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Supplementation With L-ornithine But Not L-arginine Increases Density of CD68+ and CD163+ Macrophages in Periodontitis

Sponsor
Ukrainian Medical Stomatological Academy (Other)
Overall Status
Completed
CT.gov ID
NCT05042024
Collaborator
(none)
75
Enrollment
1
Location
3
Arms
12.4
Actual Duration (Months)
6.1
Patients Per Site Per Month

Study Details

Study Description

Brief Summary

The aim of the study was to investigate whether oral administration of L-arginine or L-ornithine could modulate local representation density and ratio of macrophages in periodontitis-affected gingiva by using immunohistochemical detection of CD68+ and CD163+ macrophages in biopsies of the gingiva.

The null hypothesis tested was that L-arginine and L-ornithine have no influences on CD68+ and CD163+ macrophages densities when supplementing the treatment of periodontitis.

Materials and methods. 75 individuals with a diagnosis of generalized periodontitis at stages II-III and grade B (38 women and 37 men, 51% and 49%, respectively) were included in the study. Periodontitis was diagnosed by using the criteria of the Classification of Periodontal and Peri-Implant Diseases and Conditions 2017. 25 patients received scaling and root planing only; 25 patients additionally received L-arginine, and 25 - L-ornithine, according to instructions available in Ukraine.

For the immunohistochemical study of paraffin-embedded sections, the gingival biopsy was taken from 5 selected patients per group before treatment and after 1 month. CD68+ (cluster of differentiation 68 positive) and CD163+ cells served as a morphological equivalent of M1, M2 macrophages subpopulations, and their densities were calculated per 10000 μm2. Statistical analysis was performed by adequate power methods.

Condition or Disease Intervention/Treatment Phase
  • Procedure: Scaling and root planing
  • Drug: Oral L-arginine aspartate administration
  • Drug: Oral L-ornithine aspartate administration
 Phase 3

Detailed Description

Study design The present work was the original research study. 75 individuals with a diagnosis of generalized periodontitis at stages II-III and grade B (38 women and 37 men, 51% and 49%, respectively) were included in the study. Periodontitis was diagnosed by using the criteria of the Classification of Periodontal and Peri-Implant Diseases and Conditions 2017. Stage II of periodontitis was diagnosed in the presence of 3 to 4 mm interdental CAL at the site of greatest loss, 4 to maximum 5 mm PPD, the radiographic bone loss at the root coronal third, and no tooth loss due to periodontitis. Stage III was diagnosed in the presence of ≥5 mm interdental CAL, the radiographic bone loss extending to the middle or apical third of the root, ≤4 teeth loss due to periodontitis. In all cases, periodontitis had a generalized pattern (>30% of teeth involved) and grade B as patterns of the progression, based on indirect evidence (radiographic bone loss expressed as a percentage of root length divided by the age of the subject was from 0.25 to 1.0).

Patients were grouped by stratified randomization into three groups: the SRP Group (patients received scaling and root planing as a full-mouth procedure, n=25); the Arg Group (patients received oral L-arginine aspartate (Yuria-Pharm, Ukraine) at a dose of 1 g t.i.d. for 10 days after SRP, n=25); and the Orn Group (patients received oral L-ornithine aspartate (Farmak, Ukraine) at a dose of 3 g t.i.d. for 15 days after SRP, n=25). We used arginine and ornithine according to instructions for these medicines available for use in Ukraine. During the study, all patients were on a stable diet, without changing their rations and regiments.

For all participants, gender and age were recorded, and periodontal parameters such as periodontal pocket depth (PPD), clinical attachment level (CAL) and bleeding on probing (BoP) measurements were taken from six periodontal sites on all teeth (except for the third molars) by a single calibrated examiner using a manual periodontal probe (dental explorer tool labeled 0106.DT06.CP10, Den Tag, Italy). PPD and CAL were measured to the nearest 1 mm.

All patients were clinically examined before treatment and after 1 month ± 5 days.

Collection of gingival tissue samples For the precise immunohistochemical study, the gingival biopsy of approximately 3x3 mm was excised under local anesthesia before treatment and 1 month later in 5 selected patients per group. Biopsies were obtained in the same time-points, from a single site displaying the deepest pocket around suitable dental and periodontal procedures. Removal of these tissue biopsies did not interfere with the initial treatment plan or influence upon the expected clinical outcomes. After collection, biopsies were fixed in a 4% formalin solution for 24 hr of fixation, dehydrated, and embedded in paraffin.

Immunohistochemistry and antibodies Paraffin sections, 2-3 μm in thickness were deparaffinized and dehydrated. Heat-induced epitope retrieval in citrate buffer, power of hydrogen (pH) 6, was performed by successive heating the slides in the microwave oven, then allowed to cool, rinsed with phosphate-buffered saline (PBS), incubated with blocked reagent, rinsed, and incubated with mouse monoclonal CD68 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 Mouse/Rabbit PolyVue Plus™ HRP/DAB Detection System (Diagnostic BioSystems, The Hague, The Netherlands), and counterstained with Mayer's haemalaun. PBS was used as a negative control, the lymph node tissue - as a positive control.

Evaluation of immunohistochemical staining CD68+ and CD163+ macrophages (Mφs) were estimated by counting the number of the cells by light microscope ×400 in intensive infiltrative areas, 5 regions from each slice were selected, and all 5 counts were taken for statistics. We counted immunopositive Mφs in the areas of cell infiltration, since they are directly related to inflammation. The number of cells per 10 000 μm2 was calculated as immunopositive cell density. Photos were obtained using the light microscope Axio Lab.A1 (Carl Zeiss, Göttingen, Germany) (×400).

Statistical analysis Small sample size was justified by the expectation of a high effect size, unknown null distribution, and compensated with adequate power non parametric statistics. Precise sample size calculation was not performed.

Means of PPD and CAL were calculated for sites with PD>4mm (affected sites). Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Software, San Diego, USA) by means of descriptive statistics, chi-square test, one-way ANOVA and nonparametric ANOVA tests for multiple comparisons: Friedman - for dependent variables, Kruskal-Wallis test - for independent variables, with post-hoc analyzing. For descriptive statistics of cells numbers, the percentile ranges were also used because of non-normal distributions. The CD68+/CD163+ ratio was assessed by inter-group t-tests comparisons and Spearman correlation checking. P values of <0.05 were considered statistically significant in all of the analyses.

Study Design

Study Type:
Interventional
Actual Enrollment :
75 participants
Allocation:
Randomized
Intervention Model:
Parallel Assignment
Intervention Model Description:
Patients were grouped by stratified randomization into three groups: the SRP Group (patients received conventional periodontal therapy including scaling and root planing as a full-mouth procedure, n=25); the Arg Group (patients received oral L-arginine aspartate (Yuria-Pharm, Ukraine) at a dose of 1 g t.i.d. for 10 days after conventional periodontal therapy, n=25); and the Orn Group (patients received oral L-ornithine aspartate (Farmak, Ukraine) at a dose of 3 g t.i.d. for 15 days after conventional periodontal therapy, n=25).Patients were grouped by stratified randomization into three groups: the SRP Group (patients received conventional periodontal therapy including scaling and root planing as a full-mouth procedure, n=25); the Arg Group (patients received oral L-arginine aspartate (Yuria-Pharm, Ukraine) at a dose of 1 g t.i.d. for 10 days after conventional periodontal therapy, n=25); and the Orn Group (patients received oral L-ornithine aspartate (Farmak, Ukraine) at a dose of 3 g t.i.d. for 15 days after conventional periodontal therapy, n=25).
Masking:
None (Open Label)
Masking Description:
Open Label
Primary Purpose:
Treatment
Official Title:
Supplementation With L-ornithine Increases Representation Density of CD68+ and CD163+ Macrophages in Human Periodontitis Gingiva and Can Modulate Macrophages Phenotypes. Randomized Controlled Pilot Trial
Actual Study Start Date :
Oct 20, 2017
Actual Primary Completion Date :
Oct 2, 2018
Actual Study Completion Date :
Nov 1, 2018

Arms and Interventions

Arm Intervention/Treatment
Active Comparator: the SRP Group

Patients received conventional periodontal therapy including scaling and root planing as a full-mouth procedure, n=25.

Procedure: Scaling and root planing
Conventional periodontal therapy, non-surgical periodontal therapy
Other Names:
  • SRP

    		•
    Experimental: the Arg Group

    Patients received oral L-arginine aspartate (Yuria-Pharm, Ukraine) at a dose of 1 g t.i.d. for 10 days after conventional periodontal therapy, n=25.

    Procedure: Scaling and root planing
    Conventional periodontal therapy, non-surgical periodontal therapy
    Other Names:
  • SRP

    • Drug: Oral L-arginine aspartate administration
    The use of L-arginine aspartate as adjuncts to local conservative treatment (scaling and root planing) of periodontitis to optimize treatment by modulating local macrophage subpopulations
    Other Names:
  • Oral supplementation with Tivortin® Aspartate (Yuria-Pharm, Ukraine)

    		•
    Experimental: the Orn Group

    Patients received oral L-ornithine aspartate (Farmak, Ukraine) at a dose of 3 g t.i.d. for 15 days after conventional periodontal therapy, n=25.

    Procedure: Scaling and root planing
    Conventional periodontal therapy, non-surgical periodontal therapy
    Other Names:
  • SRP

    • Drug: Oral L-ornithine aspartate administration
    The use of L-ornithine aspartate as adjuncts to local conservative treatment (scaling and root planing) of periodontitis to optimize treatment by modulating local macrophage subpopulations
    Other Names:
  • Oral supplementation with Larnamin (Farmak, Ukraine)

    		•

    Outcome Measures

    Primary Outcome Measures

    1. Periodontal pocket depth (PPD) [Before treatment and after 1 month ± 5 days.]

      Mean changes in probing depth (PD). Measurements were taken from six periodontal sites on all teeth (except for the third molars) by a single calibrated examiner using a manual periodontal probe (0106.DT06.CP10, Den Tag, Italy) to the nearest 1 mm.

    2. Clinical attachment level (CAL) [Before treatment and after 1 month ± 5 days.]

      Mean changes in clinical attachment level.Measurements were taken from six periodontal sites on all teeth (except for the third molars) by a single calibrated examiner using a manual periodontal probe (0106.DT06.CP10, Den Tag, Italy) to the nearest 1 mm.

    3. Bleeding on probing (BoP) measurements [Before treatment and after 1 month ± 5 days.]

      Mean changes in BoP

    Secondary Outcome Measures

    1. CD68+ and CD163+ macrophages density in gingiva [Before treatment and after 1 month ± 5 days.]

      The number of cells per 10 000 μm2 was calculated as immunopositive cell density

    Eligibility Criteria

    Criteria

    Ages Eligible for Study:
    25 Years to 54 Years
    Sexes Eligible for Study:
    All
    Accepts Healthy Volunteers:
    No
    Inclusion Criteria:

    • Presence of periodontitis

    • Good general health

    • At least 19 remaining teeth

    • Written informed consent forms

    Exclusion Criteria:

    • Antibiotics or anti-inflammatory medications use within the preceding 3 months

    • Periodontal therapy within the previous 6 months

    • Purulent exudation from periodontal pockets

    • Pregnancy and breastfeeding

    • Presence of severe, uncontrolled (decompensated) diseases of the internal organs, or neuropsychiatric disorders

    • Presence of other conditions that determined the inability of the patient to understand the nature and possible consequences of the study

    Contacts and Locations

    Locations

    Site City State Country Postal Code
    1 Ukrainian Medical Stomatological Academy Poltava Ukraine 36011

    Sponsors and Collaborators

    • Ukrainian Medical Stomatological Academy

    Investigators

    • Principal Investigator: Igor P Kaydashev, Dr.hab., Ukrainian Medical Stomatological Academy

    Study Documents (Full-Text)

    None provided.

    More Information

    Publications

    Responsible Party:
    Igor Kaydashev, Honored Master of Science and Technology of Ukraine, Doctor of Medical Sciences, Professor Vice-Rector for Research and Development, Ukrainian Medical Stomatological Academy
    ClinicalTrials.gov Identifier:
    NCT05042024
    Other Study ID Numbers:
    • 0120U101151
    First Posted:
    Sep 13, 2021
    Last Update Posted:
    Sep 17, 2021
    Last Verified:
    Sep 1, 2021
    Individual Participant Data (IPD) Sharing Statement:
    No
    Plan to Share IPD:
    No
    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
    Keywords provided by Igor Kaydashev, Honored Master of Science and Technology of Ukraine, Doctor of Medical Sciences, Professor Vice-Rector for Research and Development, Ukrainian Medical Stomatological Academy
    L-arginine
    L-ornithine
    M1 M2 macrophages
    macrophages-targeted therapy
    periodontitis
    Additional relevant MeSH terms:
    Periodontitis
    N-Methylaspartate

    Study Results

    No Results Posted as of Sep 17, 2021