Design and Standardization of a Mandibular Injury Model for the Study of Craniofacial Tissue Regeneration in the Ambystoma mexicanum Model

Summary

The objective of this study was to standardize a safe and reproducible surgical approach for performing a complete transverse amputation of the mandible in Ambystoma mexicanum for the study of craniofacial tissue regeneration.

Abstract

The craniofacial complex is an anatomical region that, in humans, can be affected by developmental malformations, trauma, and pathologies such as cancer, leading to the loss of affected tissues. This creates a need to seek strategies for regenerating these lost tissues. This has promoted the study of cellular and molecular mechanisms that orchestrate the regenerative response of injured craniofacial structures in regenerating species, with Ambystoma mexicanum (A. mexicanum) being a species of great interest.

In accordance with the above, a reproducible surgical lesion model was designed and standardized in the jaw of axolotls to study the regenerative response of injured tissues and simulate a critical defect like those performed in humans. Thus, juvenile/adult animals between 12-15 cm were anesthetized with 0.1% tricaine for 20-30 min. Subsequently, a complete transverse amputation was performed using a four-handed technique on the distal third of the mandible, including the symphyseal and parasymphyseal region, removing skeletal structures corresponding to the dentary bone and Meckel's cartilage, as well as soft tissues such as muscle, connective tissue, nerves, and skin. Subsequently, bone remnants were regularized, and 0.5% sulfamerazine was applied. The tissue response was evaluated both macroscopically and by diaphanization. All procedures performed were approved by the animal experimentation committee of the University of Antioquia, Colombia. The results show that the amputated animals were able to regenerate the removed craniofacial structures morphologically, structurally, and functionally.

Of great relevance, this work provides a reproducible surgical technique with 100% survival of amputated and reamputated animals when the collection of regenerating tissues was required. Therefore, this surgical design represents a basic input for conducting comparative analyses with other vertebrates capable of regenerating craniofacial structures and for studying the cellular and molecular mechanisms underlying this regenerative response in the future.

Introduction

The ability to regenerate tissues, organs, and complex structures has been widely described throughout the animal kingdom^1,2,3. Thus, interest in understanding how these species can activate and regulate the regenerative response after injury or amputation of various structures within appendages, such as limbs and tails, has been the focus of attention in the study of the biology of regeneration^4,5,6,7,8.

Protocol

The model organism used in this project was the salamander A. mexicanum, which is endemic to Xochimilco, Mexico. This colony of animals was legally established in 2014 with the importation of parent salamanders from the Ambystoma Genetic Stock Center at the University of Kentucky, and currently has 32 parents and over 200 juvenile animals. All procedures were approved by the Animal Experimentation Ethics Committee of the University of Antioquia (UdeA) under Act No. 151 of March 27, 2023, taking into account national and international standards of ethics in animal experimentation, such as Chapter VI of Law 84 of 1989 and Title V of Resolution 8430 of 1993 of t....

Results

Considering the importance and relevance of studying the potential post-amputation regenerative response of craniofacial structures in species such as A. mexicanum²⁹, it is necessary to have a safe and reproducible surgical technique that allows not only the evaluation of the regenerative response of tissues, but also offers a low or zero mortality rate in the animals undergoing surgery. Consequently, this study presents the standardization of a successful surgical approach involving comp.......

Discussion

This paper presents the successful surgical approach to complete transverse amputation of the mandible in the A. mexicanum animal model, along with several recommendations necessary to reproduce this surgical model for monitoring the regenerative response of these structures and the potential identification of factors regulating this regenerative process.

In this study, a complete transverse amputation was performed on the distal third of the mandible of A. mexicanum, involvi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Axolotl (Ambystoma mexicanum)	Ambystoma Genetic Stock Center	N/A	15-18 cm and 16 g - 22 g wildetype
Calcium Chloride dihydrate	Duksan	1098	CaCl2 2H2O
Digital camera MotiCAM 5	MotiCAM	https://www.motic.com/upload/File/201407/2014071115335993.pdf
EDTA - Sodium salt, dihydrate	Biobasic	EB0185	C10H14N2O8Na2 . H2O
Glass petri dish	any maker	N/A	Borosilicate glass
Magnesium Sulfate 7-hydrate	ITW Reagents	131404.1211	MgSO4 7H2O
Microsurgical instruments	N/A	N/A	Forceps, clamp, 7A spatula, dentimeter and scissors
Pasteur pipette	any maker	N/A	Low-density polyethylene
Potassium Chloride	Duksan	372	KCl
Red wax pencil	any maker	N/A
Sodium Chloride	Merk	K48297604 717	NaCl
Software Motic Images Plus-Versión 2.0	Motic	https://www.motic.com/As_Support_Download/d42.html
Stereo microscope	Olympus SZX16	N/A
Sulfamerazine sodium salt	Sigma- Aldrich	S0800	4-Amino-N-(4-methyl-2-pyrimidinyl) benzenesulfonamide
Tricaine methanesulfonate	Sigma- Aldrich	E10521	Ethyl 3-aminobenzoate methanesulfonate
Tubes 1.5 mL	Axygen	MCT150C	Pure polypropylene