Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT04245982 |
| Other study ID # |
TOGU |
| Secondary ID |
|
| Status |
Completed |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
January 1, 2019 |
| Est. completion date |
December 31, 2019 |
Study information
| Verified date |
January 2020 |
| Source |
Tokat Gaziosmanpasa University |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
Objective: Diabetes and periodontitis are two chronic inflammatory diseases sharing specific
etiopathogenetic mechanisms, and both cause severe inflammation and destruction. The aim of
the present study was to determine the receptor expressions of Peroxisome
proliferative-activated receptor (PPAR)-γ, Retinoid X receptor (RXR)-α, Vitamin D receptor
(VDR), and nuclear factor kappa B (NF-κB) expressions in healthy gingiva and diseased
gingival samples with diabetes.
Methods: 45 participants as 1; healthy controls (C), 2; periodontitis group (P), and 3;
diabetes and periodontitis group (DP) were enrolled. Plaque index (PI), gingival index (GI)
and clinical attachment levels (CAL) were recorded in all participants. Two gingival biopsies
from each participant were obtained, and one underwent histological tissue processing while
the other underwent RT-PCR analysis of nuclear receptors. Inflammatory and fibroblast cell
counts, PPAR-γ, RXR-α, and VDR were evaluated.
Results: Fibroblast cells were lowest in the DP group and highest in the healthy group.
PPAR-γ, VDR, RXR, and NF-κB expressions were higher in the healthy controls in the RT-PCR
analysis and similar in the other groups. Immunohistochemistry analysis also showed similar
results.
Conclusion: Results concluded that healthy gingival samples had higher PPAR-γ, RXR, VDR, and
NF-κB expressions, and the immunohistochemistry results also supported this finding. The
healthy gingiva contained higher fibroblast cells and lower inflammatory cells.
Description:
The present study protocol was approved by the ethical committee of the Medical Ethics
Committee of Tokat Gaziosmanpasa University. The study was designed as a cross-sectional
clinical study and conducted at Tokat Gaziosmanpaşa University Faculty of Dentistry
Department of Periodontology. Informed consent was obtained from all participants, and
detailed oral and radiological examination was performed by an experienced clinician
(H.B.Y.).
Forty-two participants, 14 participants in each group, were enrolled in the study. The study
groups were as follows.
Healthy controls (C, mean age 41.05±1.80, seven men, seven women) Periodontitis patients with
stage 3 grade B, (P, mean age 42.35±1.92, seven men, seven women) Patients with diabetes and
periodontitis with stage 3 grade C, (DP, mean age 41.09±1.28, seven men, seven women)
All patients and healthy individuals went through a detailed oral examination, and
periodontal health and periodontitis diagnosis were based on the criteria defined by the
recent classification of periodontal health and diseases in the 2017 International World
Workshop for a Classification of Periodontal Diseases and Conditions [21].
All participants were never smokers. Exclusion criteria were conditions which might affect
the inflammatory state, the existence of less than ten or over missing teeth, tobacco or any
drug use, presence of pregnancy or lactation, and existence of antibiotic or periodontal
therapy within previous six months. Diabetes patients in the present study had HbA1c values
greater than 7.0 even though they were under diabetic treatment. And the diabetic patients
who had HbA1c values below 7.0 were not included.
Clinical measurements Clinical parameters were plaque index (PI), gingival index (GI) and
clinical attachment levels (CAL), and all analyses were performed before the biopsy
procedure. Full mouth measurements and the measurements of the relevant teeth were recorded
separately. CAL was measured from the cement-enamel junction to the bottom of the periodontal
pocket and Williams's type periodontal probe was used for measurements (Hu-Friedy Co.,
Chicago, IL, USA). PI and GI measurements were performed via an explorer (Hu-Friedy Co.,
Chicago, IL, USA). All analyses were performed from six points, three from buccal aspect and
three from the palatal aspect as mesial, middle, and distal sites for both teeth. A mean
value of six measurements was recorded.
Collection of Gingival Samples Gingival biopsies were obtained according to a protocol by a
previous study [22]. Two gingival biopsies were obtained from each participant. Gingival
sampling in periodontally healthy individuals was performed by crown lengthening procedure in
the routine treatment protocol or before orthodontically indicated tooth extraction. In the
periodontitis groups, biopsies were obtained from the inflamed gingival tissue with minor
surgical periodontal procedures. All biopsies were obtained from posterior maxillary teeth
(tooth #4, #5, #6 or #7).
Firstly, local anesthesia was performed, and a gingival sample was dissected with a tissue
scissor from an area with requiring either gingivectomy/crown lengthening procedures from
patients with impaired gingival topography or before tooth extraction indicated for
orthodontic treatment in healthy individuals. As for the periodontitis patients, a gingival
sample was dissected using a tissue scissor during the scaling and root planning procedure
from the gingival areas with severe gingival inflammation. One gingival sample from each
participant was immediately placed in 10% neutral buffered formalin for 48 hours and
underwent histological tissue processing. The other samples of each participant were
immediately frozen at -80⁰C until the day of analysis.
Histopathological Evaluation All histological procedures were performed by an experienced
blinded researcher (F.G.). Gingival samples which underwent histological tissue processing
were first washed and cleansed from formalin and were dehydrated with alcohol series. Then
all tissues were cleared with xylene and embedded in paraffin blocks. Each block sectioned
throughout as serial sections and three sections from each block were selected for
Hematoxylin-eosin (H&E) staining. Fibroblast and inflammatory cell infiltration were
evaluated from H&E stained slides under 1000x magnification via a light microscope .
For fibroblast and inflammatory cell evaluation, the gingival area at the connective tissue
border neighboring gingival epithelium was evaluated. A cell counting frame of 10.000 µm2
area was selected under 1000x magnification. The total inflammatory cells (neutrophil,
lymphocyte, eosinophil, and macrophage cells) within the frame were counted as inflammatory
cell counting. The fibroblasts were also counted likewise. The measurements were performed
from three different points, and the mean of these three measurements was recorded [23].
PPAR-γ, RXR-α, and VDR Immunohistochemistry Immunohistochemistry was performed as reported in
previous studies [23-26]. Three sections were chosen for each immunohistochemistry staining
from each participant. Firstly, all slides were deparaffinized and dehydrated with alcohol
series. After washing with distilled water, antigen retrieval was performed via sodium
citrate buffer (pH 6.0) for two h at 70°C, and then endogenous peroxidase activity was
suppressed with 3% hydrogen peroxide treatment. After hydrogen peroxide treatment, all slides
were incubated with normal rabbit serum for 30 min. After normal serum incubation, primary
antibody diluents were prepared and applied to the samples overnight at a humidified chamber
at 4⁰C. The primary antibodies were goat polyclonal anti-PPAR-γ antibody (1:250), anti-RXR-α
antibody (1:250), and anti-VDR antibody (1:250). After primary antibody incubation, all
slides were washed with phosphate buffer solution (PBS) three times for five minutes (3x5)
and biotinylated immunoglobulin G was applied for 30 min. Again a wash of 3x5 PBS, all
samples were exposed to a streptavidin-horseradish peroxidase-conjugated reagent for another
30 min. After washed with 3x5 PBS, all sections were treated with AEC chromogen to visualize
staining. Counterstaining was performed with Meyer's hematoxylin, and sections were mounted.
After allowing dry for two days, samples were examined under 400x magnification using light
microscopy [23, 24].
Immunohistochemical semi-quantitative H SCORE Analysis Immunohistochemistry evaluation was
performed on three different areas in each section, and three sections were evaluated for
each patient. Nine measurements were performed for each participant. All cells were marked
according to their staining as no staining '0', low staining '1', mild staining '2', and
dense staining '3'. Staining scores were converted to a numeric value as 'H score' through a
formula (∑Pi(i+l)) which is a frequently used value for immunohistochemistry [23, 24]. In
this formula, i shows the staining intensity score and Pi: indicates the percentage of the
stained cells.
RT-PCR Analysis The primers to be used for the genes were shown in table 1. The RNA isolation
and RT-PCR analysis performed according to a previous study [27]. Briefly, for RT-PCR
analysis, first gingival tissues were homogenized and subjected to sonication to reveal RNA
in the tissues and tissue supernatants were obtained. Then the supernatants were submitted to
the total mRNA isolation step. The tissues treated with Qiazole, then allowed to incubate in
the shaker for 5 minutes. At the end of 5 minutes, chloroform was added to the medium
containing the cells and incubation continued for 3 minutes at room temperature and then the
samples were centrifuged. At the end of the centrifuge, the supernatant at the top of the
tube transferred to another tube. Afterward, isopropanol was added to the supernatant and
incubated for 10 minutes at room temperature, and the centrifugation step was repeated. At
the end of the centrifugation, the pellet deposited at the bottom was washed with 75%
ethanol, remaining supernatant was centrifuged again and suspended in pure, sterile water
without RNase. mRNAs were determined by spectrophotometric method. Then using the qRT-PCR
kit, one µg mRNA were transformed into single-step cDNA using specific primers targeting the
gene of interest, later the genes were identified with the help of qRT-PCR (Verso, Thermo
Fisher Scientific, Massachusetts, USA). The results were normalized to the β-actin control
gene.
Statistical Analysis The power of the study was over 85% based on a previous study with a
similar study design [25]. IBM SPSS software was used for statistical analysis. One Sample
K-S test was used to test the normality. For the fibroblast cell counts, inflammatory cell
counts, and CAL, One Way ANOVA followed by Tukey test used. For other parameters, Mann
Whitney U and Kruskal Wallis tests were used. Data were presented as mean ± SD or percentage
as appropriate. p < 0.05 were considered statistically significant.
