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

Periodontitis Clinical Trial

Official title:

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

Clinical Trial Details — Status: Completed

Administrative data

• NCT number: NCT05506371
• Other study ID #: 0122U00049
• Status: Completed
• Phase: N/A
• Start date: September 16, 2017
• Est. completion date: June 28, 2019

Study information

• Verified date: October 2023
• Source: Ukrainian Medical Stomatological Academy
• Contact: n/a
• Is FDA regulated: No
• Study type: Interventional

Clinical Trial 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.

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.

Recruitment information / eligibility

• Status: Completed
• Enrollment: 50
• Est. completion date: June 28, 2019
• Est. primary completion date: June 28, 2019
• Accepts healthy volunteers: Accepts Healthy Volunteers
• Gender: All
• Age group: 19 Years to 54 Years

Eligibility

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

Related Conditions & MeSH terms

• Periodontitis

Intervention

Drug:
• Az+SRP
Azithromycin as an adjunct to scaling and root planning in the treatment of periodontitis
• SRP
Scaling and root planning (SRP) treatment of periodontitis
• No interventions
Diagnostocs to confirm the healthy state of the gingiva

Locations

Country	Name	City	State
Ukraine	Department of Postgraduate Education for Dentists of Poltava State Medical University,	Poltava

Sponsors (1)

Lead Sponsor	Collaborator
Ukrainian Medical Stomatological Academy

Country where clinical trial is conducted

Ukraine, 

References & Publications (15)

Allam JP, Duan Y, Heinemann F, Winter J, Gotz W, Deschner J, Wenghoefer M, Bieber T, Jepsen S, Novak N. IL-23-producing CD68(+) macrophage-like cells predominate within an IL-17-polarized infiltrate in chronic periodontitis lesions. J Clin Periodontol. 2011 Oct;38(10):879-86. doi: 10.1111/j.1600-051X.2011.01752.x. Epub 2011 Aug 31. — View Citation

Fabriek BO, Dijkstra CD, van den Berg TK. The macrophage scavenger receptor CD163. Immunobiology. 2005;210(2-4):153-60. doi: 10.1016/j.imbio.2005.05.010. — View Citation

Fujise O, Miura M, Hamachi T, Aida Y, Nishimura F. Regenerative effect of azithromycin on periodontitis with different levels of gingival inflammation: three case reports. Aust Dent J. 2014 Jun;59(2):245-51. doi: 10.1111/adj.12177. — View Citation

Garaicoa-Pazmino C, Fretwurst T, Squarize CH, Berglundh T, Giannobile WV, Larsson L, Castilho RM. Characterization of macrophage polarization in periodontal disease. J Clin Periodontol. 2019 Aug;46(8):830-839. doi: 10.1111/jcpe.13156. Epub 2019 Jun 25. — View Citation

Haffajee AD, Socransky SS, Ebersole JL. Survival analysis of periodontal sites before and after periodontal therapy. J Clin Periodontol. 1985 Aug;12(7):553-67. doi: 10.1111/j.1600-051x.1985.tb01389.x. — View Citation

Hai R, Chu A, Li H, Umamoto S, Rider P, Liu F. Infection of human cytomegalovirus in cultured human gingival tissue. Virol J. 2006 Oct 5;3:84. doi: 10.1186/1743-422X-3-84. — View Citation

Loos BG, Needleman I. Endpoints of active periodontal therapy. J Clin Periodontol. 2020 Jul;47 Suppl 22(Suppl 22):61-71. doi: 10.1111/jcpe.13253. — View Citation

Mills CD, Lenz LL, Ley K. Macrophages at the fork in the road to health or disease. Front Immunol. 2015 Feb 16;6:59. doi: 10.3389/fimmu.2015.00059. eCollection 2015. No abstract available. — View Citation

Morales A, Contador R, Bravo J, Carvajal P, Silva N, Strauss FJ, Gamonal J. Clinical effects of probiotic or azithromycin as an adjunct to scaling and root planning in the treatment of stage III periodontitis: a pilot randomized controlled clinical trial. BMC Oral Health. 2021 Jan 7;21(1):12. doi: 10.1186/s12903-020-01276-3. — View Citation

Nepokupnaia-Slobodianuk TS, Skripnikov PN. [The clinical effectiveness of long-term azithromycin in patients with chronic generalized periodontitis]. Georgian Med News. 2014 Nov;(236):27-31. Russian. — View Citation

Parnham MJ, Erakovic Haber V, Giamarellos-Bourboulis EJ, Perletti G, Verleden GM, Vos R. Azithromycin: mechanisms of action and their relevance for clinical applications. Pharmacol Ther. 2014 Aug;143(2):225-45. doi: 10.1016/j.pharmthera.2014.03.003. Epub 2014 Mar 11. — View Citation

Shapouri-Moghaddam A, Mohammadian S, Vazini H, Taghadosi M, Esmaeili SA, Mardani F, Seifi B, Mohammadi A, Afshari JT, Sahebkar A. Macrophage plasticity, polarization, and function in health and disease. J Cell Physiol. 2018 Sep;233(9):6425-6440. doi: 10.1002/jcp.26429. Epub 2018 Mar 1. — View Citation

Zhou LN, Bi CS, Gao LN, An Y, Chen F, Chen FM. Macrophage polarization in human gingival tissue in response to periodontal disease. Oral Dis. 2019 Jan;25(1):265-273. doi: 10.1111/odi.12983. Epub 2018 Oct 12. — View Citation

Zhu L, Zhao Q, Yang T, Ding W, Zhao Y. Cellular metabolism and macrophage functional polarization. Int Rev Immunol. 2015 Jan;34(1):82-100. doi: 10.3109/08830185.2014.969421. Epub 2014 Oct 23. — View Citation

Zhu LF, Li L, Wang XQ, Pan L, Mei YM, Fu YW, Xu Y. M1 macrophages regulate TLR4/AP1 via paracrine to promote alveolar bone destruction in periodontitis. Oral Dis. 2019 Nov;25(8):1972-1982. doi: 10.1111/odi.13167. Epub 2019 Oct 18. — View Citation

* Note: There are 15 references in all — Click here to view all references

Outcome

Type	Measure	Description	Time frame	Safety issue
Primary	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	The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Primary	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	The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Secondary	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)	The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Secondary	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	The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Secondary	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	The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Secondary	Change in mean level of IL1-ß in periodontitis-affected gingiva	The mean level of IL1-ß 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
Secondary	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	The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Secondary	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	The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP
Secondary	Change in mean level of TGF-ß in periodontitis-affected gingiva	The mean level of TGF-ß in the tissue culture medium, obtained from the biopsy	The pre-treatment stage (T0) and 4 weeks ±5 days after the completion of SRP