The Impact of Local vs. Systemic Adjuvant Antibiotics During Non-surgical Periodontitis Therapy on Clinical Parameters, Bacterial Count and Cytokine Levels - a Randomized Clinical Trial
Phase: NA
Conditions: Periodontitis
Sponsor: University of Belgrade

STUDY PROTOCOL  
 
 
 
 
 
 
 
 
THE IMPACT OF LOCAL VS. SYSTEMIC ADJUVANT ANTIBIOTICS DURING NON-SURGICAL 
PERIODONTITIS THERAPY ON CLINICAL PARAMETERS, BACTERIAL COUNT AND CYTOKINE 
LEVELS – A RANDOMIZED CLINICAL TRIAL 
 
 
 
 
 
NCT05608564 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Date: 15.1.2022. 
 
 
 
 
 
 
 
 
 

Background and objectives 
 
Periodontitis is a chronic inflammatory disease that leads to progressive destruction of 
periodontal tissues and tooth-supporting structures, primarily driven by microbial dysbiosis and 
an altered host immune response (1,2). The excessive accumulation of immunomodulatory cells 
due to inadequate immune regulation contributes to alveolar bone loss (3). Among key 
inflammatory mediators, tumor necrosis factor-alpha (TNF-α) plays a crucial role in initiating 
immune responses against periodontal pathogens and promoting bone resorption (4-6). 
Similarly, interleukin-17 (IL-17) has been linked to both immune defense and pathological bone 
loss through neutrophil recruitment and activation of osteoclasts (7,8). 
Non-surgical periodontal therapy (NSPT) is the first-line treatment aimed at reducing bacterial 
burden and controlling inflammation through mechanical debridement (9). Although effective, 
NSPT alone may not be sufficient for optimal outcomes, leading to the adjunctive use of systemic 
antibiotics, particularly amoxicillin, and metronidazole, which have demonstrated significant 
clinical benefits in younger individuals with generalized stage III periodontitis (10-12). The 
combination of these antibiotics has been associated with favorable shifts in the subgingival 
microbiome and long-term clinical improvements (13). However, while in vitro studies suggest 
metronidazole can inhibit TNF-α production, clinical evidence remains inconsistent (14,15). 
Additionally, local antibiotic delivery has shown mixed results, with some studies reporting 
clinical benefits while others limit its use to maintenance therapy or persistent deep pockets (16-
18). 
Given the conflicting evidence, the primary objective of this study is to compare the clinical 
outcomes of systemic versus locally delivered adjuvant antibiotics during NSPT at baseline and 
after six months. The secondary objective is to evaluate their effects on total bacterial load and 
the relative expression levels of key pro-inflammatory mediators such as TNF-α and IL-17. 
 
 
Material and methods 
 
Study design and participants 
This randomized clinical study will include 38 patients diagnosed with periodontitis stage III. The 
investigation will be conducted at the Department of Periodontology and Oral Medicine, at the 
School of Dental Medicine, University of Belgrade. The laboratory part of the research will be 
carried out at the Department of Human Genetics, at the School of Dental Medicine, University 
of Belgrade. This research initiation is approved by the Ethical Committee of the School of Dental 
Medicine, University of Belgrade (No 36/6). Patients will be divided into two groups. The test 
group will consist of 19 patients who will receive a local antibiotic (LA group), while the control 
group will consist of 19 patients who will be prescribed systemic antibiotic therapy (SA group). 
The division of patients into groups will be done using randomization envelopes after the 
diagnostic procedure. 
Participants will be eligible for inclusion if they meet the following inclusion criteria: 
• 
Individuals aged 18–40 years 
• 
Willingness to participate in the research, comply with all study protocols, and provide 
signed informed consent 
• 
Diagnosis of active periodontitis stage III according to the latest classification (1) 
• 
Non-smokers or light smokers (≤10 cigarettes/day) 
 
 

Exclusion Criteria: 
• 
Presence of systemic diseases affecting periodontal or bone metabolism (e.g., 
uncontrolled diabetes, cancer, immunodeficiency, metabolic bone diseases) 
• 
Ongoing treatment with immunosuppressive, anti-resorptive, or anti-inflammatory 
medications 
• 
Known penicillin allergy 
• 
History of periodontal therapy within the last six months 
• 
Use of local or systemic antimicrobials in the past six months 
• 
Use of oral anti-plaque mouthwash within one month before the study 
• 
Alcohol or drug abuse 
• 
Pregnancy or lactation 
 
Data collection and sampling 
Upon clinical examination, clinical periodontal parameters such as pocket probing depth (PPD), 
clinical attachment level (CAL), bleeding on probing (BOP), and plaque index (PI) will be recorded 
for each patient around every tooth. Prior to treatment, subgingival crevicular fluid will be 
sampled with paper points from periodontal pockets for laboratory analysis. 
Sampling will be performed as follows: first, a relatively dry working field will be established using 
paper water rollers and air jets. Then, three paper points (number 30 – marked in blue) will be 
applied for 30 seconds in the periodontal pocket area of the selected premolar/molar tooth with 
a depth of more than 5 mm. The procedure will be repeated immediately in the same region to 
obtain duplicate samples. After that, the paper points will be placed in Eppendorf tubes and 
immediately transported to the basic research laboratory, where they will be stored at a 
temperature of −80 degrees Celsius for subsequent microbiological and molecular analyses. 
 
Non-surgical periodontal therapy and antibiotic administration 
After sampling, all patients will undergo a non-surgical phase of periodontal therapy (NSPT) 
following a Full-Mouth Disinfection protocol (19). Six months after the NSPT, at follow-up, the 
detection of clinical parameters and the sampling procedure will be repeated. 
In the LA group, a combination of piperacillin and tazobactam in gel form (Gelcide ®, Italmed 
MedTechDental, Florence, Italy) designed for subgingival use will be applied locally. The local 
antibiotic will be administered into the periodontal pockets using a syringe and a flexible blunt 
needle, 24 hours after NSPT. The administration will be done by quadrants, following the 
principles of a dry work field, and after application, the dry field will be maintained for five 
minutes. After this procedure, the patient will be instructed not to rinse the oral cavity for 15 
minutes. 
For systemic use in the SA group, patients will receive a combination of Amoxicillin (Amoxicillin 
®, 500 mg, three times a day, for seven days) and Metronidazole (Orvagyl ®, 400 mg, three times 
a day, for seven days) for oral use after NSPT is completed (11). 
Laboratory Analyses 
Total bacterial count (TBC) and relative expression levels (REL) of proinflammatory cytokines TNF-
α and IL-17 will be detected using quantitative real-time polymerase chain reaction (qPCR) in a 
basic research laboratory at the Department of Human Genetics, at the School of Dental 
Medicine, University of Belgrade (20, 21). 
 
 
 
 

Laboratory analyses 
Total bacterial count (TBC) and relative expression levels (REL) of proinflammatory cytokines TNF-
α and IL-17 will be detected using quantitative real-time polymerase chain reaction (qPCR) in a 
basic research laboratory at the Department of Human Genetics, at the School of Dental 
Medicine, University of Belgrade (20, 21). 
 
Statistical analyses 
All statistical analyses will be performed on an intention-to-treat basis, with no expected 
dropouts during the observational period. Data analysis will be conducted using GraphPad Prism 
9.0 (GraphPad, San Diego, CA, USA) and Statistical Package for Social Science (SPSS, version 26.0; 
SPSS Inc., Chicago, IL, USA). For numerical data, mean values, medians, standard deviations (SD), 
and ranges will be used for descriptive statistics. 
The Kolmogorov–Smirnov normality test will be applied to examine the distribution of outcome 
variables. Depending on the distribution, paired Student’s t-test or Wilcoxon signed-rank test will 
be used to compare baseline and six-month follow-up measurements between treatment 
groups. Repeated measures ANOVA with Bonferroni correction or Friedman and Wilcoxon 
signed-rank test will be used for within-group comparisons. Statistical significance will be set at 
p < 0.05. 
 

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