Effects of Azithromycin as an Adjunct to Scaling and Root Planning in the Treatment of Periodontitis
Condition: Periodontitis
MeSH terms: Periodontitis
Study type: Interventional

Summary: 
      The growing interest in the possibilities of modulating macrophages in inflammatory diseases
      with therapeutic purpose has prompted the development of new approaches for the treatment of
      periodontitis.

      This randomized add-on open clinical study evaluated the short-term effects of azithromycin
      (Az) as an adjunct to scaling and root planing (SRP) in patients with chronic periodontitis.

      The investigators measured periodontal parameters, and collected gingival biopsies from
      patients with periodontitis (P group, n=50) before and 1 month after scaling and root
      planning (SRP group, n=25), after scaling and root planning with oral azithromycin
      administration (Az group, n=25), and from periodontally healthy individuals (H group, n=25).
      The last served as a reference group. Macrophage subpopulations were identified through
      immunohistochemistry as single positive CD68+ and CD163+ cells. The levels of M1-, and
      M2-related cytokines (IL1-β, IL-6, IL-10, TGF-β) assay in the tissue culture medium was
      provided by ELISA. The data were statistically analyzed by appropriate methods.

      The null hypothesis tested was that L-arginine and L-ornithine have no influences on CD68+
      and CD163+ Mφs densities.
    

Detailed description: 
      Clinical periodontal examinations Periodontal parameters were evaluated at the pre-treatment
      stage (T0) and 4 weeks ±5 days after the completion of SRP. For all participants, sex and age
      were recorded, and periodontal parameters such periodontal pocket depth (PPD), clinical
      attachment loss (CAL), and bleeding on probing (BoP) were examined. All measurements were
      conducted by one calibrated examiner using a manual periodontal probe (0106.DT06.CP10, Den
      Tag, Italy). PPD and CAL were measured at six periodontal sites around each tooth (including
      the third molars) to the nearest 1 mm, and maximal values were taken into account. For H
      group participants the signs of gingival inflammation, probing depth exceeding 3 mm and BoP
      were exclude to insure periodontal health.

      Periodontal therapy was comprised of home oral hygiene instructions and SRP. It was performed
      using ultrasonic, Gracey curettes and air-abrasive polishing. Measure for compliance control
      included counting pills and home hygiene checking.

      Collection of gingival tissue samples For precise immunohistochemical study gingival biopsy
      samples, about 3x3 mm was excised following local anesthesia from all participants without
      any harm, during appropriate dental procedures. Biopsy from periodontitis-affected gingiva
      performed at pre-treatment (T0, P Group) and 4 weeks ±5 days after the completion of
      periodontal treatment, in the same or nearest points, from a single most severe clinical
      appropriate site around common dental and periodontal procedures such was tooth extraction
      due to caries complications, and gingivectomies. Biopsy from periodontally healthy patients
      were obtained during tooth extraction due to caries complications, wisdom teeth and
      orthodontic indications. After collection, the half of biopsies were fixed in a 4% formalin
      solution for 24 hours of fixation, dehydrated, and embedded in paraffin, and another half
      were prepared for cytokines assay.

      Immunohistochemistry and antibodies Paraffin sections, 2-3 μm in thickness were
      deparaffinized and dehydrated. Heat-induced epitope retrieval in citrate buffer, pH 6, was
      performed by heating the slides in the microwave oven for 23 min, then allowed to cool,
      rinsed with phosphate-buffered saline (PBS), incubated with blocked reagent, rinsed, and
      incubated with mouse monoclonal CD68 macrophage antibodies (1:30, clone PG-M1, Diagnostic
      BioSystems, The Hague, The Netherlands) or anti-CD163 (1:100, clone 10D6, Diagnostic
      BioSystems, The Hague, The Netherlands). Then sections were stained with the 2-steps plus
      Mouse/Rabbit PolyVueTM HRP/DAB Detection System (Diagnostic BioSystems, The Hague, The
      Netherlands), and counterstained with Mayer's haemalaun. PBS was used as negative controls,
      the lymph node tissue - as a positive control.

      Evaluation of immunohistochemical staining СD68+ and CD163+ macrophages were estimated by
      counting the number of the cells by light microscope in 400 times in intensive cell
      infiltrative areas, selected 5 regions from each slice, and all 5 counts were taken for
      further statistics. Immunopositive Mφs in the areas of cell infiltration were count, because
      of directly relation to the inflammation. The number of cells per 10 000 μm2 was calculated
      as immunopositive cell density. Photos and calculation were obtained using the light
      microscope Axio Lab.A1 (Carl Zeiss, Göttingen, Germany) (×400).

      Tissue culturing Part of gingival tissue samples were cultured in a humidified incubator at
      37 °C and in the presence of 5 % CO2. Biopsies were cultured in Dulbecco's modified Eagle
      medium (DMEM) supplemented with 10 % (vol/vol) fetal bovine serum (Gibco, Thermo Fisher
      Scientific, Gibco™, US), 1 % (vol/vol) penicillin-streptomycin (Gibco™, US), and 0.2 %
      (vol/vol) fungizone amphotericin B (Gibco™, US) during 48 h [16]. For culturing, each biopsy
      sample was placed in a separate well of Costar® 24-well Plates (Corning Incorporated - Life
      Sciences, Kennebunk, USA), containing 0,4 ml of supplemented DMEM. After incubation, the
      tissues were removed and culture media were centrifuged at 800 g and 4 °C.

      Cytokine assay Cytokine assay in the tissue culture medium was provided by ELISA. IL1-β,
      IL-6, IL-10, and TGF-β concentrations were measured by commercially available kits (Vector
      Best, Novosibirsk, Russia, and MyBioSource, San Diego, USA). Cytokine concentrations were
      expressed as recommended by the manufacturer.

      Statistical analysis Statistical analysis was performed using the Prism 5 software (GraphPad
      Software, San Diego, USA) by means of nonparametric (descriptive statistics, Wilcoxon
      matched-pairs signed-rank test - for comparison of dependent results, Mann-Whitney test - for
      independent groups; nonparametric ANOVA for multiple comparisons with post-hoc tests, and
      Spearmen correlation). CD68+/CD163+ ratio was assessed by inter-group comparisons and
      Spearman correlation checking. P values of <0.05 were considered statistically significant in
      all analyses. For descriptive statistics of cell numbers, the percentile ranges were also
      used because of non-normal variables distributions.
    

Intervention name(s): Az+SRP; SRP; No interventions
Intervention type(s): Drug; Drug; Drug
Intervention description(s): Azithromycin as an adjunct to scaling and root planning in the treatment of periodontitis; Scaling and root planning (SRP) treatment of periodontitis; Diagnostocs to confirm the healthy state of the gingiva
Other intervention name(s): Azithromicyn group; Periodontal conventional treatment group; Reference/periodontal health group
Allocation: Randomized
Intervention model: Parallel Assignment
Intervention model description: Randomized controlled short-term clinical trial
Masking: Single (Outcomes Assessor)
Primary purpose: Treatment
Keywords: Azithromycin; M1 macrophages; M2 macrophages; Macrophages-targeted therapy; Periodontitis