Effects of Azithromycin as an Adjunct to Scaling and Root Planning in the Treatment of Periodontitis

October 30, 2023 updated by: Igor Kaydashev, Ukrainian Medical Stomatological Academy

Azithromycin as an Adjunct to Scaling and Root Planning Causes an Increase of CD68+, CD163+ Macrophages Density in the Gingival Tissues of Periodontitis Patients: a Randomized Controlled Short-term Clinical Trial

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.

Study Overview

Status

Completed

Conditions

Intervention / Treatment

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.

Study Type

Interventional

Enrollment (Actual)

50

Phase

  • Not Applicable

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Locations

  • Ukraine
      • Poltava, Ukraine, 36011
        • Department of Postgraduate Education for Dentists of Poltava State Medical University,

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Ages Eligible for Study

19 years to 54 years (Adult)

Accepts Healthy Volunteers

Yes

Description

Inclusion criteria for healthy volunteers:

  • Presence of more than 20 teeth (for each tooth, the probing pocket depth ≤3 mm
  • Absence of clinical gingival inflammation at intact or reduced periodontium

Inclusion Criteria:

  • The presence of at least 15 natural teeth, at least 10 sites with the periodontal pocket depth (PPD) ≥4 mm, and/or clinical attachment loss of ≥4 mm at the same site, and satisfactory general health

Exclusion Criteria:

  • Taking antibiotics or anti-inflammatory medications within the previous 3 months
  • Periodontal therapy within the previous 6 months
  • Purulent exudation from periodontal pockets
  • Pregnancy or breastfeeding
  • Severe, uncontrolled (decompensated) diseases of the internal organs, or neuropsychiatric disorders
  • Other conditions that determined the patient's inability to understand the nature and possible consequences of the study

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

  • Primary Purpose: Treatment
  • Allocation: Randomized
  • Interventional Model: Parallel Assignment
  • Masking: Single

Arms and Interventions

Participant Group / Arm
Intervention / Treatment
Experimental: Az group
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
Azithromycin as an adjunct to scaling and root planning in the treatment of periodontitis
Other Names:
  • Azithromicyn group
Active Comparator: SRP group
Periodontitis patients who underwent scaling and root planning (SRP) as one-stage full-mouth disinfection procedure.
Drug: SRP
Scaling and root planning (SRP) treatment of periodontitis
Other Names:
  • Periodontal conventional treatment group
Other: H group
Reference group. 25 healthy volunteers who underwent only diagnostics to confirm the clinical healthy state of the gingiva
Drug: No interventions
Diagnostocs to confirm the healthy state of the gingiva
Other Names:
  • Reference/periodontal health group

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Change in clinical attachment loss (CAL)
Time Frame: The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Mean CAL, measured at six periodontal sites around each tooth (including the third molars) to the nearest 1 mm
The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Change in periodontal pocket depth (PPD)
Time Frame: The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Mean periodontal pocket depth, measured at six periodontal sites around each tooth (including the third molars) to the nearest 1 mm
The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP

Secondary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Change in bleeding on probing (BOP)
Time Frame: The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
The percentage of sites per patient with bleeding measured at six sites around each tooth (including the third molars)
The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Change in density of CD68+ macrophages
Time Frame: The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
The number of single-immunopositive macrophages per 10 000 μm2 was calculated as cell density in biopsy from periodontitis-affected gingiva
The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Change in density of CD163+ macrophages
Time Frame: The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
The number of single-immunopositive macrophages per 10 000 μm2 was calculated as cell density in biopsy from periodontitis-affected gingiva
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
Time Frame: The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
The mean level of IL1-β in the tissue culture medium, obtained from the same biopsy
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
Time Frame: The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
The mean level of IL-6 in the tissue culture medium, obtained from the same biopsy
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
Time Frame: The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
The mean level of IL-10 in the tissue culture medium, obtained from the biopsy
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
Time Frame: The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
The mean level of TGF-β in the tissue culture medium, obtained from the biopsy
The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

Ukrainian Medical Stomatological Academy

Investigators

  • Principal Investigator: Igor P. Kaidashev, MD, Ukrainian Medical Stomatological Academy Poltava, Ukraine

Publications and helpful links

The person responsible for entering information about the study voluntarily provides these publications. These may be about anything related to the study.

General Publications

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Actual)

September 16, 2017

Primary Completion (Actual)

June 28, 2019

Study Completion (Actual)

June 28, 2019

Study Registration Dates

First Submitted

August 14, 2022

First Submitted That Met QC Criteria

August 17, 2022

First Posted (Actual)

August 18, 2022

Study Record Updates

Last Update Posted (Actual)

October 31, 2023

Last Update Submitted That Met QC Criteria

October 30, 2023

Last Verified

October 1, 2023

More Information

Terms related to this study

Keywords

Additional Relevant MeSH Terms

Other Study ID Numbers

  • 0122U00049

Plan for Individual participant data (IPD)

Plan to Share Individual Participant Data (IPD)?

NO

IPD Plan Description

We plan to provide all the necessary data in a future article.

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

product manufactured in and exported from the U.S.

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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