Effect of Non-chirurgical Periodontal Treatment on the Immune System From a Gender Perspective

January 13, 2026 updated by: Milagros Rocha Barajas

Effect of Non-chirurgical Periodontal Treatment on the Innate and Adaptive Immunity From a Gender Perspective: Potential Therapeutic Implications of microRNAs

The goal of this observational study is to evaluate non-surgical periodontal treatment in women and men with periodontitis with and without obesity. The main questions it aims to answer are:

  • If non-surgical periodontal treatment of patients with chronic periodontitis can modulate the innate and adaptive immune response taking into account patient gender and the coexistence of obesity
  • If there are specific miRNAs that can regulate this immune response and can be considered as suitable biomarkers and therapeutic targets.

Obese or non-obese participants with periodontitis will receive non-surgical periodontal treatment, consisting of oral health guidance and mechanical periodontal debridement throughout the mouth using an ultrasonic device and manual curettes. Researchers will compare four groups: obese women, non-obese women, obese men, and non-obese men, to clarify the involment of immune response after treatment, considering the coexistence of obesity and potential gender differences.

Study Overview

Status

Completed

Conditions

Intervention / Treatment

Detailed Description

Blood, gingival crevicular fluid (GCF), and saliva samples will be collected from patients at baseline and 12 weeks after non-surgical periodontal treatment. From fasting blood samples (12h), peripheral blood mononuclear cells (PBMCs) and neutrophil fractions will be isolated using an immunomagnetic method, following the manufacturer's protocol, and plasma and serum will be stored at -80°C until analysis. LUNA-FL will be used to determine cell count and viability (acridine orange and propidium iodide double stain) in cell samples. GCF samples will be collected in duplicate by carefully inserting sterile paper points into the periodontal pocket 1 mm for 30 s. The volume will be determined using the Periotron 8000 and stored at -80°C for future determinations. Unstimulated saliva will be collected in RNase-free tubes by expectoration, and after centrifugation, the supernatant will be stored at -80°C for microRNA (miRNA) analysis.

Clinical periodontal parameters of probing pockets depth (PD), millimetres of clinical attachment level (CAL), bleeding on probing (BOP), simplified plaque index of Silness and Löe, and simplified calculus index of Greene and Vermillion will be determined using a conventional manual periodontal probe (UNC-15 PCP). A periodontal examination will be performed to measure PD, CAL, and BOP at six sites per tooth for all teeth, excluding third molars, as previously described. Patients will be interviewed about their medical history, lifestyle habits (smoking, frequency of tooth brushing, physical activity), and sociodemographic characteristics. Weight, height, and blood pressure will be measured using standardized methods. Biochemical parameters of carbohydrate metabolism - glucose, insulin, glycated hemoglobin (A1c) -, lipid profile - total cholesterol, LDL, HDL, triglycerides (TG), apolipoproteins AI and B -, emerging inflammatory and cardiovascular risk markers - C-Reactive Protein (CRP), C3c, and retinol-binding protein 4 (RBP4) -, and complete blood count will be determined at the hospital's Clinical Analysis Service.

For detection of differences in protein expression, cells will be incubated in lysis buffer with protease and phosphatase inhibitors (RIPA Buffer) for 15 minutes at 4 degrees celsius (°C). The supernatant will be collected after centrifugation for 15 minutes at 16,000g. The total protein concentration will be quantified using a bicinchoninic acid (BCA) protein assay. Aliquots of 25 µg of protein will be resolved on 8-16% gradient SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Target proteins will be detected by incubating the membranes with anti-actin, JNK, NFkB, MCP1, GPX-1, NLRP3, ASC, procaspase 1, caspase 1, NADPH oxidase, catalase, GPX1, SOD1, Beclin, ATG5-ATG12, p62, LC3 I, LC3 II, Pink1, GRP78, eIF2alpha, IRE1 alpha, ATF6, CHOP, PGC1 alpha, mTFA, VDAC, Complex I, II, III, IV and V. The protein signal will be detected by chemiluminescence and analyzed by densitometry.

Inflammasome complex assembly (first stage of activation) through co-localization studies of NLRP3-ASC in PBMCs will be conducted using the confocal and/or fluorescence microscopy. Briefly, PBMCs will be seeded on coverslips coated with Poly-D-Lysine, fixed with paraformaldehyde (PFA) 4% for 20 minutes, permeabilized with Triton X-100 for 20 minutes and blocking with Phosphate-buffered saline buffer-Bovine serum albumn (PBS-BSA) 3% for 1 hour at room temperature. Hybridization with specific primary antibodies (diluted in PBS-BSA 1%) will be carry out overnight at 4 ºC and then, secondary antibodies conjugated with AlexaFluor fluorophores will be incubated for 1 hour in the dark at room temperature. Stained samples will be transferred the coverslip onto microscope slide and conserved in anti-fade fluorescence mounting medium.

Circulating levels of cytokines, adhesion molecules and serum oxidative stress markers will be measured in serum samples, but for secretome studies 1x10^6 PBMCs will be previously incubated in 1 mL RPMI with 10% fetal bovine serum (FBS) for 4 hours (37°C, 5% CO2) to obtain the supernatant. Both serums and supernatants will be analyzed with a Luminex® 200 analyzer system following the Milliplex® MAP Kit manufacturer's procedure. These same determinations will be carried out on GCF and serum/plasma samples from patients.

The quantification of functional autophagosomes in PBMCs will be performed using flow cytometry on our BD Accuri equipment with the CYTO-ID® Autophagy detection kit following manufacturer's procedure. In parallel, leucocyte from whole blood samples will be incubated with redox status detecting fluorescent probes for 15 minutes and then, they will be analysed using flow cytometry. Additionally, a multicolor panels for flow cytometry based on cluster of differentiation (CD) antigens detection, CD3/CD4/CD8/CD45RA/CCR7/CD38 and CD14/CD16, will be designed to analyze the percentage of lymphocyte T subpopulations (naive T cells (TN), central memory (TCM), effector memory (TEM) and terminally differentiated (TEMRA) cells) and, monocytes subpopulations (classical, non-classical, and intermediate), respectively in blood samples. In both procedures 10000 events will be acquired and single staining and FMO controls for all fluorochrome-conjugated antibodies in the panels will be performed to establish adequate compensation and define positive signals.

A parallel plate flow chamber, connected to an inverted microscope, will enable the researchers to measure neutrophil-endothelial cell interactions in vitro. Through this system, the leukocyte suspension obtained from patients will be perfused over a monolayer of immortalized endothelial cells (HUVEC/TERT 2) under conditions simulating blood flow. Videos will be analyzed afterward to determine flow, rolling velocity, and firm adhesion of leukocytes to endothelial cells, as previously described (Antioxidants. 2020 Aug 11;9(8):734).

Changes in gene expression levels will be evaluated using Nanostring® technology for nCounter®. To sum up, total RNA will be extracted from PBMCs using the GeneAllR RibospinTM total kit, and starting from 100-200 ng of total RNA, Diagnostica Longwood, S.L. will analyze the differential gene expression response, thus obtaining the multiplex metabolism panel.

For differential gene expression analysis of miRNAs (DEGs), RNA will be extracted using the miRNeasy Serum/Plasma kit (Qiagen). After short-chain RNA extraction, libraries will be prepared for sequencing using the TruSeq Small RNA library preparation kit (Illumina, Inc). Libraries will be pooled equimolarly and quantified using the KAPA SYBR FAST Universal qPCR kit with Illumina Primer Premix, and group size will be measured using a Bioanalyzer (Agilent). Finally, 2 nanomoles of the group will be sequenced on the Illumina NovaSeq6000 platform with a 1% PhiX control in the FISABIO facilities. Libraries will be sequenced using 2x100 bp chemistry in an SP flow cell (Illumina, Inc). After multiplexing, raw data will be processed using the "COMPSRA: a COMprehensive Platform for Small RNA-Seq data Analysis" pipeline.

DNA will also be extracted from plasma according to the "blood and body fluid protocol" of the QIAamp blood reagent set (QIAgen, Hilden, Germany); 400 μL of plasma will be applied to each column, DNA will be eluted in 200 μL of supplied buffer and will be stored at -20°C until use. mtDNA will be quantified with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using specific primers for obtaining circulating mtDNA levels.

Data analysis will be performed with SPSS 17.0. Groups will be compared using unpaired Student's t-tests or Mann-Whitney U tests for parametric and non-parametric data, respectively. Changes after intervention will be evaluated using paired Student's t-tests or Wilcoxon tests, depending on the variable distribution. Pearson or Spearman correlation coefficients will be used to measure the strength of association between variables. In multivariable regression models, the relationship between two or more explanatory variables (independent variables) and a response variable (dependent variable) will be evaluated by fitting a linear equation to the obtained data. Qualitative data will be expressed in percentages, and proportions will be compared using a Chi-square test. All tests will use a 95% confidence interval, and differences will be considered statistically significant when p <0.05.

Study Type

Observational

Enrollment (Actual)

100

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

  • Spain
    • Valencia
      • Valencia, Valencia, Spain, 46017
        • University Hospital Dr Peset
      • Valencia, Valencia, Spain, 46017
        • University Hospital Dr. Peset

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

  • Adult
  • Older Adult

Accepts Healthy Volunteers

Yes

Sampling Method

Non-Probability Sample

Study Population

Primary care clinic

Description

Inclusion Criteria:

  1. Men/Women with periodontitis:

    • Periodontitis will be diagnosed according to the definition of the Centers for Disease Control and Prevention / American Academy of Periodontology (CDC/AAP).

  2. Men/Women with obesity:

    • Body mass index (BMI) ≥30 kg/m2 (WHO 2000)

Exclusion Criteria:

  • Having fewer than fourteen teeth,
  • Having infectious diseases
  • Having other oral inflammatory diseases,
  • Having received periodontal treatment in the past six months or antibiotics in the previous three months, undergoing systemic anti-inflammatory treatment,
  • Pregnancy or lactation
  • Serious illnesses, congenital adrenal hyperplasia, virilizing tumors, hypothyroidism, Cushing's syndrome, prolactinomas, cardiovascular diseases, or diabetes mellitus.
  • Alcohol or drug abuse
  • Psychiatric disorders.

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

Cohorts and Interventions

Group / Cohort
Intervention / Treatment
Control Group without obesity
Control Group with periodontitis and without obesity
Procedure: non-chirurgical periodontal treatment
The non-chirurgical periodontal treatment (scaling and root planing) is the gold standard procedure in therapy for periodontitis. It involves the mechanical removal of plaque and bacterial deposits, creating a local microbial environment in harmony with periodontal health and promoting the replacement of damaged periodontal tissues with collagen-rich connective tissues. This favors a shift in the composition of the oral microbiota from a community dominated by Gram-negatives to one dominated by Gram-positives.
Other Names:
  • scaling and root planing
Obesity Group
Obesity Group with periodontitis
Procedure: non-chirurgical periodontal treatment
The non-chirurgical periodontal treatment (scaling and root planing) is the gold standard procedure in therapy for periodontitis. It involves the mechanical removal of plaque and bacterial deposits, creating a local microbial environment in harmony with periodontal health and promoting the replacement of damaged periodontal tissues with collagen-rich connective tissues. This favors a shift in the composition of the oral microbiota from a community dominated by Gram-negatives to one dominated by Gram-positives.
Other Names:
  • scaling and root planing

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Changes in inflammasome complex activation grade in patients with chronic periodontitis with and without obesity, before and after non-surgical periodontal treatment.
Time Frame: At recruitment
Relative protein expression of NLRP3, ASC, Caspase-1, AIM2, IL-1β, IL-18 and inflammatory mediators NFκB, JNK, IL6, and TNFα by Western Blot and normalized to the loading control protein, and inflammasome assembly by confocal microscopy in PBMC.
At recruitment
Changes in endothelial function in the study population.
Time Frame: At recruitment
Evaluation of leucocytes adhesion to the endothelial cell in vitro by means of a parallel flow chamber system coupled to an inverted phase-contrast microscope.
At recruitment
Changes in endothelial function at molecular level in the study population.
Time Frame: At recruitment
Evaluation of circulating levels of adhesion molecules - P-selectin, ICAM-1, and VCAM-1, and circulating cytokines (L1β, IL18, IL6 and TNFα) in serum using Luminex technique.
At recruitment
Changes in the systemic inflammation status in the study population.
Time Frame: At recruitment
Evaluation of circulating levels of mtDNA in plasma samples using RT-PCR.
At recruitment
Changes in the expression of genes related to inflammatory pathways in PBMC in the study population.
Time Frame: At recruitment
Relative expression of genes related to inflammatory pathways (TLR and NFkB) by means of nanostring technology.
At recruitment
Changes in the expression of genes related to oxidative stress levels in PBMC in the study population.
Time Frame: At recruitment
Relative expression of genes related to oxidative stress (GSR, GPX1, GPX2, SOD, CAT, TXNRD1, AKR1B10, SLC7A11, GCLC, GCLM) by means of nanostring technology.
At recruitment
Changes in the expression of genes related to cellular respiration in PBMC in the study population.
Time Frame: At recruitment
Relative expression of genes related to mitochondrial respiration (I, II, III, IV and V complexes) in PBMC by means of nanostring technology
At recruitment
Sequence miRNAs in biological fluids in patients with chronic periodontitis with and without obesity, before and after non-surgical periodontal treatment.
Time Frame: At recruitment
Analysis of differential gen expression (DEGs) of miRNAs
At recruitment
Evaluate the distribution and phenotype of different T lymphocyte and monocyte subpopulations in the study population.
Time Frame: At recruitment
Detection of T lymphocyte subpopulations with CD3/CD4/CD8/CD45RA/CCR7/CD38 multicolor panel and monocyte subpopulations with CD14/CD16 multicolor panel by flux cytometry.
At recruitment

Secondary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Changes in the protein expression of autophagy markers in PBMC in the study population.
Time Frame: At recruitment
Relative expression of intracellular proteins related to autophagy/mitophagy mechanisms (Beclin 1, ATG5, LC3II/I, p62, NRB1, PINK1, MIEAP) assessed by western blot and normalized to the loading control protein.
At recruitment
Evaluate autophagy flux in the study population.
Time Frame: At recruitment
Quantification of cationic amphiphilic tracer (CAT) probe which label vacuoles associated with the autophagy pathway by flux cytometry in PBMC.
At recruitment
Evaluate autophagosome formation in the study population.
Time Frame: At recruitment
Detection of LC3 puntae by confocal/fluorescence microscope in PBMCs.
At recruitment

Collaborators and Investigators

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

Sponsor

Milagros Rocha Barajas

Collaborators

Instituto de Salud Carlos III

Investigators

  • Principal Investigator: Milagros Rocha, PhD, FISABIO-HOSPITAL DR PESET

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)

April 15, 2024

Primary Completion (Actual)

March 1, 2025

Study Completion (Actual)

May 31, 2025

Study Registration Dates

First Submitted

January 30, 2024

First Submitted That Met QC Criteria

February 13, 2024

First Posted (Actual)

February 15, 2024

Study Record Updates

Last Update Posted (Estimated)

January 14, 2026

Last Update Submitted That Met QC Criteria

January 13, 2026

Last Verified

January 1, 2026

More Information

Terms related to this study

Keywords

Additional Relevant MeSH Terms

Other Study ID Numbers

  • PI22/1009

Plan for Individual participant data (IPD)

Plan to Share Individual Participant Data (IPD)?

NO

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

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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