Murine Model of Advanced Periodontitis Induced by Nylon Ligature in the Second Upper Molar

Summary

This study describes a modified 6-0 nylon ligation method for inducing periodontitis in mice, which is highly reproducible and represents an alternative for researchers to study periodontal disease from its development to its pathological consequences.

Abstract

Periodontal disease (PD) is an inflammatory disorder affecting the supporting tissues of teeth and is one of the most prevalent diseases worldwide. Its severe form, periodontitis, leads to the destruction of soft tissue, teeth, and bone. Animal models of periodontitis have been developed using primates, dogs, miniature pigs, and mice. Among these, the ligature-induced mouse model offers advantages such as rapid disease progression, reproducibility, predictability, and low cost while effectively replicating key aspects of human periodontitis. Mouse models using ligatures have provided valuable insights into the microbiological and immunological microenvironments of periodontal tissue, highlighting the critical role of biofilms in immune responses and their association with systemic diseases. This study presents a modified nylon ligation method for inducing periodontitis in mice. The modification involves using a nylon suture instead of a silk suture and placing it beneath the interproximal contact area rather than passing it through the contact point. This approach simplifies the technique while effectively inducing periodontitis. The detailed methodology for suture placement is graphically illustrated, and the progression of periodontitis is demonstrated through histological and histometric analyses.

Introduction

Periodontal disease (PD) is an inflammatory disorder of the supporting tissues of teeth and is among the most prevalent diseases in the world¹; the incidence of PD has been reported to range from 20%-50% worldwide². PD has different degrees of progression; its mild form, called gingivitis, affects only soft tissues, and its severe form, called periodontitis (PT), affects hard tissues such as bone³. Since PT is an inflammatory disease, it must be considered a complex immune response that can be modified by several risk factors that can alter the disease process

Protocol

All procedures involving experimental animals were conducted in strict compliance with the 'Ethical Framework for Biomedical Research on Laboratory Animals,' following the official Mexican standard NOM-062-ZOO-1999. This study was approved by the ethics committee of the Facultad de Estudios Superiores Iztacala (FES-Iztacala) under protocol CE/FESI/072024/1765. The mice were housed in animal chambers with free access to food and water in a pathogen-free environment at the FES-Iztacala animal facility. This protocol is a modification of the method previously described by Abe et al.²⁷. Six- to eight-week-old female BALB/c mice (weighing 16....

Results

This methodology allows induced mice to develop periodontitis (PT) from the second week onward. The mice were monitored weekly to verify the presence of the ligature. Euthanasia was performed on day 30. The clinical characteristics of all the groups were assessed. The control group maintained normal characteristics, such as color and the structure of the marginal gingiva, over time. Compared with those in the CTL group, the PT group tissues showed inflammation, bleeding, and periodontal pocket formation, which were obser.......

Discussion

Several animal models of periodontitis have been used to evaluate different aspects, such as microbiological and immune responses, and have some similarities with human disease⁴³. These findings provide evidence of the framework of periodontal disease, such as the role of biofilms, the immune response, and interactions with systemic conditions^44,45.

The animal models of periodontitis have different complexities .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% NaCl solution	PiSA		Rinse maxillae
6-0 nylon suture	Atramat, Internacional Farmacéutica	PE1946-N	Ligature placement
Amscope 3.7 program for digital camera	Amscope	x64, 4.11.21973.20230107	Data colection
EDTA solution	Sigma–Aldrich	E5134	Decalcification
Fine scissors	generic		To cut the 6-0 nylon suture
Fine tissue forceps	generic		6-0 nylon suture
Gauze pad	generic		To keep corporal tempature
GraphPad Prism	GraphPad Prism	Version 8.3.0	Data analysis
Hypromellose	generic		Eye lubricant
Injectable water	Pisa		Dilution of anesthetic solution
Ketamine	Anesket, Pisa		Anesthetic
Micro centrifuge tubes	Cellpro	801501	To contain tissues durin decalcification
Micropore	3M	1533	To fix mouse
Microscope digital camera	Amscope	MU1603	Histology
Microtubes	Axygen	MCT-150-C	Store anesthetic dilution
Optical microscope	UNICO	Serie G380	Histology
Orhodontic elastics	TP Orthodintics, Inc	blue	To keep the snout open
Paraformaldehyde solution	Sigma–Aldrich	158127	Tissue Fixation
Paraplast	Leica	39601006	Histology
Surgical microscope	Carl Zeiss GmbH Berlin		6-0 nylon suture
Ultra Fine Insulin Syringes 6mm U-100, 0.3 mL	BD	326385	To inject the anesthetic solution
Universal rubber bands	generic		To keep the snout open
Worktable	Generic		Wooden base to support mice
Xylazine	Porcin, Pisa		Preanesthetic