Dental

1. Periodontal Treatment Limits Platelet
Activation In Patients With Periodontitis—a
Controlled-randomized Intervention Trial
Dr. Deepthi. R
1st year Post Graduate
Department of Periodontology
Laky et al
Journal of Clinical Periodontology, 2018

2. INTRODUCTION
• Ulceration of pocket epithelium in active periodontal disease often facilitates
entrance of oral bacteria into the circulation, thereby causing transient
bacteremia and systemic inflammation. (Forner, Larsen, Kilian, &
Holmstrup, 2006).
• This bacteria-induced systemic inflammation might be the reason for the
postulated association of periodontal disease with atherosclerosis or its
major clinical complication, coronary heart disease, which has been found
in several clinical and epidemiological studies. (Bahekar, Singh, Saha,
Molnar, & Arora, 2007; Humphrey, Fu, Buckley, Freeman, & Helfand, 2008).
• These studies showed that periodontitis is associated with endothelial
dysfunction, atherosclerosis, and an increased risk of myocardial infarction
and stroke. (Scannapieco, Bush, & Paju, 2003).

3. • Platelets and their activation state are of pivotal importance to the onset
and exacerbation of atherosclerosis and (other forms of) inflammation.
• Currently, the basic treatment for patients with periodontitis is subgingival
debridement, which results in diminished burden of periodontopathogens,
which were previously shown to be responsible for platelet activation.
(Assinger, Buchberger, et al., 2011; Assinger, Laky, et al., 2011).
• Previous studies showed that periodontal therapy results in local as well as
systemic reduction of inflammation and endothelial dysfunction, regulated
systolic blood pressure and improves the lipid profile (D’Aiuto et al., 2004,
2006; Tonetti et al., 2007).

4. AIM
To evaluate the effect
of periodontal
treatment on platelet
activation and
reactivity in a
prospective
randomized
controlled
intervention trial.

5. MATERIALS AND
METHODS
• Over a period of 9 months, newly diagnosed patients with periodontitis were
consecutively recruited at the School of Dentistry, Medical University of
Vienna after initial periodontal screening.

6. INCLUSION CRITERIA EXCLUSION CRITERIA
At least one interproximal
site with a probing depth
≥5 mm and a loss of
attachment at ≥2
interproximal sites ≥5 mm;
All systemic
diseases
including
diabetes
mellitus,
chronic renal
failure or liver
cirrhosis,
Systemic
antibiotic
treatment in
the preceding
3 months,
Periodontal
treatment
within the
last 4
months,
Pregnancy or
breast feeding,
Medication
which affects
platelets.

7. • Eligible patients were invited to participate (n = 58). They underwent
baseline anamnesis of periodontal disease, microbiological assessment
and blood testing, and full medical and dental histories were collected.
• Then patients were randomly assigned to receive intensive periodontal
treatment (treatment group) or community-based periodontal care (control
group).
• Written informed consent was obtained from all study participants before
study entry.

8. • Patients of the control group donated blood on the day of diagnosis and
received community-based periodontal care, which consisted of oral
hygiene instructions but no periodontal treatment.
• Their intensive periodontal care started after the second blood donation,
which took place 3 months after diagnosis, that is after their study
participation.
• Blood samples were taken in the intervention group treatment as well as
after 3 months.

9. PERIODONTAL EXAMINATION
AND THERAPY
• Clinical attachment level (CAL), comprising both periodontal probing depth
(PPD) and recession of the gingival margin relative to the cementoenamel
junction at six sites per tooth, was examined at baseline in all patients and
after 3 months in the intervention group.
• The presence or absence of supragingival dental plaque and gingival
bleeding on probing was also recorded.
• Intensive periodontal treatment was performed by subgingival debridement
with curettes and sonic instruments generally in two to four treatment
sessions after the initial examination.

10. BLOOD COLLECTION
• Venous blood was drawn by a 20 G needle and anticoagulated with 3.8%
sodium citrate or ethylenediaminetetraacetic acid (EDTA).
• Platelet-rich plasma was obtained by centrifugation of citrated blood at 125
g for 20 min.

11. PLATELET FUNCTION TESTS
• Platelet function tests were carried out:
 to quantify platelet activation levels without any stimulus (referred to as
basal) and
 to estimate their tendency to become activated by a physiologically relevant
stimulus to test platelet reactivity.
• For measurement of basal platelet activation, phosphate-buffered saline
(PBS) was applied instead of agonists.
• For assessment of platelet reactivity, platelet agonist adenosin diphosphate
was incubated with platelets at different concentrations for 10 min.

12. • Surface expression of P-selectin(CD62P) in unstimulated PRP (basal)
represented the primary study outcome. Additionally, it was determined after
ADP-stimulation.
• GPIIb/IIIa activation was determined based on fluorescein isothiocyanate
(FITC)-labelled PAC-1 binding.
• Resting or activated PRP was incubated with the respective antibody for 20
min, before fixation with 1% formaldehyde.

13. • For analysis of reactive oxygen species (ROS) generation, native or ADP-
stimulated PRP was incubated for 10 min with dihydrorhodamine 123 at a
final concentration of 5 μM and subsequently fixed with 1% formaldehyde.
• Surface expression of CD40 ligand (CD40L) was determined after
incubation with a FITC-labelled antibody against CD40L for 30 min and
subsequent fixation (1% formaldehyde).
• Platelet RNA content was determined by staining with thiazol orange of
platelets for 10 min, which were then immediately analysed by flow
cytometry.

14. STATISTICAL ANSLYSES
• Baseline differences of metric variables between groups were assessed by t
tests, or by chi-square tests for dichotomous variables.
• Effects of treatment on basal P-selectin expression (primary endpoint) and
secondary (exploratory) parameters of platelet activation were estimated by
ANCOVA with baseline values as covariate.
• Approximate normal distribution of the dependent variables was assessed
by boxplots.
• Homogeneity of regression slopes was assessed by scatterplots

15. • To address potential confounding by the dichotomous variable gender, it
was included as additional factor in ANCOVA models.
• In case of a significant interaction with treatment, the effect of treatment
was estimated by separate ANCOVA models for each gender and is shown
in separate figures.
• To assess the influence of the potential metric confounders age, body mass
index (BMI), and smoke packs per year, they were included in additional
ANCOVA models as covariates.

16. • Periodontal data were inspected for normal distribution using box plots.
• In case of normal distribution, mean and standard deviation are shown.
Where no normal distribution was observed, data are presented as median
and 25th and 75th percentile.
• Accordingly, differences in periodontal status between the groups were
tested using t tests (normal distribution) or Mann–Whitney U tests.
• Changes between time points in the treatment group were assessed using
paired t tests (normal distributed differences) or Wilcoxon signed-rank tests.

17. • Graphical representation: Baseline and post-treatment data of each group
are presented as boxplots indicating median, 25th and 75th percentile,
minimum and maximum.
• To visualize baseline corrected treatment effects, estimated marginal means
of post-treatment values for each group are shown with respective 95%
confidence intervals (fine lined bars).
• To show the impact of the potential confounders age, BMI and smoke packs
per year, on the treatment effect, post-treatment marginal means estimated
by ANCOVA including the potential confounders as additional covariates are
also plotted with respective 95% confidence intervals (thick lines).

18. RESULTS

24. DISCUSSION
• The present study indicates that periodontal treatment prevents accelerated
platelet activation in patients with periodontitis.
• The study is able to show that the primary outcome parameter, surface
expression of CD62P, is significantly upregulated in the control group
compared to the intervention group 3 months after periodontal treatment.
• Also in response to platelet agonists the control group showed elevated
responses compared to the treatment group, which is indicative of
increased platelet hyper-reactivity.

25. • Elevated platelet activation was confirmed by analysis of other platelet
specific activation markers, including GPIIb/IIIa activation, which represents
the receptor for fibrinogen and is therefore crucial for platelet-platelet
bridging and aggregate formation.
• Moreover, the study found increased ROS production in platelets from
control subjects compared to platelets of patients that underwent
periodontal treatment.
• In line with this result CD40L, an important inflammatory mediator, which is
released in a ROS-dependent fashion, is also elevated in controls.
• Due to its central role in atherosclerosis as well as (other forms of)
inflammation and its abundance in human platelets, CD40L seems to
provide a crucial link between platelet activation, inflammation and
atherogenesis

26. • Periodontal treatment further prevented enhanced platelet aggregation in
response to the pro-thrombotic agonist collagen.
• When RNA content is analysed in platelets, elevated levels of RNA in the
control group was found compared to the treatment group, indicative for a
higher platelet turnover in the control group.
• The findings indicate that platelets in the control group had a shorter life
span and a higher percentage of reticulated platelets that are supposed to
be highly reactive.

27. • The data also suggested that male patients are more prone to suffer from
elevated levels of platelet activation and are therefore also more likely to
benefit from periodontal treatment.
• Taken together, the data indicated that periodontal treatment is able to
diminish accelerated platelet activation in patients with periodontitis.
• Therefore, treatment of periodontal disease could minimize the risk of
aggravated platelet activation in patients with periodontitis and this might
potentially diminish subsequent diseases such as cardiovascular disease in
periodontal patients.

29. CROSS
REFERENCES

30. Periodontopathogens induce soluble P-selectin release by endothelial cells and
platelets
Thrombosis Research Assinger et al
ABSTRACT
Aim: To determine the effects of periodontopathogens on the activation state of both platelets and
endothelial cells and to clarify the contribution of these cells to the release of soluble P-selectin.
Material and Methods: Soluble P-selectin was determined in 26 patients with periodontitis and 19
controls. Furthermore, human endothelial cells and platelets were investigated for their ability to
elicit soluble and surface P-selectin in response to periodontopathogens A.
actinomycetemcomitans Y4 and P. gingivalis. Moreover surface E-selectin and ICAM-1 expression
as well as NFκB translocation in response to these bacteria were determined on endothelial cells
as well as the formation of platelet-leukocyte complexes.
Results: Plasma levels of soluble P-selectin are significantly elevated in periodontitis and
correlate with severity of disease and bacterial infection. Stimulation of endothelial cells with
periodontopathogens results in rapid surface expression of P-selectin but does not induce NFκB
translocation and subsequent de novo synthesis of P-selectin, E-selectin or ICAM-1. In platelets,
bacterial stimulation leads to surface expression of P-selectin and fosters the formation of platelet-
leukocyte aggregates within minutes. P-selectin is rapidly shed from the surface of platelets and
endothelial cells and results in increased levels of soluble P-selectin.
Conclusions: Periodontopathogens are able to directly cause activation of endothelial cells and
platelets within minutes. Given that transient periodontitis-associated bacteremia commonly
occurs after tooth brushing or chewing, data suggested that reduction of periodontopathogens
might result in potential cardiovascular benefits.

31. Periodontal bacterial invasion and infection: contribution to atherosclerotic
pathology
J Clin Periodontol 2013 Reyes et al
ABSTRACT
Objective: The objective of this review was to perform a systematic evaluation of
the literature reporting current scientific evidence for periodontal bacteria as contributors to
atherosclerosis.
Methods: Literature from epidemiological, clinical and experimental studies concerning
periodontal bacteria and atherosclerosis were reviewed. Gathered data were categorized into
seven “proofs” of evidence that periodontal bacteria: 1) disseminate from the oral cavity and reach
systemic vascular tissues; 2) can be found in the affected tissues; 3) live within the affected site;
4) invade affected cell types in vitro; 5) induce atherosclerosis in animal models of disease; 6)
non-invasive mutants of periodontal bacteria cause significantly reduced pathology in vitro and in
vivo; and 7) periodontal isolates from human atheromas can cause disease in animal models of
infection.
Results: Substantial evidence for proofs 1 to 6 was found. However, proof 7 has
not yet been fulfilled.
Conclusions: Despite the lack of evidence that periodontal bacteria obtained from
human atheromas can cause atherosclerosis in animal models of infection, attainment of proofs 1
to 6 provides support that periodontal pathogens can contribute to atherosclerosis.