Effect of Non-surgical Periodontal Therapy in Hypertensive Patients With Assessment of Endocan and TNF-a Levels in Gingival Crevicular Fluid (Case Control Study)
Conditions: Periodontitis, Hypertension, TNF Alpha, Endocan, GCF
Sponsor: Ain Shams University

ID: FDASU-RECD 1293 1123 1234.
Title: Effect of non-surgical periodontal therapy in hypertensive patients
with assessment of Endocan and TNF-α levels in gingival crevicular fluid
(Case control study)
Date: 28/10/2024

Effect of non-surgical periodontal therapy in hypertensive patients
with assessment of Endocan and TNF-α levels in gingival crevicular
fluid
(Case control study)
Protocol Submitted to the Department of Oral Medicine, Periodontology and
Oral Diagnosis
Faculty of Dentistry, Ain Shams University
In partial fulfillment of the requirements of Masters’ degree in periodontology
By:
Lana Ashraf Nabil Mohamed
Master’s degree student in the Department of Oral Medicine, Periodontology & Oral
Diagnosis, Faculty of Dentistry, Ain Shams University.
BDS 2018
(Faculty of Dentistry, Misr International University)
Lana.ashraf@dent.asu.edu.eg
Under the Supervision of:
Prof. Dr. Hala Abu El Ela
Professor of Oral Medicine, Periodontology and Oral Diagnosis
Faculty of Dentistry, Ain Shams University
Dr. Hadeel Gamal Salem Al Malahy

Lecturer of Oral Medicine, Periodontology and Oral Diagnosis
Faculty of Dentistry, Ain Shams University
Dr. Sahar Abdelraouf
Lecturer of Oral Medicine, Periodontology and Oral Diagnosis
Faculty of Dentistry, Misr International University
Ain Shams University
Faculty of Dentistry
2023

Table of Contents
Topic
Page
I.
Abstract
4
II.
Background
5
III.
Research Question
9
IV.
Aims (Objectives)
10
V.
Hypothesis
11
VI.
Ethical Considerations
12
VII.
Study design
13
VIII.
Materials and Methods
●Study setting
●Sample Size
●Study Procedures
13-16
IX.
Statistical Analysis
17
X. Funding of the Study
17
XI.
References
18-20
XII.
Arabic summary
21-23

I.
Abstract
Statement of problem:
There is a strong crossway relationship between
periodontitis and hypertension. Hypertension is the most prevalent of
cardiovascular diseases and its prevalence is increased by the presence of
periodontitis. Periodontal attachment loss is higher in hypertension patients
when
compared
to
healthy
subjects.
Both
are
examples
of
non-communicable diseases (NCDs), also known as chronic inflammatory
diseases, they tend to be of long duration and are the result of a combination
of genetic, physiological and environmental factors. The lack of an
appropriate balance between these factors results in cellular changes as well
as the levels of cytokines with subsequent changes in clinical manifestations
of periodontal diseases. Both periodontitis and hypertension are associated
with inflammation and an increase in specific proinflammatory cytokines.
Aim of the study:
To investigate the correlation between the clinical
parameters and the levels of Endocan and TNF-α in gingival crevicular fluid
(GCF) before and after non-surgical periodontal therapy (NSPT) in stage II
periodontitis patients with hypertension in comparison to non-hypertensive
patients.
Materials and Methods
Clinical assessment of specific clinical parameters: plaque index, gingival
index, probing depth, clinical attachment level (CAL) as primary outcome.
Biochemical assessment of Endocan and TNF-α levels in GCF by Enzyme
Linked Immunosorbent Assay (ELISA) as well as measurement of blood
pressure at baseline and after 3 months as secondary outcomes.

II.
Background:
Hypertension and periodontitis are part of a group of diseases called
non-communicable diseases (NCDs), which are rising in prevalence due to
an increasingly aging population. NCDs comprise 71% of global death.
Hypertension is defined as repeated office SBP values ≥140 mmHg
and/or DBP ≥90 mmHg. It is a multifactorial disease, that can be caused
by endothelial dysfunction (ED), oxidative stress, and inflammation.
Periodontitis is a chronic multifactorial inflammatory condition affecting the
tooth periodontium. In periodontitis, chronic inflammation occurs in reaction
to untreated bacteria, and this is associated with continuous stimulation of
the immune system which subsequently causes damage to the tooth
supporting tissues. The damage is manifested clinically with gingival
bleeding, foul odor, clinical attachment loss, tooth mobility and may lead
eventually to tooth loss.
The interaction between bacteria and host immune responses is the
link between periodontitis and hypertension along with a number of chronic
systemic diseases, such as diabetes mellitus, other CVDs and neurological
diseases such as Alzheimer.
Patients with periodontitis often present with higher arterial BP values
and a 30% to 70% higher chance to also present with hypertension,
especially when there is active gingival inflammation.
Periodontitis patients express elevated levels of local and systemic
inflammatory biomarkers, such as C-reactive protein (CRP), TNF-α,
neutrophilic enzymes, WBCs, disparity in T-cell subtypes, neutrophil
dysfunction, which are all mechanisms resulting in vascular changes and
endothelial dysfunction (ED).

The
presence
of
periodontal
pathogens
has
been
linked
to
hypertension in epidemiological studies. Periodontal pathogens cause
prolonged T-cell activation and elicited increased levels of CRP, TNF-α, and
IL (interleukin)-1β, resulting in an increased BP.
The first study to suggest a correlation between periodontal pathogens
and
hypertension
was
INVEST
(infections
and
vascular
disease
epidemiology study) in 2012, its results concluded a direct relationship
between the levels of subgingival periodontal bacteria and both systolic
blood pressure (SBP) and diastolic blood pressure (DBP) as well as
hypertension prevalence.
Endothelial Dysfunction (ED) is one of the early signs of
hypertension, Endocan is a biomarker of ED, it also plays a role in
endothelium-dependent
pathological disorders including cardiovascular
diseases (CVDs) and periodontitis. This was the trigger to suggest Endocan
as a clinical biomarker for hypertension in 2013.
A consensus report in 2020 showed that there is evidence from
epidemiological
studies
that
periodontitis
patients
exhibit significant
endothelial dysfunction, measured by flow‐mediated dilation (FMD),
arterial stiffness (e.g., pulse wave velocity—PWV) and a significantly
greater thickness of the carotid intima media (cIMT) and elevated arterial
calcification scores. This explains why both periodontitis and hypertension
are correlated by endothelial dysfunction.
Endocan, previously known as Endothelial cell-specific molecule-1
(ESM-1) is one of the molecules secreted by endothelial cells. Endocan is a
50 kDa soluble proteoglycan that regulates many major cellular activities
like cell differentiation, cell proliferation, VCAM-1 and ICAM-1 expression,
regulation of leukocyte migration, neovascularization, and metastasis of
tumors.

Endocan was recognized of being overexpressed in inflammatory
conditions, CVDs, sepsis, cancer, and obesity along with increase in the
secretion of vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and
intercellular adhesion molecule-1 (ICAM-1). Therefore, Endocan has been
designated as a proinflammatory mediator that binds to Lymphocyte
function-associated antigen-1 (LFA-1) on human leukocytes and regulates
LFA-1 interactions with ICAM-1. Previous evidence indicates that Endocan
is increased in inflammatory diseases and inflammation has already been
shown to have an important contributing part in the pathogenetic changes in
both periodontitis and hypertension.
Serum Endocan levels were significantly higher in the HT group (P <
.001). Also, it correlated positively with carotid intima-media thickness
(cIMT),
and
high-sensitivity
C-reactive
protein
(hsCRP)
which
are
pathological indicators of atherosclerosis and hypertension.
A systematic review conducted from 20 studies in 2023, showed that
in hypertensive patients circulating Endocan levels are significantly
elevated. Thus, suggesting endocan as an easy-to-use biomarker to detect
ED in hypertension.
According to Armitage 1999, Periodontitis was classified into
chronic, aggressive, necrotizing ulcerative periodontitis and periodontitis as
a manifestation of systemic disease. However, a new system of periodontal
classification has been adopted, in which the types of disease that were
previously identified as "chronic" or "aggressive" are now classified under
one category ("periodontitis") and re-diagnosed based on many variants
(staging and grading system)
The stage is based on the severity of the disease and the complexity of
the disease management (in terms of loss of clinical attachment between
teeth, radiographic bone loss, and tooth loss), complexity and distribution.
Grading provides additional information about the biological features of the
disease, including history-based analysis of the progression rate of

periodontitis; progression risk assessment; analysis of possible adverse
effects of treatment; and risk assessment that the disease or its treatment may
adversely affect the patient's normal life.
Periodontitis is described in four stages ranging from Stage I: Initial
periodontitis (CAL 1-2mm), Stage II: Moderate periodontitis (CAL 3-4
mm), Stage III: Severe periodontitis with potential for loss of dentition due
to periodontitis (<4), and (CAL≥5mm) Stage IV: Severe periodontitis with
potential for loss of dentition due to periodontitis (≥5) and (CAL≥5mm).
Grading focuses on assessing risk factors like smoking, diabetes, and
outcomes of scaling and root surface debridement. Grade A: Slow rate of
progression (no CAL loss over 5 years), Grade B: Moderate rate of
progression (CAL loss<2mm over 5 years), and Grade C: Rapid rate of
progression (CAL loss ≥2 mm over 5 years).
Non-surgical periodontal therapy (NSPT) is the “gold standard”
therapy for periodontal diseases. NSPT aims at mechanical removal of
bacterial plaque from the tooth surface. NSPT results in reduction of the
microbial load in periodontal pockets favoring the presence of gram-positive
bacteria and reducing gram-negative bacteria. Also, NSPT results in
reducing the levels of proinflammatory cytokines.
In a study conducted by Kumar in 2020, There was a significant
reduction in the Endocan (p < 0.01) among all the groups after NSPT. This
study also suggested that Endocan levels might be increased in periodontal
disease and decreased after periodontal treatment.
On the other hand, there is limited evidence that NSPT could
improve arterial blood pressure, but many studies appear to support a
positive correlation between severe periodontitis and hypertension. Patients
with periodontitis exhibited higher mean SBP and DBP when compared with
non-periodontitis patients. Also, prevalence of hypertension is higher in
periodontitis patients. Systemic biomarkers like CRP, TNF-α and
IL
(interleukin)-1β and Endocan were elevated in patients with hypertension
and
periodontitis.
Periodontal
attachment
loss
was
higher
among
hypertensive patients in comparison to non-hypertensive patients.

To the best of our knowledge, no previous study correlated between
clinical parameters and the levels of Endocan and TNF-α before and after
NSPT in stage II periodontitis in individuals with controlled hypertension in
comparison to healthy (non-hypertension) individuals.
III.
Research Question
“What is the effect of NSPT on the clinical periodontal parameters and its
correlation to changes in GCF levels of Endocan and TNF-α in hypertensive
periodontitis patients compared to non-hypertensive patients?”
PICOTS Elements:
Patient/Problem
Stage II periodontitis patients with controlled
hypertension.
Intervention
Non-Surgical periodontal therapy.
Comparator
Stage II periodontitis healthy individuals.
Outcome
-Clinical evaluation as a primary objective.
-Biochemical
evaluation
as
secondary
objective.
-Blood pressure measurement as secondary
objective.
Time
-Baseline and 3 months (for clinical
evaluation)
-3 months (for biochemical analysis)
-3 months (for blood pressure evaluation)
Setting
Faculty of Dentistry, Ain Shams University.
IV.
Aim of the Study:
1- To correlate changes in GCF level of Endocan and TNF-α with
changes in periodontal parameters after NSPT in both groups with
clinical parameters.
2- The aim of the present study is to investigate changes in the levels of
Endocan and TNF-α in stage II periodontitis patients with controlled
hypertension in comparison to healthy subjects before and after NSPT.
3- To correlate blood pressure changes before and after NSPT.

Primary outcome:
Clinical evaluation of periodontal parameters plaque Index, gingival index,
probing depth and clinical attachment level comparing between hypertensive
and non-hypertensive groups.
Secondary outcome (s):
-
Biochemical evaluation of GCF levels of Endocan and TNF-α in
hypertensive and healthy subjects by ELISA.
-
Systolic and Diastolic Blood pressure measurements before and after
NSPT in hypertensive vs healthy individuals and check if NSPT
reduces SBP and DBP.
Clinical Relevance:
To understand the role of NSPT in stabilizing the periodontal condition as
well as reduce GCF levels of Endocan and TNF-α which will subsequently
result in better hypertension control and improve the lifestyle.
V. Hypothesis:
Hypertension influences response to NSPT and GCF levels of Endocan and
TNF-α.
VI. Ethical considerations
•Risk and discomfort of patients: This research will be conducted with
considerations of patients’ safety and with attempts to reduce patients’
discomfort by reassurance.
•Minimization of the risk: Patients’ baseline vitals will be taken before any
clinical intervention to ensure patients’ safety. Moreover, all medical
operations will be conducted under standardized infection control

procedures.
•Criteria for Discontinuation of Study/patient: Patients will be allowed to
leave the study if he/ she didn’t confer with the oral hygiene instructions,
this will be ensured by 2 weeks, 4 weeks and 6 weeks follow-up after NSPT
and assessing clinical parameters like Plaque index (PI) to determine how
compliant is the patient with oral hygiene instructions. Patients with PI 2
will be eliminated. Patients with uncontrolled hypertension (SBP greater
than or equal to 140mmHg and DBP greater than or equal to 90mmHg) will
be eliminated.
 
•Benefits to the Patients and to the Community: Patients will receive
NSPT and oral hygiene instructions with no cost. The community will
benefit from this study by having a better diagnostic modality for moderate
cases of periodontitis and preservation of dentition. NSPT is expected to
improve patients’ oral/overall health and quality of life and to be of
relevance in the management of patients with hypertension.
•Privacy: All patients’ identification including patient data and photographs
will be locked and not shared.
•Confidentiality: Patient’s data will be treated with utmost confidentiality.
No personnel other than the researchers will have access to the patients’
data.
•Data Management: All patients’ data will be saved on researcher’s
personal laptop that are not connected to any internet connections and no
copies will be made in any kind of database.
•Consent Procedures if Applicable: Any medical procedures and
photographs will have a printed written consent form.
•Patient Informed Consent Form: A proper consent form will be
formulated &signed by the patient before starting any procedure.

VII. Study Design:
Case-control study.
VIII.Materials and Methods:
●Study Setting: Faculty of Dentistry, Ain Shams University.
●Sample Size Calculation:
A power analysis was designed to have adequate power to apply a two-sided
statistical test of the null hypothesis that there is no difference would be
found between tested groups regarding clinical evaluation. By adopting an
alpha (α) level of (0.05), a beta (β) of (0.2) (i.e., power=80%) and an effect
size (d) of (1.53) calculated based on the results of a previous study; the total
required sample size (n) was found to be (26) cases. Total sample size was
increased by (25%) to account for possible dropouts during follow-up
intervals to be (34) cases (i.e., 17 cases per group). Sample size calculation
was performed using G*Power version 3.1.9.7. Increase to 20 cases per
group to compensate for dropouts. (28)
●Eligibility criteria:
Inclusion Criteria
1. Patients
with stage II periodontitis and controlled hypertension
(self-reported history of a diagnosis of HTN by a physician and blood
pressure (SBP < 140 and DBP < 90).
2. Both genders aged from 30-70.
3. Minimum 20 natural teeth excluding third molars.
4. Good compliance with the plaque control instructions following initial
therapy.
5. Availability for follow-up and maintenance program.
Exclusion criteria:
1. Smokers.
2. Pregnant and lactating females.

3. Patients with other systemic diseases, such as diabetes mellitus,
rheumatoid arthritis and cancer.
(According to Cornell Medical Index-Health Questionnaire).
4. Patients taking antibiotic, anti-inflammatory, and immunosuppressive
therapy during the preceding 3 months before the start of the trial and
during the study.
5. Patients who have undergone any periodontal therapy in the last 6
months.
Justification for Exclusions:
To reduce any confounding factors and bias that may affect the results of this
study.
Study procedures:
1- Details of the interventions, testing and follow up:
Group Case (I):
This
group
will
include
20
patients
diagnosed
with
stage II
periodontitis and controlled hypertension. They will receive NSPT and
oral hygiene measures will be instructed following treatment.
Group Control (II):
This
group
will
include
20
patients
diagnosed
with
stage II
periodontitis and no hypertension. They will receive NSPT and oral
hygiene measures will be instructed following treatment.
Study protocol and surgical steps:
1. Before enrollment, a detailed case history will be recorded.
2. A calibrated standard aneroid sphygmomanometer. The average of 3
blood pressure values (systolic blood pressure SBP and diastolic
blood pressure DBP), will be taken at 1 minute interval, this will be
used in data analyses.

(2020
International Society of Hypertension Global Hypertension
Practice Guidelines)
3. For all patients who are suitable for the study the following clinical
evaluation parameters will be measured:
a) Plaque index (PI) (Silness&Löe, 1964)
b) Gingival index (GI) (Löe&Silness, 1963)
c) Probing depth (PD) (Caton, 1980).
d) Bleeding on probing (BOP) (Ainamo, 1985)
e) Clinical attachment level (CAL) (Ramfjord, 1967). 
f) Standardized periapical radiograph. (Pananou, 2017)
Note: Full mouth assessment will be done and then the deepest site will be
evaluated for the GCF sample.
4. Baseline GCF samples will be taken the day after patients were
clinically evaluated to prevent contamination with blood related to the
probing of inflamed areas.
5. The sample areas will be insulated with cotton rolls to prevent saliva
contamination and all supragingival plaque will be eliminated. The
paper strips will be placed into the periodontal pocket and then
permitted to remain for 30 seconds.
6. All patients will undergo full mouth NSPT, this will be done using
ultrasonic and hand instruments.
7. The patients will receive oral hygiene instructions including tooth
brushing using modified bass technique. All patients will be provided
with toothbrush (soft), toothpaste (signal) and interdental cleaning
with dental floss or interdental brush.
8. Two weeks follow-up to ensure that appropriate oral hygiene
instructions are followed. The clinical parameter to be assessed will be
PI and BOP, patients with PI 2 and BOP > 10% will be eliminated
from the study.
9. Then clinical evaluation, collection of samples and measuring of SBP
and DBP will be performed at 3 months after NSPT in both groups.
Outcomes Assessment:
●Clinical assessment.
●Biochemical assessment.
●Blood pressure measurement.

For clinical assessment the following clinical parameters including PI, GI,
PD, BOP and CAL will be recorded for each individual at 1 site at baseline
and 3 months postoperatively. Clinical parameters of PI and BOP will be
assessed at 2 weeks, 4 weeks and 6 weeks follow ups to ensure proper oral
hygiene. Regarding biochemical analysis, GCF samples will be collected
preoperative and 3 months after NSPT. Blood pressure measurements will be
taken before and after 3 months of NSPT.
a) Clinical Assessment
The following clinical parameters will be taken using UNC15 Probe at
baseline and 3 months postoperative.
-
Plaque index: - (Loe and Silness ,1963)
0 = no plaque.
1 = plaque recognized only by running a probe across the tooth
surface.
2 = plaque visible to the naked eye.
3 = abundance of soft matter.
-
Gingival index: -(Loe and Silness ,1963)
0
= Normal gingiva
1
= Mild inflammation – slight edema. No bleeding on probing
2
= Moderate inflammation -redness, edema and glazing.
Bleeding on probing.
3
=
Severe inflammation – marked redness and edema.
Ulceration. Tendency for spontaneous bleeding
-
Probing depth :(Ramfjord, 1959)
Will be measured from the gingival margin to the depth of the pocket at four
points (mesio-facial, mid-facial, disto-facial and mid-lingual) to the nearest
millimeter using UNC-15 periodontal probe The average of the three facial
points will be recorded as the facial probing depth (FPD), while the
mid-lingual point will be recorded as the lingual probing depth.
-
Clinical attachment level (CAL):(Ramfjord et al., 1975). Will be
measured from the CEJ to the depth of the periodontal pocket.

b) Biochemical Assessment:
Biochemical assessment will be done at the deepest pocket at baseline, and 3
months after non-surgical periodontal therapy. Endocan and TNF-α levels
will be determined using commercial human Endocan (ESM-1) and TNF-α
ELISA
Kits.
Measurements
will
be
performed
according
to
the
manufacturer’s instructions.
c) Blood Pressure measurement:
Blood pressure measurement will be done according to, 2020 International
Society of Hypertension Global Hypertension Practice Guidelines.
IX. Statistical Analysis
The obtained results will be collected, tabulated, and subjected to
appropriate statistical analysis.
X. Funding of the Study
This study will be personally funded by the researcher.

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