Ignite Creation Date:
2024-05-06 @ 4:37 PM
Last Modification Date:
2024-10-26 @ 2:13 PM
Study NCT ID:
NCT05042024
Status:
COMPLETED
Last Update Posted:
2021-09-17
First Post:
2021-08-31
Brief Title:
Supplementation With L-ornithine But Not L-arginine Increases Density of CD68 and CD163 Macrophages in Periodontitis
Sponsor:
Ukrainian Medical Stomatological Academy
Organization:
Ukrainian Medical Stomatological Academy
Study Overview
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
Status:
COMPLETED
Status Verified Date:
2021-09
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
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
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 025 to 10
Patients were grouped by stratified randomization into three groups the SRP Group patients received scaling and root planing as a full-mouth procedure n25 the Arg Group patients received oral L-arginine aspartate Yuria-Pharm Ukraine at a dose of 1 g tid for 10 days after SRP n25 and the Orn Group patients received oral L-ornithine aspartate Farmak Ukraine at a dose of 3 g tid for 15 days after SRP n25 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 0106DT06CP10 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 130 clone PG-M1 Diagnostic BioSystems The Hague The Netherlands or anti-CD163 1100 clone 10D6 Diagnostic BioSystems The Hague The Netherlands Then sections were stained with the 2-steps MouseRabbit PolyVue Plus HRPDAB Detection System Diagnostic BioSystems The Hague The Netherlands and counterstained with Mayers 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 LabA1 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 PD4mm 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 CD68CD163 ratio was assessed by inter-group t-tests comparisons and Spearman correlation checking P values of 005 were considered statistically significant in all of the analyses
Patients were grouped by stratified randomization into three groups the SRP Group patients received scaling and root planing as a full-mouth procedure n25 the Arg Group patients received oral L-arginine aspartate Yuria-Pharm Ukraine at a dose of 1 g tid for 10 days after SRP n25 and the Orn Group patients received oral L-ornithine aspartate Farmak Ukraine at a dose of 3 g tid for 15 days after SRP n25 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 0106DT06CP10 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 130 clone PG-M1 Diagnostic BioSystems The Hague The Netherlands or anti-CD163 1100 clone 10D6 Diagnostic BioSystems The Hague The Netherlands Then sections were stained with the 2-steps MouseRabbit PolyVue Plus HRPDAB Detection System Diagnostic BioSystems The Hague The Netherlands and counterstained with Mayers 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 LabA1 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 PD4mm 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 CD68CD163 ratio was assessed by inter-group t-tests comparisons and Spearman correlation checking P values of 005 were considered statistically significant in all of the analyses
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
False
Is an FDA AA801 Violation?:
None