Supplementation With L-ornithine But Not L-arginine Increases Density of CD68+ and CD163+ Macrophages in Periodontitis
Condition: Periodontitis
MeSH terms: Periodontitis
Study type: Interventional
Phase: Phase 3

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.
    

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.
    

Intervention name(s): Scaling and root planing; Oral L-arginine aspartate administration; Oral L-ornithine aspartate administration
Intervention type(s): Procedure; Drug; Drug
Intervention description(s): Conventional periodontal therapy, non-surgical periodontal therapy; 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; 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 intervention name(s): SRP; Oral supplementation with Tivortin® Aspartate (Yuria-Pharm, Ukraine); Oral supplementation with Larnamin (Farmak, Ukraine)
Intervention MeSH terms: N-Methylaspartate
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).
Masking: None (Open Label)
Primary purpose: Treatment
Keywords: L-arginine; L-ornithine; M1 M2 macrophages; macrophages-targeted therapy; periodontitis