Volume 86 • Number 12

Gingival Crevicular Fluid Levels of Sclerostin, , and Receptor Activator of Nuclear Factor-kB Ligand in Periodontitis Umut Balli,* Ahmet Aydogdu,† Figen Ongoz Dede,* Cigdem Coskun Turer,* and Berrak Guven‡

Background: To investigate changes in the levels and rel- ative ratios of sclerostin, osteoprotegerin (OPG), and recep- tor activator of nuclear factor-kB ligand (RANKL) in the gingival crevicular fluid (GCF) of patients with periodontitis after non-surgical periodontal treatment. Methods: Fifty-four individuals (27 healthy controls and 27 patients with chronic periodontitis [CP]) were enrolled in eriodontal disease is a complex the study. Periodontitis patients received non-surgical peri- biologic process related to the in- odontal therapy. GCF sampling and clinical periodontal pa- Pteraction between groups of mi- rameters were assessed before and 6 weeks after therapy. croorganisms and the host immune/ Sclerostin, OPG, and RANKL levels were measured by enzyme- inflammatory response.1 When the bal- linked immunosorbent assay, and their relative ratios were ance between microbial challenge and calculated. host response is disturbed, periodontal Results: Total amounts and concentrations of sclerostin breakdown (clinical attachment loss [AL] were significantly higher in patients with CP than in healthy and alveolar bone resorption) can oc- individuals (P <0.025) and decreased after treatment cur.1,2 Microorganisms and their prod- (P <0.05). The RANKL/OPG ratio was significantly lower in ucts are the primary etiologic factors that healthy individuals than in patients with periodontitis before directly initiate periodontal disease. and after treatment (P <0.025), but no significant difference However, the majority of periodontal was observed in patients with periodontitis after treatment breakdown is caused by endogenous (P >0.05). The sclerostin/OPG and sclerostin/RANKL ratios proteases (matrix metalloproteinases) were significantly lower in healthy individuals than in patients and inflammatory mediators, such as < with periodontitis before and after treatment (P 0.025) prostaglandin E2 and tumor necrosis anddecreasedinpatientswithperiodontitisaftertreatment factor (TNF)-a, resulting in activation of (P <0.05). the bone resorption mechanism.2,3 The Conclusions: The GCF sclerostin level may be more reli- regulation of bone metabolism is a com- able than the RANKL/OPG ratio as a diagnostic and prognos- plicated process involving diverse signal tic marker of periodontal disease and treatment outcome. transduction pathways.4,5 Clarifying the Regulation of sclerostin levels may aid the development of different mechanisms of coupling be- new therapeutic strategies for the treatment of periodontal tween bone resorption and formation is disease. J Periodontol 2015;86:1396-1404. important for understanding and manag- KEY WORDS ing periodontal disease. The pathway of osteoclast-mediated Gingival crevicular fluid; osteoprotegerin; periodontal bone resorption is closely related to in- diseases; RANK ligand. teraction of the TNF superfamily: 1) re- ceptor activator of nuclear factor-kBligand * Department of Periodontology, Faculty of Dentistry, Bulent Ecevit University, Zonguldak, (RANKL); 2) its receptor, RANK; and 3) its Turkey. † Department of Periodontology, Faculty of Dentistry, Biruni University, Istanbul, Turkey. decoy receptor, osteoprotegerin (OPG). ‡ Department of Medical Biochemistry, Faculty of Medicine, Bulent Ecevit University. RANK is a cell-surface receptor expressed on osteoclast precursors and osteoclasts.4 RANKL is a potent osteoclastogenic factor

doi: 10.1902/jop.2015.150270

1396 J Periodontol • December 2015 Balli, Aydogdu, Ongoz Dede, Coskun Turer, Guven

expressed in , fibroblasts, lymphocytes, and ratio as a biomarker for periodontal disease activity .4-6 It binds directly to RANK on osteoclast and treatment outcomes. The correlations between lineage cells, resulting in osteoclast differentiation and biochemical markers and clinical parameters were activation and thus activating bone resorption.5,6 also tested. The primary efficacy variables were RANKL activity is regulated by OPG produced by bone changes in sclerostin, RANKL, and OPG levels and marrow stromal cells, osteoblasts, and osteocytes.4-6 the RANKL/OPG, sclerostin/OPG, and sclerostin/ OPG acts by binding to RANKL and preventing it from RANKL ratios after periodontal treatment. Second- binding to RANK, thereby inhibiting osteoclastogenesis ary outcome variables were probing depth (PD), and bone resorption.5,6 clinical attachment level (CAL), gingival index (GI), Bone metabolism is also regulated by the Wnt andbleedingonprobing(BOP). (wingless-type MMTV integration site family) signaling pathway, which is composed of 19 secreted glyco- MATERIALS AND METHODS .7,8 The Wnt pathway increases bone forma- Study Population tion and regeneration via stimulation of From December 2013 to October 2014, 54 in- development.9,10 Regulation of Wnt signaling is me- dividuals (25 males and 29 females; aged 25 to 49 diated through posttranslational modification of Wnts, years; mean age: 37.59 – 5.30 years) admitted to the receptor regulation, and antagonist binding.11,12 Periodontology Department, Faculty of Dentistry of Sclerostin, a product of the SOST , is a secreted Bulent Ecevit University, were enrolled in this study. glycoprotein that binds low-density lipoprotein All individuals provided written informed consent, and receptor-related 5 and blocks the Wnt sig- the study protocol was approved by the Ethics naling pathway.13,14 Its expression, which suppresses Committee of Bulent Ecevit University, Zonguldak, osteoblastogenesis and reduces the viability of oste- Turkey (2013-112-01/10). This study is registered at oblasts and osteocytes, leads to unbalanced bone ClinicalTrials.gov as NCT02390479. turnover in favor of bone resorption not only by an- Clinical Examinations and Intraexaminer tagonizing Wnt but also by blocking bone morpho- Reproducibility genetic protein signaling.15,16 A deficiency of PD and CAL were measured, and plaque index (PI), sclerostin leads to sclerosteosis and van Buchem GI, and BOP scores were recorded using a periodontal disease, characterized by high bone mass. Sclerostin probe.§ All clinical examinations (at six different sites is produced by osteocytes, as OPG and RANKL.14 In around each tooth: mesio-buccal, disto-buccal, a broader context, osteocytes are naturally occurring mid-buccal, mesio-lingual, disto-lingual, and mid- modulators of bone metabolism that regulate the lingual), group allocations, sampling site selections, balance between osteoclastic and osteoblastic activ- and GCF collections were performed by the same ity. Sclerostin is a marker of mature osteocytes and investigator (FOD), who was masked with respect to affects bone metabolism by inhibiting osteoblast dif- the study design. Before the actual measurement, ferentiation.17,18 It is believed to act by promoting 10 individuals were selected randomly and used to osteoclast formation via a RANKL-dependent path- calibrate the investigator. The investigator evalu- way and by interacting with osteoblasts.19 At the ated the individuals on two separate occasions, 48 molecular level, osteocytes regulate bone homeo- hours apart. The investigator’s measurements were stasis through at least three key molecules: 1) scle- considered sufficiently reproducible if those taken at rostin; 2) OPG; and 3) RANKL.6,9,20,21 baseline and at 48 hours differed by no more than The roles of OPG and RANKL in periodontal 10% at the millimeter level.22 diseasehavebeenstudiedwidely. However, to the best of the authors’ knowledge, no studies have Inclusion and Exclusion Criteria evaluated changes in sclerostin levels in the gingival Diagnoses were based on the International World crevicular fluid (GCF) of patients with chronic Workshop for a Classification of Periodontal Disease periodontitis (CP) after non-surgical therapy or have and Conditions.23 The selected individuals had investigated the relationship between the RANKL/ a minimum of 20 natural teeth, excluding third mo- OPG ratio and sclerostin in periodontal disease. lars. All individuals underwent radiography and a full- Therefore, the primary objective in this case- mouth periodontal examination including PD, CAL, control intervention study is to explore the effect PI,24 GI,25 and BOP.26 Individuals were categorized of non-surgical periodontal therapy on the GCF into two groups: 1) individuals with a clinically levels of sclerostin in patients with CP to deter- healthy periodontium (group 1; n = 27; 12 males and mine the usefulness of sclerostin as a diagnostic 15 females; aged 35.63 – 4.86 years) and 2) patients and prognostic biomarker of periodontal disease. with generalized CP (group 2A; n = 27; 13 males and As secondary objective, it was examined whether sclerostin was more reliable than the RANKL/OPG § Williams periodontal probe, Hu-Friedy, Chicago, IL.

1397 Sclerostin Levels in Periodontal Health and Disease Volume 86 • Number 12

14 females; aged 39.56 – 5.07 years). After the patients with CP had been treated with scaling and (0) (0)

† † root planing (SRP), they were considered as group

BOP (%) 2B. Inclusion criteria for the healthy group were GI = 0, PD £3 mm, and no signs of AL and bone loss

* by clinical and radiographic examination. Inclusion PI criteria for the CP group were clinical signs of in- flammation (red color and swelling of the gingival margin), GI ‡2, PD and CAL ‡5 mm, and bone loss * affecting >30% of the existing teeth on clinical and radiographic examination. Descriptive statistics of the study population are summarized in Table 1. * The exclusion criteria were as follows: 1) ag- (mm) GI CAL gressive periodontitis; 2) periapical pathologies; 3) excessive forces including mechanical forces from Sampled Sites Periodontal Examination orthodontics and occlusion; 4) systemic diseases *

PD (e.g., diabetes mellitus; cancer; human immuno- (mm)

2 (1 to 2) 2 (1 to 2) 0 (0) 0 (0 to 1) 0 deficiency virus; bone-related diseases that com- promise sclerostin, OPG, or RANKL levels; and collagen-metabolic diseases or disorders); 5) chronic high-dose steroid therapy; 6) radiation or (%)

* immunosuppressive therapy; 7) pregnancy; 8) lactation; 9) smoking within the past 5 years; or 10) BOP allergy or sensitivity to any drug. Study participants had no history of periodontal therapy or drug therapy (e.g., anti-inflammatories, antibiotics, or any other pharmacologic treatment) for at least 6 months. * PI

test (unpaired observations) and Wilcoxon signed-rank test (paired observations); Periodontal Treatment U After baseline GCF sampling had been completed,

0.36 (0.10 to 0.54) 0 (0) patients with CP received non-surgical periodontal therapy consisting of SRP (quadrant by quadrant) using manual scalers and curetsi under local an-

* esthesia. The visits were scheduled as once a week for 4 weeks, and each visit was 45 to 60 minutes long. No antibiotics were prescribed during the treatment. The patients received oral hygiene in- structions, including use of the modified Bass

Full-Mouth Periodontal Examination technique, regular toothpaste, and an appropriate

(mm) GI interdental cleaning device with dental floss and an

* interdental brush. Periodontal treatment was per- formed by the same investigator (FOD). Collection of Samples In all groups, two sites per individual were selected for sampling. GCF samples were obtained from the 0.025. = (mm) CAL mesio-buccal or disto-buccal surfaces of single-

* rooted teeth. Samples were taken from the same PD

0.05/2 sites at baseline and at 6 weeks after SRP in patients = 1.63 (1.27 to 2.31) 1.63 (1.27 to 2.31) 0 (0) 2.46 (2.10 to 2.90) 3.48 (2.49 to 4.49) 0.24 (0.00 to 0.56) 0.71 (0.31 to 0.97) 10.59 (6.18 to 15.13) 3 (2 to 3) 4 (3 to 5) 0 (0 to1) 0 (0 to 1) 0

a with CP. To prevent the contamination of GCF with blood associated with the probing of inflamed sites, all clinical examinations and sampling site selec- tions were performed 2 days before GCF samples were collected. In the healthy group, GCF samples 0.05, statistically significant difference0.05, between statistically groups. significant difference from group 2A. (healthy) (CP after treatment) < < Group 1 Group 2A (CP) 4.91Group (4.31 2B to 5.81) 5.63 (4.58 to 6.90) 2.44 (2.15 to 2.77) 2.20 (2.02 to 2.69) 87.95 (82.36 to 95.96) 6 (5 to 7) 7 (5 to 8) 2 (2 to 3) 2 (2 to 3) 100 (100) P P Bonferroni correction Table 1. Clinical Parameters (full-mouth and sampled sites periodontal examination) in the Study Groups Data are expressed as the median (range). Kruskal–Wallis/Bonferroni-adjusted Mann–Whitney i * † Hu-Friedy.

1398 J Periodontol • December 2015 Balli, Aydogdu, Ongoz Dede, Coskun Turer, Guven

were collected from sites with no clinical inflamma- It was estimated that a sample size of 21 patients in tion. In the CP group, GCF samples were collected each group would allow for a type II error level of b = from sites with the greatest clinical signs of inflam- 0.20 (80% power) and a type I error level of a = 0.05 mation (the highest GI scores with BOP) and PD, (5% probability). To account for possible dropouts, along with radiographic confirmation of alveolar bone 27 patients in each group were included. Because no loss. Before GCF sampling, the sites were isolated sample size calculation could be performed a priori, with cotton rolls, saliva was removed, and any a retrospective power was calculated. A posteriori supragingival plaque present was removed using power calculation yielded a power of 97% to detect a sterile curette. GCF was sampled with filter paper¶ differences in outcomes before and after treatment. using the intracrevicular method.27 Paper strips were The Shapiro–Wilk test was used to determine placed into the crevice until mild resistance was felt whether the data were distributed normally. Com- and were left in position for 30 seconds. Strips with parisons of the biochemical and clinical parameters visible saliva or blood contamination were discarded. were analyzed using the Kruskal–Wallis non-parametric The GCF volume of each strip was determined by test, followed by post hoc group comparisons with the electronic impedance.# The two strips from each Bonferroni-adjusted Mann–Whitney U test (group 1 participant were placed into an individual centrifuge versus group 2A and group 1 versus group 2B) after tube, pooled to make one sample, and immediately normality of data had been failed. For the Bonferroni stored at -40C until analysis. correction, a = 0.05/2 = 0.025 was considered to be statistically significant. The Wilcoxon signed-rank Quantification of Sclerostin, OPG, and RANKL test (paired observations) was used to compare the On the day of the assay, 400 µL phosphate-buffered baseline values to those after treatment. x2 analysis saline (pH 7.4) was added to each of the tubes was used to compare the BOP percentage and the containing the sample strips. The tubes were vor- proportion of sexes between groups. The Spearman texed and homogenized for 1 minute and then rank correlation test was used to detect the rela- centrifuged at 3,000 · g for 15 minutes at 4C. The tionships between GCF sclerostin, OPG, RANKL, supernatants were collected. The total amounts of and RANKL/OPG ratio with the CAL and GI of the sclerostin,** OPG,†† and RANKL‡‡ in the samples sample sites. All tests were performed using statis- were analyzed by sandwich enzyme-linked immu- tical software.§§ The median with range values were nosorbent assay using commercially available kits. calculated for each parameter, based on the patients All of the samples and standards were assayed in as the statistical unit. P <0.05 was considered to be duplicate, as suggested by the manufacturer. statistically significant. Total values of sclerostin, OPG, and RANKL were measured in picograms. The standard detection limits RESULTS of the sclerostin, OPG, and RANKL assays, as reported Clinical Findings by the manufacturer, ranged from a minimum of 125, Clinical findings are summarized in Table 1. PD, CAL, 93.7, and 78 pg/mL, respectively, to a maximum of BOP, PI, and GI measured both in the full mouth and 4,000, 6,000, and 5,000 pg/mL, respectively. The at the sample sites were significantly higher in the minimum detection limits (sensitivity) of the assay periodontitis group than in the healthy group were <15 pg/mL for sclerostin, <5 pg/mL for OPG, and (P <0.05). As expected, all clinical parameters sig- <10 pg/mL for RANKL. The intensity of the color was nificantly decreased in periodontitis patients after measured at 450 nm, and results were calculated SRP, both in the full mouth and at the sample sites using the standard curves included in each assay kit. (P <0.05). There were no significant differences in GCF sclerostin, OPG, and RANKL concentrations age or proportion of sexes between controls and were calculated by dividing the total amount of scle- patients with CP (P >0.05). rostin, OPG, or RANKL (picograms) by the volume of the GCF (microliters). Values of concentrations are Biochemical Findings expressed as picograms per microliter. Total amounts and concentration levels of sclerostin are shown in Figure 1. The total amount and con- Statistical Analyses centration of GCF sclerostin were significantly higher The primary outcome variable (change in GCF in patients with CP than in healthy individuals sclerostin levels after therapy) was used to decide the (P <0.025). The total amount and concentration of sample size calculation and determine the power of the study. However, no sample size calculation could ¶ PerioPaper, ProFlow, Amityville, NY. be performed because there was no precise infor- # Periotron 8000, ProFlow. mation available regarding GCF sclerostin levels. ** Aviscera Bioscience, Santa Clara, CA. †† Boster Bio, Pleasanton, CA. Therefore, the current estimates were based on the ‡‡ Boster Bio. pilot study, which included 12 patients in each group. §§ SPSS v.19.0, IBM, Armonk, NY.

1399 Sclerostin Levels in Periodontal Health and Disease Volume 86 • Number 12

after treatment (P <0.05), although no significant difference in the total amount of OPG was observed (P >0.05). As indicated in Figure 3, the total amount and concentration of RANKL were significantly lower in healthy individuals than in those with periodontitis, both before and after treatment (P <0.025). The concentration of RANKL increased after treatment with SRP, although the total amount of RANKL de- creased; the differences were statistically significant (P <0.05). The relative ratios of RANKL/OPG, sclerostin/ OPG, and sclerostin/RANKL are shown in Figure 4. RANKL/OPG ratios were significantly lower in healthy individuals than in those with periodontitis, both before and after treatment (P <0.025). In the periodontitis group, the RANKL/OPG ratio decreased after treat- ment, but this difference was not statistically signifi- Figure 1. cant (P >0.05). The sclerostin/OPG and sclerostin/ Total amount and concentration levels of sclerostin in GCF. *Statistically RANKL ratios were significantly lower in healthy in- significant difference from the healthy group (Kruskal–Wallis/ dividuals than in patients with periodontitis, both be- † Bonferroni-adjusted Mann–Whitney U test). Statistically significant fore and after treatment (P <0.025). In the intragroup difference between groups (Wilcoxon signed-rank test). Data are presented as box and whisker plots. The median value is indicated by comparison of quantitative variables in patients with the line within the box plot. The box extends from the 25th to the 75th periodontitis before and after treatment, the sclerostin/ percentiles. Whiskers extend to show the highest and lowest values. OPG and sclerostin/RANKL ratios were significantly decreased after SRP (P <0.05). Correlations The correlation coefficients are shown in Table 2. A statistically significant moderate positive correlation was found between sclerostin and RANKL and the RANKL/OPG ratios in all groups (P <0.05). The total amount of sclerostin was also found to be negatively correlated with OPG in all groups (P <0.05). When all clinical groups were examined together, statistically significant moderate negative correlations were found between sclerostin and OPG and between OPG and both CAL and GI. Strong positive correlations were found between sclerostin and RANKL, RANKL/OPG, CAL, and GI and also between RANKL and both CAL and GI (P <0.05).

DISCUSSION Figure 2. To the best of the authors’ knowledge, this is the first Total amount and concentration levels of OPG in GCF. *Statistically clinical study that has examined the changes in GCF significant difference from the healthy group (Kruskal–Wallis/ sclerostin levels in patients with CP after non-surgical Bonferroni-adjusted Mann–Whitney U test). †Statistically significant difference between groups (Wilcoxon signed-rank test). Data are therapy and that has also investigated the relation- presented as box and whisker plots. The median value is indicated by ship between the RANKL/OPG ratio and sclerostin in the line within the box plot. The box extends from the 25th to the 75th patients with periodontal disease. percentiles. Whiskers extend to show the highest and lowest values. GCF constituents reflect local cellular response in gingival tissue, and collection of GCF is a minimally sclerostin in patients with periodontitis decreased invasive alternative to gingival biopsies. Moreover, GCF after SRP (P <0.05). The total amount and concen- is suitable for evaluating both periodontal disease status tration of OPG were significantly higher in healthy and the outcome of therapy.28 Together, in the present controls than in patients with periodontitis before and study, the levels of sclerostin, OPG, and RANKL were after treatment, as is shown in Figure 2 (P <0.025). A analyzed in GCF samples. The GCF was sampled with significant reduction in OPG concentration was found filter paper strips using the intracrevicular method. The

1400 J Periodontol • December 2015 Balli, Aydogdu, Ongoz Dede, Coskun Turer, Guven

advantages of this technique are that it is quick and easytouse,canbeappliedtoindividualsites,andis possibly the least traumatic when used correctly.27 Data presented as total amount of a constituent in the GCF instead of its concentration are more appropriate when examining the relationship between the GCF constituents and periodontal diseases.29,30 Increasing GCF volumes in diseased sites would dilute the con- centration of the GCF constituent when it is primarily locally secreted in the GCF.27,30 Moreover, the volume of GCF by which concentration is directly affected is a dependent variance; hence, data presented as con- centration become dependent variables in the quanti- fication of a constituent of the GCF.30 Therefore, reporting total biomarker amounts per sampling time in theGCFmaybeabetter,valid,andreliableindicatorfor diagnostic purposes. The present study is based mainly on total amount data, although the total amounts and Figure 3. concentrations are both calculated. Total amount and concentration levels of RANKL in GCF. Bone metabolism is controlled by a delicate bal- *Statistically significant difference from the healthy group (Kruskal– Wallis/Bonferroni-adjusted Mann–Whitney U test). †Statistically ance between osteoblasts and osteoclasts. Sclerostin significant difference between groups (Wilcoxon signed-rank test). is a biomarker that negatively regulates bone for- Data are presented as box and whisker plots. The median value is mation by antagonizing osteoblastogenesis and the indicated by the line within the box plot. The box extends from the . Suppression of sclerostin 25th to the 75th percentiles. Whiskers extend to show the highest leads to increased bone mineral density, bone vol- and lowest values. ume, bone formation, and bone strength in mice.31 The administration of antisclerostin to ovariectomized rats and non-ovariectomized mon- keys has been associated with increased bone mass and strength on trabecular, periosteal, endocortical, and intracortical surfaces.32,33 Moreover, humanized monoclonal antibody has an anabolic effect on bone metabolism, with marked increases in bone mineral density and bone formation and a decrease in bone resorption.34-36 There is evidence that ligature- induced alveolar bone loss is associated with increased expression of RANKL (enhanced osteoclast forma- tion) and sclerostin (suppressed osteoid forma- tion).37 A study by Taut et al.38 suggested that local injection of sclerostin had a limited regenerative ef- fect on bone volume in an experimental periodontitis model, whereas subcutaneous sclerostin adminis- tration effectively improved bone healing. In a recent report, sclerostin levels were higher in patients with peri-implantitis than in patients with perimucositis Figure 4. 39 The relative ratios of RANKL/OPG, sclerostin/OPG, and sclerostin/ and healthy peri-implant tissues. In the present RANKL in GCF. *Statistically significant difference from the healthy study, sclerostin levels in GCF were higher in patients group (Kruskal–Wallis/Bonferroni-adjusted Mann–Whitney with CP than in healthy individuals. Only one clinical U test). †Statistically significant difference between groups (Wilcoxon study has explored the involvement of sclerostin in signed-rank test). Data are presented as box and whisker plots. The periodontitis. In that study, sclerostin was upregu- median value is indicated by the line within the box plot. The box extends from the 25th to the 75th percentiles. Whiskers extend to lated in the gingival tissue samples of patients with show the highest and lowest values. CP; this is similar to the findings in the group 2A patients. It was also reported that sclerostin levels were correlated positively with PD and CAL.40 This is consistent with the finding that GCF sclerostin levels had a strong positive correlation with CAL and GI.

1401 Sclerostin Levels in Periodontal Health and Disease Volume 86 • Number 12

The OPG/RANKL/RANK pathway plays an * * * * important role in the regulation of osteoclasto- * * 41

GI genesis. Although OPG and RANKL play a ma- 0.838 0.02 0.001 0.464

< jor role in the regulation of bone metabolism, the RANKL to ratio of RANKL/OPG or OPG/RANKL is consid- * * * * ered to better reflect bone resorption or turnover

GI than does the level of either of these factors in- 0.498 0.09 0.02 0.333 0.448 0.009 0.001 0.494 42 OPG to - - < - dividually. Several clinical studies have explored the contribution of the RANKL/OPG system to * * * * alveolar bone loss in periodontal disease. Evi- * * dence indicates that the ratio of RANKL/OPG in CAL 0.899 0.163 NA NA 0.435 0.02 0.001 0.411 0.03 - < GCF is elevated in patients with periodontitis and RANKL to that this increased ratio indicates the existence of periodontitis.43-46 The present study results are * * * * *

* consistent with these findings; the authors ob-

CAL served an increased RANKL/OPG ratio in patients 0.646 0.395 0.001 0.585 0.001 OPG to - - < - with CP. The effect of non-surgical periodontal treat- * * * ment on sclerostin levels and RANKL/OPG ratios * in GCF was also examined. A direct comparison GI NA 0.79 0.42 NA NA 0.862 0.385 0.047 0.04 0.001 0.424 0.03

< with other studies is not possible, because no

Sclerostin to other studies have reported the effect of non- surgical periodontal treatment on sclerostin. In the * * * * * * present study, sclerostin levels in GCF decrease after non-surgical periodontal therapy. Previously CAL 0.949 0.30 0.206 NA 0.054 0.668 0.001 0.001 0.545 0.003 < < published studies of the prognostic significance of

Sclerostin to RANKL/OPG have reported conflicting results. Decreased, unchanged, and increased RANKL/ OPG ratios after periodontal treatment have all been reported in patients with CP.47-49 These data * * * * * * *

* obtained from clinical trials indicate that this biomarker may not be completely reliable for 0.808 0.001 0.591 0.566 0.001 0.002 0.457 0.02 RANKL < evaluating periodontal disease activity and treatment outcomes.50 In this study, RANKL/OPG Sclerostin to OPG/ ratios remain unchanged after non-surgical peri- odontal therapy, despite a significant improve- ment in clinical parameters. However, sclerostin/ * * * * * * *

* OPG and sclerostin/RANKL ratios were higher in patients with periodontitis than in healthy in- 0.933 0.001 0.628 0.620 0.001 0.001 0.437 0.02 ) Among Groups With Respect to Total Amount of Sclerostin, OPG, RANKL, and RANKL < <

r dividuals. Both ratios were also significantly de- Sclerostin to creased after periodontal therapy. Given these findings, it may be speculated that the ratios of sclerostin/OPG and sclerostin/RANKL may * * * * * * * * have potential diagnostic values for periodontitis. OPG 0.674 0.01 0.471 0.001 0.542 0.003 0.418 0.03 Moreover, the decreased GCF sclerostin/OPG and - - < - -

Sclerostin to sclerostin/RANKL ratios after SRP indicate that these ratios also have prognostic potential for periodontal treatment outcomes. This study showed that the RANKL/OPG ratio and sclerostin levels in GCF are elevated in peri- odontal disease. Non-surgical periodontal therapy resulted in decreased levels of sclerostin but had no effect on the RANKL/OPG ratio, despite an im-

treatment) provement in clinical parameters. In addition, not applicable. groups 0.05, statistically significant. r r P P r r P P = < reduction of the level of sclerostin is directly Group 1 (healthy) Group 2A (periodontitis) Group 2B (after All P Table 2. The Spearman Rank Correlation ( NA * Sampled Site CAL and GI associated with improved clinical outcomes.

1402 J Periodontol • December 2015 Balli, Aydogdu, Ongoz Dede, Coskun Turer, Guven

Considering the levels of sclerostin in patients with 13. Baron R, Rawadi G. Targeting the Wnt/beta-catenin periodontitis before and after treatment, it is possible pathway to regulate bone formation in the adult skeleton. to speculate that sclerostin is involved in alveolar Endocrinology 2007;148:2635-2643. 14. Silverman SL. Sclerostin. J Osteoporos 2010;2010: bone loss and that measurement of this protein is 941419. useful for monitoring the response to non-surgical 15. Winkler DG, Sutherland MK, Geoghegan JC, et al. periodontal treatment. control of bone formation via sclerostin, a novel BMP antagonist. EMBO J 2003;22:6267- CONCLUSIONS 6276. 16. Devarajan-Ketha H, Craig TA, Madden BJ, Robert The results suggest that the GCF sclerostin level is Bergen H 3rd, Kumar R. The sclerostin-bone protein more reliable than the RANKL/OPG ratio as a di- interactome. Biochem Biophys Res Commun 2012; agnostic and prognostic marker of periodontal dis- 417:830-835. ease and treatment outcome. Considering the results 17. Poole KE, van Bezooijen RL, Loveridge N, et al. Scle- of the present study in conjunction with those of rostin is a delayed secreted product of osteocytes that inhibits bone formation. FASEB J 2005;19:1842-1844. previous reports, sclerostin may prove to be a valu- 18. Atkins GJ, Rowe PS, Lim HP, et al. Sclerostin is a locally able indicator of the prognosis of periodontal disease, acting regulator of late-osteoblast/preosteocyte differ- as well as a potent strategy to protect bone and entiation and regulates mineralization through a MEPE- a potential target for more effective therapy. ASARM-dependent mechanism. J Bone Miner Res 2011;26:1425-1436. ACKNOWLEDGMENT 19. Wijenayaka AR, Kogawa M, Lim HP, Bonewald LF, Findlay DM, Atkins GJ. Sclerostin stimulates osteocyte The authors report no conflicts of interest related to support of osteoclast activity by a RANKL-dependent this study. pathway. PLoS One 2011;6:e25900. 20. 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and bone strength in a rat model of postmenopausal crevicular fluid of patients with periodontitis. J Dent Res osteoporosis. JBoneMinerRes2009;24:578-588. 2004;83:166-169. 33. Ominsky MS, Vlasseros F, Jolette J, et al. Two doses of 44. Lu HK, Chen YL, Chang HC, Li CL, Kuo MY. Identifica- sclerostin antibody in cynomolgus monkeys increases tion of the osteoprotegerin/receptor activator of nu- bone formation, bone mineral density, and bone clear factor-kappa B ligand system in gingival crevicular strength. J Bone Miner Res 2010;25:948-959. fluid and tissue of patients with chronic periodontitis. 34. Padhi D, Jang G, Stouch B, Fang L, Posvar E. Single- J Periodontal Res 2006;41:354-360. dose, placebo-controlled, randomized study of AMG 45. Bostanci N, Ilgenli T, Emingil G, et al. Gingival crev- 785, a sclerostin monoclonal antibody. J Bone Miner icular fluid levels of RANKL and OPG in periodontal Res 2011;26:19-26. diseases: Implications of their relative ratio. J Clin 35. McClung MR, Grauer A, Boonen S, et al. Romosozumab Periodontol 2007;34:370-376. in postmenopausal women with low bone mineral 46. Bostanci N, Ilgenli T, Emingil G, et al. Differential density. N Engl J Med 2014;370:412-420. expression of receptor activator of nuclear factor- 36. Recker RR, Benson CT, Matsumoto T, et al. A randomized, kappaB ligand and osteoprotegerin mRNA in periodon- double-blind phase 2 clinical trial of blosozumab, a sclero- tal diseases. J Periodontal Res 2007;42:287-293. stin antibody, in postmenopausal women with low bone 47. Buduneli N, Buduneli E, Ku¨tu¨kcxu¨ler N. Interleukin-17, mineral density. JBoneMinerRes2015;30:216-224. RANKL, and osteoprotegerin levels in gingival crev- 37. Kim JH, Lee DE, Cha JH, Bak EJ, Yoo YJ. Receptor icular fluid from smoking and non-smoking patients activator of nuclear factor-kB ligand and sclerostin ex- with chronic periodontitis during initial periodontal pression in osteocytes of alveolarboneinratswithligature- treatment. J Periodontol 2009;80:1274-1280. induced periodontitis. J Periodontol 2014;85:e370-e378. 48. Dereka XE, Markopoulou CE, Fanourakis G, Tseleni- 38. Taut AD, Jin Q, Chung JH, et al. Sclerostin antibody Balafouta S, Vrotsos IA. RANKL and OPG mRNA level stimulates bone regeneration after experimental peri- after non-surgical periodontal treatment. Inflammation odontitis. J Bone Miner Res 2013;28:2347-2356. 2010;33:200-206. 39. Rakic M, Struillou X, Petkovic-Curcin A, et al. Estima- 49. Bostanci N, Saygan B, Emingil G, Atilla G, Belibasakis tion of bone loss biomarkers as a diagnostic tool for GN. Effect of periodontal treatment on receptor acti- peri-implantitis. J Periodontol 2014;85:1566-1574. vator of NF-kB ligand and osteoprotegerin levels and 40. Napimoga MH, Nametala C, da Silva FL, et al. In- relative ratio in gingival crevicular fluid. J Clin Peri- volvement of the Wnt-b-catenin signalling antagonists, odontol 2011;38:428-433. sclerostin and dickkopf-related protein 1, in chronic 50. Belibasakis GN, Bostanci N. The RANKL-OPG system periodontitis. J Clin Periodontol 2014;41:550-557. in clinical periodontology. J Clin Periodontol 2012;39: 41. Boyce BF, Xing L. Functions of RANKL/RANK/OPG in 239-248. bone modeling and remodeling. Arch Biochem Bio- phys 2008;473:139-146. Correspondence: Dr. Umut Balli, Department of Periodon- 42. Kwan Tat S, Amiable N, Pelletier JP, et al. Modulation of tology, Faculty of Dentistry, Bulent Ecevit University, OPG, RANK and RANKL by human chondrocytes and 67600 Kozlu, Zonguldak, Turkey. Fax: 90-372-2613403; their implication during osteoarthritis. Rheumatology e-mail: [email protected]. (Oxford) 2009;48:1482-1490. 43. Mogi M, Otogoto J, Ota N, Togari A. Differential Submitted April 23, 2015; accepted for publication June expression of RANKL and osteoprotegerin in gingival 28, 2015.

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