Accepted: 7 July 2017

DOI: 10.1111/jre.12492

ORIGINAL ARTICLE

Role of toll-­like receptor 2 in inflammation and alveolar bone

loss in experimental peri-­implantitis versus periodontitis

X. Yu1,2,* | Y. Hu2,* | M. Freire3 | P. Yu4 | T. Kawai2 | X. Han2

1
Department of Periodontology, The Affiliated
Hospital of Qingdao University, College of
Stomatology, Qingdao University, Qingdao,
Shandong, China
2
Department of Immunology and Infectious
Diseases, The Forsyth Institute, Cambridge,
MA, USA
3
Department of Applied Oral Sciences, The
Forsyth Institute, Cambridge, MA, USA
4
State Key Laboratory of Oral Diseases, West
China Hospital of Stomatology, Sichuan
University, Chengdu, Sichuan, China
Correspondence
Xiaozhe Han, Department of Immunology and
Infectious Diseases, The Forsyth Institute,
Cambridge, MA, USA.
Email: xhan@forsyth.org
Funding information
NIH NIDCR, Grant/Award Number:
DE023807 and DE025255
Background and Objective: Peri-­implantitis and periodontitis are different entities in im-
mune characteristics even though they share similar features in clinical and radiologic signs.
Toll-­like receptor 2 (TLR-2), one of the key pathogen-­recognition receptors in the innate
immune system, plays an important role in the progression of periodontitis. However, the
role of TLR-2 in peri-­implantitis remains unclear. The objective of this study was to investi-
gate the role of TLR-2 in inflammation and alveolar bone loss in a murine model of ligature-­
induced peri-­implantitis and to compare it with ligature-­induced periodontitis.
Material and Methods: Smooth-­surface titanium implants were placed in the alveolar
bone of the left maxillary molars of wild-­type (WT) and Tlr2 knockout (Tlr2-­KO) mice
6 weeks after tooth extraction. Silk ligatures were applied to the left implant fixtures
and the right maxillary second molars to induce peri-­implantitis and periodontitis
4 weeks after implant placement. Two weeks after ligation, bone loss around the im-
plants and maxillary second molars was analysed by micro-computed tomography
(micro-CT), and inflammation around the implants and maxillary second molars was
assessed at the same time point using histology and TRAP staining, respectively.
Expression of mRNA for proinflammatory cytokines (interleukin-­1β [Il1β], tumor ne-
crosis factor-­
α [Tnfα]), an anti-­
inflammatory cytokine (interleukin-­
10 [Il10]) and
osteoclastogenesis-­related cytokines (Rankl, osteoprotegerin [Opg]) were evaluated, in
gingival tissue, using real-­time quantitative PCR (RT-­qPCR).
Results: The success rate of implant osseointegration was significantly higher in Tlr2-­KO
mice (85.71%) compared with WT mice (53.66%) (P = .0125). Micro-­CT revealed signifi-
cantly decreased bone loss in Tlr2-­KO mice compared with WT mice (P = .0094) in peri-­
implantitis. The levels of mRNA for Il1β (P = .0055), Tnfα (P = .01) and Il10 (P = .0019) in
gingiva were significantly elevated in the peri-­implantitis tissues of WT mice, but not in
Tlr2-­KO mice, compared with controls. However, the gingival mRNA ratios of Rankl/Opg
in peri-­implant tissues were significantly upregulated in both WT (P = .0488) and Tlr2-
KO (P = .0314) mice. Ligature-­induced periodontitis exhibited similar patterns of bone
loss and inflammatory cytokine profile in both groups of mice, except that the level of
Il10 was elevated (P = .0114) whereas the Rankl/Opg ratio was not elevated (P = .9755)
in Tlr2-­KO mice compared with control mice. Histological findings showed increased
numbers of TRAP-­positive cells and infiltrated inflammatory cells in ligature-­induced
peri-­implantitis in both WT (P < .01) and Tlr2-­KO mice (P < .05), and the numbers of
both types of cell were significantly higher in WT mice than in Tlr2-­KO mice (P < .01).

*These authors contributed equally.

98  |  wileyonlinelibrary.com/journal/jre
© 2017 John Wiley & Sons A/S. J Periodont Res. 2018;53:98–106.
Published by John Wiley & Sons Ltd
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      99

Conclusion: This study suggests that TLR-2 mediates bone loss in both peri-­implantitis
and periodontitis. However, different molecular features may exist in the pathogenesis
of the two diseases.

KEYWORDS
bone resorption, inflammatory cytokine, peri-implantitis, periodontitis, toll-like receptor 2

1 | INTRODUCTION through differential induction of the nuclear factor of activated T-­cells,

Dental implants have become widely used for edentulous and partially
dentate patients because of their high predictability and high success
1
rate. However, approximately 30% of patients with dental implants
develop peri-­implantitis.2 Peri-­implantitis is characterized by infection
of soft tissue and loss of the surrounding bone, and eventually leads
to implant loss.3 It is considered to occur as a result of biofilm for-
mation on the implant surface analogous to periodontitis, 4,5
leading
to the activation of host immune and inflammatory responses around
the implant, which is essential in the pathogenesis of peri-­implantitis.6
Although peri-­implantitis and periodontitis have many features
in common in clinical and radiologic signs, they represent distinct
entities 7–9
because of their different anatomic and histologic envi-
ronments, core microbiomes 10
and immune characteristics. 8,11
Peri-­
implantitis displays unique features12 and the destruction of tissues
in peri-implantitis appears to be of significantly greater severity than
occurs in periodontitis. 13,14
Quantitative transcriptome analysis indi-
cated that peri-­implantitis lesions primarily differ from periodontitis
in cell-­to-­cell adhesion, wound healing, complement activation and
7
innate immune responses. It was reported that among 208 tran-
scripts from gingival soft tissue closely related to peri-­
implantitis
and/or periodontitis, transcripts associated with innate immune re-
sponses and defense responses were expressed more strongly in peri-­
implantitis tissue, while in periodontitis tissues, bacterial response
genes dominated.7 Some research also revealed differences between
periodontitis and peri-­implantitis in innate immune responses of the
soft connective tissue. Granulation tissue from peri-­implantitis sites
exhibits stronger expression of mRNA for proinflammatory cytokines
compared with granulation tissue from periodontitis sites. 15
Moreover,
clinical studies have suggested that peri-­implantitis represents a more
elevated proinflammatory state, with higher levels of interleukin-­6,
interleukin-­
8, macrophage inflammatory protein-­
1b and TIMP-­
1 , 11
and a larger number of CD138-­, CD68-­and myeloperoxidase-­positive
cells16, compared with periodontitis.
Toll-­like receptors (TLRs) are a family of at least 13 proteins that
function as key pathogen-­recognition receptors in the innate immune
system, responding to diverse microbial products and injury-­induced
endogenous products.17–19 TLR-­2 not only mediates cellular responses
to a wide variety of pathogens and their products20,21 but also inter-
acts with a wide array of microbial molecules derived from commensal
bacteria.22 Previous studies have demonstrated that TLR-­2 is required
for inflammatory bone loss in periodontitis and TLR-­
2-­
dependent
osteoclastogenesis can be modulated by Porphyromonas gingivalis
cytoplasmic 1 protein and nuclear factor-­kappaB.23,24 Furthermore,
production of TLR-­2-­dependent tumor necrosis factor (TNF) is re-
quired in macrophage-­elicited osteoclastogenesis in response to bac-
terial stimulation.25,26 However, the role of TLR-­2 in peri-­implantitis
remains unknown. We hypothesized that TLR-­2 signaling plays an im-
portant role in the pathogenesis of peri-­implantitis tissue inflammation
and bone loss.
The coexistence of peri-­implantitis and periodontitis has become
increasingly observed clinically in patients with periodontal disease.
It is essential to develop an animal model to incorporate these two
diseases in one susceptible host in order to compare their common
characteristics with those that are different in the context of microbi-
ota and host immune responses.
The aim of this study was to determine the role of TLR-­2 on the
inflammation and bone loss in a murine model of experimental peri-­
implantitis and to compare it with ligature-­induced periodontitis. We
developed ligature-­
induced experimental peri-­
implantitis and peri-
odontitis in the same animal, attempting to simulate the clinical situa-
tion of simultaneous development of the two diseases.
2 | MATERIAL AND METHODS
2.1 | Mice
Wild-­type (WT) C57BL/6 and Tlr2 knockout (Tlr2-­KO) mice (B6.129-­
Tlr2tm1Kir/J) were purchased from the Jackson Laboratory (Bar Harbor,
ME, USA). Experiments using these mice were approved by the
Institutional Animal Care and Use Committee of the Forsyth Institute.
All mice used in the study (4 weeks old; male:female = 2:1) were main-
tained in specific pathogen-­free units of the Forsyth Institute Animal
Facility. Mice were fed a soft diet ad libitum for the duration of the
experiment.
2.2 | Tooth extraction, implant placement and
ligature-­induced experimental peri-­implantitis and
periodontitis
The complete procedures are shown in Figure 1. The maxillary first and
second molars were extracted on the left side in all mice under general
anesthesia by intraperitoneal administration of ketamine (100 mg/kg)
and xylazine (5 mg/kg), and the extraction sites were allowed to heal
for 6 weeks. Mice were given antibiotics (sulfamethoxazole and tri-
methoprim, 850 μg/170 μg per mL), dissolved in their drinking water,
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100       YU et al.

F I G U R E   1   Clinical observations of the procedures of implantation and ligation. (A) Six weeks postextraction: the tooth-­extraction socket had
healed well with a smooth gingival surface. Implant day 0, implants were placed in alveolar bone without flap elevation. Four weeks post-­implant
placement, before ligation, there was no visible sign of inflammation. Ligature day 0, 7-­0 ligatures were applied under the fixture head. Two
weeks post-­ligature application, peri-­implant mucositis was visible with obvious edema and gingivitis. (B) Schematic diagram depicting the timing
of the experimental design. Tlr2-­KO, toll-­like receptor-­2 knockout; WT, wild type

for 2 weeks to facilitate healing of the extraction sites. Six weeks
later, gingival tissue corresponding to the extraction site was punched
manually using a blunt 25G needle (Becton Dickinson Corporation,
San Jose, CA, USA). The right maxillary molars were used as a spa-
tial reference. Implants were placed as previously described.27 Briefly,
the osteotomy was started by using a 0.3-­mm-­diameter carbide micro
hand drill (D. P. Machining Inc, La verne, CA, USA) by manual rotation
into alveolar bone approximately 1 mm in depth. A smooth-­surface,
screw-­shaped titanium implant (1 mm in length and 0.5 mm in diam-
eter; D. P. Machining Inc) was screwed clockwise into the maxillary
bone. Implants were allowed to heal for 4 weeks, during which the
antibiotics and powder food were given to mice as described above.
Four weeks after implant placement, experimental peri-­implantitis and
periodontitis were initiated. Briefly, a 7-­0 silk ligature (Fisher Scientific,
Waltham, MA, USA) was placed subgingivally around each implant im-
mediately apical to the implant head on the left side of the maxilla.
The right maxillary second molar was tied with a 7-­0 silk ligature sub-
gingivally. Two weeks after ligature placement, all mice were killed by
CO2 inhalation. All the procedures, including tooth extraction, implant
placement and ligature placement, were performed under optical mi-
croscopy (S6D Stereozoom; Leica, Baffalo Grove, IL, USA).
2.3 | Sample preparation
To assess the position and osseointegration of the implant using
micro-­computed tomography (micro-­CT), five mice were killed imme-
diately after implant placement and five mice were killed at the 4-­
week post-­implantation (before ligation) time point in the WT group.
Five mice in the WT group and five mice in the Tlr2-­KO group were
fed for 6 weeks post-­implantation without ligation. For each experi-
mental group (ligated or unligated), 10 mice were killed 2 weeks after
ligation and were prepared for micro-­CT and gene-­expression analy-
ses. An additional four mice from each group were prepared for histo-
logical evaluation. Maxillae were harvested, the skin and muscle were
removed and gingival tissues at the palatal side were collected under
surgical microscopy. The gingival tissues were stored at −80°C for fu-
ture use. The maxillae were defleshed by a dermestid beetle colony.
After bleaching with 3% hydrogen peroxide, the bone was scanned
with micro-­CT. Additional samples were fixed with 10% paraform-
aldehyde overnight, then decalcified in 10% EDTA for 3 weeks at
4°C with agitation. After complete demineralization, implants were
removed manually by rotating counterclockwise. All tissue samples
were immersed in 10%, 20% and 30% sucrose solution and then em-
bedded in OCT solution (Tissue-­Tek, Sakura Finetek, Torrance, CA,
USA). Frozen samples were cut into 8-­μm-­thick sections along the me-
sial–distal plane in a cryostat and were collected on Superfrost-­plus
slides (Fisher Scientific) for histological analysis.
2.4 | Micro-­CT analysis
Mice maxillae were scanned using a high-­resolution scanner (mCT-­40;
Scanco Medical, Wayne, PA, USA). Samples were exposed to poly-
chromatic X-­rays on a rotating stage at a steep angle of 0.18° over
360°. Measurements were taken at an operating voltage of 70 kVp
and 114 mA current and 6 mm isotropic voxel resolution, with an
exposure time of 200 ms, and five frames were averaged per view.
Quantitative three-­
dimensional measurements of the implants or
teeth were performed using Seg3D software (NIH center for inte-
grative biomedical computing, Salt lake city, UT, USA). Briefly, the
same volume of interest (VOI) was chosen for each sample around
the second maxillary molar or the implants. A cylinder with a diam-
eter of 1.0 mm and a height of 1.0 mm is defined as VOI from the top
surface of implants or a similar position of the natural tooth. A three-­
dimensional morphometric analysis was conducted to determine the
architecture of the bone according to the following: total VOI vol-
ume (TV) and total bone volume (BV). The empty space volumes (ESV)
surrounding teeth or implants were calculated as TV minus BV. The
micro-­CT images of implants and natural teeth were converted and
collected using Amira software (FEI Visualization Sciences Group,
Hillsboro, OR, USA).
YU et al.
2.5 | Real-­time quantitative PCR
Palatal gingival tissues were isolated from around both ligatured
teeth and ligatured implants, as well as their controls (nonligatured),
and were homogenized in lysis buffer using a tissue homogenizer
(Omni International, Kenesaw, NE, USA). Total RNA was extracted
using PureLink® RNA Mini Kit (Ambion, Austin, TX, USA). cDNA
was synthesized using the SuperScript III Reverse Transcriptase kit
(Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s pro-
tocol. The expression of interleukin-­
1β, Tnfα, interleukin-­10, Rankl
and osteoprotegerin (Opg) mRNAs in gingiva were determined by
reverse transcription–quantitative real-­
time PCR (RT-­
qPCR) using
LightCycler® SYBR Green I Master and LightCycler® 480 Instrument
(Roche) systems. The sequences of primers are shown in Table 1. The
glyceraldehyde-­3-­phosphate dehydrogenase (Gapdh) gene was used as
an internal control.
2.6 | Histological analysis
Sections of 8 μm thickness were produced in the mesial–distal plane
for hematoxylin and eosin and TRAP staining. Histologic images were
captured (DMLS; Leica) and analysed by Image-­
J software (NIH,
Bethesda, MD, USA). For hematoxylin and eosin staining, the number
of inflammatory cells in four unit squares (50 μm × 50 μm) of peri-
odontal connective tissue was counted at an objective magnification
of 40× and then averaged. For TRAP staining, tissue sections were
T A B L E   1   Primers and sequences used for PCR
Primers Sequences
IL-­1β Forward: 5′-­ATGCCTTCCCCAGGGCATGT-­3′
Reverse: 5′-­CTGAGCGACCTGTCTTGGCCG-­3′
TNF-­α Forward: 5′-­CAACGCCCTCCTGGCCAACG-­3′
Reverse: 5′-­TCGGGGCAGCCTTGTCCCTT-­3′
IL-­10 Forward: 5′-­GACCAGCTGGACAACATACTGCTAA-­3′
Reverse: 5′-­GATAAGGCTTGGCAACCCAAGTAA-­3′
RANKL Forward: 5′-­CAT GTG CCA CTG AGA ACC TTG AA-­3′
Reverse: 5′-­CAG GTC CCA GCG CAA TGT AAC-­3′
OPG Forward: 5′-­AGCAGGAGTGCAACCGCACC-­3′
Reverse: 5′-­TTCCAGCTTGCACCACGCCG-­3′
GAPDH Forward: 5′-­CCCCAGCAAGGACACTGAGCAA-­3′
Reverse: 5′-­GTGGGTGCAGCGAACTTTATTGATG-­3′
GAPDH, glyceraldehyde-­ 3-­phosphate dehydrogenase; IL, interleukin;
OPG, osteoprotegerin; TNF-­α, tumor necrosis factor-­α.
T A B L E   2   Success rate (SR) of
osseointegrated implants 4 weeks after
Variable
implant placement
Wild-­type group
Tlr2-­KO group
Tlr2-KO, toll-­like receptor 2 knockout.
|
      101
stained using an acid phosphatase kit (378A; Sigma, St. Louis, MO,
USA). After counterstaining with hematoxylin, TRAP-­
positive cells
with three or more nuclei were considered as osteoclasts. A region
of interest (ROI) was applied to peri-­implantitis samples. It was a
1.5 mm × 1 mm rectangular area of which the longer side aligned with
the central long axis of the implant and covered the whole implant
length from head to tip. For periodontitis samples, the ROI was the
area from the gingiva papilla to the root apex between the second
molar and adjacent molars. Each section was acquired under light mi-
croscopy at an objective magnification of 40×. Osteoclast numbers
either around the implant surface or at the infiltrated connective tis-
sue within the ROI were quantified manually by Image-­J.
2.7 | Statistical analysis
The results are presented as mean ± standard error. The unpaired
Student’s t test was used to analyse differences between the con-
trol group and the ligature group in WT or Tlr2-­KO mice. One-­way
ANOVA was used to analyse differences among multiple groups.
Results with P < .05 are considered statistically significant.
3 | RESULTS
3.1 | Postsurgical observation
Four weeks after implant placement, the success rate of implant os-
teointegration was significantly higher in Tlr2-KO mice (85.71%) than
in WT mice (53.66%) (Table 2). The difference in implant success rate
between these two groups was statistically significant (P = .0125).
Once integrated, no implant was lost 2 weeks after ligation in any
mouse.
3.2 | Alveolar bone resorption
The volumes of bone loss around implants were significantly increased
around ligatured implants in both WT mice (P = .0001) and Tlr2-KO
mice (P = .0006) compared with non-­ligated controls (Figure 2A,B).
Furthermore, the bone loss volume around ligatured implants was
significantly higher in WT mice than in Tlr2-KO mice (P = .0094)
(Figure 2B). These results indicate that Tlr2 deficiency effectively
ameliorates bone resorption in experimental peri-­implantitis. Similarly,
ligature-­induced periodontal bone resorption was significantly higher
in WT mice than in Tlr2-KO mice (P = .0056) (Figure 2C,D). Also, the
percentages of ligature-­induced bone loss were significantly higher
in peri-­implantitis than in periodontitis in both WT mice and Tlr2-KO
mice (Fig. S1).
Total SR
implants Lost Loose Osseointegrated SR (%) P-value
41 15 4 22 53.66 .0125
21 3 0 18 85.71
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102       YU et al.
3.3 | Gingival mRNA expression of
inflammatory cytokines
In WT mice, the levels of Il1β mRNA expressed in gingiva were signifi-
cantly increased in the ligated side compared with the control side in
both peri-­implantitis (P = .0055) and periodontitis (P = .0069), whereas
in Tlr2-KO mice, the levels of Il1β mRNA expressed were not signifi-
cantly changed after ligation compared with controls (Figure 3A,B).
The levels of Il1β mRNA expressed in gingiva on the ligated site were
significantly higher in WT mice than in Tlr2-KO mice in peri-­implantitis
(Figure 3A) and periodontitis (Figure 3B). The expression of Tnfα
mRNA was similar to that for Il1β mRNA (Figure 3C,D). The levels of
Tnfα mRNA expressed in the ligated site were significantly higher in
WT mice than in Tlr2-KO mice in peri-­implantitis (Figure 3C) and peri-
odontitis (Figure 3D). Meanwhile, the levels of Il10 mRNA expressed in
gingiva on the ligated side were significantly higher than those on the
control side in WT mice in both peri-­implantitis (P = .0019) (Figure 3E)
and periodontitis (P = .0218) (Figure 3F). In Tlr2-KO mice, there was
no significant difference in the level of Il10 mRNA in gingival tissues
around ligatured versus non-­ligatured implants (P = .7899) (Figure 3E),
but there was a significant increase in the level of Il10 mRNA in gin-
gival tissues around ligatured teeth compared with those around
non-­ligatured teeth (P = .0114) (Figure 3F). There were no significant
differences of Il10 mRNA expression levels in the ligated site in WT
mice compared with Tlr2-KO mice in peri-­implantitis (Figure 3E) and
periodontitis (Figure 3F). The gingival Rankl/Opg relative ratio in peri-­
implant tissue was significantly elevated after ligation in both WT mice
(P = .0488) and Tlr2-KO mice (P = .0314), compared with controls
(Figure 3G). The Rankl/Opg ratio in periodontal tissue was significantly
increased in WT mice (P = .01331) but was not changed in Tlr2-KO
mice compared with the control (P = .9755) (Figure 3H). Also, there
were no significant differences of Rankl/Opg ratios on the ligated side
in WT mice compared with Tlr2-KO mice in peri-­implantitis (Figure 3G)
F I G U R E   2   Peri-­implant and periodontal
bone loss measured using micro-­computed
tomography (micro-­CT). (A) Micro-­CT
images of mesial–distal bone of implants
in wild-­type (WT) and toll-­like receptor-­2
knockout (Tlr2-­KO) mice. (B) The volumes
between the head and the bone crest
around the implants. (C) Micro-­CT images
of the mesial–distal bone of natural
teeth in WT and Tlr2-­KO mice. (D) The
volumes between the cemento–enamel
junction (CEJ) and the bone crest around
natural teeth. Data are shown as the
mean ± standard error of the difference
between two means (SED) (n = 6).
**P < .01. 2D, two dimensional; 3D, three
dimensional
and periodontitis (Figure 3H). Compared with WT mice, a signifi-
cantly decreased gingival level of Il1β mRNA (P = .0025) (Figure 3A)
and Tnfα mRNA (P = .0092) (Figure 3C), but an unchanged level of
Il10 mRNA (P = .9730) (Figure 3E) and Rankl/Opg ratio (P = .2139)
(Figure 3G), were observed in Tlr2-KO mice after ligature-­
induced
peri-­implantitis. Similar results were observed in ligature-­induced peri-
odontitis, demonstrating significantly decreased gingival levels of Il1β
mRNA (Figure 3B) and Tnfα mRNA (Figure 3D) but an unchanged level
of Il10 mRNA (Figure 3F) and Rankl/Opg ratio (Figure 3H). In Tlr2-KO
mice, but not in WT mice, the relative level of gingival Il1β mRNA ex-
pressed was significantly higher in ligature-­induced periodontitis than
in ligature-­induced peri-­implantitis (Fig. S2A). No difference was ob-
served in relative gingival Tnfα mRNA level when comparing ligature-­
induced peri-­implantitis with periodontitis (Fig. S2B). Compared with
those from ligature-­
induced peri-­
implantitis tissues, gingival Il10
mRNA was significantly increased in ligature-­induced periodontitis tis-
sues in both WT and Tlr2-KO mice (Fig. S2C), whereas the Rankl/Opg
ratio was significantly decreased in Tlr2-KO mice (Fig. S2D).
3.4 | Histological findings
The number of TRAP-­positive cells was significantly increased at the
ROI in peri-­implantitis compared with control in both WT (P < .0001)
and Tlr2-KO (P = .0175) mice (Figure 4A,C). Moreover, the increase
in the number of TRAP-­positive cells in WT mice was significantly
greater than that in Tlr2-KO mice (P < .0001) (Figure 4C). The same
results were observed in the periodontitis sites (Figure 4B,D) in WT
mice (P = .0008) and Tlr2-KO mice (P = .0489), where osteoclas-
togenesis were also greater in periodontal tissues in WT mice than
in Tlr2-KO mice (P = .0019) (Figure 4D). Also, the relative number of
TRAP-­positive cells at the ligation side was significantly higher in peri-­
implantitis tissues than in periodontitis tissues in WT mice but not in
Tlr2-KO mice (Fig. S3).
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      103

F I G U R E   3   Expression of inflammatory molecules in gingival tissues. The implant and the maxillary second molar at the opposite side were
ligatured for 2 weeks in wild-­type (WT) and toll-­like receptor-­2 knockout (Tlr2-­KO) mice. Gingival tissues around ligatured implants and teeth
were excised and processed for quantitative PCR (qPCR) analyses to determine expression of interleukin-­1beta (Il1β) (A, B), tumor necrosis
factor-­α (Tnfα) (C, D) and interleukin-­10 (Il10) (E, F) mRNAs and the RANKL/osteoprotegerin (Rankl/Opg) ratio (G, H). Data are mean ± standard
error of the difference between two means (SED) (n = 4 mice for control groups, n = 10 mice for ligatured groups) analysed using an unpaired
t test. *P < .05, **P < .01, N.S., no significant difference

Compared with controls, a larger number of inflammatory cells, in-
cluding plasma cells, macrophages and polymorphonuclear leukocytes,
had infiltrated into the periodontal connective tissues in peri-­implantitis
in WT mice (P = .0215) and Tlr2-KO mice (P = .0143) (Figure 5A,C). A
larger inflammatory infiltrate was found in WT mice than in Tlr2-KO mice
(P = .0144) (Figure 5C). The same results were observed in the periodonti-
tis sites (Figure 5B,D) in WT mice (P < .0001) and Tlr2-KO mice (P = .0014),
demonstrating a denser inflammatory infiltration in WT mice compared
with Tlr2-KO mice (P = .0047) (Figure 5D). Moreover, there were no sig-
nificant differences in the relative number of inflammatory infiltrating cells
in ligature-­induced peri-­implantitis tissue compared with ligature-­induced
periodontitis tissue in both WT and Tlr2-KO mice (Fig. S4).
4 | DISCUSSION
This study showed that alveolar bone loss was alleviated in Tlr2-KO
mice compared with WT mice following ligature-­induced infection
in peri-­implantitis, similarly to that observed in periodontitis. This is
the first report in a murine model to demonstrate that peri-­implantitis
bone resorption is associated with TLR-­
2 signaling analogous to
periodontitis.
The Rankl/Opg relative ratio is considered as an accurate diagnostic
method for assessing periodontal disease activity.28,29 TLR-­2 has been
shown to inhibit osteoclastogenesis by downregulating the expression
of RANKL.24,30,31 In the present study, we found that the Rankl/Opg
mRNA ratio in gingiva was elevated 2 weeks after ligation in peri-­
implant tissues in both WT mice and Tlr2-KO mice when compared with
controls (Figure 3G). These results indicate that TLR-­2-­mediated peri-­
implantitis bone loss does not occur through the RANKL/RANK/OPG
axis. Indeed, some clinical studies reported that the soluble RANKL/
OPG ratio in peri-­implant crevicular fluid showed no significant differ-
ences between peri-­implantitis and healthy controls.32–34 In contrast,
in Tlr2-KO mice, no significant difference between ligatured teeth and
control was observed (Figure 3H). These results are consistent with
our previous findings indicating that TLR-­
2-­
mediated periodontal
 |
104       YU et al.

F I G U R E   4   Histological images of
TRAP staining of peri-­implant tissues and
periodontal tissues. Bone resorption was
confirmed by the presence of osteoclasts.
Yellow arrows: TRAP-­positive osteoclasts
(pink color). TRAP-positive osteoclasts
were arrayed along the bone edge adjacent
to gingival connective tissues along the
implant (A) or along the alveolar bone
surface (B). Osteoclast numbers within the
region of interest (ROI) in wild-­type (WT)
and toll-­like receptor-­2 knockout (Tlr2-­KO)
mice were analysed in peri-­implantitis (C)
and periodontitis (D). Data are shown as
the mean ± standard error of the difference
between two means (SED) (n = 4). *P < .05,
**P < .01

F I G U R E   5   Histological images of
hematoxylin and eosin (H&E) staining
of peri-­implant tissues and periodontal
tissues. A large number of inflammatory
cells infiltrated into the connective tissues
from coronal to alveolar bone. H&E-­
stained images of peri-­implantitis (A) and
periodontitis (B). The number of infiltrated
inflammatory cells within the region of
interest (ROI) of peri-­implantitis (C) and
periodontitis (D) in wild-­type (WT) and
toll-­like receptor-­2 knockout (Tlr2-­KO)
mice were statistically analysed. Data are
shown as the mean ± standard error of
the difference between two means (SED)
(n = 4), *P < .05, **P < .01

bone loss is RANKL dependent.23 Histological findings further con- ratio were not determined, which are warranted to be verified in fu-
firmed the molecular profiles we found in this study (Figures 4 and 5). ture studies.
It is noted that because of the limited amount of gingival tissues col- Secretion of proinflammatory cytokines is closely related to the
lected, the protein levels of inflammatory cytokines and RANKL/OPG progression of peri-­implantitis and periodontitis. Clinical studies have
YU et al.
demonstrated that the levels of interleukin-­1β,35,36 TNF-­α35,37,38 and
35
interleukin-­10 in peri-­implant crevicular fluid with peri-­implantitis
were higher than the levels in healthy controls. Our present findings,
using a murine model, showed that the expression of Il1β and Tnfα
mRNAs were upregulated significantly after ligation in peri-­implant
tissues (Figure 3A,C) and periodontal tissues (Figure 3B,D) of WT
mice but not in those of Tlr2-­KO mice, suggesting that the expres-
sion of interleukin-­1β and TNF-­α are commonly regulated through
TLR-­2 signaling in both peri-­implantitis and periodontitis. This ob-
23
servation was supported by our previous study in periodontitis
and by other research.25,26 Interestingly, upregulated expression of
Il10 mRNA in gingiva was observed in WT mice but not in Tlr2-­KO
mice in ligature-­
induced peri-­
implantitis (Figure 3E). However, Il10
mRNA was upregulated in both WT mice and Tlr2-­KO mice in ligature-­
induced periodontitis (Figure 3F). These results suggest that the anti-­
inflammatory interleukin-­
10 response in peri-­
implantitis is TLR-­
2
dependent, whereas in periodontitis such a response occurs through a
TLR-­2-­independent pathway.
Previous studies have indicated that in osteoblasts TLR-­
2 sig-
naling was required for cell apoptosis and calcification induced by
Staphlylococcus aureusis39 and the inhibitory effect of P. gingivalis lip-
ids on osteoblast differentiation.40 Moreover, TLR-­2 activation sig-
nificantly inhibited the migratory response of murine bone marrow
stem cells.41 In agreement with these studies, we found that the os-
seointegration in WT mice (53.66%) was significantly lower than that
in Tlr2-KO mice (85.71%). Our result suggests that knockout of TLR-­2
might benefit implant osseointegration through increasing osteoblast
differentiation and downregulating local inflammatory cytokine levels.
Furthermore, implant survival was 100% for implants with ligatures
in both genotype groups. Pirih et al27 reported an osseointegration
rate of 82% in WT mice and, at the end of the experiment, implant
survival rate was 60%. The difference of implant survival for ligatured
implants in WT mice is probably a result of the duration of the experi-
mental time period after ligation. In this study, samples were collected
2 weeks after ligation but it was 12 weeks in Pirih’s study.
In the present study, we developed a novel method to evaluate
the alveolar bone in peri-­implantitis and periodontitis using micro-­CT.
We defined the VOI as a cylinder (1.0 mm in diameter and 1.0 mm in
height) around the implants or natural teeth to standardize the eval-
uation of bone loss. This method is more accurate for quantifying the
irregularity of intrabony bone loss compared with measuring the bone-­
level value from the implant head27,42 in peri-­implant and periodontal
diseases and can be modified to fit experimental periodontitis models
in other species.
Moreover, our study provided a novel animal model to compare
peri-­implantitis and periodontitis by including the two diseases in the
same host environment. The data showed that bone loss was sig-
nificantly more severe in peri-­implantitis than in periodontitis in WT
and Tlr2-KO mice (Fig. S1), and the number of TRAP-­positive cells
was significantly higher in peri-­
implantitis than in periodontitis in
WT mice but not in Tlr2-KO mice (Fig. S3). These results indicate that
peri-­implantitis may induce more extensive osteoclastogenesis and
bone destruction than occurs in periodontitis under the same host
|
      105
environment and that TLR-­2 signaling plays an important role in this
process.
In conclusion, the data presented herein demonstrate that TLR-­2
signaling mediates bone loss in both peri-­implantitis and periodontitis
through upregulation of interleukin-­1β and TNF-­α. However, different
molecular features may be involved in TLR-­2-­mediated bone loss in
peri-­implantitis (interleukin-­10 cytokine response) versus periodonti-
tis (RANKL dependent). We provide a useful animal model to dissect
pathogenic mechanisms of peri-­implantitis and periodontitis simulta-
neously, and to explore specific therapeutic targets for each disease.
AC KNOW L ED G EM ENTS
This study was supported by NIH NIDCR grant DE023807 and
DE025255 to X. Han. The authors declare no potential conflicts of in-
terest with respect to the authorship and/or publication of this article.
O RC I D
X. Han   http://orcid.org/0000-0001-8357-1471
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S U P P O RT I NG I NFO R M AT I O N
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porting information tab for this article.     
How to cite this article: Yu X, Hu Y, Freire M, Yu P, Kawai T,
Han X. Role of toll-­like receptor 2 in inflammation and alveolar
bone loss in experimental peri-­implantitis versus periodontitis. J
Periodont Res. 2018;53:98–106. https://doi.org/10.1111/
jre.12492