Volume 86 • Number 12 Gingival Crevicular Fluid Levels of Sclerostin, Osteoprotegerin, 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 osteoblasts, fibroblasts, lymphocytes, and ratio as a biomarker for periodontal disease activity osteocytes.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 proteins.7,8 The Wnt pathway increases bone forma- Study Population tion and regeneration via stimulation of osteoblast 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 gene, 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 protein 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),