An interventional study of Az+SRP and SRP in Periodontitis, sponsored by Ukrainian Medical Stomatological Academy. Completed at 1 site in Ukraine. Open to participants aged 19 Years to 54 Years, including healthy volunteers. Per ClinicalTrials.gov, last updated 2023-10-31.
Sponsored by Ukrainian Medical Stomatological Academy · Not applicable, Interventional, and Treatment
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.
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.
1,664 studies on the registry are indexed under Periodontitis; 328 are open to participants now.
This study's enrollment of 50 is above the median of 45 across 1,206 interventional studies indexed under Periodontitis.
Browse Periodontitis studies →Ukrainian Medical Stomatological Academy is the lead sponsor of 4 studies on the registry; 1 is open to participants now.
Counted across the registry records on this site, refreshed daily.
Inclusion criteria for healthy volunteers:
Inclusion Criteria:
Exclusion Criteria:
Periodontitis patients, who underwent scaling and root planning (SRP) as one-stage full-mouth disinfection procedure and additionally received oral azithromycin ("Health" Pharmaceutical Company, Kharkiv, Ukraine) in a dose of 500 mg q.d. for 7 days, then 500 mg q.w. for the next 3 weeks
Drug: Az+SRP
Periodontitis patients who underwent scaling and root planning (SRP) as one-stage full-mouth disinfection procedure.
Drug: SRP
Reference group. 25 healthy volunteers who underwent only diagnostics to confirm the clinical healthy state of the gingiva
Drug: No interventions
Azithromycin as an adjunct to scaling and root planning in the treatment of periodontitis
Also known as: Azithromicyn group
Scaling and root planning (SRP) treatment of periodontitis
Also known as: Periodontal conventional treatment group
Diagnostocs to confirm the healthy state of the gingiva
Also known as: Reference/periodontal health group
Change in clinical attachment loss (CAL)
Mean CAL, measured at six periodontal sites around each tooth (including the third molars) to the nearest 1 mm
Time frame: The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Change in periodontal pocket depth (PPD)
Mean periodontal pocket depth, measured at six periodontal sites around each tooth (including the third molars) to the nearest 1 mm
Time frame: The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Change in bleeding on probing (BOP)
The percentage of sites per patient with bleeding measured at six sites around each tooth (including the third molars)
Time frame: The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Change in density of CD68+ macrophages
The number of single-immunopositive macrophages per 10 000 μm2 was calculated as cell density in biopsy from periodontitis-affected gingiva
Time frame: The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Change in density of CD163+ macrophages
The number of single-immunopositive macrophages per 10 000 μm2 was calculated as cell density in biopsy from periodontitis-affected gingiva
Time frame: The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Change in mean level of IL1-β in periodontitis-affected gingiva
The mean level of IL1-β in the tissue culture medium, obtained from the same biopsy
Time frame: The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Change in mean level of IL-6 in periodontitis-affected gingiva
The mean level of IL-6 in the tissue culture medium, obtained from the same biopsy
Time frame: The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Change in mean level of IL-10 in periodontitis-affected gingiva
The mean level of IL-10 in the tissue culture medium, obtained from the biopsy
Time frame: The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Change in mean level of TGF-β in periodontitis-affected gingiva
The mean level of TGF-β in the tissue culture medium, obtained from the biopsy
Time frame: The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Plan to share: No — We plan to provide all the necessary data in a future article.
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Ukrainian Medical Stomatological Academy