Received: 2 August 2017 Revised: 9 October 2017 Accepted: 20 October 2017

DOI: 10.1002/JPER.17-0475

ORIGINAL ARTICLE

The evaluation of peri-implant sulcus fluid osteocalcin,

osteopontin, and osteonectin levels in peri-implant diseases
Oya Turkoglu Cakal1 Candan Efeoglu2 Emir Bozkurt3

1 Department of Periodontology, School of

Dentistry, Ege University, Izmir, Turkey
2 Department of Oral Surgery, School of
Dentistry, Ege University, Izmir, Turkey
3 Department of Biology, Celal Bayar
University, Manisa, Turkey
Correspondence
Oya Turkoğlu Cakal, Ege University, School
of Dentistry, Department of Periodontology,
Bornova-35100, Izmir, Turkey.
Email: oya_t@yahoo.com
Abstract
Background: Peri-implant mucositis is an inflammation of the soft tissues surround-
ing an implant. Peri-implantitis refers to a process characterized by peri-implant
bone loss along with an inflammation of the soft tissues. Osteocalcin, osteopon-
tin, and osteonectin proteins are related to bone remodeling. The aim of the present
study was to investigate peri-implant sulcus fluid (PISF) osteocalcin, osteopontin, and
osteonectin levels in peri-implant mucositis and peri-implantitis.
Methods: Fifty-two implants with peri-implantitis, 46 implants with peri-implant
mucositis, and 47 control implants were included in the study. Clinical parameters
including probing depth, modified sulcus bleeding index and modified plaque index
were recorded. PISF osteocalcin, osteopontin, and osteonectin levels were analyzed
by ELISA kits.

Results: There were no significant differences in PISF osteocalcin, osteopontin, and

osteonectin total amounts between healthy controls, peri-implant mucositis and peri-
implantitis groups (P > 0.05). Probing depths were not correlated with PISF osteo-
calcin, osteopontin, and osteonectin levels in the study groups (P > 0.05).

Conclusions: Soft tissue inflammation around dental implants does not cause a
change in osteocalcin, osteopontin, and osteonectin levels in PISF. Also, peri-
implantitis does not seem to give rise to an increase in PISF levels of osteocalcin,
osteopontin, and osteonectin.

KEYWORDS
osteocalcin, osteonectin, osteopontin, peri-implantitis

1 I N T RO D U C T I O N similar to those in chronic periodontitis and healthy controls,

Peri-implant mucositis is an inflammation of the soft tissues
surrounding a dental implant in function.1 Peri-implantitis
refers to an inflammatory process characterized by peri-
implant bone loss.2 Plaque accumulation is the most impor-
tant etiological factor for peri-implant diseases.3 However,
possible risk factors can affect the occurrence of disease.4
Poor oral hygiene, history of periodontitis and smoking has
been considered as risk factors at the Sixth European Work-
shop on Periodontology.3 Monje et al.5 have stated that sur-
vival rate of dental implants in aggressive periodontitis is
418 © 2018 American Academy of Periodontology
however success rate of implants is lower in patients with
aggressive periodontitis than those of chronic periodontitis
and controls. In a meta-analysis, it is stated that smoking
increases peri-implant bone loss by 0.16 mm per year.6
Osteocalcin is the most abundant non-collagenous
calcium-binding protein of mineralized tissues,7 and is mainly
synthesized by osteoblasts and odontoblasts.8 It plays a role
in bone resorption and mineralization.8 It is shown that serum
osteocalcin level is a marker of bone turnover and a clinical
diagnostic marker of metabolic bone diseases.8 Osteopontin
is also an important component of the bone, however studies
wileyonlinelibrary.com/journal/jper J Periodontol. 2018;89:418–423.
CAKAL ET AL.
demonstrated that osteopontin is present in non-mineralized
tissues.9 During early bone formation, osteopontin levels
are the highest in preosteoblastic cells. This protein plays an
important role in bone resorption and mineralization.8
Researchers demonstrated that no significant amounts of
osteocalcin in gingival crevicular fluid (GCF) were detected
in gingivitis patients.10 However, significant amounts of GCF
osteocalcin were detected in periodontitis patients and the lev-
els of GCF osteocalcin were significantly correlated with clin-
ical periodontal parameters.10 It was postulated that osteo-
calcin levels might reflect the severity of the periodontal
inflammation.10 Nakashima et al.11 also showed that GCF
osteopontin levels are associated with the progression of peri-
odontal disease and that non-surgical periodontal treatment
decreases the levels of GCF osteopontin levels.
Osteonectin, a component of bone matrix, has a role in
cell-matrix interaction in remodeling of the tissues.8,12 As
well as osteoblasts, endothelial cells and fibroblasts secrete
osteonectin.8,12 Since active osteoblasts have osteonectin this
bone component has been considered as a bone formation
marker,8,12 however its function is still unclear.
There are several studies investigating osteocalcin and
osteopontin levels in different periodontal diseases.9,11,13 It
is known that osteocalcin, osteopontin, and osteonectin pro-
teins are significantly related to bone remodeling.8 In the
present study, we hypothesized that the level of these pro-
teins might be affected by peri-implant inflammation, espe-
cially in peri-implantitis since the balance between bone
apposition and resorption is disturbed in favor of bone resorp-
tion during peri-implantitis. Therefore, the aim of the present
study was to investigate the PISF osteocalcin, osteopontin,
and osteonectin levels in peri-implant diseases to shed light
on universal attempts to identify relevant clinical markers of
peri-implant diseases.
2 M AT E R I A L S A N D M E T H O D S
2.1 Participant recruitment
A total of 144 dental implants (47 healthy control, 46 peri-
implant mucositis, and 52 peri-implantitis) in 111 participants
were included in the study. All participants were recruited
from the Department of Periodontology, School of Den-
tistry, Ege University between June 2006 and February 2008.
Study protocol was explained and written informed consent
was obtained from all participants. Ethics Committee of the
School of Medicine, Ege University approved the study pro-
tocol. Exclusion criteria were existence of uncontrolled sys-
temic diseases, immunological disorders, hepatitis, and HIV
infections, medications that could affect bone turnover and
mucosal healing, pregnancy or lactation. Former smokers and
patients aged < 18 years were also excluded from the study.
419
None of the participants had received antibiotics within the
last 3 months or periodontal treatment within the last 6 months
prior to enrolling this study. All oral implants were installed
for a minimum of 12 months and functionally loaded for a
minimum of 6 months.
2.2 Clinical examination
Examinations were performed by a calibrated examiner (OT).
A plastic periodontal probe was used to measure probing
depth (PD) at six sites per implant. Modified sulcus bleed-
ing index (MSBI)14 and modified plaque index (MPI)14 were
recorded. Existence of suppuration was also evaluated. A peri-
apical radiograph was taken from each implant. Addition-
ally, whole mouth PD, clinical attachment level (CAL), bleed-
ing on probing15 and plaque index16 were recorded to define
the periodontal state of the participant. The intraexaminer
reliability was high as revealed by an intraclass correlation
coefficient of 0.87 and 0.85 for PD and CAL measurements,
respectively.
2.3 Study groups
2.3.1 Peri-implant healthy group
Implants in this group were diagnosed by the absence of BOP
with no evidence of radiographic bone loss beyond normal
remodeling.
2.3.2 Peri-implant mucositis group
Implants in this group were diagnosed by the presence of BOP
with no evidence of radiographic bone loss beyond normal
remodeling.17
2.3.3 Peri-implantitis group
Implants in this group were diagnosed by BOP at least one
surface of the implant, PD ≥5 mm and radiographically
detectable bone loss > 2 mm from the implant platform after
initial remodeling.17 The presence of 2 mm of bone loss alone
without BOP was not diagnosed as peri-implantitis.17
2.3.4 Collecting PISF samples
The participants of the present study were recalled for PISF
sampling a day after the clinical examination. GCF samples
have been collected from the heathy sites in healthy controls,
sites with mucosal inflammation in peri-implant mucositis
group and sites with peri-implantitis in peri-implantitis group.
Sampling sites were selected from interproximal site of the
implant. Site was approached from buccal aspect for sam-
pling. Prior to sampling, supragingival plaque was removed
from the interproximal surfaces and surfaces were gently
dried and isolated by cotton rolls. PISF samples were taken
420 CAKAL ET AL.

with paper strips∗ . Paper strips were inserted 1 mm into the TABLE 1 Demographic data of the study groups
crevice and left for 30 seconds. Strips contaminated with Peri-implant Peri-
blood were discarded. The absorbed PISF volume of each strip Healthy mucositis implantitis
was measured by a calibrated electronic device† and placed group group group
into a sterile polypropylene tube and kept at -80◦ C. The read- Demographics (n = 47) (n = 46) (n = 52)
ings from this electronic device were converted to an actual Age (mean ± SD) 51 ± 9 50 ± 10 55 ± 10
volume (microliters) by reference to the standard curve. Sex
M (%) 13 (28) 15 (32) 21 (40)
2.3.5 Laboratory analysis Smoker (%) 8 (17) 10 (21) 15 (28)
PISF samples were eluted from two paper strips by incu- Time between 8±3 8±2 9±3
bating them in 100 𝜇L 0.5% Tween containing 0.1 M placement of
phosphate buffered saline solution (PBS-T, pH 7.4) for 15 the prosthetic
restoration and
minutes. After 100 𝜇L of PBS-T addition samples were
examination
centrifuged at 13,000 g for 5 minutes. Osteocalcin, osteopon- date (year)
tin, and osteonectin levels were measured with the enzyme- (mean ± SD)
linked immunosorbent method using the commercial ELISA
kits‡, § , ¶ in accordance with the manufacturer's instructions.
The minimum detectable limits of osteocalcin, osteopontin
and osteonectin are 0.5 ng/mL, 5 ng/mL, 0.6 ng/mL. The lev- of smokers and non-smokers was also similar in study groups
els of bone markers in each sample were calculated based on (P > 0.05). No significant difference was observed regarding
the dilutions and the results were expressed as total amount in the time between placement of the prosthetic restoration and
30 seconds in PISF sample. the date of examination among the study groups (P > 0.05).

2.3.6 Statistical analysis

All data were analyzed by a statistical package# . One-way
analysis of variance (ANOVA) test was used for comparison
of age, and Chi square test was used for comparison of gen-
der and smoking state among the study groups. Comparisons
of clinical parameters of sampling sites were performed by
analysis of variance or Kruskal-Wallis tests as appropriate.
Post hoc two-group comparisons were assessed by the Dun-
nett T3 or Mann-Whitney U tests. Similarly, PISF osteocalcin,
osteopontin, and osteonectin levels were analyzed by Kruskal-
Wallis test. Spearman rank correlation analysis was used to
evaluate the correlation between probing depth of sampling
sites and bone markers investigated.
3 RESULTS
3.1 Demographic data
The demographic data of the study participants are presented
in Table 1. There were no significant differences in age and
gender among the study groups (P > 0.05). The distribution
∗ Periopaper, ProFlow, Inc., Amityville, NY
† Periopaper, ProFlow, Inc., Amityville, NY
‡ Human Osteopontin (OPN) ELISA kit, Mybiosource
§ Human Osteocalcin/Bone gla protein (OT/BGP) ELISA kit, Mybiosource
¶ Human Osteopontin ELISA kit, Mybiosource
# SPSS Inc., ver. 17.0, Chicago, IL
3.2 Clinical data
Table 2 demonstrates clinical data of the sampling sites. As
expected, PD value of the peri-implantitis group was sig-
nificantly higher compared to peri-implant mucositis and
healthy groups (P < 0.05). Peri-implant mucositis group
had significantly higher PD value than healthy control
(P < 0.05). Similarly, MPI and MSBI scores were sig-
nificantly higher in peri-implantitis groups compared with
the other groups (P < 0.05). There were significant dif-
ferences in MPI and MSBI scores between the healthy
group and peri-implant mucositis group (P < 0.05). PISF
volume was significantly higher in peri-implantitis group
than peri-implant mucositis and healthy control groups
(P < 0.05).
3.3 Laboratory findings
Table 3 presents the PISF osteocalcin, osteopontin, and
osteonectin total amounts in the study groups. There were
no significant differences in PISF osteocalcin total amounts
between healthy controls, peri-implant mucositis and peri-
implantitis groups (P > 0.05). No significant differences
were observed in PISF osteopontin total amounts between
controls, peri-implant mucositis and peri-implantitis groups
(P > 0.05). Similar results were obtained for PISF osteonectin
total amounts (P > 0.05). No significant correlation was
observed between PD of sampling sites and PISF osteocalcin
(P = 0.835, r = 0.017), osteopontin (P = 0.308, r = −0.084),
and osteonectin levels (P = 0.888, r = −0.012).
CAKAL ET AL.
TABLE 2 Clinical parameters of sampling sites
Healthy group
Clinical parameters (n = 47)
PD (mean ± SD) 2.7 ± 0.9
MPI, med (min to max) 0 (0 to 1)
MSBI, med (min to max) 0 2 (1 to 2)b
PISF volume, med (min to max) 0.14 (0.01 ± 0.8)
a
significant difference from other groups.
b
significant difference from control group.
TABLE 3 PISF osteocalcin, osteopontin and osteonectin levels in the study groups
Healthy group
Bone related proteins (n = 47)
Osteocalcin (ng/site) (mean ± SD) 1.26 ± 0.29
Osteopontin (ng/site) (mean ± SD) 4.35 ± 0.79
Osteonectin (ng/site) (mean ± SD) 0.78 ± 0.22
4 DIS CUSSI O N
Up to date, several studies have examined different bio-
logic mediators such as interleukin 1 beta, prostoglandin,
matrix metalloproteinases, and proteolytic enzymes in peri-
implant diseases.18–23 However, there are very limited num-
ber of studies evaluating PISF osteocalcin, osteopontin, and
osteonectin levels in different peri-implant diseases. The
results of the present study demonstrated that PISF osteo-
calcin, osteopontin, and osteonectin total amounts were not
different between healthy controls, peri-implant mucositis,
peri-implantitis groups. Additionally, probing depths were
not found correlated with PISF osteocalcin, osteopontin, and
osteonectin levels.
In the present study, PISF volume in peri-implantitis sites
was significantly higher than sites with peri-implant mucosi-
tis and healthy sites. This finding is in accordance with
the study by Tumer et al.24 and Murata et al.25 Increased
levels of serum osteocalcin have been demonstrated during
rapid bone turnover periods. Although osteocalcin levels have
been evaluated in periodontal diseases, a very limited num-
ber of studies have investigated osteocalcin levels in peri-
implant diseases.10,11,23–25 Kunimatsu et al.10 demonstrated
that GCF osteocalcin levels were increased in periodontitis
while no significant GCF osteocalcin amount was detected
in gingivitis. Nakashima et al.26 found that GCF osteocal-
cin total amounts in sites with periodontitis were significantly
higher than those of gingivitis and healthy sites. Researchers
stated that elevated GCF osteocalcin levels might have diag-
nostic values for periodontal diseases.11 On the other hand,
Wohlfahrt et al.23 investigated PISF osteocalcin levels in peri-
implant diseases by Luminex. Researchers showed that no
significant decrease in PISF osteocalcin levels was obtained
421
Peri-implant mucositis group Peri-implantitis group
(n = 46) (n = 52)
4.7 ± 0.9b 6.8 ± 1.6a
b
2 (1 to 3) 3 (2 to 3)a
2 (2 to 3)a
0.41 (0.03 to 0.92) 0.74 (0.15 to 0.99)a
Peri-implant mucositis group Peri-implantitis group
(n = 46) (n = 52)
1.22 ± 0.23 1.23 ± 0.27
4.26 ± 0.47 4.42 ± 0.63
0.79 ± 0.15 0.73 ± 0.25
following surgical treatment of peri-implantitis.23 Similarly,
Moheng and Feryn27 stated that serum osteocalcin levels
were not predictive for implant loss. However, Tumer at al.24
examined PISF osteocalcin levels in peri-implant diseases
by radioimmunassay technique and they have revealed that
PISF osteocalcin levels were higher in peri-implantitis sites
than healthy sites. Murata et al.25 stated that PISF osteocal-
cin levels were significantly higher in peri-implant mucositis
sites than healthy sites. Researchers also showed that PISF
osteocalcin levels in peri-implantitis were not different from
either peri-implant mucositis or healthy sites.25 In the present
study, our results did not show any significant difference in
PISF osteocalcin levels between healthy and diseased groups.
These conflicting results might be caused by different study
protocols used in those studies. In the present study, only
four of the implants with peri-implantitis had suppuration.
Although peri-implantitis sites had bleeding on probing, it
might be suggested that disease was not in progression stage
at the time of sampling:therefore, PISF osteocalcin levels
remained unchanged. It is stated that GCF osteocalcin levels
might reflect the inflammation of periodontal tissues:however,
the role of PISF osteocalcin in peri-implant diseases is not
well understood yet.
Peri-implantitis has irreversible nature as well as periodon-
titis, therefore early diagnosis is important for being protected
from onset and the progression of the disease. Osteopontin has
a dual function in bone mineralization and resorption, and it
might serve as a diagnostic marker.8 It has been demonstrated
that GCF osteopontin levels were increased in sites with peri-
odontitis, and periodontal treatment gives rise to decrease in
GCF osteopontin levels.11 However, Wohlfahrt et al.23 found
no significant change in PISF osteopontin levels following
surgical periodontal treatment. In accordance with the study
422
by Wohlfahrt et al.23 no significant differences in PISF osteo-
pontin levels were observed among the study groups in the
present study.
Osteonectin has been considered as a bone formation
marker8,12 however its function is still unclear. To the best
of our knowledge, there are very limited numbers of studies
investigating osteonectin protein in periodontal diseases28,29
and none of them questioned its role in peri-implant dis-
eases. In the present study, we were expecting a change
in PISF osteopontin levels in especially implants with peri-
implantitis because of its role in bone formation. How-
ever, our results demonstrated no difference between control
implants and implants with peri-implantitis. Similarly, Baeza
et al.28 stated that there was no significant difference in GCF
osteonectin levels between patients with chronic periodonti-
tis and healthy controls. Moreover, Taylor30 stated that osteo-
calcin and osteonectin proteins are uncertain biomarkers for
periodontitis because the data about salivary osteocalcin and
osteonectin proteins are inconsistent though both have a role
in bone metabolism.
The present study has some limitations. First, PISF sam-
pling has been performed from implant sites with prosthetic
restorations. These restorations may interfere with the appro-
priate collection of PISF samples to some extent. Another
limitation might be the cross-sectional design of the present
study. This study design might prevent to determine the exact
nature of unchanged levels of osteocalcin, osteopontin, and
osteonectin levels in PISF. PISF sampling was performed in
sites with peri-implant inflammation however the existence
of peri-implant inflammation does not mean that there is a
progression of peri-implantitis. Additionally, if more markers
related to connective tissue and bone metabolism have been
investigated in the present study, the work would have given
more detailed information about the inflammatory process of
the diseases. Therefore, the studies evaluating the parame-
ters such as MMP, TIMP, RANKL, and OPG would give bet-
ter understanding about the pathogenesis of the peri-implant
diseases.
5 CONC LU SI ON S
Mucosal inflammation around dental implants does not have
an influence on osteocalcin, osteopontin, and osteonectin lev-
els in PISF. Additionally, peri-implantitis characterized by the
peri-implant tissue loss does not seem to cause an increase
in PISF levels of osteocalcin, osteopontin, and osteonectin.
Studies that performed PISF sampling following removal of
the fixed partial denture would give more precise information
about PISF osteocalcin, osteopontin and osteonectin levels in
peri-implant diseases. The unchanged pattern of chosen bone
markers could not present any conclusive evidence regarding
their role in peri-implant diseases.
CAKAL ET AL.
ACKNOW LEDGMENTS
The authors greatly appreciate the statistical advice received
from Professor Mehmet Orman, Department of Biostatistics
and Medical Informatics, School of Medicine, Ege University,
Izmir. This study was supported by a grant from Ege Univer-
sity Research Foundation. The authors declare no conflicts of
interest related to the present study.
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How to cite this article: Cakal OT, Efeoglu C,
Bozkurt E. The evaluation of peri-implant sulcus
fluid osteocalcin, osteopontin, and osteonectin levels
in peri-implant diseases. J Periodontol. 2018;89:418–
423. https://doi.org/10.1002/JPER.17-0475