Table 1.

The most widely used models to generate experimental periodontitis in mice.

Experimental Periodontitis Model	Description	Requirements	Advantages and Biological Approaches		Limitations	References
			Technical Advantages	Biological Application and Plausibility
Oral gavage/oral infection	Inoculation of live human bacteria, such as Pg, Aa, and/or Fn, via an oral-esophageal-gastric cannula or a micropipette into the mouse digestive system. • Gut microbiota dysbiosis and bacteremia favors chronic low-grade inflammation similar to periodontitis.	1) Anaerobic/capnophilic culture and bacteria-compatible animal facilities. 2) Constant monitoring and standarization of bacteria MOI. There is no consensus regarding the ideal concentration or quantity of inoculated bacteria.	1) Allows the precise enteric administration of bacteria. 2) It can be performed without anesthesia.*	1) Promotes periodontal inflammation and progressive alveolar bone resorption consistent with a chronic form of periodontitis. 2) Allows the bacterial invasion of mice oral tissues and bacteremia during a relatively long period of time (4-8 weeks). 3) The sustained systemic microbial challenge and low-grade systemic inflammation resembles the chronicity of periodontitis. 4) Enables the study of the association between systemic conditions and periodontitis-associated bacterial strains. Depending on the MOI and bacterial strain, it can also favor gut dysbiosis, joint inflammation, atheroma formation, and neuroinflammation.	1) Not fully effective to induce periodontal lesions. It generates less alveolar bone loss compared with other models. 2) Multiple inoculations in a long period of time (4-8 weeks) are needed until disease development. Increased animal stress and risk of esophageal lesions. 3) Effectiveness depends on the used bacterial strain and its virulence. 4) Not fully compatible with immunocompromised mice strains and humanized mice models.	(7, 25, 33–39)
Periodontal inoculation of bacteria	Localized microinjection of live human bacteria into mouse vestibular or palatal mucosa. • Mucosal infection and local bacterial challenge induce a local immune response capable of generating periodontal lesions.	1) Anaerobic/capnophilic culture and bacteria-compatible animal facilities. 2) Constant monitoring and standardization of bacteria MOI. 3) Constant anesthesia supplementation and post-intervention animal surveillance. There is no consensus regarding the ideal concentration or quantity of inoculated bacteria.	1) Semi-precise local administration of bacteria.	1) Promotes periodontal inflammation and alveolar bone resorption consistent with a chronic form of periodontitis. 2) Enables the study of specific periodontal host-bacteria interactions associated to infection, such as PRR-antigen interaction. 3) Useful for the study of virulence/immunogenic/pathogenic differences between periodontitis-associated bacteria.	1) Not fully effective to induce periodontal lesions. It generates less alveolar bone loss and inflammatory response compared with other models, such as ligature. 2) Repetitive injection regimen (2-3 per week) and mid-long experimental period until disease development (20-45 days).	(6, 18, 35, 40–42)
Oral and anal inoculation of periodontitis-associated bacteria	Topical administration of a mixture of 3%CMC and periodontitis-associated bacteria, such as Pg, into the mouse oral cavity and anus. • Mice coprophagia promotes continuous re-infection and establishment of chronic oral microbial challenge.	1) Anaerobic/capnophilic culture and bacteria-compatible animal facilities. 2) Constant monitoring and standarization of bacteria MOI.	1) Minimal or no trauma to the mouse mucosa. 2) It can be performed without anesthesia.*	1) Allows the bacterial invasion of mice oral tissues and bacteremia during a relatively long period of time (4-8 weeks). 2) Promotes periodontal inflammation and alveolar bone resorption consistent with a chronic form of periodontitis. 3) The sustained systemic microbial challenge and low-grade systemic inflammation resembles chronicity of periodontitis. 4) Enables the study of the association between systemic affections and periodontitis-associated bacterial strains.	1) Unprecise administration of bacteria. 2) Gut dysbiosis and faecal bacteria can be confounding factors. 3) Consecutive application regimen (8 days) and mid-long experimental period until disease development (8 weeks). 4) Not fully compatible with immunocompromised mice strains and humanized mice models.	(43–45)
Periodontal inoculation of isolated bacterial antigens	Local microinjection of known bacterial components, derived or not from periodontitis-associated bacteria, such as LPS. The inoculation is carried out into mouse vestibular or palatal mucosa. • Bacterial antigens directly elicit the mouse immune response.	1) Skilled operator. 2) Constant anesthesia supplementation and post-intervention animal surveillance. There is no consensus regarding the ideal concentration or quantity of inoculated bacterial antigen for the model.	1) It does not need bacterial culture† or their inoculation. 2) Compatible with immunocompromised mice strains and humanized mice models.	1) Promotes periodontal inflammation and alveolar bone resorption consistent with an acute/aggresive form of periodontitis. 2) Induces low-grade systemic inflammation (in the case of LPS) when applied for at least 2 weeks. It can provoke cortical lesions, neuroinflammation, and arthritic lesions. 3) Enables the study of the specific interaction of PAMPs with the host immune response.	1) Repetitive injection regimen (2-3 per week). 2) It does not emulate bacteria-host interaction, essential during periodontitis.	(18, 46–50)
Chemically-induced periodontitis	TNBS and/or DSS are orally delivered weekly and/or biweekly. • DDS targets the innate immune response by undermining the epithelial barrier and inducing ROS production. TNBS induces a T-cell mediated response.	1) Experiment can last between 7 to 18 weeks, with weekly or biweekly interventions.	1) It does not need anesthesia nor bacterial culture. 2) Compatible with immunocompromised mice strains and humanized mice models.	1) Promotes periodontal inflammation and progressive alveolar bone resorption consistent with a chronic form of periodontitis. 2) Induces low-grade systemic inflammation, including colon and liver lesions. 3) Allows the study of the association between gut mucosal and oral mucosal inflammation.	1) It does not emulate bacteria-host interaction, essential during periodontitis. 2) Not fully effective to induce periodontal lesions. It generates less alveolar bone loss compared with other models, such as ligature.	(51, 52)
Ligature-induced periodontitis	Placement of a retentive ligature, usually silk, around or at the interproximal spaces of the mouse tooth. • Accumulation of bacterial biomass favors the development of a dysbiotic microbiota capable of inducing a local mucosal immune response and periodontal lesions.	1) Highly skilled operator, with optional magnification devices. 2) Most models use silk sutures around maxillary second molars, though there is no consensus regarding the place or width/length of the ligature or the need of its renewal.	1) Compatible with immunocompromised mice and humanized mice models. 2) Minimal trauma to the mouse mucosa. 3) Allows the collection of mouse gingival crevicular fluid.	1) Promotes acute periodontal inflammation and rapid alveolar bone resorption resembling an acute/aggresive form of periodontitis. 2) Compatible with the current oral dysbiosis-associated periodontitis pathogenesis paradigm. 3) Induces low-grade systemic inflammation, also compatible with periodontitis definition. 4) Allows the study of local immune response against inespecific bacterial challenge and alveolar bone regeneration after ligature removal. 5) When combined with oral gavage or periodontal inoculation, it can be useful for the study of virulence/immunogenic/pathogenic differences between periodontitis-associated bacteria.	1) Risk of mechanical trauma if not performed by calibrated operator. 2) Animals need to be constantly checked for ligature position. 3) No sustained bone loss after prolonged periods of time, unless combined with bacteria inoculation or gavage; thus, not resembling periodontitis chronicity.	(20, 23, 29, 32–35, 53, 54)
Calvaria inoculation of periodontitis-associated bacteria	Subcutaneous inoculation of periodontitis-associated bacteria, mostly Pg, at the skull midline between the ears and eyes. • The injection induces the formation of an abscess, acute local inflammation, and adjacent bone resorption.	1) Anaerobic/capnophilic culture and bacteria-compatible animal facilities. 2) Constant monitoring and standarization of bacteria MOI. There is no consensus regarding the ideal concentration or quantity of inoculated bacteria.	1) Semi-precise local administration of bacteria. 2) Does not require a skilled operator.	1) Promotes acute subcutaneous inflammation and rapid alveolar bone resorption, resembling an acute/aggresive form of infection/inflammation-induced bone resorption. 2) Allows the study of the immunogenic and pathogenic potential of bacteria.	1) Abscess formation does not resemble a periodontitis lesion. 2) Not fully compatible with immunocompromised mice strains and humanized mice models.	(55–58)

Aa, Aggregatibacter actinomycetemcomitans; CMC, carboxymethyl cellulose; DSS, dextrane sulfate sodium; Fn, Fusobacterium nucleatum; LPS, lipopolysaccharide; MOI, multiplicity of infection; PAMPs, pathogen-associated molecular patterns; Pg, Porphyromonas gingivalis; PRR, pattern recognition receptor; ROS, reactive oxygen species; TNBS, 2,4,6-Trinitrobenzene sulfonic acid.

*The use of isofluorane anesthesia is recommended in some publications to reduce the provoked stress and the incidence of esophageal lesions during oral gavage.

^†Some authors would prefer to extract bacterial components, such as LPS, from their own bacteria cultures.