Archives of Oral Biology 143 (2022) 105528

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Archives of Oral Biology

journal homepage: www.elsevier.com/locate/archoralbio

Local and systemic effects produced in different models of experimental

periodontitis in mice: A systematic review
Yasmin Dal Acqua a, 1, 2, Cristhiam Hernández a, 1, 2, Mariana Fogacci b, 2, Davi Barbirato c, 2,
Daniela Palioto a, *, 2
a
Department of Oral & Maxillofacial Surgery and Periodontology, Ribeirão Preto Dental School, University of São Paulo, Ribeirão Preto, SP, Brazil
b
Department of Clinical and Preventive Dentistry, Division of Periodontics, Federal University of Pernambuco, Pernambuco, PE, Brazil
c
Department of Oral & Maxillofacial Surgery, Pernambuco Dental School, University of Pernambuco, Recife, PE, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Objective: The objective of this study was to summarize and compare the local and systemic impact of different
Periodontitis induction models of experimental periodontitis in mice.
Chronic periodontal disease Design: After defining the PICO strategy, was performed a search for articles in English following the PRISMA
Mouse models
guidelines in PubMed, Embase, Scopus, Web of Science, Cochrane and LILACS databases. The search strategy
Ligature
provided 8815 articles from which were selected experimental studies comparing the local and/or systemic
Gavage
Alveolar bone resorption effects of two or more models for the induction of periodontitis in mice according to the inclusion and exclusion
criteria.
Results: After selection, 7 articles that met the inclusion criteria for the review. Were analyzed differences be­
tween the following experimental models of periodontitis in mice: ligature, gavage, injection of bacteria, ligation
embedded in bacteria and association between them.
Conclusion: Although most experimental models of periodontitis are efficient in causing alveolar bone loss, there
are differences in their characteristics. In ligature, an acute process is established and the host tends to repair
itself, decreasing the significance of this loss over time. In models where bacterial challenge is added bone loss
appears to be significant with longer induction time, indicating the presence of a chronic condition. Regarding
systemic outcomes, gavage showed greater potential for modulating the host’s response with systemic inflam­
matory changes, proving to be more promising between periodontitis and chronic systemic diseases.

1. Introduction regions (GBD, Viewpoint Collaborators, 2019, 2020), characterized by

Periodontal disease has been substantial and growing in the last
three decades. Estimate that in 2019 there were 1.1 billion prevalent
cases of severe periodontitis worldwide. From 1990–2019, the age-
standardized prevalence rate of severe periodontitis increased by 8.44
% (6.62–10.59 %), being highest among less developed countries and
an immunoinflammatory pathogenesis that affects the supporting tis­
sues of the tooth (Chen et al., 2021; Jurdziński et al., 2020; Kassebaum
et al., 2017) being dependent on and sustained by the oral microbial
biofilm (Jurdziński et al., 2020; Lamont et al., 2018). Even though the
presence of key pathogens, such as Porphyromonas gingivalis, reflects a
dysbiotic state, it is now accepted that the host immune modulation may

Abbreviations: PICO,, Patient, Intervention, Comparison and Outcome; SWiM,, Synthesis without meta-analysis; SYRCLE,, Systematic Review Center for Labo­
ratory animal Experimentation; PCR,, Polimerase chain reaction; ELISA,, Enzyme-Linked Immunosorbent Assay; RANKL,, Receptor activator of nuclear factor-kappa
beta ligand; TNFα, tumor necrosis factor alpha; TLR 2,, 4, Toll-like receptor 2,4; TGFβ,, transforming growth factor beta; DNMT3b,, DNA Methyltransferase 3 Beta;
BTLA,, B and T lymphocyte attenuator; MMP9,, Matrix Metallopeptidase 9; BAFF, B-cell activating factor; RANTES,, Regulated upon Activation, Normal T Cell
Expressed and Presumably Secreted; VEGF,, Vascular endothelial growth fator.
* Correspondence to: Department of Oral & Maxillofacial Surgery and Periodontology, Ribeirão Preto Dental School, University of São Paulo - USP, Avenida do
Café, s/n - FORP-USP, Ribeirão Preto 14040-904, SP, Brazil.
E-mail address: dpalioto@forp.usp.br (D. Palioto).
1
Both the authors have contributed equally to the work.
2
Executing institution: Ribeirão Preto Dental School, University of São Paulo.

https://doi.org/10.1016/j.archoralbio.2022.105528
Received 29 March 2022; Received in revised form 16 August 2022; Accepted 18 August 2022
Available online 22 August 2022
0003-9969/© 2022 Published by Elsevier Ltd.
Y.D. Acqua et al.
play an important role in this breakdown transmuting commensal ho­
meostatic microbiota into a polymicrobial disease (Hajishengallis &
Lamont, 2012; Tonetti et al., 2018) - activation of an systemic immu­
noinflammatory response is not only dependent on specific periodontal
pathogens but an exacerbation of inflammatory condition that leads to
underlying tissues destruction.
Despite a considerable number of investigations, the mechanisms
involved in the pathogenicity of periodontal disease still need to be
determined (Borges et al., 2020) as well as its correlation with systemic
pathologies, which makes the study of the subject of paramount
importance. For the study of periodontal disease, several methods are
used such as the stimulation of periodontal disease by inducing patho­
genic microorganisms through injections of inactivated bacteria or
bacterial lipopolysaccharide (Klausen, 1991), through oral gavage
(Fiehn et al., 1992), ligature (Rovin et al., 1966) and ligature soaked in
bacteria (Kimura et al., 2000).
The great advantage of using experimental animal models are Ethical
considerations in conducting studies in humans make it necessary to
conduct them in animals and the benefit of allowing the longitudinal
study of the disease from the initial onset to the progression of the lesion
in the various tissues of the organs that make up the analysis (Graves
et al., 2008; Graves et al., 2012). However, there are many uncertainties
about which models are suitable for the induction of periodontitis, in
part due to the issues of disease onset, progression, and the events that
lead to the irreversible degradation of periodontal tissues, which allow
them to maintain their chronic nature as in humans (Graves et al., 2008;
Kinane & Hajishengallis, 2009; Oz Hs & Puleo DA 2011; Palioto et al.,
2019).
Taking into account the need for more studies that help to under­
stand the effectiveness of these models and analyze their local and
systemic outcomes, this systematic review seeks to compare and help to
better understand experimental models of periodontitis in mice.
2. Materials and methods
This study was conducted according to The Enhancing the QUAlity
and Transparency Of health Research (EQUATOR network) recom­
mendations, including the Preferred Reporting Items for Systematic
Reviews and Meta-Analyses PRISMA (Page et al., 2021); PRISMA 2020
checklist available as Supplemental Material S1. The synthesis of qual­
itative results followed the SWiM reporting guideline (Campbell et al.,
2020). Risk of bias within studies were assessed using specific risk of bias
and methodological quality assessment tools for SYRCLE’s tool for
studies in animals [Systematic Review Center for Laboratory animal
Experimentation] (Hooijmans et al., 2014). The review protocol at The
Open Science Framework Repo was registered (https://osf.io/pbq7r/):
DOI 10.17605/OSF.IO/PBQ7R.
2.1. Focused question
Based on the PICO principle—the Population: mice as an animal
model of experimentally induced periodontitis; the Intervention and the
appropriate Control (or comparator): different methods of induction of
experimental periodontitis in mice (e.g., oral inoculation, gavage, liga­
ture or combination between them); the Outcomes of interest: local and
systemic effect, such as alveolar bone loss, parameters/biomarkers of
local (gingival tissue) and systemic inflammation, bacterial colonization
confirmation, histological, immunohistochemical, immunofluorescence
and immunocytochemistry analyses of maxilla—the following focused
question was proposed: “What are the local and systemic effects of
different methods of induction of experimental periodontitis in mice?”.
2.2. Study selection criteria
Inclusion criteria: Experimental studies comparing the local and/or
systemic effects of two or more techniques/protocols for the induction of
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Archives of Oral Biology 143 (2022) 105528
experimental periodontitis in mice.
Exclusion criteria: i. studies of induction of periodontitis in humans
or other animal models other than mice; ii. studies that describe only one
type of induction technique/protocol of periodontitis; iii. lack of
experimentally induced periodontitis confirmation, review studies and
absence of outcomes of interest; iv. studies in which outcomes of interest
were not available for analysis and the original values could not be
retrieved after contacting the original authors; and v. unavailability of
full paper copy. No data or language restrictions were applied.
2.3. Information sources
Searches were performed in the MEDLINE using the PubMed search
engine (http://www.ncbi.nlm.nih.gov/sites/pubmed), Scopus (http://
www.scopus.com) and Embase (https://www.embase.com) through
Elsevier (https://www.elsevier.com), Web of Science – WOS (https://
www.webofknowledge.com) accessed through the Clarivate Analytics
(https://clarivate.com), Cochrane Library (https://www.cochraneli­
brary.com), LILACS and BBO (https://bvsalud.org), for articles pub­
lished until January 2022. Other sources were consulted through Google
Scholar (https://scholar.google.com.br) and System for Information on
Grey Literature in Europe (SIGLE) through OpenGrey (www.opengrey.
eu) databases. The protocol registration databases included Clin­
icalTrials.gov and ReBEC (Registro Brasileiro de Ensaios Clínicos).
Hand-searches were also performed in specialized periodicals (Step 1):
Journal of Periodontology; Journal of Clinical Periodontology; Journal
of Periodontal Research; Periodontology 2000; Journal of Periodontal &
Implant Science; The International Journal of Periodontics & Restor­
ative Dentistry; The Journal of American Dental Association; Journal of
Oral Pathology & Medicine; Oral Surgery, Oral Medicine, Oral Pathol­
ogy and Oral Radiology; and in reference lists of selected articles (step
2). There was no query to the database of register for experimental
protocol using animal. Experts were identified using expertscape.com
(https://expertscape.com) and contacted for other data sources.
2.4. Search strategy
The search strategy was created using MeSH terms, DeCS/MeSH
terms, Emtree terms and other free terms, combined by the Boolean
operators "OR" and "AND" (Table S1). The electronic search was per­
formed in August 2020. Databases alerts were created to identify studies
published after the time of the search, until January 2022.
Other sources were accessed by consulting the Google Scholar
(https://scholar.google.com.br), System for Information on Grey Liter­
ature in Europe (SIGLE) through OpenGrey (www.opengrey.eu) data­
base, and Open Science Framework – OSF (https://osf.io). Hand-
searches were performed in specialized journals ([Step 1] Journal of
Clinical Periodontology; Journal of Periodontal Research; Journal of
Periodontology; Journal of Dental Research; Periodontology 2000;
Clinical Oral investigations; The Journal of the American Dental Asso­
ciation; and Journal of Dentistry) and in reference lists of selected ar­
ticles (step 2). There was no query to the database of register for
experimental protocol using animal.
2.5. Selection process
The retrieved articles were exported to rayyan® reference manager
(https://www.rayyan.ai) and duplicates were removed by the program
and manually. Authors of studies that were not retrieved in full text were
contacted by e-mail up to five attempts. If two studies had sample
overlapping, and the same methodology criteria assessed, the least
complete study was excluded. The selection process was conducted in
two phases: Phase 1, two researchers (Y.S.D.A. and C.H.) independently
examined the titles and abstracts of all identified references, applying
the including process (blind process); and Phase 2, the same two re­
viewers independently applied the exclusion criteria to the other
Y.D. Acqua et al.
studies, based on reading the full text (blind process). Inter-reviewer
reliability in the study selection process was determined by the Cohen
κ test, assuming an acceptable threshold value of 0.80 (Landis & Koch,
1977). The disagreement at any stage was resolved by discussion and
mutual decision (consensus meeting) with two other reviewers (M.F.F.
and D.B.P.B.). The final decision/selection was always based on the full
text of the publication. Authors of studies not retrieved in full text were
contacted by e-mail up to five attempts.
2.6. Data collection process
The full texts were evaluated and judged in the entire document.
Authors were contacted through electronic mail, during five consecutive
weeks, when necessary to obtain details on study design and data clar­
ification. Data were extracted by two independent reviewers (Y.S.D.A.
and C.H.) from the included studies and described in the paper at a
consensus meeting with the two other reviewers (M.F.F. and D.B.P.B.).
When there were unclear or missed information, lack of data or when
the full text was not available, weekly attempts were made for up to five
weeks to contact the authors. In case there were no return from the
authors to identify data in graphs, it was used the digital program
WebPlotDigitizer® online (https://automeris.io/WebPlotDigitizer/).
The accuracy of extracted data was confirmed by another author (M.F.F.
and D.B.P.B.). In addition, if two or more studies had overlapping
samples and used similar methodologies, the most complete study was
included. Google translator program was used in case of studies in a
foreign language not provided by the researchers (https://translate.
google.com.br/?hl=pt-BR).
2.7. Data items and synthesis
Data were independently extracted by two reviewers (Y.S.D.A. and
C.H. [blinded process]) using a standardized sheet, as recommended by
the Cochrane Collaboration’s handbook for systematic review (Higgins
et al., 2022); the accuracy of extracted data was confirmed by another
authors (M.F.F. and D.B.P.B.). From the selected articles, the following
data were extracted: Author/Year, animal model, experimental perio­
dontitis/sample size, intervention, follow-up time, method of diagnosis,
result (alveolar bone loss) and secondary outcomes (Tables 2 and 3).
Descriptive results were presented in the form of text, figure and tables
in the order of the PICO strategy. The synthesis of qualitative results
followed the SWiM reporting guideline (Campbell et al., 2020).
Missing data or data available only in graphs were retrieved by
contacting the corresponding authors via e-mail and/or social media up
to five attempts. In case of no response, data available only in graphs
were extracted by the program WebPlotDigitizer® version 4.4 (https://
automeris.io/WebPlotDigitizer/), if necessary. In addition, if two or
more studies had overlapping samples and used similar methodologies,
the most complete study was included.
2.8. Risk of bias within studies
The methodological quality of the articles included in this review
was assessed by two reviewers (Y.S.D.A. and C.H.) using the SYRCLE’s
tool for studies in animals (Systematic Review Centre for Laboratory
animal Experimentation) to assess the risk of bias (Hooijmans et al.,
2014). The following items were evaluated: sequence generation,
baseline characteristics, allocation concealment, random housing,
blinding, random outcome assessment, blind outcome assessor, incom­
plete outcome data, selective outcome reporting and other sources of
bias. For each domain of bias, the trials were classified as representing
low, unclear or high risk of bias. The studies were independently eval­
uated by two authors, and agreement was reached in a consensual
meeting with a third (M.F.F.) and fourth author (D.B.P.B.). as needed.
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Archives of Oral Biology 143 (2022) 105528
3. Results
3.1. Study selection
8815 potentially relevant articles were identified through database
searching (Fig. 1), of which 848 after removing duplicates were
excluded. Of the 7967 selected studies, were excluded 7946 while
reading titles and abstracts. After assessing the full text or study design
of the remaining 21 publications, subject excluded 14 studies and 7
studies were included in the review (Table 1).
3.2. Study characteristics
There are many uncertainties about which models are appropriate
due to open questions about the onset and progression of periodontitis.
It is recognized that the host response plays an important role in the
degradation of periodontal tissue and that it is likely to involve innate
and acquired immunity. One of the most important uncertainties
regarding periodontal disease is its chronic nature. Therefore, to try to
elucidate these uncertainties, the characteristics of the included studies
are shown in Tables 2 and 3.
All studies performed comparison of different techniques for
inducing experimental periodontal disease; all included ligature groups,
ligature with Porphyromonas gingivalis (Lapérine et al., 2016;
Saadi-Thiers et al., 2013), oral gavage with Porphyromonas gingivalis (de
Molon et al., 2014, 2016; Duan et al., 2016; Palioto et al., 2019;
Saadi-Thiers et al., 2013), oral gavage with Porphyromonas gingivalis
associated with Fusobacterium nucleatum (de Molon et al., 2014, 2016).
One study performed the combination of two models of induction of
periodontal disease gavage and ligature (Palioto et al., 2019) of all
studies two evaluated the induction model Heat-killed Porphyromonas
gingivalis ATCC 33277 injected into the palatal mucosa (de Molon et al.,
2014; Lin M et al., 2017).
Ligature placement for local biofilm retention varied between the
maxillary first molar (de Molon et al., 2014, 2016; Lapérine et al., 2016;
Saadi-Thiers et al., 2013) and the second molar (Duan et al., 2016; Lin
et al., 2021; Palioto et al., 2019). Regarding the experimental time, the
longest were 67 (Saadi-Thiers et al., 2013) and 60 days (Palioto et al.,
2019) but Saadi-Thiers et al. (2013) compared the outcomes in two more
times (15 and 45 days). Lin et al. (2021) was the study that made a single
assessment in the shortest time (14 days), unlike Duan et al. (2016) who
chose a longer time to assess periodontitis (42 days). They were shorter,
medium and long: 7.15 and 30 days (de Molon et al., 2014) and Lapérine
et al. (2016) at 4,14 and 28 days.
When designing studies that assess the various steps required for
infection and periodontal disease, several parameters need to be
considered. It is clear that the infectious process occurs in several
distinct but interconnected stages. Examining each step independently
and interdependently can provide a unique window into the disease
process.
Bone homeostasis is maintained by a continuous physiological pro­
cess called bone remodeling. This process requires the balanced activity
of osteoblasts and osteoclasts. Excessive osteoclast activity leads to
pathological bone resorption, including periodontitis. Since periodontal
models cause alveolar bone destruction, they have been used in many
studies to reveal the pathological mechanisms or to discover a treatment
strategy for periodontitis.
3.3. Analysis of alveolar bone loss
Morphometric analysis of μCT was performed to assess trabecular
and cortical bone parameters at oral skeletal sites, this analysis was
preformed in most studies [n = 4] (de Molon et al., 2014, 2016;
Lapérine et al., 2016; Palioto et al., 2019), making it possible to obtain
two-dimensional results: from the cemento-enamel junction to the bone
crest alveolar and three-dimensional: bone volume of a region of interest
Y.D. Acqua et al. Archives of Oral Biology 143 (2022) 105528

Fig. 1. PRISMA flowdiagram.

observation regarding the evaluation of bone resorption in these studies

Table 1
was the tendency of this process to become chronic when added some
Study selection process based on full text assessment.
bacterial challenge, which can be seen through the ligature Porphyr­
Selection process (Phase 2) References omonas gingivalis groups (Lapérine et al., 2016; Saadi-Thiers et al., 2013)
Total full texts analyzed (n, 21) Barros et al. 2011; de Molon et al. 2013; in the Porphyromonas gingivalis injection group (de Molon et al., 2014)
de Molon et al. (2014);de Molon et al. and gavage induction (Palioto et al., 2019). In contradiction to de Molon
(2016);Duan et al. (2016);Graves et al.
et al. (2016) was the only one that did not find bone resorption by
(2008);Graves et al. (2012); Ideguchi
et al. 2019;Kimura et al. (2000);
gavage in a longer experimental time (45 days).
Lapérine et al. (2016);Lin et al. (2017); Biological evidence in the periodontal tissue allowed the evaluation
Maekawa et al. 2014;Marchesan et al. of the degradation of collagen fibers due to the chronic inflammation of
(2018);Oz and Puleo (2011);Palioto periodontitis, allowing the understanding of the reduction of the alve­
et al. (2019); Polak et al. 2010;
olar bone that eventually leads to tooth loss.
Saadi-Thiers et al. (2013); Sulijaya et al.
2019; Sun et al. 2020; Yoneda et al.
2001; Zhuang et al. 2019
Excluded (n, 14) Barros et al. 2011a; de Molon et al. 3.4. Histological analysis
a
Studies that describe only one type of 2013b;Graves et al. (2008)b;Graves et al.
induction technique/protocol of (2012)b;Kimura et al. (2000)a; Maekawa Were performed by two studies (de Molon et al., 2014, 2016) with
periodontitis et al. 2014c;Marchesan et al. (2018)a;Oz the objective of evaluating, respectively, the presence of inflammatory
b
Studies that were a literature review and Puleo (2011)b; Polak et al. 2010c;
c
Studies that performed some type of Sulijaya et al. 2019c; Sun et al. 2020c;
infiltrates in different regions of the maxilla and the degree of bone
procedure prior to the induction of the Yoneda et al. 2001a; Zhuang et al. 2019c resorption.
disease In de Molon et al. (2014) significant differences in gingival tissue
Included (n, 7) de Molon et al. (2014);de Molon et al. inflammation were reported at 7, 15 and 30 days after induction of
(2016);Duan et al. (2016);Lapérine et al.
periodontitis by ligature, while in the Injection-bacterial model local
(2016);Lin et al. (2017);Palioto et al.
(2019);Saadi-Thiers et al. (2013) inflammation was evident from day 15 and persisted over time. How­
ever, de Molon et al. (2016) did not observe inflammation or alveolar
bone resorption 45 and 60 days after periodontitis induction in both
that differed according to each study. In the remaining studies, these gavage with Porphyromonas gingivalis and gavage with Porphyromonas
data were obtained by digital stereomicroscopy (Lin et al., 2021), gingivalis and Fusobacterium nucleatum groups, unlike the local effects of
dissection microscope (Duan et al., 2016) and histomorphometry assay ligature represented by increased inflammatory infiltrate and increased
(Saadi-Thiers et al., 2013). They only presented two-dimensional cemento-enamel junction distance.
(linear) results measured from the cemento-enamel junction to the Saadi-Thiers et al. (2013) evaluated bone resorption by histo­
bone crest alveolar. morphometry. The authors reported an increase in the number of oste­
After the analysis, it was possible to observe that in most studies bone oclastic cells in the ligature with Porphyromonas gingivalis 15 and
losses were significant in the initial periods of induction in the ligature ligature15 groups, fifteen days after the intervention, compared with the
group, with the exception of Lapérine et al. (2016) that did not induce control, gavage and gavage positive control groups. In addition, bone
significant bone loss compared to the control group. Another important loss was time-dependent and more pronounced in ligature than ligature

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Y.D. Acqua et al. Archives of Oral Biology 143 (2022) 105528

Table 2
Summary of characteristics of included animal studies.
Author/ Animal Comparison Intervention Follow- Method of diagnosis Result (alveolar bone Secondary
Year Model groups up time loss) outcomes

Palioto C57BL/6 Control (Sham) silk ligatures around maxillary 60 days Micro-computed Was found that the Multiplex immunoassay
et al. male mice n=5 second molars was placed for 5 tomography (μCT) alveolar bone loss was Mice with LIG and GAV groups
(2019) 6–8 wk Ligature (LIG days. greater in the gavage induced significantly higher
group) n = 5 Oral inoculation of 109 P. and in the combination serum inflammatory mediators,
(Lig 5 days) gingivalis (ATCC 33277) model. in contrast to LIG+GAV, which
P. gingivalis After 5 days, the Ligature was was lower.
gavage (Pg- removed and the animals were Immunofluorescence
GAV) n = 5 inoculated with Pg. staining.
The A greater number of
combination of inflammatory and epigenetic
the two models mediators was observed in the
(LIG+GAV) periodontium of the GAV and
n=5 LIG + GAV group than in the
LIG group alone.
In the intestine: GAV greater
number of these markers
compared to LIG and LIG+GAV
Lin M C57BL/6 J Control 10 μL of live P. gingivalis ATCC 14 days ImageJ software The LIG group Real-time PCR and ELISA
et al., - WT P. gingivalis- 33277 (1 × 1010/mL in PBS) in revealed significantly assay
2017 -TLR2(KO) injection the sulcus of the maxillary greater bone loss in all The increased RANKL mRNA
-TLR4(KO) Ligature second molars. For 4 types of mice and protein expression induced
-TLR2 consecutive days after initial compared to the by P. gingivalis was diminished
&4KO infection control. when both TLR2 and TLR4 were
mice 8–10 silk ligatures around maxillary The P. gingivalis lacking. Moreover, the ligation-
wk second molars for 2 weeks infection group had induced increase of RANKL
significantly greater protein expression was TLR2-
bone loss compared to and TLR4-independent.
the WT and TLR2 KO
control group, but not
all groups were TLR4
dependent.
Laperine CD1 Swiss Ligature soaked 6.0 silk ligature wih Pg (ATCC 4, 14 Micro-computed LIGPg induced Imunohistoquímica (IHC)
O et al., mice in P. gingivalis 33277) 109 CFU/mL and tomography (μCT) significant alveolar RT-qPCR
2016 (Janvier) (LIGPg) 6.0 silk ligature around the 28 days bone loss that persisted ELISA for IL33
with 12wk Ligature first maxillary molar. until 28 days, LIG by LIG and LIG-Pg showed high
Ligature (sham) incision in the sulcular itself did not achieve levels of IL-33 at 4 and 14 days,
epithelium the same result. but for continuous expression
(at 28 days) this significant
increase was maintained only
in the LIG-Pg group.
de Molon C57BL/6 J Ligature (LIG) Around the first maxillary 45 and Micro-computed All groups showed Stereometric analysis
RS female P. gingivalis- molar. 60 days tomography (μCT) normal bone Only the LIG group showed a
et al., mice with gavage. (GAV) Inoculation of 1 × 109 CFU of architecture with the significant decrease in
2016 8 wk. P. gingivalis P. gingivalis (ATCC 33277 exception of the LIG fibroblasts and a significant
n = 48 with 100 μL of bacteria (50 μL of group increase in inflammatory cells
F. nucleatum - 1 × 109 CFU/mL of at 45 and 60 days compared to
gavage. P. gingivalis (ATCC 33277) and the other groups.
(GAVPgFn) 50 μL of 1 × 109 CFU of Histometric analysis
Control F. nucleatum (ATCC 25586)] Inflammatory infiltrate and
resorption of the interproximal
bone was observed only in the
LIG group
Duan X C57/BL6 P. gingivalis- 109 CFU P. gingivalis (ATCC 42 days Dissecting Females developed q- PCR analysis
et al., mice with gavage (GAV) 33277) microscope (×40 more bone loss than Infection GAV causes a low
2016 8–10wk. (female) Around the maxillary second magnification) male mice in the GAV level of inflammation and did
females (male) molar. Alveolar bone loss was model, although not not present a significant
and males Ligature (LIG) examined 7 days later. significant. difference between the sexes,
(female) Female mice are more but in LIG there was a greater
(male) susceptible to bone presence of cytokines and pro-
Control gavage loss in the LIG model. inflammatory proteins in
(CONTGAV) females resulting in more
severe periodontitis.
de Molon Female Control (CONT) Around the first maxillary 7–15– Micro-computed Alveolar bone loss was Histologic Analyses
RS C57BL/6 Ligature (LIG) molar and tomography (μCT) observed in the LIG The LIG model for 7, 15, and 30
et al., wild-type P.gingivalis- Oral inoculation of 1 × 109 30–days model on days 7 and days induced an inflammatory
2014 mice. 8wk gavage (GAV) CFU of Pg (ATCC 33277) 15, with a decrease in response an intense infiltration
n = 108 P.gingivalis with directly into the oral cavity of the intensity of bone of inflammatory cells, CT
F.nucleatum mice with a micropipette. loss during the 30-day attachment loss (AL), and
gavage Pg (1 × 109 CFU cells per mL) period, whereas in the alveolar bone resorption. The
Heat-killed Pg was mixed with an equal heat-killed Pg injection heat-killed Pg model displayed
injected into volume of Fn (ATCC 25586) model from day 15 an increased influx of
the palatal 0.5 μL 1 × 1010 CFU/mL heat- there was significant inflammatory cells and alveolar
mucosa killed Pg diluted in PBS. bone loss from day 15.
(continued on next page)

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Y.D. Acqua et al. Archives of Oral Biology 143 (2022) 105528

Table 2 (continued )
Author/ Animal Comparison Intervention Follow- Method of diagnosis Result (alveolar bone Secondary
Year Model groups up time loss) outcomes

(i-LPS) loss that continued to Quantitative Analyses of

Phosphate- progress over time mRNA Expression
buffered saline All cytokines were more
injected into pronounced in the LIG model
the palatal with a reduction on day 15. In
mucosa the heat-killed Pg model group,
this increase was visible during
the 15-day period, but with no
significant difference between
the groups.
Saadi- C57BL/6 J Ligature (LIG) In the first maxillary molars. 15–45 Histomorphometry Alveolar bone loss was Clinical and histologic
Thiers male mice (15 days) Sterilized black braided 6.0 and 67 and osteoclast TRAP increased in LIGPg 15 examination.
K et al., with (45 days) silk threads were incubated in days. assay. compared to LIG15, Significant epithelial degrowth
2013 6–12wk Ligature soaked culture medium containing Pg but was significantly observed in LIGPg15 and LIG15
n = 50 in P. gingivalis ATCC 33277 in an anaerobic higher compared to compared to CONT15, GAVtest
(LIGPg) chamber for 1 day. CONTROL15, GAVtest and GAVcontrol.
(15days) 109 UFC Pg suspended in and GAVcontrol. Blood Cytokine Levels
(30 days) 100 μL PBS containing 2% Osteoclastic cells were The amount of IL-6 was more
P.gingivalis- carboxymethylcellulose. more numerous in the than twice as elevated in LIGPg
gavage. LIGPg15 group than in mice compared to other groups.
(GAVtest) the LIG15 group.
(GAVcontrol)
Control
(15 days)
(45 days)

Table 3
Analysis methods used in the studies.
Analysis methods used in the studies Palioto et al. MeinLin et al. Laperine O de Molon RS Duan X et al., de Molon RS Saadi-Thiers K
(2019) (2017) et al., 2016 et al., 2016 2016 et al., 2014 et al., 2013

Alveolar bone loss + + + + + + +

(micro-ct) In vivo * **
Levels of serum cytokines + – + – – – +
PCR(bacterial colonization confirmation) – – – + + – –
PCR gingival tissue – + + – + + –
(inflammatory biomarkers)
Enzyme-linked immunosorbent assay – + – – – – –
(ELISA)- gingival tissue
Immunohistochemical analysis – maxilla – – + – + – –
Histological analysis – – – + – + +
Maxilla
Immunofluorescence analysis + – – – – – –
Intestinal tissue
Immunofluorescence analysis + – – – – – –
Maxilla
Immunocytochemistry analysis – – – – – – +

* analysis of bone loss made by dissection microscope

* * histomorphometric analyses

with Porphyromonas gingivalis mice.

Table 4
Periodontal disease is a prevalent disease that results in the loss of
Inflammatory biomarkers per study.
gingival tissue, which provides the first line of defense against various
environmental and microbial irritants. In particular, epithelial cells are Biomarkers References

interconnected by tight junctions, adherence junctions, desmosomes, IL1β, IL6, RANKL, OPG de Molon et al. (2014)
and gap junctions, forming an epithelial barrier at the surface layer of IL1β, IL6, IL10, IL17, TNFα, TGFβ Duan et al. (2016)
IL1β, IL10, TNFα and Lin et al. (2017)
the gingival tissue.
RANKL(ELISA e PCR)
Three studies evaluated local inflammation by the PCR method (de
Molon et al., 2014; Duan et al., 2016; Lin et al., 2021). Among them, one Footnote: IL1β, interleukin 1 beta; IL6, interleukin 6; IL10, interleukin 10; IL17,
study evaluated the expression level of RANKL also by ELISA (Lin et al., interleukin 17; TNFα, tumor necrosis factor alpha; TGFβ, transforming growth
factor beta; RANKL, receptor activator of nuclear factor-kappa beta ligand; OPG,
2021). The biomarkers evaluated in each study are summarized in Ta­
osteoprotegerin.
bles 4 and 5.
It was possible to notice that the induction time is a factor capable of
influencing the behavior of the biomarkers, interleukin 6, 1β, RANKL all markers in 15 days (de Molon et al., 2014).
and osteoprotegerin. Presenting more expressive levels in the ligature In Lin et al., 2021 who compared groups: control, LPS injection and
group 7 days after induction of periodontitis with a significant reduction ligature; in 4 mouse strains: WT, TLR2&4 gene knockout, TLR2 gene
after 15 days. However, when observing the other experimental group knockout and TLR4 gene knockout by analyzing their results we
(Porphyromonas gingivalis infection) there was a tendency to increase for observed that each biomarker may be able to express itself in different
ways in the same type of induction of periodontitis. In the case of the

6
Y.D. Acqua et al. Archives of Oral Biology 143 (2022) 105528

Table 5
Risk bias assessment.
Items Risk of Bias Palioto et al. MeinLin et al. Laperine O et al., de Molon RS et al., Duan X et al., de Molon RS et al., Saadi-Thiers K et al.,
(2019) (2017) 2016 2016 2016 2014 2013

Sequence generation – – – – – – –
Baseline characteristics ± – – – – – ±
Allocation concealment ± ± ± – ± – ±
Random housing ± – – – + – –
Blinding + + + + + + +
Random outcome + + + + + + +
assessment
Blind outcome assessors ± – ± – ± – –
Incomplete outcome ± – – – + – –
data
Selective outcome – – – – – – –
reporting
Other sources of bias – – – – – – –

Keys
þ High risk of bias
- Low risk of bias
± Unclear risk of bias

Porphyromonas gingivalis group, the interleukin10 marker was signifi­
cantly higher in WT mice compared to the control, but the same did not
occur with TNF-α and interleukin 1β, which did not obtain significant
differences compared to the control group for any of the 4 types of mice.
When verifying within the ligature group the cytokine interleukin 1β
was the most expressive compared to the control group for the 4 types of
animals, whereas the opposite happened with TNF-α. However, inter­
leukin 10, despite not being significant in 3 types of mice: WT, TLR2
gene knockout and TLR4 gene knockout, showed significantly lower
levels in TLR2 & 4 gene knockout (Lin et al., 2021).
It was also possible to note in this study that a single biomarker may
present itself differently for each induction model. As shown by inter­
leukin 1β that in the ligature group showed significantly higher levels
compared to the control group for the 4 types of mice. However, in the
Porphyromonas gingivalis infection group, its levels did not change
significantly for any type of mouse selected in the study (Lin et al.,
2021).
When the RANKL protein (ELISA) was analyzed, it also behaved
differently, depending on the type of each animal and on the induction
model: in Porphyromonas gingivalis-induced periodontitis, its expression
was lower in the absence of TLR2 and TLR4, but in ligature, there was a
greater expression being independent of TLR2 and TLR4. In the mRNA
analysis method (qPCR), its levels were significantly higher in the 2
models, compared to the control for some types of mice (Lin et al.,
2021).
Furthermore, the sex of C57BL/6 mice can also interfere with the
expression of inflammatory or anti-inflammatory markers such as
interleukin 1β, 6, 10, 17, TNF-α and TGF-β. Among these, interleukin
17,1β and 6 were higher in females compared to males and TNF-α, TGF-β
and interleukin 10 did not have significant differences between sexes in
the ligature model. In the experimental model of gavage, low levels of
expression of these markers were found, not inducing a statistical dif­
ference between the sexes (Duan et al., 2016).
This systematic review forces an interesting shift in the paradigm
that chronic periodontitis needs a systemic disturbance and not just a
local challenge to develop the complex pathology of periodontal disease
and its possible relationship to systemic diseases. However, the under­
lying mechanism is still unclear and it is difficult to demonstrate the
mechanisms by epidemiological and/or interventional studies in
humans. In this section, we review the relationship of pro-inflammatory
immune mediators as well as the expression of some epigenetic markers.
3.5. Immunohistochemical analysis
immunohistochemistry in two studies (Duan et al., 2016; Lapérine et al.,
2016). Duan et al. (2016) performed histochemical experiments after
the discrepancy found by PCR in the expression of interleukin 17 A
compared to other cytokines. Thus, they looked for activities of inter­
leukin 17 A, 1β and 6 in gingival tissues in mice from the ligature group
compared to the control group. As expected, there was confirmation that
interleukin 17 A is the most expressing protein and its levels were higher
in female mice than in male mice. The other two cytokines (inter­
leukin1β and 6) were also elevated after periodontitis induced by liga­
ture compared to the control, but there was no significant difference
between genders (Duan et al., 2016).
The other study only evaluated the presence of interleukin 33, this
marker was elevated after periodontitis induction in both the ligature
and Porphyromonas gingivalis-embedded ligature groups compared to the
positive control group, but this level remained elevated for a longer time
(28 days) only in the ligature with Porphyromonas gingivalis group
compared to the ligature group (Lapérine et al., 2016).
3.6. Immunofluorescence and Immunocytochemistry analysis
Cell activity in maxillary samples was evaluated in C57BL/6 mice by
immunofluorescence (Palioto et al., 2019) and immunocytochemistry
(Saadi-Thiers et al., 2013). In the first study, immunofluorescence was
performed in order to analyze epigenetic (DNMT3b) and inflammatory
(BTLA and interleukin18R1) markers, all of which were found in greater
quantities in the group that associated ligature with gavage and in the
group with only gavage compared to the ligature alone group (Palioto
et al., 2019). In the other study, three experimental groups (gavage,
ligature and ligature with Porphyromonas gingivalis) and two control
groups (ligature positive control and gavage positive control) evaluated
the expression of CATB and MMP-9. Regarding CATB, this was not
detected in the ligature group, 15 days after ligature placement, but it
was expressed in the same period in the group in which Porphyromonas
gingivalis was added to the ligature; in the gavage group, this marker was
detected in only a few cells (Saadi-Thiers et al., 2013).
MMP-9 mainly detected in connective tissue and periodontal liga­
ment cells in the ligature15 and ligature Porphyromonas gingivalis 30
groups, but it can be found in leukocytes and osteoclasts in all experi­
mental models (Saadi-Thiers et al., 2013). A note on the immunocyto­
chemical analysis of this study: it appears that their purpose was only to
describe the effects or confirm the effectiveness of the models to induce
periodontitis in mice lacking statistical data comparing the models
(Saadi-Thiers et al., 2013).

The local expression of cytokines was evaluated by

7
Y.D. Acqua et al.
3.7. Analysis of systemic inflammation
Lapérine et al. (2016), Palioto et al. (2019) and Saadi-Thiers et al.
(2013) investigated the systemic impact of periodontitis. Serum levels of
cytokines and other immunoinflammatory markers were evaluated in
each study. Such as: BAFF, IP-10, RANTES, RANKL and VEGF (Palioto
et al., 2019), interleukin 6 and 1β (Saadi-Thiers et al., 2013) and
interleukin 33 (Lapérine et al., 2016). Furthermore, Palioto et al. (2019)
evaluated by immunofluorescence the expression of BTLA and inter­
leukin 18R1 in the intestine (Palioto et al., 2019).
The gavage induction model led to a higher expression of BTLA and
interleukin 18R1 in the intestine compared to the ligature and ligature
+ gavage groups. In serum, both ligature and gavage significantly
increased serum levels of the biomarkers BAFF, interleukin 10, RANTES,
RANKL and VEGF. However, the association of ligature+gavage resulted
in lower serum levels of these markers compared to the methods used
alone (Palioto et al., 2019).
Saadi-Thiers et al. (2013) reported twice as high serum levels of
interleukin 6 in the ligature Porphyromonas gingivalis group compared to
ligature and gavage. However, the authors observed a reduction in the
concentration of this cytokine during the experiment, except in the
ligature group, in which there was a significant increase over time. In
that same study, another inflammatory cytokine (interleukin 1β)
behaved in the opposite way, increasing its levels over time
(15–30 days) in the ligature Porphyromonas gingivalis model and
decreasing in the ligature group (15–45 days) (Saadi-Thiers et al.,
2013).
Lapérine et al. (2016) after analysis by ELISA did not find increased
levels of interleukin 33 in serum in any of the experimental groups
(ligature Pophyromonas gingivalis and ligature) suggesting that perhaps
this cytokine is not involved in a systemic inflammatory process
(Lapérine et al., 2016).
3.8. Summary
The pathological mechanisms underlying periodontitis have not
been fully elucidated. Here, model mice of ligature, gavage, injection of
bacteria, ligation embedded in bacteria and association between them
were evaluated to examine the effects of periodontitis on periodontal
tissues; however, these studies focused primarily on individual tissues
and cells. Furthermore, no studies have investigated the effect of peri­
odontitis on cementum, bone, ligament, epithelium to find out from
which tissue the disturbance among the surrounding tissues originates.
As a result, ligation-induced periodontitis appears to be a model of acute
periodontitis and cannot reflect long-term bone loss and inflammatory
infiltration in chronic periodontitis.
3.9. Risk of bias analysis
When analyzing the 10 items necessary for the analysis of risks of
bias (Table 5), in three items: sequence generation, selective outcome
reporting and other sources of bias, all articles included in the review
(n = 7) presented low risk. The opposite can be observed in the items:
blinding and random outcome assessment where all articles pointed to
high risk. The topic “allocation concealment” was the one that presented
the most unclear risks (n = 5) (Duan et al., 2016; Lapérine et al., 2016;
Lin et al., 2021; Palioto et al., 2019; Saadi-Thiers et al., 2013); “baseline
characteristics” mostly showed low risk (n = 5) and only two articles
presented unclear risk (Palioto et al., 2019; Saadi-Thiers et al., 2013).
When analyzing the items “random housing” and incomplete
outcome data, only Duan et al. (2016) presented high risks for both
items and Palioto et al. (2019) unclear risks; the other articles (n = 5)
showed low risk. Finally, “blind outcome assessors”, were identified
only three studies with unclear risks (Duan et al., 2016; Lapérine et al.,
2016; Palioto et al., 2019) the others classified as low risk.
8
Archives of Oral Biology 143 (2022) 105528
4. Discussion
This systematic review was carried out to compare different experi­
mental models of periodontitis in mice and to sort through their local
and systemic outcomes. Animal models are widely used to establish
cause and effect relationships and mechanistic signaling via. Their se­
lection will depend on the purpose of each study. However, it would be
important to consider the ability to simulate a chronic immunoin­
flammatory process, similar to what is observed in periodontitis in
humans (Graves et al., 2008; Usui et al., 2021).
Rats and mice are the most used to investigate biological mecha­
nisms in periodontitis. In this article, we chose to include only studies
with mice, as these animals have greater availability of transgenic
strains, are relatively inexpensive, have faster metabolism, establishing
a better cause-and-effect relationship between periodontal disease and
the host’s immune response (Graves et al., 2012; Liang et al., 2010).
In view of the existence of several models of experimental peri­
odontitis, the present article included two main types that induce peri­
odontal disease through different pathways, one through local challenge
ligature and another through systemic microbial challenge gavage. As
well, their combinations ligature+gavage and associations ligature
bacteria and injection-bacteria were included. These different induction
mechanisms raise the question of how much they may differ in terms of
the chronicity of periodontal disease, the host response pathways and
the duration or intensity of stimuli.
The ligature model involves some questions despite of the fact that
the rapid and expressive alveolar bone loss can be observed around
seven days (de Molon et al., 2014) - an acute picture is established and it
might reflect a completely different event comparing to a chronic
pathogenesis that is characteristic of periodontitis (Lin et al., 2021).
Although Abe et al., (2013) report that ligature placement in mice causes
alveolar bone loss by the accumulation of endogenous bacteria (Abe &
Hajishengallis, 2013), others argue that ligature placement invariably
leads to traumatic injury, and only secondarily, it acts as a biofilm
retention factor (Li et al., 2020; Marchesan et al., 2018). There is also the
claim that the traumatic nature of its placement would be an important
factor in the production of periodontal tissue destruction (Jiao et al.,
2013).
Although some studies try to keep the ligature in position for a longer
period so that a constant and expressive induction of the disease occurs
(de Molon et al., 2016; Wu et al., 2020), there is the idea that bone loss
cannot be sustained over time. Requiring combination with bacteria to
establish a chronic condition of periodontitis (Lin et al., 2021; Palioto
et al., 2019).
Following this idea, gavage models are seen as a good option when
one wants to achieve chronicity and host modulation (Boyer et al.,
2020). However, there are still certain contradictions between the re­
sults obtained and the amount of inoculation performed during the
experimental period (Hariyani et al., 2021). This discrepancy can be
observed through two studies included here (de Molon et al., 2014;
Palioto et al., 2019). de Molon et al. (2014) with five inoculations found
less bone loss compared to ligature. Nonetheless, only bone measures
were taken and no approach to distinguish the models for systemic in­
flammatory process was done. Palioto et al. (2019) found significant
bone resorption after 6 inoculations (gavage of Porphyromonas gingivalis)
every other day, during two consecutive weeks and the results were
maintained until a period of 42 days from the start of the induction. The
study characterized the inflammatory and epigenetic outcomes in the
maxilla and in the intestines as being diverse between gavage and
ligature.
When comparing the gavage model and its combination with ligature
versus ligature, a longer time to establish bone loss (4–6 weeks) is noted
(Hart et al., 2004),but unlike the ligature group, this result can still be
observed even after withdrawal of the stimulus in the groups in which
the periodontal pathogen was added. This suggests that the systemic
microbial challenge (gavage) or its combination with the local challenge
Y.D. Acqua et al.
(ligature+gavage) may be good and perhaps the most similar strategies
to model human periodontal disease.
However, an interesting fact to be highlighted is the controversial
results found by Palioto et al. (2019) in which the combination of
models (Ligature+Gavage) modified the systemic (serum) expression of
inflammatory markers differently than the two models by itself - when
gavage was associated to ligature a more pro-resolutive process took
place. The authors speculates that the ligature removal would elicitate a
pro-resolutive process that counteracts the proinflammatory processes
of gavage itself.
Regarding the local association of bacteria and ligature, it was
possible to observe that the bacterial presence can aggravate the in­
flammatory response to a mechanical injury, in addition to generating a
more intense systemic response than ligature or gavage alone (Saa­
di-Thiers et al., 2013). This condition can be seen in the study by
Lapérine et al. (2016), as through this model they managed to obtain
significant bone loss that persisted up to 28 days after periodontitis in­
duction, unlike the ligature model that did not induce relevant loss
compared to a group control (Lapérine et al., 2016).
de Molon et al. (2014) and Lin et al. (2021) inducted periodontitis by
Injetion-bacteria among their experimental groups. It was possible to
observe the efficiency of this model as both were able to induce bone
loss, being significant from 15 days of initial induction continuing to
progress with time - the gingival tissues expressed a high peak of
inflammation in the first days, but this condition was not maintained
throughout the experiment (de Molon et al., 2014). The same was
observed in Lin et al. (2021) where the results were quite heterogeneous
and most inflammatory mediators were not significantly expressed.
When cellular activities were observed by immunofluorescence and
immunocytochemistry in the maxilla, inflammatory (BTLA and IL-18R1)
and epigenetic (DNMT3b) markers were more expressive in the gavage
and Ligature+Gavage models compared to the ligature group alone
(Palioto et al., 2019). The increased presence of these markers on the
surface of the alveolar bone shows an intense innate and adaptive im­
mune response (IL-18R1), in addition to identifying possible systemic
inflammation (BTLA) (Shubin, Monaghan, Heffernan, Chung, & Ayala,
2013) and DNA methylation (DNMT3b) contributes to a deregulated
inflammatory process and is a sign of recognition of changes in the
epigenetic framework (Benakanakere, Finoti, Palioto, Teixeira, & Kin­
ane, 2019).
Regarding the systemic impacts that some induction models, espe­
cially those of oral infection and gavage, cause on the host, it is possible
to create a bridge between studies of periodontitis and systemic diseases
(Mata et al., 2021; Parvaneh et al., 2021; Tsuzuno et al., 2021). Studies
suggest that oral administration of Porphyromonas gingivalis, the main
periodontopathogenic bacterium used in these models, alters the intes­
tinal microbiota and increases the levels of inflammatory markers in
systemically compromised mice, which can worsen the host’s systemic
condition (Hamamoto et al., 2020; Sato et al., 2017). Among all the
studies included in this article, only Palioto et al. (2019) evaluated the
effects of experimental periodontitis on the intestine, and showed that,
when a systemic bacterial challenge was chosen there was an increased
rates of inflammatory and epigenetic markers when compared to local
induction and also, with association of local induction and bacterial
challenge as well. Considering that the intestinal microbiota commu­
nicates with peripheral organs and its microbial dysbiosis directly im­
pairs host modulation influencing the development of several diseases
such as obesity, inflammatory and immunological diseases (Schroeder &
Bäckhed, 2016), the hypothesis is that gavage models can be the main
candidates when studying the association between periodontitis and
systemic diseases (Kato et al., 2018; Tsuzuno et al., 2021).
During the search for data for this review, it was possible to conclude
that in the literature, there are many methodological variations between
the induction models and they differ in terms of the animals used bac­
terial inoculations, number of days of ligature in position.
After this comprehensive search, many methodological variations
9
Archives of Oral Biology 143 (2022) 105528
were observed between the induction models that differ in relation to
the animals used, bacterial inoculations and number of days of ligation
in position. Regarding the extraction of data on the characteristics of
each article included here (Table 2) we could observe that were more
used C57BL/6 J mice and the most present induction methods were
ligature and gavage. In ligature models, this was preferentially placed
around the maxillary first molar; in gavage the bacterium Porphyr­
omonas gingivalis (ATCC 33277) was the most used. Moreover, the most
used evaluation method for alveolar bone loss results was computed
micro tomography.
The main strengths of this work included a systematic review
methodology with a reproducible search strategy. However, the review
was more limited, as the search was performed for studies that used and
compared more than one method of inducing periodontitis and, only in
mice, selection bias languages, in addition to the difficulties in obtaining
numerical data from the different selected studies, which made difficult
to carry out a meta-analysis.
Despite the small number of selected articles (n = 7), when
analyzing the items for risk of bias according to SYRCLE’s, most of the
included studies showed a low risk of bias for all topics, leaving some­
thing to be desired only in the blinding and random outcome re­
quirements; assessment (Table 5) showing to be quality and reliable
studies.
When analyzing the items “random housing” and "incomplete
outcome data", only Duan et al. (2016) presented high risks for both
items and Palioto et al. (2019) unclear risks; the other articles (n = 5)
showed low risk. Finally, regarding “blind outcome assessors”, only
three studies showed unclear risks (Duan et al., 2016; Lapérine et al.,
2016; Palioto et al., 2019) and the others were classified as low risk.
These risks of bias do not represent a systematic distortion of the effi­
ciency of experimental models of periodontitis.
The domain “random housing” does not represent selection bias or
confounding, as the primary studies used isogenic and homogeneous
animals in terms of age, body weight, male/female ratio and health
status (healthy animals).
The domain "incomplete outcome data" corresponds to data not
shown, which may be requested from the authors. However, these data
do not compromise the evaluation of the experimental models of
periodontitis.
The lack of blinding outcome assessors in animal model experiments
implies a considerable risk of observer bias towards subjective results
(Bello et al., 2014), which does not apply to this systematic review. In
addition, the three studies were classified as uncertain risk of bias
because they did not provide a precise description of the measures used
to blind outcome assessors from knowing which intervention each ani­
mal received. Thus, as non-subjective quantitative results were
compared, evaluated by systematic methods, this condition should not
be considered a confounding bias.
Therefore, despite the variability of the models found in the litera­
ture, few studies evaluate and compare the temporal evolution and the
local and systemic results of experimental periodontitis through
different induction models.
This review sought, as far as possible, to present these questions in an
attempt to assist in choosing an experimental model that is the most
appropriate to answer the question posed by each type of study so that
its results are more assertive.
5. Conclusion
This review provided ample evidence related to the models of in­
duction of periodontitis in mice and their outcomes, suggesting that
although most of them are efficient in causing alveolar bone loss, there
are differences in their characteristics. In cases of ligature is more pre­
sent the acute process, as the reabsorption is noticeable in a short period,
but after the removal of the local stimulus, the host tends to repair itself,
decreasing the significance of this loss over time. When a bacterial
Y.D. Acqua et al.
challenge is added (Ligature Porphyromonas gingivalis, Gavage or
Injetion-bacteria), bone loss appears more significant with longer in­
duction time, establishing a chronic process over time. Regarding sys­
temic outcomes, gavage showed greater potential for modulating the
host response with systemic inflammatory changes, showing to be more
promising when studying the relationship between periodontitis and
chronic systemic diseases.
Ethical statement
All authors declared that this research has follow the ethical
procedure.
Funding
This paper was supported by University of São Paulo (USP) and the
Federal University of Pernambuco (UFPE) for all the support, and the
following Brazilian funding agencie: São Paulo Research Foundation
(FAPESP N◦ 2019/20103-5).
CRediT authorship contribution statement
Yasmin DalAqua and Cristhiam Hernández: Contributed to the
conception and design of the study, acquisition of data, analysis and
interpretation of data, writing the original draft and final approval of the
version to be sub-mitted. Mariana Fogacci: Contributed to the analysis
and interpretation of data, revising it for important intellectual content
and final approval of the version to be submitted. David Barbirato:
Contributed to the conception and design of the study, drafting the
article, revising it critically and final approval of the version to be
submitted. Daniela Palioto: Contributed to the conception and design
of the study, analysis and interpretation of data, drafting the article and
critically revising it for intellectual content and final approval of the
version to be submitted.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors acknowledge the financial support provided by the
following Brazilian funding agencies: Coordination for the Improvement
of Higher Education (CAPES), National Council for Scientific and
Technological Development (CNPq) and the São Paulo Research Foun­
dation (FAPESP N◦ 2019/20103–5). We are also grateful to the Uni­
versity of São Paulo (USP) and the Federal University of Pernambuco
(UFPE) for all the support.
Compliance with ethical standards
Ethical approval was not required for this study.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.archoralbio.2022.105528.
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