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Journal of Prosthodontic Research 56 (2012) 249–255

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Original article
Matrix metalloproteinase-8 is the major potential collagenase
in active peri-implantitis
Hikaru Arakawa DDS, PhD, Junji Uehara DDS, PhD, Emilio Satoshi Hara DDS,
Wataru Sonoyama DDS, PhD, Aya Kimura DDS, PhD, Manabu Kanyama DDS, PhD,
Yoshizo Matsuka DDS, PhD, Takuo Kuboki DDS, PhD*
Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan
Received 21 January 2012; received in revised form 13 July 2012; accepted 30 July 2012

Abstract
Purpose: Current clinical procedures to control or regenerate bone loss due to peri-implantitis are not predictable neither accomplish complete
resolution. Therefore, early detection of the onset and the active periods of bone loss are crucial for prevention of extensive peri-implant bone
resorption. This study aimed to determine a possible association between the presence of collagenases (MMP-1, MMP-8 and MMP-13) in peri-
implant sulcular fluid (PISF) and active periods of bone loss by annually adjusted vertical bone loss (AVBL) measurements.
Methods: Intended sample consisted of 76 consecutive patients who received oral implant treatment at the Fixed Prosthodontic Clinic of Okayama
University Hospital from 1990 to 2000. Twelve subjects were lost to follow-up or refused to participate. Consequently, the actual sample consisted
of 64 patients who were followed-up for at least one year. Those patients with AVBL > 0.6 mm were included in the severe peri-implantitis group,
and randomly selected, age-, gender- and implantation site-matched healthy patients (AVBL < 0.3 mm) comprised the control group. PISF
samples were collected from both groups and further analyzed by western blot for detection of collagenases.
Results: Four patients presented severe peri-implantitis. MMP-8 was the only collagenase detected in peri-implant sites with ongoing bone loss.
PISF samples from control group showed no positive reactions to any collagenase.
Conclusion: This study showed MMP-8 as a possible marker for progressive bone loss in peri-implantitis.
# 2012 Japan Prosthodontic Society. Published by Elsevier Ireland. Open access under CC BY-NC-ND license.

Keywords: Dental implant; Peri-implantitis; Peri-implant sulcular fluid; Matrix metalloproteinase (MMP)

1. Introduction the peri-implant epithelial seal, pocket formation, purulence,

Implant-supported oral rehabilitation has gained worldwide
popularity throughout the last decades due to its efficient
clinical success rate and substantiated improvement of
individual’s quality of life [1,2]. After complete osseointegra-
tion, however, one of the major remaining concerns is peri-
implantitis disease which has been reported to occur in 6–10%
of the installed implants, and eventually can lead to implant
mobility and loss [3–6]. Peri-implantitis is defined as an
inflammatory process affecting soft and hard tissues surround-
ing an osseointegrated implant associated with breakdown of
* Corresponding author at: Oral Rehabilitation and Regenerative Medicine,
Okayama University Graduate School of Medicine, Dentistry and Pharmaceu-
tical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
Tel.: +81 86 235 6680; fax: +81 86 235 6684.
E-mail address: kuboki@md.okayama-u.ac.jp (T. Kuboki).
and progressive bone loss [7,8]. Although several clinical
procedures have been recently tried to restrain and regenerate
peri-implantitis progressive bone loss (e.g., bone graft
transplantations and/or laser irradiation after surgical curettage
of inflammatory tissue), the present techniques to fully recover
and re-osseointegrate the bone tissue around implant body are
not predictable neither accomplish complete disease resolution
[9–13]. Therefore, early detection and diagnosis of the onset
and ongoing periods of bone loss are essential to prevent future
extensive peri-implant bone loss. However, such biological
markers have not been reported yet.
Despite particular anatomical and histological characteristics
around the implant, the etiopathology of peri-implantitis has
been considered to be similar to periodontitis, with comparable
bacterial-type colonization and immune cell exudates. More
recently, studies have associated the irreversible peri-implant
connective tissue destruction with upregulation of matrix

1883-1958 # 2012 Japan Prosthodontic Society. Published by Elsevier Ireland. Open access under CC BY-NC-ND license.
http://dx.doi.org/10.1016/j.jpor.2012.07.002
250 H. Arakawa et al. / Journal of Prosthodontic Research 56 (2012) 249–255

Fig. 1. The preliminary validation study on PISF sampling method. (A) Examination protocol for the PISF sampling method. The preliminary study showed that the
most appropriate method (lane 1) was to carefully remove visible supra-gingival plaque and mildly dry the sampling site (mesio-buccal corner) by air spray in order to
remove saliva or blood from the sampling site. Paper strips were then gently inserted approximately 1 mm into the peri-implant sampling sites and kept in place for
30 s; GC, gingival crevice. (B) Collected samples were separated by SDS-PAGE, and detected by sliver staining. Sampling efficacy was higher in the conditions
utilizing a PBS-T solution (lanes 1, 2, 5 and 7) compared to PBS (lanes 3, 4 and 6). Numerous bands were observed in lanes 5 and 7 due to contamination of saliva and
blood in the PISF sampling process.

metalloproteinases (MMPs) [14–17]. MMP family comprises
five subgroups (collagenases, gelatinases, stromelyins, matrily-
sins and membrane-type MMPs) and is known by its capability to
degrade almost all extracellular matrix and basement membrane
molecules both in physiologic tissue repair and pathologic tissue
destruction [18–24].
The collagenase subgroup is composed mainly by MMP-1,
MMP-8 and MMP-13, and is of particularly relevance in both
periodontitis and peri-implantitis conditions due to their
capability to cleave native fibrillar collagens (types I, II, III,
and V) as well as various non-collagenous molecules
[20,22,23]. Previous studies have reported an upregulation
of these collagenases in gingivitis, early-onset (juvenile)
periodontitis, adult periodontitis and peri-implantitis [25–36].
However, most studies on peri-implantitis were cross-
sectional, lacked control group with healthy-implant patients,
or did not measure progressive/ongoing peri-implant bone
loss. Therefore, the purpose of this longitudinal study was to
analyze if MMP-1, MMP-8 and MMP-13 are detectable in
peri-implant sulcular fluids (PISFs) of healthy and diseased
sites, as well as to investigate the association between the
possible presence of these aforementioned MMPs and ongoing
bone loss measured by annually adjusted vertical bone loss
(AVBL) measurements.
2. Materials and methods
2.1. Patients and AVBL
Intended sample consisted of 76 consecutive patients who
received implant treatment (IMZ implants, Friatec1,
Friedrichsfeld, Germany; or Brånemark system implant,
Nobel Biocare1, Gothenburg, Sweden) at the Fixed
Prosthodontic Clinic of Okayama University Dental Hospital
from 1990 to 2000. Exclusion criteria were subjects who
received prosthesis (or functional loading) for less than one
year prior to the study onset, unwilling to participate, with
systemic diseases such as diabetes, immunodeficiency and
osteoporosis, and who had undergone irradiation therapy. In
response, 12 patients were excluded and the remaining 64
subjects constituted the actual study sample. All subjects
were explained about the objectives of this study and signed
the informed consent. The Ethics Committee of Okayama
University Graduate School of Medicine, Dentistry and
Pharmaceutical Sciences approved the research protocol
(#729).
All patients were followed-up for at least one year at a
frequency of 6–12 months. In order to assess periodically bone
levels, radiographic images were taken from all patients by
well-trained technicians using the parallel method at every
follow-up return. Bone level measurements (radiographic
image analyses) were calculated by three pre-calibrated
experienced dentists in a blind manner, and determined by
the distances between the alveolar bone crest and the respective
tooth cusp. Measurements were performed for the distal and
mesial sites, and were represented by the average of these two
values.
The annually adjusted vertical bone loss (AVBL) was
measured by subtraction of two consecutive bone level
measurements, i.e., current minus immediately previous bone
level. Positive AVBL values indicated progressive peri-implant
bone loss, whereas negative AVBL values corresponded to
progressive peri-implant bone regeneration. Each implant was
classified into three groups according to the AVBL measure-
ments: severe peri-implantitis group (AVBL > 0.6 mm), mild
peri-implantitis group (0.3 mm < AVBL < 0.6 mm) and
healthy group (AVBL < 0.3 mm). For calculation of peri-
implantitis incidence, in case of multiple implantation sites, the
 H. Arakawa et al. / Journal of Prosthodontic Research 56 (2012) 249–255 251

Table 1
Demographic information of the peri-implantitis and control patients.
Subject Age Sex Functioning Implant site Lane Baseline AVBL at the first AVBL at the second Total protein
(years) period (year) number AVBL MMP analysis MMP analysis (mg/mL)
Peri-implantitis patients
A 68 M 6.83 R-Man-1st 1 0.23 0.14 0.60 306
6.83 R-Man-2nd 2 0.66 1.98 0.51 425
B 65 F 4.83 L-Man-1st 3 1.16 0.81 0.01 438
4.83 L-Man-2nd 4 0.23 0.58 0.58 278
C 63 F 1.67 R-Max-1st 5 2.40 8.55 8.55 525
1.67 R-Max-2nd 6 0.84 0.50 0.50 312
D 78 M 7.42 R-Man-2nd 7 0.59 0.30 – 247
4.25 L-Man-1st 8 0.85 0.14 – 308
4.25 L-Man-2nd 9 0.68 0.35 – 375
Mean 68.50 4.73 0.84* 357.11*
S.D. 6.65 2.09 0.65 90.20

Control patients
E 66 M 1.00 L-Man-2nd 10 0 – – 227
1.00 L-Man-3rd 11 0 – – 240
F 59 F 4.42 L-Man-1st 12 0 – – 191
4.42 L-Man-2nd 13 0.07 – – 237
G 65 F 2.75 R-Man-1st 14 0.11 – – 187
2.75 R-Man-2nd 15 0 – – 224
H 74 M 2.42 R-Man-1st 16 0.25 – – 207
2.42 R-Man-2nd 17 0 – – 125
Mean 66.00 2.64 0.05* 204.75*
S.D. 6.16 1.30 0.09 37.83
AVBL, annually adjusted vertical bone loss; R, right; L, left; Max, maxillary; Man, mandibular; 1st, first molar; 2nd, second molar; 3rd, third molar; –, data not taken.
Severe peri-implantitis cases (AVBL > 0.6 mm) are highlighted in bold numbers.
*
p < 0.05 (t-test) relative to control group.

worst peri-implant site condition determined patient’s peri-
implantitis severity.
2.2. Preliminary validation of PISF sampling procedure
Prior to actual PISF sampling, a preliminary validation study
was carried out to determine the best clinical sampling strategy
(Fig. 1). PISF samples were collected from one patient according
to the clinical steps shown in Fig. 1A. The optimal sampling
efficacy (Fig. 1B) was obtained when utilizing a phosphate-
buffered saline containing Tween-20 (PBS-T) solution (lanes 1,
2, 5 and 7) compared to PBS solution. On the other hand,
numerous bands were observed in lanes 5 and 7 probably due to
saliva and blood contamination during the sampling process.
Therefore, the most appropriate sampling method (lane 1) was to
carefully remove any visible supra-gingival plaque, mildly dry
the sampling site (mesio-buccal corner) by air-spray in order to
remove saliva or blood from the peri-implant site, and
subsequently insert paper strips (Diadent1, Seoul, Korea) gently
into approximately 1 mm of the mesio-buccal corner of the peri-
implant sulcus, keeping it in place for 30 s.
Collected samples were separated by sodium dodecyl
sulfate-polyacrylamide (SDS-PAGE) and detected by a silver
stain kit (BIO-RAD, Hercules, CA, USA), further dissolved in a
300 mL solution of PBS-T and stored at 20 8C for further
analysis. The total amount of protein was assessed using a BCA
Protein Assay Reagent kit (PIERCE, Rockford, IL, USA).
2.3. PISF sample collection and analysis
PISF sampling was performed at two different time-points
by three distinct dentists. Initial PISF samples were collected
from all implant sites of peri-implantitis patients and compared
to randomly-selected healthy controls matched by age, gender
and the implantation site. Peri-implantitis patients were then
submitted to mechanical peri-implant scaling and anti-
microbial treatment by a subgingival application of minocy-
cline hydrochloride (Periocline1, Sunstar, Osaka, Japan), and,
subsequently, to second PISF sampling for re-assessment of
possible changes in the presence/absence of collagenases.
Collagenase presence were analyzed by western blotting
with anti-human monoclonal antibodies specific for MMP-1,
MMP-8 and MMP-13 with positive controls [37]. In brief,
samples were initially electrophoresed in SDS gel (10%) under
reducing conditions. Proteins were transferred onto a poly-
vinilidene-difluoride membrane and blocked for 1 h with 10%
skim milk in PBS-T. The blocked membrane was incubated
with anti-human MMP-1 (1 mg/mL), MMP-8 (10 mg/mL) and
MMP-13 (2 mg/mL) monoclonal antibodies (Fuji Chemical
Industry, Takaoka, Japan), diluted with 10% skim milk in PBS-
T and incubated overnight. The membrane was washed 3 times
with PBS-T, and incubated with peroxidase-conjugate anti-
mouse IgG (1/2000 dilution in PBS-T, Amersham Life Science,
Little Chalfort, Buckinghamshire, England) for 1 h at 37 8C.
Finally, the membrane was washed 3 times with PBS-T,
252 H. Arakawa et al. / Journal of Prosthodontic Research 56 (2012) 249–255
Fig. 2. Total accumulated bone loss (TABL) of peri-implantitis implants. (A)
Left charts show the total amount of accumulated bone loss (TABL) with
periods of active and inactive bone loss after implant loading. Solid and dotted
arrows indicate the time-points of the first and second PISF sampling, respec-
tively. (B) Radiographic images show notable vertical bone loss around the
implant bodies with active severe peri-implantitis at the first PISF sampling.
developed by ECL plus reagent (Amersham Life Science, GE
Healthcare, USA) and exposed to ECL Hyperfilm1 (Kodak,
Tokyo, Japan).
3. Statistics
Baseline data comparison between peri-implantitis and
healthy control groups was performed by t-tests for indepen-
dent samples, using SPSS version 17.0 (SPSS Japan Inc.).
Significant level was set as a = 0.05.
Fig. 3. Initial PISF analysis for MMP-8. The 85 kDa band corresponding to
latent MMP-8 was observed in lanes 2, 3 and 5 of patients A, B and C,
respectively. However, the 64 kDa band corresponding to active MMP-8 was
observed only in lane 3 (patient B). No band equivalent to MMP-8 was observed
either in the lanes of patient D or in control group.
4. Results
4.1. Baseline data and peri-implantitis incidence
Among the total of 64 patients (male/female: 31/33; mean
age: 68.5  6.7 years; 162 implants) that fulfilled the selection
criteria, only 4 subjects (male/female: 2/2; mean age:
68.5  6.7 years; 9 implants) presented baseline AVBL higher
than 0.6 mm and were included in the severe peri-implantitis
group (Table 1). Among the 9 implants installed in the 4 peri-
implantitis patients, six of them (implants 2, 3, 5, 6, 8 and 9)
presented severe peri-implantitis condition, while three cases
(implants 1, 4 and 7) had normal bone levels, which enabled an
intra-individual control group. At the first PISF sampling,
however, implants 7, 8 and 9 in patient D presented normal
AVBL levels (Table 1).
No patient was classified in the mild peri-implantitis group.
The healthy group comprised a total of 60 patients, among
which 4 subjects (male/female: 2/2, mean age: 66.0  5.8
years, 8 implants) were randomly selected and matched to the
peri-implantitis group by age, gender and implantation site.
Despite of sample matching, mean functional duration was
significantly ( p = 0.029) longer in the peri-implantitis group
(5.0  2.3 years) compared to control group (2.7  1.4 years).
Additionally, mean amount of collected protein from the peri-
implantitis group (357.11  90.2) was also significantly
( p = 0.001) higher than the control group (204.75  37.83)
(Table 1).
Based on these results, the incidence of peri-implantitis was
of 6.25% of patients and 3.7% of implants.
4.2. Radiographic findings
Fig. 2 shows radiographic images of the 4 peri-implantitis
subjects with notable vertical bone loss around the active peri-
implantitis sites (implants 2, 3, 5, 6, 8 and 9) compared to
inactive peri-implantitis sites (implants 1, 4 and 7). Charts
demonstrate the total accumulated amount of bone loss (TABL)
with notable periods of active bone resorption.
4.3. PISF sample analysis
Fig. 3 shows western blotting results of the first PISF
sampling. The 85 kDa band corresponding to pro-MMP-8 was
 H. Arakawa et al. / Journal of Prosthodontic Research 56 (2012) 249–255
Fig. 4. Initial PISF analysis for MMP-1 and MMP-13. MMP-1 and MMP-13 were not detected in any PISF sample.
Fig. 5. Second PISF analysis for MMP-8 after the anti-microbial treatment. At
the second PISF sampling analysis, an 85 kDa band corresponding to latent
MMP-8 was observed only in implant 5 of patient C who could not receive
mechanical and anti-microbial treatment. Half-a-year following the second
PISF sampling, the implant 5 in patient C was lost.
observed in implants 2, 3 and 5 of patients A, B and C,
respectively. On the other hand, the 64 kDa band corresponding
to active-MMP-8 was observed only in implant 3 of patient B.
No band equivalent to MMP-8 was observed in the control
group (Fig. 3). MMP-1 and MMP-13 were not detected in any
lane (Fig. 4).
After the first PISF sampling, patients A and B received peri-
implant mechanical plaque removal and anti-microbial treat-
ment, however, patient C was hospitalized for cerebral
infarction and was then unable to receive any treatment. The
second PISF sampling analysis showed a pro-MMP-8 band
only in implant 5 of patient C, who had the respective implant
lost 6 months later due to severe peri-implantitis (Fig. 5).
5. Discussion
In this longitudinal study, the incidence of peri-implantitis
was of 6.25% of patients and 3.7% of implants, which is
consistent with previous literature [5,6,38]. One particularity of
this investigation was the classification of peri-implantitis
condition according to the ongoing bone loss (annually adjusted
vertical bone loss; AVBL), which enabled a dynamic evaluation
of the peri-implant tissue condition with periods of stability,
bone loss (positive AVBL) or even bone regeneration (negative
AVBL). For example, remarkable bone loss levels were
253
observed within the first and third year after implant
installation, which could possibly indicate the peri-implantitis
onset-period (Fig. 2). On the other hand, bone regeneration in
peri-implantitis cases was detected specially after mechanical
and anti-microbial peri-implant treatment. The implant number
2 in patient A presented mild bone regeneration, whereas
implant 3 in patient B showed stabilization of bone loss within
normal values (AVBL < 0.3 mm). Patient D was a clear
example that presented active periods of bone resorption (initial
AVBL > 0.6 mm) followed by periods of stability (AVBL at
the first PISF sampling < 0.3 mm) without being submitted to
any treatment. These observations indicated that peri-implan-
titis presented episodic and site-specific tissue destruction,
similar to chronic periodontitis [26,35].
In this study, we were also able to compare the peri-
implantitis cases (implants 2, 3, 8 and 9) with both intra-
individual (implants 1, 4 and 7) and inter-individual (implants
10 to 17) healthy implant controls. In the first PISF sampling,
MMP-8 was the only collagenase observed in peri-implantitis
sites (Fig. 3). Conversely, no collagenase could be detected
either in the intra-individual or inter-individual healthy sites.
These results are consistent with previous cross-sectional
studies which demonstrated the detectability of MMP-8 in peri-
implantitis PISF samples [28,29,35,36].
In particular, no collagenase was detected in PISF samples
of patient D in the peri-implantitis group. A possible
explanation could be that although this patient presented
initial AVBL higher than 0.6 mm, bone resorption activity was
stabilized within normal values (AVBL < 0.3 mm) in all three
implant bodies (implants 7, 8 and 9) at the exact PISF sampling
time. This dynamic alveolar bone turnover could also explain
the reason why some MMPs were not found in PISF samples in
previous reports due to probable latent period in bone loss at the
time of sample collection [21,39].
It should also be emphasized that detection of MMP-8 was
not dependent on the total accumulated amount of bone loss
(TABL) level. For example, although implants 7 and 8
presented higher TABL compared to implant 5 (Fig. 2), bone
loss turnover at the exact PISF sampling time was active only in
254 H. Arakawa et al. / Journal of Prosthodontic Research 56 (2012) 249–255
the latter case, and consequently, MMP-8 was detected only in
this case.
After mechanical and anti-microbial peri-implant treatment,
AVBL measurements and PISF samples were again collected
from patients A, B and C. Calculation of AVBL measurements
demonstrated normalization of bone loss in patients A and B.
On the other hand, patient C, unfortunately, was unable to
attend the peri-implantitis treatment follow-up returns due to
hospitalization. Consequently, patient C continued presenting
with severe peri-implantitis in implant 5, which was lost 6
months after the second PISF sampling, whereas implant 6
presented AVBL < 6 mm.
This situation, however, unexpectedly enabled inter-indivi-
dual comparison between this particular patient and the other
two that received peri-implantitis treatment. Western blot
analysis of the second PISF samples again detected MMP-8
only in implant 5 of patient C (Fig. 3). MMP-8 could not be
detected either in the intra-individual implant control (implant
6) or in the implants of patients A and B (Table 1 and Fig. 3).
These results demonstrated that MMP-8 was the only
collagenase associated with severe peri-implantitis during
ongoing periods of bone loss. This association, however, does
not necessarily imply that MMP-8 directly degrades the peri-
implant tissues in situ, because the western blot results showed
MMP-8 mostly in its pro-active (latent) form.
As seen in Fig. 3, active MMP-8 was observed only in
implant 3 of patient B, which coincided with a terminal phase of
bone resorption (Fig. 2). On the other hand, latent MMP8 was
detected in implants 2 and 5 of patients A and C, respectively,
and corresponded to active phase of bone resorption. A possible
reasoning for active MMP8 being detected in terminal phase is
based on data of recent studies that demonstrated that MMP8
could also act as a protector of bone resorption [40]. Kuula et al.
demonstrated that MMP8 knock-out mice presented more
extensive alveolar bone resorption caused by bacterial infection
compared to wild type controls [40]. Therefore, MMP8 could
act as an inhibitor of bacterium-induced peri-implant tissue
destruction, and therefore be in its active form in the terminal
phase of bone resorption.
MMP-1 and MMP-13 could not be detected in any PISF
sample (Fig. 4). This is in corroboration with previous reports
using western blotting which also showed negative MMP-1
detectability in peri-implant tissues [21]. On the other hand,
immunohistochemistry of peri-implantitis and healthy peri-
implant granulation tissues showed significant difference with
respect to quantitative immunolabelling for MMP-13, but for
MMP-1 [25]. Nevertheless, that study utilized granulation
tissues as subject’s samples, which definitely could attain great
concentrations of MMPs; however, in terms of biomarker utility
in a clinical setting, the authors believe that PISF sampling is
more convenient, simple and harmless compared to intra-
granulation tissue sampling procedure.
Other reasons for undetectability of MMP-1 and MMP-13
could be associated with the limited sample size as well as the
solutions utilized in this present investigation. A previous study
reported that MMP detectability was very sensitive to methods
and materials, especially medium and/or buffer solutions which
could possibly mask the exact bands corresponding to MMPs
[39]. Therefore, research is ongoing to determine the solutions
for optimal detection of several types of MMPs.
Another point to consider in this study is that classification
of peri-implantitis groups was based exclusively on radio-
graphic images, which are subjected to intrinsic distortions as
well as sensitivity of the techniques applied. Nevertheless,
Esposito et al. [41] reported that well-performed and well-
analyzed radiographic images are more reliable than other
clinical parameters such as peri-implant probing depth or
bleeding. For example, probing depth may be difficult to be
performed around implant screws, especially in severe bone
loss cases in which the threads have already been reached [41].
Additionally, other clinical classifications such as bleeding and
gingival index have been reported not to directly correlate with
the severity of bone loss [31].
6. Conclusion
This study showed that MMP-8 is the major collagenase
present in PISF of active peri-implantitis sites. Future studies
with larger study samples are necessary to confirm this possible
role of MMP-8 as a predictor for active periods of peri-
implantitis alveolar bone loss. Additionally, analysis of the risk
factors for MMP-8 unbalance, as well as a deeper insight into
the molecular interactions among MMPs, other inflammatory
cytokines, microorganisms and peri-implant tissues should also
be investigated.
Conflict of interest
All authors have no conflict of interest.
Acknowledgements
This study was partially supported by a Grant-in-Aid
(#13672031, #15592049, #17592025) from the Japan Society
for the Promotion of Science.
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