Optimization of the Ligature-induced Periodontitis Mouse Model for Preclinical Research

Summary

The ligature-induced periodontitis mouse model is an effective and increasingly popular tool for studying periodontal disease. Detailed guidance is presented for implementing a simplified version of the model focused on interproximal alveolar bone destruction, including laboratory study design, 3D-printed tool production, and bone quantification using μCT analysis.

Abstract

The experimental model of ligature-induced periodontitis in mice has played a fundamental role in understanding periodontal disease pathogenesis; yet, it has not been fully leveraged for translational and therapeutic applications. Similar to human disease, ligature-induced periodontitis in mice develops via a series of distinct microbial, inflammatory, and osteo-immunological events. The ligature-induced alveolar bone destruction progresses in a reasonably short period and is easily quantifiable, making it a cost-effective disease model for preclinical studies.

To overcome the technical challenge of the classic ligature model, the establishment of the simplified ligature-induced periodontitis mouse model was curated. Rather than inducing disease in the entire circumference of two adjacent teeth, the simplified disease model is centered on inflammatory bone destruction only in the interproximal region of two posterior teeth, which is also the region that common periodontal disease initiates in humans. Because it employs three dimensional (3D)-printed tools for ligature placement, the approach is fast and effective  leading to a procedural time of 5-10 min per mouse. While osteoclastogenic signaling is detected after 3 days of ligature placement, bone destruction consistently develops at 9 days post ligation.

Here, the simplified ligature model protocol is elaborated by highlighting important elements specific to study design (i.e., power calculation, procedure standardization, calibration), development of the 3D printed tools (i.e., the "mouse dental bed" and "ligature holder"), and alveolar bone quantification using μCT analysis. Gingival gene expression of Rank, Rankl, and Opg, a master regulatory triad of osteoclastogenesis, is used as indirect evidence of therapeutic effectiveness. The methods presented in this paper will guide investigators' implementation of the simplified ligature-induced periodontitis model to test innovative periodontal therapeutics in the preclinical setting.

Introduction

Periodontal disease is a complex inflammatory process that leads to the destruction of tooth-supporting structures including the gingiva, the periodontal ligament, the cementum, and the alveolar bone. For periodontal bone loss to develop, the breakage of the gingival barrier and bacterial invasion need to occur, in addition to the recruitment of inflammatory cells, and the transition from an innate-dominated to an acquired-dominated immune response¹. The final imbalance between osteoblasts and osteoclasts will result in alveolar bone loss via its destruction². The hallmark phenotypic finding that differentiates the diagn....

Protocol

This protocol adhered to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines^22,23. All experimental procedures described in this protocol were approved by the IACUC of the University of North Carolina at Chapel Hill.

1. Experimental design

1. Sample-size calculation
   NOTE: Because of the translational nature of the research program, the power calculation presented here is based on alveolar bone measurements of 8-12-week-old wild-type C57Bl6 mice.
   1. Analyze bone based on the linear measurements between the cementum-ename....

Results

aRepresentative data analysis of linear alveolar bone measurements from WT female and male mice at baseline (health) and 9 days post ligation are presented in Figure 1A. The power calculation indicates that a total of 10 mice (4-5 mice per group) are needed to detect differences between these groups if the primary outcome considered is linear bone levels measured by μCT (Figure 1B). However, a power calculation for preclinical studies requires that both groups o.......

Discussion

The present study provides insightful information and detailed, step-by-step guidance on the simplified ligature-induced periodontitis mouse model protocol previously presented¹⁰. The details included in the current paper can dramatically impact reproducibility and are supported by the group's experience with this specific ligature-induced periodontitis mouse model^11,12,13,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Blade	Integra	4-315C	15C blade
Ceramic beads	VWR	10158-608	0.5 mL x 1.4 mm Ceramic beads
Glue/resin material	The Gorilla Glue Company	8401519
Ligature Holder (With Notch)	UNC at Chapel Hill BeAM Design Center facility core	not applicable	118.01 mm x 13 mm x 13.74 mm
Ligature Holder Lock	UNC at Chapel Hill BeAM Design Center facility core	not applicable	17.5 mm x 14.57 mm x 17.8 mm
Lulzbot mini v1.0 3D printer	Lulzbot (Fargo Additive Manufacturing Equipment 3D, LLC)	not applicable	Aleph Objects, Lulzbot Mini v1.0 model
MicroView Software	Parallax Innovations	2.5.0-3139	Software used for bone measurements
Mouse Dental Bed (With Notches)	UNC at Chapel Hill BeAM Design Center facility core	not applicable	103.86 mm x 196.85 mm x 50.99 mm
Mouse Dental Bed Top	UNC at Chapel Hill BeAM Design Center facility core	not applicable	99.8 mm x 77 mm x 21.5 mm
Polylactic acid (PLA) filament	Verbatim	55259	3.0 mm PLA
Rapidpure RNA Tissue Kit	MP Biomedicals	112721050
Scalpel blade handle	Bard-Parker	371030	15C scalpel blade handle
Suture-silk	Roboz Surgical Instrument	SUT-15-1
Taqman qPCR primers	Thermo Fisher	Rank (Mm00437135_ m1), Rankl (Mm00441906_ m1), Opg (Mm00435452_ m1), Gapdh (Mm99999915_g 1)
VX-765	InvivoGen, San Diego, USA	#inh-vx765-1	Caspase-1 inhibitor used for host modulation