Impact of Periodontal Treatment on the RANKL/OPG Ratio in Crevicular Fluid
Condition: Periodontal Diseases; Biomarkers; Bone Resorption
MeSH terms: Bone Resorption; Periodontal Diseases
Study type: Observational

Summary: 
      Objectives: Alveolar resorption is one of the most important events in periodontitis.
      Osteoclast activity is regulated by the ratio between receptor activator of NF-κB ligand
      (RANKL) and osteoprotegerin (OPG). The aim of this study was to evaluate changes in the
      RANKL/OPG ratio in crevicular fluid after periodontal treatment.

      Material and methods: A total of 15 patients with periodontitis were included in the study
      group. Samples were collected from an area with active periodontitis and a healthy area. The
      RANKL and OPG levels were measured before and after periodontal scaling and root planing
      (SRP) treatment. The study group was compared to the control group, which included 10
      patients without periodontitis.
    

Detailed description: 
      Material and methods Study design A case-control study was proposed. A group of periodontally
      healthy subjects was compared to a group of patients with periodontitis before and after
      scaling and root planing.

      Two sites were studied in each subject with periodontitis: one affected site (with
      periodontitis) and one healthy site (without periodontal disease); the latter site served as
      a control. Thus, the study units were the healthy and pathological sites before and after SRP
      treatment.

      The masking was triple blind, including the researcher in charge of sample collection, the
      laboratory technician and the statistician.

      Due to the absence of pre-established reference values for the RANKL and OPG concentrations
      in periodontal health, the results obtained were compared to a control group of subjects with
      periodontally healthy conditions during the second part of the study.

      This protocol was approved by the ethics committee of the University of Valencia (Spain)
      according to the Declaration of Helsinki. Written informed consent was obtained from the
      study subjects.

      Sample selection The sample size was determined in relation to the main variable (the RANKL
      and OPG concentrations); currently, no reference values are available for the concentrations
      of these parameters under healthy or periodontal disease conditions. Therefore, to determine
      the sample size, the investigators used the results of previously published studies. Bostanci
      et al. (2011) and Buduneli et al. (2009) (sample sizes of 27 and 20 subjects, respectively)
      analysed the concentration changes of these parameters in the CVF before and after basic
      periodontal treatment.

      Therefore, 25 subjects were included in the final sample in our study. The subjects were
      recruited consecutively at the Periodontics Unit at the Faculty of Medicine and Odontology of
      the University of Valencia.

      The control group included 10 periodontally healthy subjects without signs or symptoms of
      periodontal disease.

      The study group included 15 subjects diagnosed with mild or advanced chronic periodontitis.
      Each periodontal patient had one non-affected single-rooted tooth (healthy site) and another
      single-rooted tooth with periodontitis (periodontitis site) with the following features:

      Healthy site: a single-rooted tooth with probing depths below or equal to 3 mm without
      recession and without bleeding on probing.

      Periodontitis site: another single-rooted tooth from the same patient with clinical
      attachment loss equal to or greater than 6 mm and bleeding upon probing.

      The exclusion criteria were as follows: subjects with aggressive periodontitis; systemic
      diseases or consumption of drugs affecting bone metabolism; antibiotic, anti-inflammatory or
      contraceptive treatment for the last three months prior to study initiation; antiplatelet
      therapy for the last 7 days prior to study onset; primary or secondary occlusal trauma;
      periapical or periodontal abscess in some of the teeth included in the study; any type of
      periodontal treatment within the last 6 months; under orthodontic treatment; smoking; and
      pregnant or breastfeeding.

      Periodontal clinical examination:

      The examination was performed by only one explorer using a Williams type manual periodontal
      probe (PQ-OW 208 396, Hu-Friedy®, USA). The probing depth, recession and attachment loss were
      measured for six sites per tooth. The "Gingival Bleeding Index" of the six sites per tooth
      was used to assess bleeding upon probing. The Silness and Loe index was used to assess dental
      plaques.

      A complete radiographic series (18 radiographs) was performed for the study group with the
      ORing paralleling system (Dentsply®) and digital phosphor plates (Durr dental ®). Two
      bitewing horizontal radiographs were taken in the control group.

      Periodontal treatment In the study group, periodontal scaling and root planning treatment of
      the 4 quadrants was performed for two consecutive weeks without the use of antiseptics or
      antibiotics, and the patients were instructed in oral hygiene, including both brushing
      techniques and proximal hygiene. Four weeks after the last SRP, the patients were examined
      clinically, and samples of crevicular fluid were collected again.

      Study sequence Day 0- Subjects were chosen based on the inclusion and exclusion criteria.
      Crevicular fluid samples were collected.

      Day 7- CVF samples were collected from the selected sites in both groups, and the samples
      were processed.

      Day 14- Periodontal treatment (study group): scaling and root planing of 2 quadrants and
      provision of oral hygiene instructions.

      Day 21- Periodontal treatment (study group): scaling and root planing of the 2 remaining
      quadrants and provision of oral hygiene instructions.

      Day 49- Control group: clinical record and crevicular fluid sample collection and processing.

      Collection of crevicular fluid The Periotron® 8000 was calibrated prior to sample collection
      [27]. In the study group, 4 CVF samples were collected, 2 for each single-rooted tooth with
      the clinical conditions described above. Two samples from one single-rooted tooth were
      collected from the control group.

      Supragingival plaque was removed with a sterile curette, sluiced, isolated with cotton rolls
      and dried with air to avoid contamination with saliva. The tip of the Periopaper® was
      inserted into the sulcus or periodontal pocket until resistance was found and maintained for
      30 seconds; tips dyed with blood were discarded. Subsequently, the CVF volume was measured by
      the Periotron 8000®.

      In the following step, each sample was allocated into a sterilised Eppendorf tube with 100
      microlitres of buffer containing phosphate-buffered saline (PBS) with protease inhibitors
      (C.N. P8340 Sigma, MO, USA). The Eppendorf tubes were centrifuged at 15,000 rpm for 5 min;
      then, another 100 microlitres of buffer was added, and the tubes were centrifuged for 5 min
      at 15,000 rpm. Finally, 200 microlitres of each sample was stored at -80°C prior to
      processing.

      Sample processing RANKL and OPG were quantified using an enzyme-linked immunosorbent assay
      (ELISA). The technical specifications of the RANKL (total Human ELISA, BioVendor) and OPG
      (Bender MedSystems) kits were followed. The total RANKL and OPG concentrations were assessed.
    

Intervention name(s): periodontal treatment
Intervention type(s): Procedure
Intervention description(s): In the study group, periodontal scaling and root planning treatment of the 4 quadrants was performed for two consecutive weeks without the use of antiseptics or antibiotics, and the patients were instructed in oral hygiene, including both brushing techniques and proximal hygiene.
Observational model: Case-Control
Time perspective: Prospective
Sampling method: Probability Sample
Study population: 
        The study population is the classic refreshing race of the population of Spain.
      
Keywords: RANKL; OPG; periodontal biomarkers; alveolar bone loss; chronic periodontitis