Stabilizing a Femur Osteotomy with a Plate Fixation in Ambystoma mexicanum

Summary

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.

Abstract

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). ≥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.

Introduction

The axolotl (Ambystoma mexicanum) is an important model for organ regeneration, including the tail, spinal cord, brain, heart, gills, and limbs^1,2,3,4,5. 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 skeleton^6,7, the axolo....

Protocol

The following procedure was performed with approval from the Magistrate of Vienna (GZ: MA 58-65248-2021-26). 5-8 years old, ≥ 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 .......

Discussion

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.......

Materials

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