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ABSTRACT Periodontal disease (PD) is a common dental disease associated with the interaction between dysbiotic oral microbiota and host immunity. It is a prevalent disease, resulting in loss

of gingival tissue, periodontal ligament, cementum and alveolar bone. PD is a major form of tooth loss in the adult population. Experimental animal models have enabled the study of PD

pathogenesis and are used to test new therapeutic approaches for treating the disease. The ligature-induced periodontitis model has several advantages as compared with other models,

including rapid disease induction, predictable bone loss and the capacity to study periodontal tissue and alveolar bone regeneration because the model is established within the periodontal

apparatus. Although mice are the most convenient and versatile animal models used in research, ligature-induced periodontitis has been more frequently used in large animals. This is mostly

due to the technical challenges involved in consistently placing ligatures around murine teeth. To reduce the technical challenge associated with the traditional ligature model, we

previously developed a simplified method to easily install a bacterially retentive ligature between two molars for inducing periodontitis. In this protocol, we provide detailed instructions

for placement of the ligature and demonstrate how the model can be used to evaluate gingival tissue inflammation and alveolar bone loss over a period of 18 d after ligature placement. This

model can also be used on germ-free mice to investigate the role of human oral bacteria in periodontitis in vivo. In conclusion, this protocol enables the mechanistic study of the

pathogenesis of periodontitis in vivo. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through

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support SIMILAR CONTENT BEING VIEWED BY OTHERS TRANSCRIPTOMIC PHASES OF PERIODONTITIS LESIONS USING THE NONHUMAN PRIMATE MODEL Article Open access 29 April 2021 THE IL-33/ST2 AXIS IS

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a saliva-conditioned flow cell. _Appl. Environ. Microbiol._ 70, 4340–4348 (2004). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS This study was supported by grants

T90DE021986 and F32DE026688 (Y.J.), an IBM Junior Faculty Development Award from the University of North Carolina at Chapel Hill and grants K01DE027087 (J.M.), 5R01DE023836 (S.O.) and

5R01DE025207 (M.H.S.). We thank W.V. Giannobile of the University of Michigan School of Dentistry for constructive suggestions and helpful discussion. We thank B. Huang and J. Ashley of the

Histology Research Core Facility at UNC for histological embedding and sectioning. We thank the Microscope Core facility for the images. We thank the UNC Biomedical Research Imaging Center

for microCT scanning. We thank the Germ Free & Gnotobiotic Mouse Facilities at the University of Michigan and the Gnotobiotic Rodent Core Facility at the University of North Carolina for

assistance with the human bacterial infection experiment. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. AUTHOR

INFORMATION AUTHORS AND AFFILIATIONS * Department of Periodontology, School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Julie Marchesan, Mustafa S.

Girnary, Li Jing, Shaoping Zhang, Lu Sun, Thiago Morelli, Steven Offenbacher & Yizu Jiao * Department of Oral and Craniofacial Health Sciences, School of Dentistry, University of North

Carolina at Chapel Hill, Chapel Hill, NC, USA Michael Zhe Miao & Lu Sun * Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Mark H. Schoenfisch *

Department of Pathology, University of Michigan, Ann Arbor, MI, USA Naohiro Inohara Authors * Julie Marchesan View author publications You can also search for this author inPubMed Google

Scholar * Mustafa S. Girnary View author publications You can also search for this author inPubMed Google Scholar * Li Jing View author publications You can also search for this author

inPubMed Google Scholar * Michael Zhe Miao View author publications You can also search for this author inPubMed Google Scholar * Shaoping Zhang View author publications You can also search

for this author inPubMed Google Scholar * Lu Sun View author publications You can also search for this author inPubMed Google Scholar * Thiago Morelli View author publications You can also

search for this author inPubMed Google Scholar * Mark H. Schoenfisch View author publications You can also search for this author inPubMed Google Scholar * Naohiro Inohara View author

publications You can also search for this author inPubMed Google Scholar * Steven Offenbacher View author publications You can also search for this author inPubMed Google Scholar * Yizu Jiao

View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Y.J. and J.M. designed the experiments. Y.J., J.M. and M.S.G. performed the in vivo

experiments. L.J. performed H&E and MPO staining and analysis. M.Z.M. performed TRAP staining and analysis. M.S.G. performed qRT-PCR and analysis using the RT2 Profiler Array. S.Z. and

L.S. performed the microCT analysis. Y.J., J.M., T.M. and M.S.G. developed the murine dental bed and ligature holder. J.M. and Y.J. wrote and edited the manuscript together. S.O., N.I. and

M.H.S. helped to develop the model and edited the manuscript. All authors approved the final version of the manuscript. CORRESPONDING AUTHOR Correspondence to Yizu Jiao. ETHICS DECLARATIONS

COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published

maps and institutional affiliations. RELATED LINKS KEY REFERENCE USING THIS PROTOCOL 1. Jiao, Y. et al. _Cell Host Microbe_ 13, 595–601 (2013): https://doi.org/10.1016/j.chom.2013.04.005

INTEGRATED SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 Typical colony morphology for _S_. _gordonii_, _F_. _nucleatum_ and _V_. _parvula_ on a TSA plate. SUPPLEMENTARY INFORMATION

SUPPLEMENTARY TEXT AND FIGURES Supplementary Figure 1 REPORTING SUMMARY SUPPLEMENTARY MANUAL Detailed top view of blueprints required for manufacture of the murine dental bed RIGHTS AND

PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Marchesan, J., Girnary, M.S., Jing, L. _et al._ An experimental murine model to study periodontitis. _Nat Protoc_

13, 2247–2267 (2018). https://doi.org/10.1038/s41596-018-0035-4 Download citation * Published: 14 September 2018 * Issue Date: October 2018 * DOI: https://doi.org/10.1038/s41596-018-0035-4

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