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 ...

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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+axolotls+can+regenerate+original+neuronal+diversity+in+response+to+brain+injury.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+spinal+cord+following+ablation+in+urodele+larvae.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectoderm+to+mesoderm+lineage+switching+during+axolotl+tail+regeneration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+regeneration+in+Ambystoma+mexicanum+after+surgical+injury.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancements+to+the+axolotl+model+for+regeneration+and+aging.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphibians+as+research+models+for+regenerative+medicine.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+axolotl+limb+blastema:+Cellular+and+molecular+mechanisms+driving+blastema+formation+and+limb+regeneration+in+tetrapods.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+healing:+Mechanisms+and+interventions).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+specialist+in+regeneration-the+Axolotl-a+suitable+model+to+study+bone+healing.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+axolotl+fibula+as+a+model+for+the+induction+of+regeneration+across+large+segment+defects+in+long+bones+of+the+extremities.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+analysis+of+intra-articular+interzone+tissue+in+axolotl+salamanders.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+generation+of+closed+femoral+fractures+in+mice:+A+model+to+study+bone+healing.">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/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> 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+protocol+for+a+standardized,+femoral+osteotomy+model+to+study+fracture+healing+in+mice.">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/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>Manassero, 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=Establishment+of+a+segmental+femoral+critical-size+defect+model+in+mice+stabilized+by+plate+osteosynthesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+review+of+mouse+diaphyseal+femur+fracture+models.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postembryonic+development+and+aging+of+the+appendicular+skeleton+in+Ambystoma+mexicanum.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medicine+and+surgery+of+amphibians.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+I-based+scaffolds+negatively+impact+fracture+healing+in+a+mouse-osteotomy-model+although+used+routinely+in+research+and+clinical+application.">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 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.66 mm Gigly wire saw&#160;</td>
			<td>RISystem</td>
			<td>RIS.590.120</td>
			<td></td>
		</tr>
		<tr>
			<td>7.0 Optilene suture&#160;</td>
			<td>Braun</td>
			<td>C3090538</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzocaine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E1501</td>
			<td>dilute to 0.03%&#160; 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&#160; 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&#160;</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-&#38;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 &#38; 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.

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

Stabilizing a Femur Osteotomy with a Plate Fixation in Ambystoma mexicanum

We study tetrapod regeneration and use the axolotl as a top-tier regenerative species, comparing it to frogs and mice to discover cellular and molecular mechanisms of regeneration. The development of transgenics in genome editing, assembly of the axolotl genome, and application of RNA-Seq, ATAC-Seq, spatial transcriptomics and proteomics allow us to deeper understand the regeneration microenvironment and compare it to less regenerative species, including humans. We established the cellular contribution to tissues in the regenerating limb and spinal cord, and major regulators of initial cell proliferation and positional identity in the blastema.In a recent paper, we showed axolotl bone healing going through endochondral ossification, and the cellular level was similar to that of mammals. In contrast to previous techniques, where the axolotl fracture was either unfixed or the neighboring bone served as a support, in the current protocol, the bone is fixated with a plate, thus allowing to create a reproducible and aligned fracture and enabling a sound comparison to the studies in mice. The protocol allows for stable fracture with fixed gap size, broadening the studies on plate fixated fractures to amphibians.Despite their extreme regeneration abilities and full limb restoration upon amputations, axolotl surprisingly cannot heal large bone fractures with critical size defects. We aim to define the pro-regenerative blastema factors to treat bone non-union in critical size defects.

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

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.

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

Left Hind Limb Osteotomy in Ambystoma mexicanum

To begin, place the sedated ambystoma Mexicanum with the ventral side down on wet paper towels soaked in 0.03%Benzocaine solution. Cover the animal with benzocaine-soaked paper towels. Use a pair of ring forceps to stretch out the operative hind limb.With a scalpel, make a lateral longitudinal incision above the femur bone spanning the whole thigh in the upper hind limb. Carefully displace the muscles and nerves from the surgery site without cutting. Gently put Bode forceps under the femur to expose it for the surgery.Now place a rigid 7.75 millimeter four-hole fixator plate along the femur diaphysis without touching the joints and secure it in the aligned position with forceps. Use four two-millimeter titanium screws to attach the bone to the plate. Under irrigation, with 0.7 times PBS, and 1%penicillin streptomycin, manually drill to create the first hole in the bone for screw insertion.Then place the first screw in the hole. Apply the saw-guiding device onto the first screw and align it with the bone and plate. Use the saw-guiding device for drilling and inserting the remaining screws.After ensuring alignment of the plate with the bone, remove the saw guide, and use a screwdriver to break off the handles from the screws. Place a piece of sterilized plastic film under the femur to prevent soft-tissue damage during the osteotomy process. Then place a Gigli wire saw between the bone and protection film.Cut the bone using a Gigli wire saw creating a single 0.7 millimeter cut in the femur. Remove the saw and the protection film and irrigate the surgery site with 0.7 times PBS and 1%Penicillin streptomycin. Cover the top of the plate and screws with sterile bone wax to protect skin and muscles from irritation by the edges of the screws.Place muscles and skin on top of the wax. Close the incision site with a size 7.0 synthetic suture using simple interrupted stitches. After surgery, to reawaken, place the animal in a fresh artificial-pond-water tank supplemented with penicillin, streptomycin, and butorphanol.Micro CT and histological analysis facilitated the visualization of bone, fragment positions and gap sizes and the healing state. Notably, ambystoma Mexicanum showed no callous formation three weeks post-surgery contrasting with mammals. A cartilaginous callous was visible at three months and an ossified bony callus appeared at six months post-surgery.

375 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.

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

Summary