Received: 22 March 2022 Revised: 4 October 2023 Accepted: 15 October 2023

DOI: 10.1111/jcpe.13896

ORIGINAL ARTICLE

Clinical, immunological and microbiological evaluation of

experimental peri-implant mucositis and gingivitis in subjects
with Grade C, stage III/IV periodontitis background

Tamires Pereira Dutra 1,2 | Mabelle Freitas Monteiro 1 |

1
Isabela Lima França-Grohmann | Renato Corrêa Viana Casarin 1 |
Márcio Zaffalon Casati 1 | Karina Gonzalez Silvério Ruiz 1 | Purnima S. Kumar 2 |
Enílson Antônio Sallum 1

1
Department of Prosthodontics and
Periodontics, Division of Periodontics, Abstract
Piracicaba Dental School, University of
Aim: To compare individuals with a periodontitis background (Grade C, stage III/IV—
Campinas, Piracicaba, São Paulo, Brazil
2
Department of Periodontics and Oral
formerly generalized aggressive periodontitis) (H-GAP) with periodontally healthy
Medicine, University of Michigan – School of subjects (H-Health) in terms of molecular changes (immunological/microbiological)
Dentistry, Ann Arbor, Michigan, USA
accompanying experimental peri-implant mucositis and gingivitis.
Correspondence Materials and Methods: H-GAP and control (H-Health) subjects were recruited, and
Tamires Pereira Dutra, 1011 N. University
Avenue, Ann Arbor, MI 48109-1078, USA.
experimental mucositis/gingivitis was induced around a single screw-retained implant
Email: [email protected] and one contralateral tooth. Participants refrained from oral hygiene for 21 days in

Funding information
the selected areas, followed by professional prophylaxis and hygiene instructions for
Brazilian National Council for Scientific and 21 days. Clinical parameters, immunological markers (multiplex analysis) and microbial
Technological Development (CNPq),
Grant/Award Number: 301635/2019-6; data (16S rRNA gene sequencing) were collected at baseline, during induction
Fundação de Amparo à Pesquisa do Estado de (7, 14 and 21 days) and following remission (42 days).
São Paulo, Grant/Award Numbers:
2018/06115-8, 2019/01522-7
Results: Clinically, no significant differences were observed between the groups
(n = 10/each group) (H-GAP vs. H-Health) (p > .05, Mann–Whitney test) and the
type of site (tooth vs. implant) (p > .05, Wilcoxon test) at the time of onset and reso-
lution, or severity of gingival/mucosal inflammation. H-GAP displayed lower concen-
trations of the cytokines interleukin (IL)-1B, IL-4, IL-17, tumor necrosis factor-α and
interferon-γ around implants than H-Health at baseline and during induction of
mucositis (p < .05, Mann–Whitney test). In both groups, implants showed signifi-
cantly higher inflammatory background at baseline and all subsequent visits when
compared with teeth (p < .05, Wilcoxon test). Alpha and β-diversity metrics showed a
significant shift in the microbiome composition and abundances of core species dur-
ing induction and resolution of peri-implant mucositis and gingivitis (p < .05,
restricted maximum likelihood method of Shannon and Bray–Curtis indices, respec-
tively). Differences were not significant for these parameters between the H-Health
and H-GAP groups when the periodontal and peri-implant microbiomes were

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© 2023 The Authors. Journal of Clinical Periodontology published by John Wiley & Sons Ltd.

J Clin Periodontol. 2023;1–13. wileyonlinelibrary.com/journal/jcpe 1

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2 DUTRA ET AL.

compared separately; however, at each time point, the peri-implant microbiome dif-
fered significantly from the periodontal microbiome.
Conclusions: Within the limitations of this pilot study (e.g. low power), it can be con-
cluded that different microbial shifts contribute to the onset and progression of
inflammatory responses around teeth and implants and that history of periodontal
disease experience plays an additional role in modulating the immune response of
peri-implant and periodontal tissues to biofilm accumulation.

KEYWORDS
dental implants, gingivitis, inflammation, microbiome, periodontal disease

Clinical Relevance
Scientific rationale for study: Microbiological/immunological features associated with experimen-
tal peri-implant mucositis/gingivitis in patients with Grade C, stage III and IV periodontitis back-
ground are not fully understood.
Principal findings: Implants present with a higher inflammatory background than teeth even in
health. Pro-inflammatory responses of the peri-implant mucosa to progressive biofilm accumula-
tion are of higher magnitude than those of gingiva. History of periodontitis predisposes to a
dampened inflammatory response to biofilm accumulation around implants.
Practical implications: Implants differ from teeth (considering molecular changes) in individuals
with a periodontitis background, emphasizing the need for further studies on long-term care
and maintenance of implants, especially in patients with periodontitis.

1 | I N T RO DU CT I O N validated to study the development and resolution of inflammation in

the peri-implant sulcus of periodontally healthy subjects (Clever
Tooth loss is a serious health problem that affects the quality of life et al., 2019; Meyer et al., 2016; Pontoriero et al., 1994; Ribeiro
(GBD, 2020; WHO Global Oral Health Status Report, 2022). There- et al., 2018; Schincaglia et al., 2017; Schwarz et al., 2014; Zitzmann
fore, the introduction of dental implants to replace missing teeth has et al., 2001). Additionally, it has been reported that in experimental
become one of the great achievements in dentistry. Despite the high gingivitis trials, patients with a history of periodontitis show a higher
success and survival rates of implant therapy, complications associ- rate of plaque accumulation and earlier development of redness and
ated with infection and inflammation do occur. bleeding on probing when compared with periodontally healthy indi-
According to the literature, the factors associated with peri- viduals (Abbas et al., 1986; Johnson et al., 1997; Van der Velden
implant diseases are poor oral hygiene, cigarette smoking, diabetes, et al., 1985). Furthermore, in periodontally healthy patients, the pro-
alcohol consumption and genetic traits (Heitz-Mayfield, 2008; gression events of peri-implant mucositis were found to be similar to
Pimentel et al., 2018; Sgolastra et al., 2015), with a history/ those of experimental gingivitis but led to different clinical, immuno-
background of periodontitis considered the strongest predictor of logical and microbiological responses (Nagasawa et al., 2022). How-
peri-implantitis (Anderson et al., 2020). Patients with a history of ever, to our knowledge, there is no study comparing tooth and dental
aggressive periodontitis (Armitage, 1999) are reported to have higher implant in patients with different periodontal backgrounds using a
incidences of bone loss and peri-implantitis and lower rates of implant controlled and reversible method that enables examining the clinical,
success and survival (De Boever et al., 2009; Mengel et al., 2007; microbiological and host-response shifts.
Mengel & Flores-de-Jacoby, 2005; Swierkot et al., 2012; Theodoridis Hence, the primary aim of this investigation was to compare indi-
et al., 2017). Therefore, although implant therapy is a reliable and safe viduals with a periodontitis background (Grade C, stage III/IV, previ-
technique, rehabilitation of patients with a history of aggressive peri- ously called generalized aggressive periodontitis) (H-GAP) with
odontitis remains a challenge for researchers and clinicians and there periodontally healthy subjects (H-Health) in terms of molecular
is an urgent need to better understand the factors that contribute to changes (immunological/microbiological) accompanying experimental
peri-implant disease in these individuals. peri-implant mucositis and gingivitis. The secondary aim was the com-
Experimental gingivitis has been long established as a model to parison of dental implant versus tooth (split-mouth) in terms of micro-
examine the dynamics of biofilm-induced inflammation (Theilade biological, immunological and clinical events associated with
et al., 1966; Löe et al., 1967). This model has also been extended and experimental mucositis/gingivitis.
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DUTRA ET AL. 3

2 | MATERIALS AND METHODS stent was fabricated to cover approximately 2 mm of the soft tissue
but with no contact with it in the test areas. In order to begin the sec-
This was a single-centre experimental gingivitis/mucositis pilot study ond phase (Day 0) and receive the customized stents with instructions
registered in the U.S. National Institutes of Health Clinical Trials Reg- of use, all participants had to have an angulated bleeding score (ABS)
istry (NCT03713567), with parallel and split-mouth comparisons. All (Van der Weijden et al., 1994) equal to zero. The participants wore
participants were recruited between November 2017 and February removable stents to cover the test areas (one tooth and one dental
2018 from patients previously treated and under periodontal/peri- implant) during daily brushing. The periodontal/peri-implant parame-
implant maintenance at the Piracicaba Dental School. The participants ters, namely modified gingival index (mGI) (Trombelli et al., 2004),
were informed about the procedures and signed the informed angulated bleeding score (ABS) (Van der Weijden et al., 1994), PI
consent form. (Silness & Loe, 1964), gingival crevicular fluid (GCF) and peri-
implant crevicular fluid (PICF) volume were recorded. Crevicular
fluid and plaque samples were also collected weekly at Days 0, 7,
2.1 | Participant enrolment and clinical protocol 14 and 21 by a single calibrated examiner (ILFG). The examiner's
intra-class correlation coefficient was .945. At the beginning of the
The study design compared the changes occurring around implants resolution phase, the subjects received a professional prophylaxis
and teeth during the experimental mucositis and gingivitis trial in and instructions to correctly clean the tooth and implant. The sub-
patients with Grade C, stage III and IV periodontitis background and jects were further monitored weekly for safety, and a professional
those with no history of periodontitis. The study inclusion criteria prophylaxis was performed. At Day 42, the periodontal exams,
were age ≥18 years and good general health (American Society of GCF/PICF volume and plaque sampling were repeated followed by
Anesthesiologists [ASA] I or II). Individuals who were previously diag- professional prophylaxis.
nosed with generalized aggressive periodontitis (Armitage, 1999) and
who presented with ≥20 teeth at the time of recruitment as well as
those under semi-annual supportive periodontal/peri-implant care 2.2 | Sample collection
were included in the H-GAP group. According to the new classifica-
tion of periodontal diseases, the clinical and radiographic characteris- The procedure for sample (plaque, GCF and PICF) collection was kept
tics of the H-GAP group of patients is now assigned as periodontitis consistent throughout the trial (Days 0, 7, 14, 21 and 42), and the same
Grade C, stage III or IV. Periodontally healthy subjects, with no history tooth and dental implant were included in the study. To get the most
of periodontitis, were included in the H-Health group. Exclusion cri- representative scenario of the host–bacteria interaction without disturb-
teria were current or immunosuppressive therapy; use of antibiotics, ing the biofilm, the sample collection was performed on an undisturbed
anti-inflammatory drugs, or any medication known to affect periodon- (nonrepeated) surface (buccal, mesio-buccal, disto-buccal, lingual, mesio-
tal status within 3 months of enrolment; patients with major systemic lingual, disto-lingual) at each time point of the trial. All test sites were
diseases such as diabetes mellitus and cancer; smokers; and pregnant isolated with cotton rolls and dried. Samples of GCF and PICF were col-
and lactating women. For the implant selection, one single implant lected with paper filter strips (Periopaper, Oraflow, Smithtown, NY). The
(Straumann system) rehabilitated with a screwed metal-ceramic crown Periopaper was left in place for 30 s. The fluid volume was determined
in function for at least 6 months was included. For the tooth site, one (Periotron 8000, Oraflow, Plain view, NY, USA), and the paper filters
tooth located at the same arch in the corresponding position of the were stored in a microtube (coded for each participant) at 20 C until
implant was included. Both (tooth and implant) included sites pre- analysis. The subgingival biofilm was collected using sterile paper cones
sented probing depth <4 mm, clinical attachment level <5 mm, no (Tanariman Industrial LTDA, Manacapuru, AM, Brazil). The paper cones
interproximal alveolar bone loss and plaque index (PI) <20% (Silness & were inserted to the bottom of the sulcus, left in place for 30 s and then
Loe, 1964). Sites with peri-implantitis (Lang et al., 2011) and/or peri- removed and stored in a microtube at 20 C until analysis.
odontitis (Armitage, 1999) in possible test areas were excluded.
Because of the novelty of the present pilot study, with strict inclusion
and exclusion criteria, the sample size was estimated based on previ- 2.3 | Immunological analysis
ous investigations with similar parameters (Meyer et al., 2016; Salvi
et al., 2012; Schincaglia et al., 2017). However, post hoc power analy- The cytokine levels of interleukin (IL)-10, IL-1β, IL-4, tumour necrosis
sis using paired t-test at H-GAP and H-Health with the delta of the factor (TNF)-α, interferon (INF)-γ, IL-6, IL-17 and osteocalcin (OC),
modified gingival index revealed a statistical power of 33.8% osteopontin (OPN), osteoprotegerin (OPG) and RANK-L in the GCF
(Supplementary Material). and PICF were determined via a multiplexed fluorescent bead-bead
Figure 1 shows the flow diagram for the three phases of the immunoassay (HCYTOMAG-60K-07, HBNMAG-51K-03 and
study. During the preparatory phase, informed consent and medical/ HRNKLMAG-51K-01 kits, Millipore Corporation, Billerica, MA, USA).
dental history were obtained, following which supportive periodontal/ Assays were carried out according to the manufacture's recommenda-
peri-implant therapy (SPT) including polishing and oral hygiene tions using the Magpix instrument (MiraiBio, Alameda, CA, USA). All
instructions was provided to the participants. A personalized acrylic cytokine values were expressed as a concentration (pg/mL).
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4 DUTRA ET AL.

F I G U R E 1 Flow diagram of the study design. ABS, angulated bleeding score; GCF, gingival crevicular fluid; GR, gingival recession; mGI,
modified gingival index; PD, probing depth; PI, plaque index; PICF, peri-implant crevicular fluid.

2.4 | Microbiome analysis abundance of the most important selected species were analysed
using LDA. For a more detailed methodological description of the bac-
Bacterial DNA was isolated using a Qiagen MiniAmp kit (Valencia, CA, terial analysis, see Supplementary Material.
USA) according to the manufacturer's instructions and stored at
20 C. The hypervariable V4 (515/806) region of the 16S rRNA gene
was amplified and sequenced on the Illumina Miseq platform to pro- 2.5 | Data analysis
duce 250-bp paired-end sequences.
Bioinformatic analysis was performed using the QIIME 2 2019.7 GraphPad Prism software 9.3.0 (1992–2021 GraphPad Software, LLC)
(Bolyen et al., 2019). Taxonomy assignment of amplicon sequence was used to perform all demographic, clinical, and immunological data
variants (ASVs) was performed using the expanded Human Oral analyses. Data distribution was analysed using the Shapiro–Wilk test.
Microbiome (eHOMD, V15.2) Database (Escapa et al., 2020). Comparison between groups (H-GAP vs. H-Health) at a single time
α-Diversity was analysed using the Shannon metric (Spellerberg & point was done with unpaired samples via the Mann–Whitney test.
Fedor, 2003) and β-diversity analysis was performed using composi- Differences between tooth and implant sites at a single time point
tional tensor factorization (Martino et al., 2020) and visualized with were evaluated via paired-sample Wilcoxon tests. The responses of
principal coordinate analysis (PCoA). Furthermore, Bray-Curtis, Jac- tooth and implant sites during experimental phases (0, 7, 14, 21 and
card, unifrac weighted, and unifrac unweighted metrics were used to 42) were evaluated with the Friedman test. To evaluate two-time
estimate the β-diversity of each group/site across time, separately. points, the Dunn test was performed. All tests considered
Linear discriminant analysis (LDA) was used for dimensionality reduc- alpha = 5%. Since microbiome data showed variances over time, all
tion. LDA plots were generated with PhyloToAST (PCoA.py). The core microbial community parameters (e.g. α- and β-diversity) were statisti-
microbiome was considered as species presented in at least 50% of cally tested with the restricted maximum likelihood method (REML) to
the samples from a group. The cytokine concentration and the relative reduce biases in variance and co-variance.
 TABLE 1 Clinical parameters before (Day 0), during (Days 7, 14, 21) and after (Day 42) the induced biofilm accumulation, expressed as median and inter-quartile range.
DUTRA ET AL.

H-GAP H-Health H-Health versus H-GAP

p-Value (tooth p-Value (tooth Tooth Implant

Tooth Implant vs. implant) Tooth Implant vs. implant) p-value p-value
PI Day 0 0 (0.0–1.0) 0 (0.0–1.0) .250 0 (0.0–1.0) 0.5 (0.0–1.0) .687 .521 >.99
a a a,b a
Day 7 1 (1.0–2.0) 1 (1.0–1.0) .250 2.0 (1.0–2.0) 1.5 (1.0–2.0) .500 .178 .140
Day 14 2a,b (1.37–2.0) 1a (1.0–2.0) .109 2.0a,b (2.0–2.0) 1.5a (1.0–2.0) .125 .791 .649
a,b a,b a,b a,b
Day 21 2 (1.87–2.0) 1.75 (1.0–2.0) .125 2.00 (2.0–2.0) 2.00 (1.75–2.0) .625 .945 .499
Day 42 0 (0.0–0.0) 0 (0.0–0.0) >.99 0 (0.0–0.0) 0 (0.0–0.0) >.99 >.99 >.99
p-Value <.0001 <.0001 <.0001 <.0001
mGI Day 0 1 (0.37–1.0) 0.75 (0.0–1.0) .437 0 (0.0–1.0) 0 (0.0–1.0) >.99 .169 .432
a a,b a,b
Day 7 1 (1.0–2.0) 1 (1.0–1.0) .250 1 (1.0–1.25) 1 (1.0–2.0) >.99 .628 .582
Day 14 1a (1.0–2.0) 1a,b (1.0–1.12) .500 2a,b (1.0–2.0) 1a (1.0–2.0) .250 .293 .582
Day 21 2.0a (1.37–2.0) 1.75a,b (1.0–2.0) .250 2a,b (1.0–2.0) 2a,b (1.0–2.0) >.99 .742 .649
Day 42 0 (0.0–0.0) 0 (0.0–0.50) .500 0 (0.0–0.25) 0 (0.0–0.25) >.99 .473 >.99
p-Value <.0001 <.0001 <.0001 <.0001
ABS Day 0 0 (0.0–0.0) 0 (0.0–0.0) >.99 0 (0.0–0.0) 0 (0.0–0.0) >.99 >.99 >.99
Day 7 0 (0.0–1.0) 0 (0.0–0.25) .500 0 (0.0–1.0) 0 (0.0–1.0) >.99 >.99 .628
Day 14 0 (0.0–1.0) 0 (0.0–0.50) .375 1 (0.0–1.0) 0 (0.0–1.0) .500 .656 .442
Day 21 1 (0.37–1.0) 0.75 (0.0–1.0) .250 1 (0.0–1.0) 1 (0.0–1.0) >.99 0.828 .6499
Day 42 0 (0.0–0.0) 0 (0.0–0.0) - 0 (0.0–0.0) 0 (0.0–0.0) - >.99 >.99
p-Value .0014 .0025 .0037 .0026
GCF PICF p-Value (GCF vs. PICF) GCF PICF p-Value (GCF vs. PICF) GCF PICF
CF (μl) Day 0 0.20a (0.11–0.33) 0.31 (0.19–0.52) .310 0.13 (0.09–0.16) 0.12 (0.05–0.31) .503 .146 .092
Day 7 0.18a (0.11–0.43) 0.29 (0.08–1.37) .492 0.17 (0.13–0.22) 0.26 (0.17–1.66) .250 .985 .668
Day 14 0.16 (0.10–0.25) 0.21 (0.12–0.96) .314 0.35a (0.21–0.44) 0.49 (0.32–1.40) .322 .060 .210
a a a
Day 21 0.30 (0.12–1.66) 1.26 (0.27–1.87) .312 0.35 (0.13–0.82) 0.44 (0.21–1.67) .232 .752 .301
Day 42 0.07 (0.05–0.09) 0.10 (0.05–0.21) .117 0.07 (0.06–0.12) 0.10 (0.06–1.17) .539 .694 .927
p-Value .0090 .0079 .0005 .0183

Note: Mann–Whitney test for comparison between groups (H-GAP vs. H-Health) at a single time. Differences between tooth and implant sites at a single time point were evaluated via paired-sample Wilcoxon
tests. The longitudinal analysis (0, 7, 14, 21 and 42) was carried out with Friedman test, and to evaluate two time points, the Dunn test was performed.
Abbreviations: ABS, angulated bleeding score; GCF, gingival crevicular fluid volume; H-GAP, H-generalized aggressive periodontitis; mGI, modified gingival index; PI, plaque index; PICF, peri-implant crevicular
fluid volume.
a
Significant difference with difference with Day 42 scores ( p < .05) (Friedman test, and to evaluate two time points, the Dunn test).
b
Significant difference with baseline scores ( p < .05).
5

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6 DUTRA ET AL.

3 | RESULTS 3.3 | Induction and resolution of experimental

inflammation: Tooth versus implant
Twenty subjects (10 per group) gave informed consent and were
enrolled in the study. During the clinical protocol, no complications or In both H-GAP and H-Healthy groups, teeth and implants showed sig-
adverse events were reported. Among the 20 implants included, none nificantly different microbial shifts ( p < .05, REML test, Figure 3) and
presented mucosal recession. Among the 20 teeth included, small gin- immunological responses ( p < .05, Friedman test, Figure 2) to these
gival recessions were observed (≤3 mm/RT1 A-) (Cairo et al., 2011). shifts during induction and resolution of inflammation.
The clinical parameters at baseline are shown in Table S1. Among the During the induction phase, the concentrations of INF-γ, IL-17,
20 individuals who started the study, none was excluded or dropped IL-4 and TNF-α around implants were significantly higher than around
out. The H-GAP (n = 10) and H-Health (n = 10) groups were not dif- teeth in both groups ( p > .05, two-sample Wilcoxon test, Figure 2),
ferent demographically ( p > .05, two-sample t-test, Table S1). while in the H-Health group, IL-10 and IL-1β were also significantly
higher at implants compared with teeth ( p > .05, two-sample Wil-
coxon test, Figure 2).
3.1 | Baseline characteristics of teeth and implants Microbiologically, Figure 3 shows the changes in the microbial diver-
in H-GAP versus H-Health individuals sity for tooth and implant, in both groups. Figure 3a shows a significant
variation with time of the α-diversity (Shannon index) in the teeth sites
At baseline, implants and teeth did not differ clinically ( p > .05, of both groups. No significant difference was observed over time in
Mann–Whitney test, Table 1) or microbiologically ( p > .05, REML test, implants. Regarding the β-diversity analysis, statistically significant shifts
Figure 3) between H-GAP and H-Health groups. However, implants (p < .05 REML test, Figures 3b–d) across time was observed in both
from the H-GAP group showed lower levels of INF-γ, IL-10, IL-17, IL- teeth and implants. PCoA (Figure 3b) shows the microbiological dissimi-
1β, IL-4 and TNF-α compared with the H-Health group ( p < .05, larities during biofilm accumulation and resolution phases in teeth and
Mann–Whitney test, Figure 2); however, teeth from both groups did implants of both groups. The changes with time in teeth were more evi-
not show significant differences in the measured cytokines. Moreover, dent than in implants, regardless of the group (Figure 3b,d-A–P). Addi-
implants showed higher concentrations of cytokines when compared tionally, Figure 3c presents the longitudinal β-diversity variation (first
with teeth in both groups (p < .05, two-sample Wilcoxon test, axis, PC1), showing that both implant and tooth changed significantly
Figure 2). with time. However, during the induction phase, implants showed a sig-
nificantly lower diversity (p < .05, REML, Figure 3b) compared with teeth
(CTF metric). Following clinical resolution of inflammation (day 42), the
3.2 | Induction and resolution of experimental peri-implant microbiome returned to a similar profile as the baseline, but
peri-implant mucositis: H-GAP versus H-Health the periodontal microbiome reset to a significantly lower diversity (CTF
metric) than baseline (p < .05, REML, Figure 3c).
Despite the similar trends in immunological response during the
induction and resolution of experimental mucositis (Figure 2), signifi-
cant differences were observed between H-GAP and H-Health 3.4 | Inflammatory and microbiological
groups. During the induction phase, INF-γ, IL-10, IL-17, IL-4, TNF-α characteristics of tooth and implant sites
and IL-1β were significantly lower at implants of the H-GAP group
compared to the H-Health group ( p < .05, Mann–Whitney test, Figure 4 shows the summary association between the bacterial (spe-
Figure 2). Additionally, during the resolution phase of experimental cies level) and cytokine levels of tooth and implant sites using LDA at
mucositis, IL-17 and IL-1β were also lower at implants of subjects clinical health (Days 0 and 42) and clinical inflammation (Days 7, 14,
from the H-GAP group ( p < .05, Mann–Whitney test, Figure 2). These and 21) irrespective of the history of periodontitis. The analysis
differences in immune markers were not accompanied by differences showed a different inflammatory and microbial pattern between tooth
in clinical parameters during the induction and resolution of experi- and implants, which were mostly discriminated by the inflammatory
mental mucositis, and no significant difference was observed between markers, where the tooth was related to IL-17, IL-10 and TNF-α
H-GAP and H-Health at any of the indices analysed (PI, mGI, ABS, CF) and implant to INF-γ. IL-4. Additionally, the implant site showed an
( p > .05, Mann–Whitney test, Table 1). Similarly, in the analysis based overlap of clinical health and mucositis, while a more evident dis-
on α-diversity (Shannon metric), no difference between H-GAP and crimination between clinical health and gingivitis was observed in
H-Health was observed (Figure 3a, REML test, p > .05). Furthermore, tooth sites.
in the analysis based on β-diversity, statistically significant difference
among the communities of each time point was observed (Figure 3b,
CTF, Compositional Tensor Factorization; d-A–D, Bray-Curtis; E–H, 4 | DI SCU SSION
Jaccard; I–L, unifrac unweighted; M–P, unifrac weighted); however,
no significant difference between the groups H-GAP and H-Health The present pilot study explored the differences between individuals
was observed ( p > .05, REML test, Figure 3b,c). with a background of periodontitis and those with periodontal health
1600051x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcpe.13896, Wiley Online Library on [14/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
7

pg/mL pg/mL
pg/mL pg/mL
pg/mL

H-GAP versus H-Health

H-GAP versus H-Health
H-GAP versus H-Health

H-GAP versus H-Health

H-GAP versus H-Health

pg/mL pg/mL
pg/mL pg/mL
pg/mL
(g)

(h)

(k)
(i)

(j)
pg/mL pg/mL pg/mL pg/mL pg/mL

Legend on next page.

pg/mL

H-GAP versus H-Health

H-GAP versus H-Health

H-GAP versus H-Health
H-GAP versus H-Health
H-GAP versus H-Health

H-GAP versus H-Health

FIGURE 2
DUTRA ET AL.

pg/mL
(a)

(b)

(d)

(e)
(c)
pg/mL pg/mL pg/mL

(f)
pg/mL
pg/mL
 1600051x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcpe.13896, Wiley Online Library on [14/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
8 DUTRA ET AL.

who were submitted to experimental plaque accumulation around one inflammatory markers may be due to variability across individuals in
dental implant and tooth. Therefore, in addition to group comparisons, the concentrations of inflammatory markers (high, low and slow
the design of the study allowed comparisons between different types responders) (Bamashmous et al., 2021) and the number of participants
of site (implant vs. tooth) in the same individuals (split-mouth). Within (Ghallab et al., 2018), and therefore should be cautiously considered.
the limitations of the experimental conditions (e.g. low power), it was In the present study, significant differences between Day 42 and
observed that the immune response of peri-implant and periodontal the plaque accumulation phase were observed for some cytokines.
tissues may be driven by site and periodontal background while the Indeed, the participants went through very rigid plaque control after
microbiological response was affected by the type of site. However, the induction phase, including two sessions of prophylaxis within
no significant clinical differences between tooth and implant and 21 days, resulting in complete control of any clinical inflammatory
between groups were seen. In this respect, it can be noted that a signs. It should be recognized that this professional plaque control
recent systematic review of similar studies presented a number of may have influenced the clinical, immunological and microbiological
included participants up to 25 (Nagasawa et al., 2022). In spite of that, response and should be considered while comparing the present
care should be exercised when interpreting the results of this pilot results with those of earlier studies. Regarding the increase in concen-
study because post hoc power analysis revealed low power. The tration of some cytokines at Day 42, it is interesting to note that a
approach of matching different periodontal diagnoses and different study exploring the microbiota and inflammatory characteristics dur-
sites under strict inclusion criteria favoured the comparisons but also ing the resolution of naturally occurring gingivitis did not observe
was expected to limit the number of eligible participants. Therefore, changes in the inflammatory mediators after the hygiene phase and
the lack of significant differences and disagreements with previous professional treatment (Stone et al., 2019). Thus, the results showed
studies should be viewed with caution and be addressed in larger that the time course of cytokine release—and the reduction of it—
studies. Nevertheless, concerning the comparison between the type during inflammation establishment and resolution needs further clari-
of sites, the absence of clinical differences is consistent with the exist- fication. It is possible that there is a time dependence between the
ing literature (Pontoriero et al., 1994; Schincaglia et al., 2017; stimulus and changes in GCF/PICF local levels and the role of each
Zitzmann et al., 2001), which also showed no significant differences in cytokine acting as a stimulus for other inflammation player during dis-
the clinical parameters between tooth and implant after biofilm accu- ease occurrence. Moreover, the biological relevance of the local con-
mulation. On the other hand, previous studies had reported that centration of each cytokine also remains poorly understood and
patients with a periodontitis diagnosis (compared with future studies are needed to better address these aspects.
non-periodontal subjects) showed an increased rate of plaque accu- Immunological analysis showed a difference between the responses
mulation associated with early development of redness, bleeding on of subject-matched implant and tooth sites and between H-Health and
probing and a higher gingival crevicular fluid volume during the exper- H-GAP subjects. Implants of both groups showed higher concentration
imental gingivitis (Abbas et al., 1986; Johnson et al., 1997; Trombelli of inflammatory and anti-inflammatory cytokines compared to teeth.
et al., 2006; Van der Velden et al., 1985). Recently, Serichetapongse et al. (2022) proposed that the presence of
Although inflammation-associated clinical signs occurred, there implant and abutment materials may influence the level of inflammatory
were no significant increases in the cytokines\ levels during biofilm cytokines in PICF. Therefore, it can be hypothesized that the materials
accumulation. Although this result may seem contradictory, Offenba- and anatomical differences between implants and teeth may have an
cher et al. (2010) reported a decrease in levels of several cytokines influence on the concentration of cytokines. Consequently, these results
during experimental gingivitis. The authors suggested that this phe- should be further explored in a bigger population with different peri-
nomenon may represent a degradation of cytokines within the sulcus odontal backgrounds, aiming to understand the individual variability
or a difference or shift in chemokine compartmentalization in tissue observed in the literature and the impact of the titanium surface, as well
versus the crevicular fluid. Additionally, an in vitro study with Porphyr- as the anatomy and prosthetic materials in the crevicular fluid content. In
omonas gingivalis demonstrated that the bacterium may be able to addition, a significantly lower concentration of most of the cytokines
release an endotoxin that may suppress chemokine synthesis com- analysed was observed at implants of the H-GAP group when compared
pared to the stimulatory activity of enteric LPS (Lipopolysaccharide) to H-Health, which may suggest a depressed and maybe less complete
species (Darveau et al., 1998). It should be noted that the absence of inflammatory response of the H-GAP group, in response to a similar clini-
statistically significant differences or discrepancies in the cal stimulus and microbiological community. Hence, these results should

F I G U R E 2 Changes in the inflammatory (a–g) and bone (h–k) cytokines in the crevicular fluid in teeth and implants of H-Health and
H-generalized aggressive periodontitis (GAP) groups. Box plots represent the interquartile range, with the middle line representing the median
and the whiskers showing the range. The upper brackets show p-values from statistical tests that evaluated differences during the experimental
protocol. The box below each graph shows comparisons between groups (H-GAP vs. H-Health) using the Mann–Whitney test. Differences
between tooth and implant in the same group were calculated using the Wilcoxon test, and the differences are highlighted in the graphs (*p < .05;
**p < .01; ***p < .001). δ indicates a significant difference in values compared to Day 0 and ℇ indicates a significant difference in values compared
to Day 42 (longitudinal analysis – Friedman and Dunn's tests). Significance level 95% (p < .05).
1600051x, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcpe.13896, Wiley Online Library on [14/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
9

Legend on next page.

FIGURE 3
DUTRA ET AL.
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10 DUTRA ET AL.

F I G U R E 4 Linear discriminant
analysis of cytokine levels and relative
abundance of the top 10% differentials, in
tooth and in implant, and during the
healthy and inflammatory phase.

be further verified to see whether this apparently depressed immunologi- corroborates with the α- and β-diversity analysis, suggesting that the
cal response should be one of the reasons for the susceptibility of peri- periodontal history of the subject did not affect the microbiological
odontal patients to peri-implants diseases. Similarly, Zein et al. (2017) response under the conditions of the present study.
observed significantly lower levels of cytokines in the GCF and plasma of The difference in the β-diversity (CTF metric) between sites cor-
individuals with generalized aggressive periodontitis individuals com- roborates with Heuer et al.'s (2012) study, which identified different
pared with healthy controls. The authors suggested that this large indi- bacterial compositions at the inflamed implant and tooth sites in the
vidual variability of cytokines observed in plasma may be due to same individual, with a high diversity in teeth. Similarly, Schincaglia
differences in the ability to establish an adequate inflammatory response. et al. (2017) observed a more evidently increased microbiome diver-
Therefore, a proper balance between the destructive and protective roles sity with time in teeth than in implants. Thus, the peri-implant envi-
of cytokines in periodontitis may protect the periodontal tissue (Garlet ronment seems to respond differently, with more resistance to
et al., 2010). The biological meaning of the differences in cytokine levels change than the tooth during the establishment of inflammation.
observed in the present study needs further evaluation in larger studies Therefore, when comparing the changes around dental implants and
addressing individual variability and possible impact of factors such as teeth, in the community, one could suggest a resilience in the microenvi-
titanium surface, anatomical differences and patient periodontal back- ronment of the tooth compared with higher resistance in the peri-
ground in this complex immunological process. implant tissues. Although the reason for this phenomenon is still not fully
It was interesting to note that, despite the lack of significant increase known, it can be speculated that the implant titanium surface, as well as
in the CF content during mucositis/gingivitis induction, a shift in the bac- the anatomical, physiological and inflammatory differences of peri-
terial community and an increase in the microbiome core of species at implant environment compared with the periodontium, can significantly
Day 21 were observed at all sites and groups compared with Days 0 and interfere in this process (Heuer et al., 2012). These dissimilarities driven
42. This result suggests maturation of the microbial community during by the local characteristics of each site could impact on their behaviour
inflammatory induction and the increase in complexity, with the emer- during the induction of inflammation and its resolution.
gence of later colonizers. Additionally, we could note the elevated per- Additionally, integrated microbiome–inflammatory data analysis
centage of species shared between sites, despite the groups, which highlighted the difference between teeth and implants sites during

F I G U R E 3 (a) α-Diversity (Shannon index) of tooth and implant, of both groups before (Day 0), during (Days 7, 14, 21) and after biofilm
accumulation. (b) β-Diversity (CTF, Compositional Tensor Factorization) represented by the principal coordinate analysis distance of tooth and
implant of both groups with time. (c) β-Diversity volatility (first axis—PC1) across time. (d) Linear discriminant analysis (LDA) of β-diversity indices
(A–D, Bray-Curtis; E–H, Jaccard; I–L, unifrac unweighted; M–P, unifrac weighted) over time in H-Health and H-generalized aggressive
periodontitis (GAP) implants and teeth. No significant differences between groups (H-GAP vs. H-Health) were observed. *Represents significant
difference between tooth and implants of the same group at that time point. If present, the differences among periods (longitudinal analysis) of
each site are indicated by different letters (restricted maximum likelihood method test, p < .05).
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DUTRA ET AL. 11

the clinically healthy and inflamed phases. Implant sites presented a tissues to biofilm accumulation is driven by the site and
more stable response during experimental peri-implant mucositis even periodontal history, while the microbiological changes are affected
when the microbiome and cytokines were evaluated together. On the mainly by the type of site (implants vs. teeth).
contrary, teeth sites presented an evident response to the clinical phase.
Interestingly, the most descriptive variables to discriminate the sites and AUTHOR CONTRIBU TIONS
clinical phase were the cytokines, IL-10, IL-17 and TNF-α being the most Tamires Pereira Dutra and Enílson Antônio Sallum conceived/
discriminant of the tooth and INF-γ and IL-4 for the implant. These designed the study, contributing to data collection, analysis/interpre-
results emphasize the importance of inflammation in modulating the sub- tation, drafting, editing and revising the manuscript. Isabela Lima
gingival/peri-implant environment. At the same time, they demonstrate França-Grohmann obtained all the clinical parameters and performed
how only species/genera were not good descriptors of the subgingival/ sampling. Mabelle Freitas Monteiro, Renato Corrêa Viana Casarin and
peri-implant site. Interestingly, earlier studies showing a higher preva- Purnima S. Kumar contributed to data analysis/interpretation, prepa-
lence of inflammatory disorders around implants installed in periodontal ration of figures and editing/revising the manuscript. Márcio Zaffalon
patients led us to hypothesize an elevated concentration of cytokines in Casati and Karina Gonzalez Silvério Ruiz contributed to data interpre-
the PICF environment during mucositis in H-GAP compared with individ- tation/discussion and editing/revising the manuscript. All authors
uals with no history of periodontitis. However, under the conditions of approved the final version of the manuscript.
the present study, the analysis showed what seems to be a depressed
inflammatory response in the H-GAP group that could not be attributed FUNDING INF ORMATI ON
to the differences in microbiological composition. In addition, it also This study was funded by the São Paulo Research Foundation, Grant
showed a consistent effector aspect of the implant on the local environ- numbers: 2018/06115-8 and 2019/01522-7. Dr. Sallum thanks the
ment over the immunological and microbiological shifts. Brazilian National Council for Scientific and Technological Develop-
It is important to highlight that previous studies and the present ment (CNPq) for a research scholarship (301635/2019-6).
one considered the 1999 Classification of Periodontal Diseases
(Armitage, 1999) to select the subjects, which was based on the evi- CONFLIC T OF INTER E ST STATEMENT
dence that subjects with a diagnosis of GAP display a specific family- The authors report no conflicts of interest related to this study.
related immunological and microbiological profile. However, in light of
the newest classification of periodontal diseases, and considering clin- DATA AVAILABILITY STAT EMEN T
ical and radiographic characteristics of the individuals, we adapted the The data that support the findings of this study are available from the
term to a new one in favour of the presentation and current under- corresponding author upon reasonable request.
standing of the results observed.
In spite of unprecedented associations in the comparisons of peri- ET HICS S TAT E MENT
odontal backgrounds and between teeth and dental implants, suggesting The study was approved by the Ethical Committee of the Piracicaba
that the peri-implant tissue in individuals with periodontitis background Dental School – State University of Campinas, Piracicaba, Brazil
responds differently to microbial plaque than periodontally healthy indi- (CAAE #70803817.1.0000.5418) and conducted in accordance with
viduals, it is worth highlighting the exploratory nature and limitations of the Helsinki Declaration of 1975, and the observational studies in epi-
this study. It can be assumed that the differences between teeth and demiology (STROBE) guidelines.
implants (e.g. surface roughness, free energy, chemical composition and
implant abutments) are limitations, even with the effort to standardize OR CID
the type of implant and clinical parameters around the teeth and Tamires Pereira Dutra https://orcid.org/0000-0003-0325-4439
implants. So the heterogeneity in the subject-specific and site-specific (e. Mabelle Freitas Monteiro https://orcid.org/0000-0001-9333-4349
g. presence of small gingival recession) characteristics associated with a Renato Corrêa Viana Casarin https://orcid.org/0000-0003-1743-
well-known variability across individuals in the microbiome profiles and 5855
concentrations of inflammatory markers should be recognized. In addi- Purnima S. Kumar https://orcid.org/0000-0001-5844-1341
tion, the strict inclusion criteria and sample size (study power) might have
influenced the results. Hence, a larger study would be necessary to con- RE FE RE NCE S
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