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University of Medicine and Pharmacy "Gr. T. Popa"
Faculty of Dental Medicine

ASSESSMENT OF PATHOPHYSIOLOGICAL MECHANISMS
IN ETIOPATHOGENY OF CHRONIC AND AGGRESSIVE
PERIODONTAL DISEASE

DOCTORATE THESIS
- Abstract -

SCIENTIFIC COORDINATOR

PROF. DR. NORINA CONSUELA FORNA

PROF. DR. MARCEL COSTULEANU

PH.D. ASPIRANT
dr. BUCTARU ( MRU) IOANA

IAI
- 2013 -

STATE OF KNOWLEDGE

INTRODUCTION. MOTIVATION BASIS
CHAPTER 1. ETIOPATHOGENIC FACTORS INVOLVED IN CRONIC AND
AGRESSIVE FORMS OF PERIODONTAL DISEASE

CHAPTER 2. PATHOPHYSIOLOGICAL MECHANISMS IN PERIODONTAL DISEASE

CHAPTER 3. CLINICAL AND PARACLINICAL DIAGNOSTIC ELEMENTS IN
CHRONIC AND AGGRESIVE PERIODONTITIS

PERSONAL CONTRIBUTIONS

CHAPTER 4. MICROBIOLOGICAL STUDIES REGARDING IDENTIFICATION OF
BACTERIAL AGENTS INVOLVED IN PERIODONTAL ETIOLOGY
STUDY 1. MOLECULAR ANALISYS OF THE PREVELANCE OF
PERIODONTOPATHOGENIC AGENTS WITH AGGRESIVE PERIODONTITIS
4.1.1. INTRODUCTION
4.1.2. PURPOSE OF STUDY
4.1.3. MATERIALS AND METHODS
4.1.3.1. ALLOCATION OF STUDY GROUPS
4.1.3.2. MICROBIOLOGICAL EVALUATION
4.1.3.3. STATISTICAL ANALYSIS
4.1.3. RESULTS
4.1.4. DISCUSSION
4.1.5 CONCLUSIONS
STUDY 2. SPECTROMETRIC ANALYSIS OF BIOLOGICAL MACROMOLECULES
PATHOGENS CULTURE - SPECTROMETRY MALDITOF
4.2.1. AIM OF THE STUDY
4.2.2.1. COMPOSITION STUDY GROUPS
4.2.3. RESULTS
4.2.3.1 PERIODONTAL CLINICAL PARAMETERS
4.2.3.2 MICROBIOLOGIC RESULTS
4.2.4. DISCUSSIONS
4.2.5. CONCLUSIONS

CHAPTER 5. ANALYSIS OF OXIDATIVE STRESS MARKERS IN PERIODONTAL
DISEASE
5.1. INTRODUCTION
5.2. PURPOSE OF STUDY
5.3. MATERIALS AND METHODS
5.3.1 EVALUATION OF BIOCHEMICAL MARKERS OF DYSLIPIDEMIA
5.3.2 PERIODONTAL STATUS EXAMINATION
5.3.3 ANALYSIS OF OXIDATIVE STRESS MARKERS IN CREVICULAR FLUID
5.4. ANALYSIS OF CLINICAL-RESULTS
5.4.1 DEMOGRAPHIC EVALUATION
5.4.2 PERIODONTAL CLINICAL ASSESSMENT
5.4.3 RESULTS GPX, SUPEROXIDE DISMUTASE AND MALONIDIALDEHIDA
5.5. DISCUSSIONS
5.6. CONCLUSIONS

CHAPTER 6. STUDY REGARDING THE ROLE OF CYTOKINES IN THE
PATHOGENY OF PERIODONTAL CHRONIC AND AGGRESSIVE PROCESSES
6.1 INTRODUCTION
6.2 THE CUANTIFICATION OF IL-8, TNF-, IFN- CYTOKINES USING THE ELISA
TECHNIQUE
6.2.1 THE OBJECTIVES OF THE STUDY
6.2.2 THE PURPOSE OF THE STUDY
6.2.3.1 COLLECTION AND PROCESSION OF THE BIOLOGIC MATERIAL
6.2.3.2 THE ELISA ENZYME IMMUNOASSAY SANDWITCH TYPE PRINCIPLE
6.2.3.3. THE ASSESSMENT OF SERUM/PLASMA IL-8 LEVELS IN PATIENTS WITH
AGGRESSIVE PERIODONTITIS, CHRONIC PERIODONTITIS AND IN THE
CONTROL GROUP
6.2.3.4 THE ASSESSMENT OF SERUM/PLASMA IFN- IN PATIENTS WITH
CONTROL GROUP
6.2.3.5 THE ASSESSMENT OF SERUM/PLASMA TNF-ALPHA IN PATIENTS WITH
CONTROL GROUP
6.2.4 RESULTS
6.2.4.1 THE RESULTS OF IL-8 ASSESSMENT ON PATIENTS WITH AGGRESSIVE

AND CHRONIC PERIODONTITIS, WHEN COMAPRED WITH CONTROL GROUP
6.2.4.2 THE RESULTS OF TNF-ALPHA ASSESSMENT ON PATIENTS WITH
AGGRESSIVE AND CHRONIC PERIODONTITIS, WHEN COMAPRED WITH
CONTROL GROUP
6.2.4.3 THE RESULTS OF IFN- ASSESSMENT ON PATIENTS WITH AGGRESSIVE
AND CHRONIC PERIODONTITIS, WHEN COMAPRED WITH CONTROL GROUP
6.4 DISCUSSIONS
6.5 CONCLUSIONS

FINAL CONCLUSIONS
ORIGINALITY STUDY
BIBLIOGRAPHY

ASSESSMENT OF PATHOPHYSIOLOGICAL MECHANISMS IN ETIOPATHOGENY OF CHRONIC AND
AGGRESSIVE PERIODONTAL DISEASE
DOCTORATE THESIS- Abstract

1
INTRODUCTION

PURPOSE AND MOTIVES FOR RESEARCHING THE
TOPIC

The most common diseases of periodontal tissues are inflammatory
processes of the gums and its periodontal support. Periodontal diseases are
polymicrobial infections associated with the local accumulation of bacterial plaque,
subgingival pathogenic flora, and calculus.
Periodontal diseases are a major cause of tooth loss in adults and they cover:
*gingivitis- localized inflammation, frequent in children and adolescents, and
*periodontitis- characterized by the extension of inflammatory infiltrate towards the
subjacent gingival mucosa, the formation of periodontal pockets and the eventual loss
of alveolar bone.
Cronic and aggresive periodontitis both present the clinical characteristics of
bone and attachement loss as a response to the bacterial biofilm colonisation of tooth
surfaces. Accepted definitions of these two conditions have changed regularly, while
the understanding of disease etiology process has evolved. The classification will
without a doubt be modified again, once our understanding of cellular and subcelluar
mechanisms which are the basis of these diseases, improves.Some are innate defects
such as leukocyte adhesion deficiency, others are acquired upon exposure to
medications such as agranulocytosis inductors. Correct medical history confirming
systemic issues needs to be included in diagnosing these diseases.

CHAPTER 1

ETIOPATHOGENIC FACTORS INVOLVED IN
CRONIC AND AGRESSIVE FORMS OF
PERIODONTAL DISEASE

Aggresive periodontitis is a distructive and more rare periodontal disease,
and is characterised by the following: fast attachment loss and bony distruction found
in pacients appearing to be clinically healthy with microbial deposits regardless of
disease severity and familial aggregation.

2
The disease tends to show up in the first decades of a pacients life. It has
been classified in two types: localised and generalised. The international seminar on
periodontal disease classification has indicated the presence of specific
microorganisms, while Aggregatibacter actinomycetemcomitans represents one of the
secondary characteristics of AgP.
Microbiological conclusions have no influence on how the initial treatment is
administered, hence the microbiological test can be carried out upon completion of
the first stage of treatment.
It should be noted however, that reducing the bacterial load can increase the
possibility of negative false results when intense microbiological tests are used.
As such, keeping in mind the insufficient knowledge on the etiopathogenic
process, there is no viable universal treatment for all patients.

CHAPTER 2

PATHOPHYSIOLOGICAL MECHANISMS IN
PERIODONTAL DISEASE

The oral cavity can very much be considered an ecosystem which
encompasses a multitude of microbial species that carry out antagonistic activities. As
long as this antagonistic activity is maintained in a stabile equilibrium, the health of
the periodontium and oral tissues are maintained at a normal state. However when this
equilibrium destabilises and the virulence of certain bacteria escalates, or the oral
tissues decline in their defence mechanisms, the disease onset appears.
More importantly, internal host factors cause a patient to become more
susceptible to disease. Due to continuous research, these factors have become known
to be more numerous while their relationships and functions better understood. Host
risk factors have been found to affect every line of defence.
To be noted however, is that recent data suggests that a decrease in local
antioxidant activity in the crevicular fluid happens as a result of periodontal
inflammation, and therefore, traditional non-surgical therapies that aim to eliminate
plaque and reduce inflammation, re-stabilise antioxidant capacity levels to a normal
level in comparison to control patients. [105]


3
CHAPTER 3

CLINICAL AND PARACLINICAL DIAGNOSTIC
ELEMENTS IN CHRONIC AND AGGRESIVE
PERIODONTITIS

In a general sense, chronic and aggressive periodontitis share numerous
clinical characteristics, however the specific details of those characteristics are not
necessarily identical to both forms of the disease. Nonetheless, its been established
that both are complex infections that appear in susceptible hosts and are determined
by the bio film that forms on tooth surfaces. In both cases, disease inducing biofilms
include microorganisms that are part of the normal oral microbiota. In addition,
inflamatory/immune reactions of the host are mainly responsible for periodontal
attachment and alveolar bone loss that sustain the teeth.
In conclusion, while the majority of clinicians agree that the aggressive
forms of periodontitis exist as separate clinical entities, the clear cut clinical
distinction between the aggressive and the chronic (especially the generalised) forms
cannot be made.

CHAPTER 4

MICROBIOLOGICAL STUDIES REGARDING
IDENTIFICATION OF BACTERIAL AGENTS
INVOLVED IN PERIODONTAL ETIOLOGY

4.1 STUDY 1

MOLECULAR ANALISYS OF THE PREVELANCE OF
PERIODONTOPATHOGENIC AGENTS WITH AGGRESIVE
PERIODONTITIS

4.1.1 INTRODUCTION

Establishing family involvement in cases relies on a combination of history
and clinical examination of family members of those affected.

4
At this stage there is not a more evident and correct way in establishing
familial involvement of those affected.
It should be mentioned that in the majority of AgP cases, the level of
periodontal destruction seems to be higher that initially estimated, due to an
accumulation of local factors. The observation can be incorrect in some cases.
It is underlined however, that diagnosing by way of laboratory tests is
essential for a AgP but should be correlated with the other evaluations. [13]

4.1.2 PURPOSE OF STUDY

The purpose of this study was to analyze the genotype of periodontal
pathogen agents, Agreggatibacter actinomycetemcomitans, Porphyromonas
gingivalis, Prevotella intermedia, Tannerella forsythensis, Campylobacter
rectus, Eikenella corrodens, Fusobacterium nucleatum, Veillonella parvula,
Capnocytophaga ochracea i Fusobacterium spp. on lots of patients with
aggresive periodontitis AgP, and chronic periodontitis CrP.

4.1.3 MATERIALS AND METHODS
4.1.3.1. ALLOCATION OF STUDY GROUPS

The study was done at Innsbruck Medical University- Department of
molecular mechanisms of infectious diseases, analysis and investigations
were carried out at the department of Hygiene and Medical
Microbiology.
The patients were recruited from the Dental Medicine and OMF
Surgery clinic Zahnmedizen of Tyrol Innsbruck-Department of Oral
Prevention- Periodontology Department.
A total of 45 patients with AgP were included in epidemiological
analysis. All of the patients were previously treated and were monitored
between 2007-2012.


5
GROUP I- AgP PATIENTS
The patients were diagnosed according to the advanced clinical and
radiographic signs five years before the study commenced, and were treated,
however revaluation found persistence of aggressive periodontal disease, while
also considering their age and history of periodontal issues.
Patients with CrP or those that were administered anti-inflammatory or
antimicrobial therapy in the last 6 months were excluded from the study.
The average age or the AgP patients was 34.7 years.
Before any therapy was administered, four samples of subgingival
periodontal plaque were harvested from every patient and preferably from every
quadrant, from the deepest periodontal pockets(4mm), which bled upon probing.
Additionally, samples from clinically unaffected sites were harvested.
After eliminating the supragingival plaque with a sterile chiurette, three
sterile paper cones were introduced in every site [ISO 35; Becht, Offenburg,
Germania].

GROUP II- CrP PATIENTS
The second study group- used for evaluation comparison- consisted of 21
patients with CrP.

Iclussion criteria was as follows:
- Age 35 years
- At least 20 teeth present
- No history of severe periodontitis
- No site with a PD> 5 mm

Criteria of exclusion was the presence of
gingivitis, severe periodontitis,
chronic systemic diseases
anti-inflammatory or antimicrobial therapy administered
within the last 6 months.
patients that had not received any periodontal treatment in the
past.

6


For the microbiological evaluation, harvested patient samples were
taken from the deepest periodontal pockets(for more relevance)
The cones were immersed in the transfer tubes/containers, either all in one
tube or in different tubes.
The tubes were processed at the research lab of the Microbiology Institute
Tyrol Innsbruck.
All of the samples were tested for the presence of Agreggatibacter
actinomycetemcomitans, Porphyromonas gingivalis, T.forsythia, T.denticola i
Prevotella intermedia .

4.1.3.3. STATISTICAL ANALYSIS

When we compared the colonization of periodontal pockets and control sites
of PAG patients, we chose an arbitrary periodontal bag and a shallow site per patient
which were analyzed and evaluated using chi-square test.Evaluating the prevalence of
species in periodontal pockets from PAG patients versus sites in samples of the ones
with PC was made for four sites per subject and using chi-square test.

4.1.4. RESULTS

We have analyzed a total of 224 subgingival samples from 45 patients
scaling with PAG and 84 samples from 21 patients with PC.
For all patients with PAG and the ones with PC have been identified species
of the genus Fusobacterium, however, Fusobacterium nucleatum / F. periodonticum
were detected in 91% of patients with PAG, but significantly less than [57%], in
patients with PC.
Tannerella forsythensis occurred in 95.5% of patients with PAG and in
85.7% of patients with PC so there is a significant difference between the two groups.
The organism seems to be a common colonizer even in healthy individuals.
Prevalence of Porphyromonas gingivalis and Prevotella intermedia in
patients with PAG was 63.6% and 70.5%, respectively.

7
In patients with PAG to 36.4% was identified Agreggatibacter
actinomycetemcomitans. In contrast, only two of 21 subjects were settled PC by that
species.
Compared with the control group, significantly more patients with PAG were
colonized by Eikenella corrodens [75%] and Campylobacter rectus [56.8%] than
people with PC. Prevalence of Veillonella parvula was moderate in both groups [PAG
25.4%, PC 42.9%], with no statistically significant difference.
A very important important difference [p <0.0001] was observed for
Capnocytophaga ochracea.
As more to 95.2% of patients with PC positive outcomes have been
identified for this species, while the prevalence in the population with PAG was only
15.9%. Calculations suggest evidence of colonization by target species in both
groups.

Figure 4.1 One of those species prevalence in 44 patients PAG (PAG patient was
excluded because of missing control site) and 21 with PC. A patient was considered
positive when at least one site to insulate almost species. The significance of
differences between groups was calculated using the chi-square test.

We evaluated also the frequency of detection of bacteria in periodontal
lesions and healthy control sites in 44 of patients with PAG.
Were included in the analysis a periodontal bag aleas arbitrarily and a site
of. Patients had a large number of bags colonized by Fusobacterium spp. (97.7%),
Fusobacterium nucleatum / F.p. (70.5%), Tannerella forsythensis gingivalis (88.6%)
and Porphyromonas gingivalis (59%).
Prevotella intermedia, Eikenella corrodens and Campylobacter rectus were
identified in 30% -40% of the bags. Comparison of bags and positive control sites

8
showed a highly significant difference (p <0.001) for Fusobacterium. spp,
Fusobacterium nucleatum, Porphyromonas gingivalis and Tannerella forsythensis.
These species and Campylobacter rectus and Prevotella intermedia, were more
frequently detected in diseased sites as compared to clinical healthy sites. Detecting
low frequency periodontal lesions were observed for Agreggatibacter
actinomycetemcomitans (20.5%), Veillonella parvula (6.8%) and Capnocytophaga
ochracea (2.4%). However, all the species investigated (with the exception of
Veillonella parvula) could be identified less frequently than other species.

4.1.5. DISCUSSION

The current chapter emphasizes that periodontitis is a polymicrobial infection
and microbial population screening, rather than isolating single members of
subgingival flora, should give a more comprehensive research than in pathogenesis of
various forms of periodontitis.
Microbiological tests for detecting suspected periodontal pathogens are not
diagnostic criteria for periodontal disease, periodontitis as a consequence of
opportunistic infections caused by microorganisms belonging to the resident
microflora. Obviously, the influence of host immunity changes in clinical outcome
measures high.
Patients with an altered inflammatory response may be less able to tolerate
the presence of specific organisms. People with low apparent risk of developing
destructive periodontal disease may have a protective established the so-called
beneficial subgingival flora. [9, 18]

4.1.6. CONCLUSIONS

1. The aim of the current research was to analyze the prevalence of microorganisms
associated with periodontitis in patients with generalized aggressive periodontitis and
chronic periodontitis using molecular biological detection methods as the eubacterian
one. In despite of improved microbiological methods for detection of bacterial actual
size and their mutual relations in a oral ecosystem is still unclear.
2. Currently, no definitive answer can be given to whether the expression of
aggressive etiologic agents (involving infection with a highly virulent microbiota) or a
high level of individual susceptibility to periodontal disease, or a specific combination
of both factors is crucial in aggressive periodontitis pathogenesis.


9
4.2 STUDY NO 2
SPECTROMETRIC ANALYSIS OF BIOLOGICAL MACROMOLECULES
PATHOGENS CULTURE - SPECTROMETRY MALDITOF

Plaque composition was studied for decades, using different microbiological
and molecular techniques.
All efforts have been supported by the utopia that once discovered the
causative agent of periodontitis will develop a clinical risk rapid microbial
identification and periodontal disease may be treated as any infection.
Bacterial community should be understood as a group of organisms that
coexist in synergistic relationship and are able to exaggerate their potential virulent as
individual bacterial species.
Given the low prevalence in the population of patients with PAG, cost-
effective detection of cases requires sensitive screening approach, namely the
application of diagnostic approaches able to correctly identify most cases of the
disease. [34, 54]

4.2.1 AIM OF THE STUDY

The aim of the study was to estimate the prevalence of oral streptococci and
between species of periodontal bacteria in aggressive periodontitis patients diagnosed
after 1 year of childbearing re-evaluation and grupuilui: Being the control-group of
patients with chronic periodontitis.


4.2.2.1. COMPOSITION STUDY GROUPS

The study was conducted at Innsbruck Medical University - Department
of Molecular mechanism of infectious diseases, microbiological analyzes
and investigations were made at the Department of Hygiene and
Medical Microbiology.

Patients were recruited from the clinic of Dental Medicine and Surgery
UMF Zahnmedizen of Tyrol Innsbruck - Department of Oral
Prevention - disicipline of Periodontology.

The study patients were divided into two study groups:
A group of patients with chronic periodontitis , 56 patients
B group - 31 patients with aggressive periodontitis patients.

10
4.2.2.2 MICROBIOLOGICAL EVALUATION

MALDI-TOF spectrometers
MALDI-TOF spectrometers are formed by two or three levels:

1. MALDI Matrix Assisted Laser Desorbtion Ionization Stanard
2. TOF Reflectron Time Of Flight Standard
3. CID II Collision Induced Disociated Generaia II Standard TOF-TOF
From each pocket we collected 3 cones per patient in a transportation
environment: 12 samples in 3 transportation environment.
The sub-gingival plaque was previously removed to avoid the contamination;
the contamination by saliva was also prevented.
During the collection, the paper cones were maintained in the situs for 10
seconds. The samples were maintained in a solution of glycol thiolate.
The samples for the Maldi Tof analysis were immediately transported at the
Dental Medicine and Surgery, UMPh UMF Zahnmedizen of Tyrol Innsbruck Oral
Prevention Department Periodontology.

4.2.3 RESULTS

4.2.3.1 PERIODONTAL CLINICAL PARAMETERS

The observation sheets were recorded for all the patients, including personal
data, general and dental medical history. We assessed the possible correlations
between the local impairment degree and the type of periodontal disease.
The patients evaluation revealed the following:
Comparison between chronic periodontitis patients versus aggressive
periodontitis patients The diagnosis of aggressive generalized periodontitis implies
that the patients age is under 30 years old (but even older), when compared to
chronic periodontitis which affects older patients (over 30 years old).
The assessment of oral hygiene indices
The necessity of a complete evaluation by using odontal-periodontal indices
is determined after the setting of a certain diagnosis and is included in the treatment

11
planning, with the purpose to quantify for the disease and to monitor the odontal-
periodontal status.
The monitoring is specially addressed to bacterial plaque and gingivitis.
With the purpose to identify the manner in which the oral hygiene indices
indicate the onset and the evolution of aggressive and chronic periodontitis, we traced
their prevalence for the two study groups.
If the scanning detects other significant characteristics of the periodontal
disease, the monitoring is conducted according to local and systemic factors.

Figure 4. 1 The sub-gingival calculus reported to age prevalence percentage
from the total number of subjects

Figure 4. 2 The sub-gingival calculus reported to age prevalence percentage
from the total number of teeth
The results of our study demonstrate a direct relationship between the degree
of calculus formation and the age and the oral hygiene of the studied groups but there
is a high variance in the chronic and aggressive periodontitis groups.

12
We observed that the prevalence of the total and sub-gingival calculus
deposal rises with the age. For the 20-29 years olds with conservative treatment the
prevalence was 38% and for the surgical treatment group- of 40 %. In the age goup of
30 39 years old, the prevalence was of 19,43% for the A group and 19.78% for the
B group.After the data analysis, we observed that the prevalence for A and B groups
is rises with the age. For the 60-69 years olds - 89.94% for A group and 89.94% and
90% for B group.As for the total calculus prevalence reported to the age groups,
89.6% from A group and 90% for the B group presented calculus deposits.
The evaluation of periodontal indices
Having in mind the low prevalence of aggressive periodontitis patients in the
general population, an effective cost of case detection necessitates a sensitive
screening, like a diagnosis process able to correctly identify the cases. The evaluation
of the prevalence and of the attachment loss degree (gingival recession, periodontal
pockets depth) in chronic periodontitis patients demonstrates a direct relationship
between the disease and the calculus prevalence.
From these data we can conclude that there is a direct relationship between
the prevalence and the attachment loss and the presence of calculus, for A and B
groups. Therefore, on 30-39 years olds the gingival recession is similar, of 37,8%, the
affected teeth percentage is 8,6%, while for the 60 69 years olds 39,8%. We
observed that the percentage of affected subjects is almost two times higher while the
affected teeth number being 5 times higher. Regarding the periodontal pockets depth,
we observed a high prevalence and a high number of affected teeth for both groups,
with a mean value of 62% and an affected teeth number higher than 18%.

Figure 4. 3 The prevalence and the percentage of teeth with gingival bleeding
reported to the age groups

13
Periodontal pockets
We measured the periodontal pockets with a depth of at least 2mm. the
surfaces with gingival recessions were significantly higher for the B group than for
the A group (4,3+/-4,2, 1,4+/-2,5, respectively).Approximately 66% of the subjects in
the A group and 32% of the subjects in the B group did not present gingival
recessions; 7% of the patients with chronic periodontitis and approximately 36% of
aggressive periodontitis patients had more than 5 surfaces with gingival recessions.

4.2.3.2 MICROBIOLOGIC RESULTS

The analysis of microbiologic profiles of aggressive periodontitis was limited
to a series of presumptive pathogenic agents:
Staphylococcus aureus,
Streptococcus salivarius,
Streptococcus sanguinis,
which were previously associated with the disease etiopathogeny.
Nevermore, the disease process is related to a complex microbiota, in a
circumstance of an indefinite number of microorganisms could also have a role.
The relative proportion for each pathogenic agent was also summarized.
Aggressive periodontitis patients have significant lower level of Streptococcus
salivarius than chronic periodontitis patients. The identified microorganisms
associated with the periodontal pocket are part of indigenous microorganisms which
protect the health of the host.

Figure 4. 4 The detection frequencies of Streptococcus salivarius in periodontal
samples for the chronic periodontitis patients

14

Figure 4. 5 The detection frequencies of Streptococcus salivarius in periodontal
samples for the aggressive periodontitis patients

Figure 4.6 The detection frequencies of Streptococcus salivarius according to the
periodontal pockets depths

Figure 4.5 The detection frequencies for Streptococcus salivarius and
Staphylococcus aureus in periodontal pockets samples for chronic periodontitis
group

15

Figure 4.6 The detection frequencies for Staphylococcus aureus reported to the
periodontal pockets depth

The loading of these species were significantly higher for chronic
periodontitis group.

4.2.4 DISCUSSIONS

Another purpose of this study was to conduct clinical and microbiologic
studies based on personal data regarding the health status and periodontal impairment
on susceptible or affected subjects by estimating the prevalence between oral
streptococcus species and periodontal pathogenic bacteria.
Having in mind the low prevalence of aggressive periodontitis patients in the
general population, an effective cost of case detection necessitates a sensitive
screening, like a diagnosis process able to correctly identify the cases.The relatively
simple technique for sample preparation, the accuracy and the speed of data obtaining
make the MALDI-ToF MS cell analysis a strong instrument, specially conceived for
the detection of biologic agents. Moreover, the simplicity of the procedure reduces the
risk of accidents during the sample processingA reduced number of microorganism
and even fewer morphologic types can be found in the healthy gingival sulcus. The
affected situses present a complex microbiota, with a high proportion of anaerobic
gram negative micro-organisms.

4.2.5 CONCLUSIONS

1. The microbiologic diagnosis can be useful in different phases of the treatment
plan as a part of the initial diagnosis, on re-assessment and after the

16
maintaining phase.
2. The need of microbiologic information depends on the general treatment strategy.
3. By using the identification technology above-mentioned, the present study
demonstrates that aggressive periodontitis seems to be associated to high loss of
bone tissue and also with quantitative lack of Streptococcus sangvinis, mutans,
oralis, intermedis, involved in the protection against periodontal pathogens.
4. An early microbiologic detection of the periodontal involvement is essential for
the intervention and prevention of the tissue destruction by the aggressive
periodontal disease.

CHAPTER 5

ANALYSIS OF OXIDATIVE STRESS MARKERS IN
PERIODONTAL DISEASE

5.1 INTRODUCTION

Oxidative stress can lead to periodontal tissue damage either directly by
oxidation of important biomolecules or indirectly through activation of transcription
factors sensitive to redox reactions [ie, nuclear factor B (NF-B)] leading to a
remote expression possibly reflected systemic proinflammatory genes.

5.2 AIM OF STUDY

Our study targeted a comparative investigation of the mediators of oxidative
status of enzymetic type and non-enzymetic in the gingival fluid in patients with
chronic periodontitis and aggressive periodontitis patients versus the healthy
periodontal patients.

5.3 MATERIALS AND METHOD
For the study we realized we took into account a representative group of 42
patients, aged between 24 and 55 years, selected from the patients of the
Periodontology Clinic, UMF "Gr.T.Popa" University, and they have been examined
between May 2012-November 2012.

17
These patients were divided into three study groups:
o Group A of patients with chronic periodontitis, 16 patients
o Group B-11 patients with aggressive periodontitis patients
o Group C periodontal healthy patients -15 patients

5.3.1 EVALUATION OF BIOCHEMICAL MARKERS OF DYSLIPIDEMIA
Biochemical assessment was performed after the collection of blood by
venous puncture in heparin lithium vacumtainere as a stabilizer. Samples were
centrifuged and the sera obtained were then processed using an automatic analyzer
COBAS INTEGRA type 400-700-800. There were determined: cholesterol,
triglycerides, HDL-cholesterol (high density cholesterol, drpt considered good
cholesterol), LDL (low density cholesterol is considered as bad cholesterol).

5.3.2 PERIODONTAL STATUS EXAMINATION
Clinical examination of periodontal tissues included measurements and
scoring indices variations on surfaces of all teeth except the third molar.
We always took into account:plaque index (PI), bleeding index (SI), gingival
index (GI), plaque index (IT), attachment loss (AL),registration of exogenous local
factors (PII, CI).

5.3.3 ANALYSIS OF OXIDATIVE STRESS MARKERS IN CREVICULAR
FLUID
5.3.3.1 COLLECTION OF CREVICULAR FLUID

We analyzed the enzymatic markers of stress in all 3 groups of patients:
superoxide dismutase, glutathione peroxidase, malondialdehyde (in collaboration
with the Department of Immunology and Pharmacy Grigore T. Popa Iai) using
crevicular fluid (GCF) as biological material.

5.3.3.2 DETERMINATION OF GPX (GLUTATHIONE PEROXIDASE), AND
SUPEROXIDE DISMUTASE (SOD)
Measurement of glutathione peroxidase - GPx Ransel GPx was performed
using the kit according to the manufacturer's protocol.

18
For superoxide dismutase - SOD were acquired and processed Ransod kits and the
results were analyzed and interpreted using a spectrophotometer model Cobas 6000.
Determination of glutathione peroxidase activity (GPx) in crevicular fluid
samples was performed as described by Paglia and Valentine, using Randox kits.

5.3.3.2 DETERMINATION OF MALONDIALDEHYDE
To assess lipid peroxidation, a major mechanism for the development of
cellular lesions involving the formation of radical species and their products
consecutive oxidation of unsaturated fatty acids, we used the malondialdehyde
(MDA).
This compound resulted from lipid peroxidation (as an alternative way to
physiological degradation by beta oxidation of fatty acids) can be dosed in many
biological samples, which is why I thought it would be relevant to the determination
of its value and crevicular fluid as an expression periodontal lipid peroxidation level
in aggressive periodontitis compared with chronic periodontitis.
Malonildialdehida was determined by liquid chromatographic techniques
(HPLC) isolated from the supernatant centrifuged in a row crevicular fluid, by the
method described in Merendino et al [53].

5.4 ANALYSIS OF CLINICAL-RESULTS
5.4.1 DEMOGRAPHIC EVALUATION
Evaluation of patients in the study revealed the following: 26% of the
patients studied were diagnosed with aggressive periodontitis with or without some
form of systemic involvement (diabetes or dyslipidemia), 38% with chronic
periodontitis not associated with systemic involvement, and 15 patients representing
38% had no periodontal problems, they were included in the study as controls in the
subsequent evaluation of oxidative stress.
36%
38%
26%
parodontita
agresiva
parodontita
cronica
clinica sanatosi

Figure 5. 1 Comparison between the group of patients with chronic periodontitis
(group A) vs. patients with aggressive periodontitis group (group B) and patients
healthier in terms of periodontal (group C).

19
5.4.2 PERIODONTAL CLINICAL ASSESSMENT
A tighter periodontal examination is of critical importance in the collection
of data necessary to arrive at a diagnosis and to create a treatment plan.
At the mouth were considered:
gingival status. Mystery Shopping assignment WHO periodontal probe,
causing the 4 units gum (mesial, distal, buccal and lingual). It was followed
experiencing bleeding, inflammatory phenomena in the gums
We examined each tooth individually characteristic symptoms of
periodontal disease: bleeding, recession and the presence of periodontal pockets, tooth
mobility and expanding presence tarts
Patients were first informed of the need periodic monitoring, the implications
of changes in triggering periodontal serious systemic diseases.

Parameter Minimum
value
Maximum
value
standard
deviation
variation-%
Age 24 55 10,72 114,94.
Plaque index (%) 8,9 65,2 21,27 452,72
Calculus index(%) 7,9 60,57 18,28 334,28
Bleeding index(%) 30,12 85,6 18,29 334,87
Gingival index 1,23 2,85 0,51 0,27
Table 5. 1 Clinical parameters of the study group

From the point of view of oral hygiene, the statistical data on the study group
a mean plaque index 34.96 6.72, with a very high peak of 65.20% and a mean
plaque index of 34.21 5.78 and here the maximum being 60.57%.
Both peaks were recorded for patients in rural areas.
Statistical analysis of periodontal indices showed a strong correlation in
terms satatistic between these indices. Statistical analysis was done using SPSS
software.
Most patients were submitted to clinical accusing aesthetic disorders,
bleeding or pain.
Aesthetic disorders prevailed between the ages of 30-49 years, female
patients, mainly due gingival present in one or more teeth.
Pain was charged 27.78% of patients, being present mainly in the morning,
medium intensity, often accompanied by a slight feeling EGRES tooth. Medical
history have sought to identify possible risk factors of chronic marginal periodontitis
versus aggressive periodontitis.

20
5.4.3 RESULTS GPX, superoxide dismutase AND
MALONIDIALDEHIDA
This study reveals significant differences between most parameters of
oxidative stress in crevicular fluid analyzed.Statistical analysis showed differences
between groups analyzed, the statistical significance was achieved especially those
with aggressive periodontitis, less in those with chronic periodontitis compared with
controls.
Malonildialdehida

Figure 5.2 Distribution of mean values malonildialdehida millimolar
concentrations in gingival crevicular fluid in groups of patients with aggressive
or chronic periodontitis as compared to the control group

A separate analysis of malonildialdehida values in the group of patients with
chronic periodontitis in SPSS test showed statistical significance of values in patients
with chronic periodontitis compared with healthy patients group (P <0.0001) and
those with aggressive form of the disease (P <0.0001), but found no statistical
significance between the two types of periodontal problems.

Figure 5. 3 Graphical representation of the values of superoxide dismutase
(SOD) in the group of healthy patients versus those who had chronic or
aggressive periodontitis

21
Statistical analysis using SPSS in the three study groups with mean values
and coefficient of variation values for superoxide dismutase (SOD) in crevicular fluid.
SOD values in crevicular fluid decreased both in group and in chronic
periodontitis aggressive periodontitis group compared to the control group of patients.
Correlation between SOD values in control group of patients with aggressive
periodontitis and chronic periodontitis, shows that there is a strong correlation
between SOD values in patients with aggressive periodontitis and control group and a
weak correlation in those with periodontitis chronic compared with controls.
SOD values, comprativ between the control group and with aggressive
periodontitis SOD also between the values of patients with aggressive periodontitis
and control group, no statistically significant differences.

Figure 5. 4 Mean values of glutathione peroxidase (units / milliliter) in
gingival crevicular fluid in form study groups with aggressive or chronic
periodontitis compared to control group

Mean glutathione peroxidase activity in crevicular fluid were: 742.8 units /
ml for healthy patients, 736.8 units / ml for aggressive periodontitis group and 736.75
units / ml for aggressive periodontitis.
We found statistically significant differences between the three groups of
patients in this analysis.
Even if there is a strong correlation between glutathione peroxidase values in
all 3 groups studied, we observed that there are significant differences between GPx
values in the group of patients with aggressive periodontitis, chronic periodontitis and
the control group.

5.5 DISCUSSIONS
Inheritance of susceptibility to chronic PAG or is likely insufficient for
disease development: environmental exposure to potential pathogens specific
virulence factors is a necessary step.

22
This data gives partial information required for a definite diagnosis of Pag or
chronic, however, requires an additional parameter anamnesis and clinical
investigation with more advanced means to enable accurate diagnosis, therapy and
monitoring of these diseases.. Understanding that overall disease may be associated
with aggressive or chronic periodontitis is essential. Some are relatively common
disorders such as poorly controlled diabetes or dyslipidemia, others are hereditary.
Some are inherent defects that leukocyte adhesion deficiency, others are acquired
after exposure to the inducing medications as agranulocytosis.
Patients with diabetes whose blood sugar is kept under control usually shows
significantly fewer periodontal pockets greater than or equal to 4mm compared with
patients whose diabetes is not under control. This indicates a pronounced effect on
periodontal patients with uncontrolled.

5.6 CONCLUSIONS
1. Contributions in reducing oxidative stress appear to be involved both in the
production decrease oxidative stress and antioxidant protection in improving
enzymatic equipment.
2. In this study we observed some correlation with the severity peroxidation of
end product forms of periodontal disease, and an inverse relationship
between the level of antioxidant enzymes studied and advanced forms of
periodontal destruction.

CHAPTER 6

STUDY REGARDING THE ROLE OF
CYTOKINES IN THE PATHOGENY OF
PERIODONTAL CHRONIC AND AGGRESSIVE
PROCESSES

6.1. INTRODUCTION

Periodontitis is an inflammatory disease determined by periodontal
pathogens. Usually, periodontitis is classified in aggressive periodontitis and chronic
periodontitis.

23
The onset age for aggressive periodontitis is younger than for chronic
periodontitis and the aggressive forms are accompanied by severe rapid destructions
of the periodontal tissues.
The correlation between aggressive periodontitis and the neutrophils
dysfunction, the familial history, single nucleotide polymorphism, specific immune
response to infectious agents and stress was questioned.
The exposure of the chemotactic mechanism is useful for the research of the
neutrophils dysfunction on aggressive periodontitis patients.

6.2. THE CUANTIFICATION OF IL-8, TNF-, IFN- CYTOKINES USING
THE ELISA TECHNIQUE

6.2.1. THE OBJECTIVES OF THE STUDY:

1. Having in mind the previous studies, we proposed an evaluation of the IFN-
serum levels in patients with aggressive periodontitis, when compared to
chronic and periodontally healthy patients.
2. Along with this parameter, we also proposed the evaluation of cytokines like
TNF- (tumor necrosis factor) and
3. The assessment of cytokines like IL-8 and establishing a correlation in the
aggressive versus chronic periodontitis.

6.2.2. THE PURPOSE OF THE STUDY

The purpose of this study was to assess the differences between the serum
levels of inflammatory biomarkers like cytokines (IL-8, TNF-, IFN-) in healthy,
aggressive and chronic periodontitis patients and also to establish a correlation
between the levels of these markers and the patients age.


6.2. 3.1. COLLECTION AND PROCESSION OF THE BIOLOGIC MATERIAL

The study was conducted on a number of 42 representative patients, with the
age between 24 and 55 years old, selected from the patients in the Periodontology

24
Clinic of UMPh Gr. T. Popa Iai, examined between May 2012 and November
2012.
We collected venous blood from the patients, on transportation environment
with heparin and we determined the IL-8, TNF- and IFN- levels using the ELISA
enzyme immunoassay, with ELISA R&D Systems kit and a semi-automatic ELISA
line Sanofi Pasteur.

6.2.3.2. THE ELISA ENZYME IMMUNOASSAY SANDWITCH TYPE
PRINCIPLE

ELISA (Enzyme Linked Immunosorbet Assay) represents one of the most
used methods in the identification and quantitative appreciation of antibodies,
antigens, cytokines and a variety of other molecules, including synthetic peptides.
For this study we used ELISA sandwich tests.
6.2.3.3. THE ASSESSMENT OF SERUM/PLASMA IL-8 LEVELS IN PATIENTS
WITH AGGRESSIVE PERIODONTITIS, CHRONIC PERIODONTITIS AND
IN THE CONTROL GROUP

For the quantitative assay of the serum/plasma total level of IL-8 in the
patients included in the study, we used the ELISA de R&D Systems, Inc. USA kit
which quantifies the serum/plasma levels of IL-8 of 0 2000 pg/ml.
With the purpose of establishing a standard curve, the lyophilized IL-8
solution was reconstructed with the proper diluent provided by the manufacturer and
we conducted successive dilutions. The diluent served as a 0 standard and the
standard solution as the most concentrated standard (2000 pg/ml).
All the incubations were made on room temperature. The used enzyme was
HRP (horseradish peroxidase), with a substrat of 30% H
2
O
2
, combined with TMB
(3,3,5,5 - tetramethyilbenzidine).
The enzyme reaction to the substrate generates the blue color. The reaction is
stopped with sulphuric acid 2N and generated a yellow soluble product, quantified by
spectrophotometry with a wavelenght of 450nm, with a wavelenght correction of
540nm. The color intensity is proportional with the quantity of IL-8 bound after the
first incubation of serum on the pad.The concentration of unknown products was
determined with the aid of the standard curve, constructed immediately by projecting
the calibrators extinction (Y) to their known concentration (X).

25
6.2. 3.4. THE ASSESSMENT OF SERUM/PLASMA IFN- IN PATIENTS
WITH AGGRESSIVE PERIODONTITIS, CHRONIC PERIODONTITIS AND
IN THE CONTROL GROUP

The principle in the assay of IFN- is similar to the method of detecting IL-8.

6.2.3.5. THE ASSESSMENT OF SERUM/PLASMA TNF-ALPHA IN
PATIENTS WITH AGGRESSIVE PERIODONTITIS, CHRONIC
PERIODONTITIS AND IN THE CONTROL GROUP

The working protocol in serum determination of TNF- was similar to the
ones of IL-8 and IFN-, except the step of adding the Biotine-TNF conjugate.
In this step, the incubation is conducted on 37
0
C and the incubation time is
of 2 hours. Therefore, the total time is of 3 hours.
Although the sensitivity of the kit is high, the minimal values registered are
of 0,09pg/ml.

6.2.4. RESULTS

From the 42 patients in the group, 22 were males and 20 were females.
These patients were divided in 3 study groups:
1. Group A: chronic periodontitis patients -16 patients
2. Group B: aggressive periodontitis patients- 11 patients
3. Group C: periodontally healthy patients- 15 patients.
We obtained the informed consent for each patient regarding the therapy,
clinical and therapeutic implications.
The exclusion criteria that we used from the beginning were:
immunosuppressed patients, pregnancy and lactation, patients under orthodontic
treatment; patients under odonto-periodontal therapy in the last 12 months, patients
who received antibiotic therapy in the last 6 months.
We collected crevicular fluid and saliva from the periodontally impaired
patients at the beginning of the study to assess the various bacterial types and their
level.


26

6.2.4.1. THE RESULTS OF IL-8 ASSESSMENT ON PATIENTS WITH
AGGRESSIVE AND CHRONIC PERIODONTITIS, WHEN COMAPRED
WITH CONTROL GROUP
The 42 patients were divided in 3 age groups:
o Patients with the age of 24-30 years old
o Patients with the age of 30-40 years old
o Patients with the age of more than 40 years old
The percentage of patients with the age of 24-30 years old was the lowest,
being of 14,28 %, while 28,57 % of the patients were between 30 and 40 years old.
The higher percentage was registered for patients with the age higher than 40 years
old, being of 57,14 %.
The statistical analysis of the results for IL-8 on the control group revealed a
mean value of 0,54 0,21pg/ml, while the maximum concentration was of 0,87pg/ml.
The mean value of IL-8 in the aggressive periodontitis patients group was 0,62
0,23pg/ml, with a maximum value of 0,96pg/ml, while in the chronic periodontitis
group the registered mean value was 0,47 0,18, with a maximum value of
0,89pg/ml. The statistical analysis for IL-8 results for the three study groups did not
reveal a significant difference between the healthy patients group and the chronic
periodontitis group. Moreover, there were no significant differences between the
aggressive periodontitis group and the other two groups.

WITH CONTROL GROUP

TNF- is known as a pro-inflammatory cytokine which induces the
collagenase secretion by fibroblasts, bone and cartilage resorption.
Therefore, this cytokine was involved in the alveolar bone destruction.
The identification of TNF- in serum revealed values between 0 i 2,4 pg/ml
for healthy patients, with a mean value of 1,3 pg/ml and a standard deviation of 0,54.
The same cytokine in chronic periodontitis patients presented a mean value of 2,4
pg/ml, with a maximum value of 4,8 pg/ml; standard deviation was of 1,21. The
mean value for TNF- in aggressive periodontitis patients was of 3,2 pg/ml, with a
standard deviation of 0, 72. The maximum value for this group was of 5,7 pg/ml.


27
The statistical analysis of the results for the 3 groups revealed a significant
difference between the healthy subjects and the periodontitis groups.
The chronic periodontitis mean values were almost two times higher than the
control group values.
The TNF- values in the aggressive periodontitis group were the highest,
with a maximum value of 5,7 pg/ml.
The analysis of TNF- values on age groups revealed a maximum value for
this cytokine in the group of patients older than 40.
Lower values, close even to the ones in the control group, were registered for
patients with the age between 24 and 30 years old.

6.2.4.3. THE RESULTS OF IFN- ASSESSMENT ON PATIENTS WITH
WITH CONTROL GROUP

IFN- is a cytokine released in the early and further steps of the immune
response by natural killer cells (NK) and activated T cells, being involved in a number
of immune response aspects.
The statistical analysis of IFN- serum values revealed for the healthy
patients group a mean value of 67,32 pg/ml, with a standard deviation of 12,34. For
the aggressive periodontitis patients the mean value was of 85,69pg/ml 8,9 and for
the chronic periodontitis patients- 99,56pg/ml 24,56.
The analysis of the results revealed a significant difference for the aggressive
and chronic periodontitis groups.
Moreover, the highest values were registered for chronic periodontitis
patients-121,3pg/ml.
When comparing the result between the chronic and aggressive periodontitis
patients, we observed a significant difference between these two groups.
The analysis of the correlation between the age of the patients and the IFN-
values show significant higher values for the patients with the age higher than 40
years old and significant lower for the patients with the age up to 30 years old.

6.4. DISCUSSIONS

In our study the aggressive periodontitis patients presented IL-8 levels
slightly higher than the chronic periodontitis and healthy groups but these values were
not statistically significant.

28
Therefore, we could not observe a significant change in the serum values of
this cytokine in patients with periodontal impairments, when compared to healthy
subjects. We could not draw a final conclusion regarding the implication of IL-8 in
the periodontal disease, a further assessment for this cytokine in the crevicular fluid
being necessary [15, 16].
Our results sustain the already published ones, with also high levels of serum
and crevicular TNF- in patients with aggressive and chronic periodontitis.
The statistical analysis of TNF- results for the 3 groups revealed a
significant difference between the healthy subjects and the periodontitis groups.
The chronic periodontitis mean values were almost two times higher than the
control group values. The TNF- values in the aggressive periodontitis group were
the highest, with a maximum value of 5,7 pg/ml.
These results suggest a positive correlation between the periodontal disease
and high levels of serum TNF-. Therefore, we can support the possibility of using
TNF- as a marker of the periodontal disease.
In our study we could observe significant differences of IFN- values
between the 3 study groups. The analysis of the result revealed significant differences
for both chronic and aggressive periodontitis groups. Moreover, the highest values
were registered for chronic periodontitis patients-121,3pg/ml.
When comparing the result between the chronic and aggressive periodontitis
patients, we observed a significant difference between these two groups.
The analysis of the correlation between the age of the patients and the IFN-
values show significant higher values for the patients with the age higher than 40
years old and significant lower for the patients with the age up to 30 years old.

6. 5. CONCLUSIONS

1. The biologic activity of a variety of cytokines can be relevant for the
physiopathological mechanism of the periodontal disease onset and for the
associated clinical signs, like attachment loss, collagen destruction and
alveolar bone resorption.
2. Regarding our results, we can conclude that the periodontal disease is a result
of the interactions between the periodontal micro-organisms and the various
responses of the host.
3. In conclusion, the cytokine profile analysis should be continuously explored,
to distinguish periodontal diseases patients from healthy subjects and also to
be used as a periodontal risk assessment method in early phases.

29

FINAL CONCLUSIONS

1. Currently, no definitive answer can be given to whether the expression of
aggressive etiologic agents (involving infection with a highly virulent microbiota) or a
high level of individual susceptibility to periodontal disease, or a specific combination
of both factors is decisive in the pathogenesis of aggressive periodontitis.
2. The aim of the current research was to analyze the prevalence of
microorganisms associated with generalized aggressive periodontitis (PAG) and
chronic periodontitis (CP) using molecular biology methods such as chain
amplification (PCR) technique and MALDI-TOF mass spectrometry.
3. Presumed pathogens involved in periodontitis: Tannerella forsythensis,
Porphyromonas gingivalis, Fusobacterium nucleatum and Campylobacter rectus can
be suggested as the key bacteria in patients with aggressive periodontitis.
Agreggatibacter actinomycetemcomitans could be detected in only a few patients, the
importance of reducing these bacteria, is suspected in the pathogenesis of generalized
aggressive periodontitis.
4. It could be observed only a weak association for Prevotella intermedia and
Eikenella corrodens in patients with aggressive periodontitis or periodontal health.
The results support previous findings that generalized aggressive periodontitis are
associated with a more complex microbiota.
5. The study using the identification technology, high specific protocols such as
MALDI-TOF Mass spectroscopy, accredit the idea that aggressive periodontitis is
likely associated with deficiency of some bacterial agents (Streptococcus sanguis,
mutans, oralis, intermedius) involved in protection against periodontal pathogens.
6. The method of identification by mass spectrometry MALDI-TOF technology
has real valences fast, targeted in identifying the specific pathogenicity aggressive
periodontitis associated pathological processes and / or chronic.

30
7. In the territory of periodontal deterioration the contributions of therapeutic
act in modulating oxidative status seem to be directed towards a reduction in
oxidative stress mediated specifically by increasing antioxidant capacity through
high-specific enzyme activity of superoxide dismutase.
8. Results of the study on oxidative stress reveals significant differences in the
oxidative status in the crevicular fluid, which indicates a significant modulation in
both the enzymatic parameters (SOD) and nonenzimatici (MDA).
9. The average values of glutathione peroxidase activity in crevicular fluid were
relatively close in range: 742.8 units / ml for healthy patients, 736.8 units / ml for
aggressive periodontitis group and 736.75 units / ml for chronic periodontitis. We
found no statistically significant differences between the three groups of patients in
this analysis.
10. Superoxide dismutase-SOD in crevicular fluid showed low levels both in the
group with chronic periodontitis and aggressive periodontitis group compared to the
control group of patients prior to initiating any treatment plan.
11. Although according to some data that result from specialized studies in the
literature, our findings indicate a different curve amending markers of oxidative stress
and antioxidant depletion especially in conditions of impaired periodontal territories.
12. Nevertheless in this study we observed some correlation with the severity
peroxidation end product forms of periodontal disease, and an inverse relationship
between the level of antioxidant enzymes studied and advanced forms of periodontal
destruction.
13. Contributions in reducing of the oxidative stress appear to be involved both
in production decrease of the oxidative stress and in improving the enzymatic
antioxidant protection equipment.
14. Recordings of the values IL-8 among patients healthy, among those with
chronic periodontitis and among those with aggressive periodontitis did not show a
statistically significant increase in any of the groups of patients, however, the levels of

31
these cytokines in aggressive periodontitis were slightly higher than in the control
group.
15. TNF- showed elevated levels in both serum and gingival fluid in patients
with periodontal disease in the chronic periodontitis group values almost twice as
high as control and aggressive periodontitis patients, TNF- values were the highest
being recorded a value of maximum 5.7 pg / ml.
16. Our results suggest a positive correlation between periodontal disease and
increased levels of TNF- in serum. It can be concluded therefore that there is the
possibility of using TNF- as a "marker" of periodontal disease.
17. IFN- showed higher values in the group of patients diagnosed with chronic
and aggressive periodontitis, the highest values occurring in chronic periodontitis
18. The analysis of the correlation between patient age and IFN- values show
significantly higher values among patients aged 40 years and much lower values in
patients under 30 years.
19. Given our results we conclude that periodontal disease and periodontal
microflora resulting from the interaction of various host responses regarding
aggressive or chronic pathogenic event.

ORIGINALITY STUDY

This paper aims to be a starting point and a connection bridge research in future
concerns of doctors and therapists of different specialties to holistic address the
patient and focuse attention on the other methods of recording and interpreting
the pathophysiological mechanisms of periodontal disease .
The research points out that the study of the determinants of health, oral health
trends and determinant risk factors of periodontal disease as well as of the
methodology necessary to implement oral health programs targeted at the risk

32
group are of real importance to establish effective methods for improving oral
health status of the targeted population affected by marginal periodontitis.
The identification Method MALDI-TOF Mass spectroscopy, has a real valence
of a fast and targeted technology, for the identification of the specific
pathogenicity associated the pathological processes of aggressive and / or
chronic periodontitis.
Another element of originality is bringing to the current level of knowledge of
this topic in order to understand the involvement of IL 8 in periodontal disease
and a positive correlation between periodontal disease and increased levels of
TNF- in serum and the posibility to use TNF- as a "marker" of periodontal
disease.
Cytokine profile analysis should be further explored to distinguish patients with
periodontal disease by those who are healthy and also to be used as a method of
determining the risk of periodontal disease in the early stages.
Another element of originality of this thesis consists in detecting and monitoring
the effect of antioxidants on periodontal status (information useful in periodontal
treatment to prevent the development and occurrence of pathological
phenomena) and the correlation of this information to the influence on systemic
lipid status, which can take an important aspect in detecting of some issues in the
development of cardiovascular pathology.

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