As expected, this simple technique permits rapid postoperative rehabilitation in animals. The correct position of the internal fixation was confirmed by an x-ray or micro-CT scanning analysis. The range of motion was significantly restricted in the immobilized knee joint than that observed in the contralateral knee joint demonstrating the effectiveness of this model. Besides, histological analysis revealed the development of fibrous deposition and adhesion in the posterior-superior knee joint capsule over time. Thus, this mini-invasive model may be suitable for mimicking the development of immobilized knee joint contracture.

Here, we present a protocol to describe a minimally invasive technique for knee joint immobilization in a rat model. This reproducible protocol, basing on muscle-gap separation modus and the mini-incision skill, is suitable for studying the underlying molecular mechanism of acquired joint contracture.

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to ...

Developmental Biology

Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.

One constraint of preclinical research in the field of bone repair is the lack of experimental control over the local mechanical environment within a healing bone lesion. We report the design and use of an external fixator for bone repair with the ability to change fixator stiffness in vivo.

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there ...

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 ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

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 ...

Combination of Anatomical Plates and Rotator Cuff Reinforcement for Minimally Invasive Treatment of Greater Tuberosity Fractures of the Humerus

Greater tuberosity fractures of the humerus can be challenging to manage due to their complex anatomy and the potential for compromised shoulder function. We present a novel technique for treating greater tuberosity fractures utilizing specialized anatomical plates and rotator cuff reinforcement. The technique involves the use of an anatomically T-shaped plate designed specifically for the greater tuberosity region of the humerus, allowing for precise fixation and stability. Additionally, rotator cuff reinforcement is performed using sutures to enhance structural integrity and promote early mobilization. The simplified process is as follows: After administering anesthesia, a 3 cm incision is made along the lateral aspect of the shoulder to precisely expose the fracture site of the greater tuberosity. A suture of size 5 is skillfully threaded through the tough rotator cuff tendon to securely attach the proximal humerus to the anatomical plate after the greater tuberosity has been reduced. Intraoperative fluoroscopy is utilized to verify the accurate positioning of plates and screws. After ensuring everything is correct, the surgery concludes.

Here, we present a protocol to utilize anatomical plates alongside rotator cuff reinforcement in a minimally invasive surgery to efficiently treat humerus greater tuberosity fractures, promising quicker healing and improved stability.

Greater tuberosity fractures of the humerus can be challenging to manage due to their complex anatomy and the potential for compromised shoulder function. ...

Stabilizing a Femur Osteotomy with a Plate Fixation in Ambystoma mexicanum

Summary

Explore More Videos

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

A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats

Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models

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

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

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

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

Combination of Anatomical Plates and Rotator Cuff Reinforcement for Minimally Invasive Treatment of Greater Tuberosity Fractures of the Humerus