Rezumat Engleza Ioana ( Martu) Bucataru

Investeşte în oameni !

Proiect cofinanţat din Fondul Social European prin Programul Operaţional Sectorial Dezvoltarea Resurselor Umane 2007 – 2013

Axa prioritară „Educatia si formarea profesională în sprijinul cresterii economice si dezvoltării societătii bazate pe cunoastere”

Domeniul major de intervenţie 1.5 „Programe doctorale si post-doctorale în sprijinul cercetării”

Titlul proiectului: „Burse doctorale pentru cresterea competitivitatii in domeniul medical si farmaceutic”

Numărul de identificare al contractului: POSDRU/88/1.5/S/58965

Beneficiar : Universitatea de Mdicina si Farmacie „Gr. T. Popa” Iasi

Partener : Universitatea de Medicina si Farmacie „Iuliu Hatieganu” Cluj Napoca

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. BUCĂTARU ( MÂRŢU) IOANA

IAŞI

- 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-α, IFN-γ 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-γ 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- γ 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 pacient’s 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, it’s 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 şi 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 şi

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 aleasǎ 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 Generaţia 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 κB (NF-κB)] 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 Iaşi) 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-α, IFN-γ 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-γ

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-α (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-α, IFN-γ) in healthy,

aggressive and chronic periodontitis patients and also to establish a correlation

between the levels of these markers and the patient’s 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” Iaşi, 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- α and IFN-γ 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 ½ 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% H2O2, 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-γ IN PATIENTS

WITH AGGRESSIVE PERIODONTITIS, CHRONIC PERIODONTITIS AND

IN THE CONTROL GROUP

The principle in the assay of IFN-γ 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-α was similar to the

ones of IL-8 and IFN-γ, except the step of adding the Biotine-TNF conjugate.

In this step, the incubation is conducted on 370C 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-α 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-α in serum revealed values between 0 şi 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- α 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-α values in the aggressive periodontitis group were the highest,

with a maximum value of 5,7 pg/ml.

The analysis of TNF-α 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- γ ASSESSMENT ON PATIENTS WITH


WITH CONTROL GROUP

IFN-γ 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-γ 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-γ

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-α in patients with aggressive and chronic periodontitis.

The statistical analysis of TNF-α 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-α 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-α. Therefore, we can support the possibility of using

TNF-α as a „marker” of the periodontal disease.

In our study we could observe significant differences of IFN-γ 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-γ

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-α 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-α 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-α in serum. It can be concluded therefore that there is the

possibility of using TNF-α as a "marker" of periodontal disease.

17. IFN-γ 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-γ 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-α in serum and the posibility to use TNF-α 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.

BIBLIOGRAPHY

1. Agarwal S, Suzuki JB, Piesco NP, Aichelmann-Reidy MB. [1994] Neutrophil

function in juvenile periodontitis: induction of adherence. Oral Microbiol

Immunol: 9: 262–271.

2. Agarwal S, Suzuki JB, Riccelli AE. [1994] Role of cytokines in the

modulation of neutrophil chemotaxis in localized juvenile periodontitis. J

Periodontal Res: 29: 127–137.

3. Appay V, Dunbar PR, Cerundolo V, McMichel A, Czaplewski L, Rowland-

Jones S. [2000] RANTES activates antigen-specific cytotoxic T lymphocytes




33

in a mitogen-like manner trough cell surface aggregation. Int

Immunol;12:1173-1182

4. Ahmed N., [2005], Advanced glycation endproducts—role in pathology of

diabetic complications, Diabetes Res Clin Pract 67 pp. 3–21.

5. Anwen S. Williams, et al. [2007]; Interferon- γ Protects Against the

Development of Structural Damage in Experimental Arthritis by Regulating

Polymorphonuclear Neutrophil Influx Into Diseased Joints. Arthritis &

Rheumatism 56: 2244-2254

6. Ahmed N., S. Battah, N. Karachalias, et al., [2003] ―Increased formation of

methylglyoxal and protein glycation, oxidation and nitrosation in

triosephosphate isomerase deficiency, Biochimica et Biophysica Acta,]; 2:

121–132.

7. Akalın FA, Baltacıoğlu E, Alver A, Karabulut E [2007] Lipid eroxidation

levels and total oxidant status in serum, saliva and gingival crevicular fluid in

patients with chronic periodontitis. J Cin Periodontol 34:558–565

8. Albandar JM, Kingman A, Lamster IB. [1998] Crevicular fluid level of b–

glucuronidase in relation to clinical periodontal parameters and putative

periodontal pathogens in earlyonset periodontitis. J Clin Periodontol: 25: 630–

639.

9. Albandar JM, Winn DM, et al. [2001]. Associations between serum antibody

levels to periodontal pathogens and early-onset periodontitis. J Periodontol ;

72/11: 1463- 1469..

10. ALLHAT Officers and Coordinators for the ALLHAT Collaborative Research

Group, [2002], The Antihypertensive and Lipid-Lowering Treatment to

Prevent Heart Attack Trial Major outcomes in moderately

hypercholesterolemic, hypertensive patients randomized to pravastatin vs.

usual care: The Antihypertensive and Lipid-Lowering Treatment to Prevent

Heart Attack Trial (ALLHAT-LLT), JAMA 288 pp. 2998–3007.

11. Altman LC, Page RC, Vandesteen GE, Dixon LI, Bradford C. [1985]

Abnormalities of leukocyte chemotaxis in patients with various forms of

periodontitis. J Periodontal Res: 20: 553–563.

12. Arakawa Hidetoshi, Masako Maeda, SachieOkubo, Tadakatsu Shimamura,

.[2004] Role of Hydrogen Peroxide in Bactericidal Action of Catechin, Biol.

Pharm. Bull; 27(3) 277—281

13. Armitage GC. [2004] Periodontal diagnosis and classification of periodontal

diseases Periodontol 2000; 34: 9-21




34

14. Sozzani, S., Agwu, D.E., Ellenburg, M.D., Locati, M., Rieppi, M., Rojas, A.,

Mantovani, A. and McPhail, L.C. [1994] Activation of phospholipase D by

interleukin-8 in human neutrophils. Blood 84, 3895–3901

15. Asman B, Bergstrom K, Wijkander P, Lockowandt B. [1988] Peripheral PMN

cell activity in relation to treatment of juvenile periodontitis Scand. J Dent

Res: 96: 418–420.

16. Bagby G Jr, Heinrich M [2000], - Growth Factors, Cytokines and the Control

of Hematopoiesis. In: Hoffman R (eds.) Hematology. Basic Principles and

Practice, 3rd ed., Churchill Livingstone, Philadelphia, 154-202.

17. Baltacioğlu E, Akalin FA, Alver A, Değer O, Karabulut E [2008] Protein

carbonyl levels in serum and gingival crevicular fluid in atients with chronic

periodontitis. Arch Oral Biol 53:716–722

18. Bannerman D. D. and Goldblum S.E. [2003] ,Mechanisms of bacterial

lipopolysaccharide-induced endothelial apoptosis AJP - Lung Physiol, vol. 284

no. 6, L899-L914

19. Batista AC, Silva TA, Chun JH, Lara VS [2002], Periodontal Diseases:

Pathogenesis Nitric oxide synthesis and severity of human periodontal disease,

Oral Diseases 8, 254–260

20. Bera, D. Lahiri and A. Nag, [2006] Studies on a natural antioxidant for

stabilization of edible oil and comparison with synthetic antioxidants, Journal

of Food Engineering 74, pp. 542–545.

21. Berger A: Th1 and Th2 responses: what are they? BMJ 2000; 321: 424.

22. Berry, C.A. Hamilton, M.J. Brosnan, F.G. Magill, G.A. Berg and J.J.

McMurray et al., [2000] Investigation into the sources of superoxide in human

blood vessels: angiotensin II increases superoxide production in human

internal mammary arteries, Circulation 101, pp. 2206–2212.

23. Beswick R.A., A.M. Dorrance, R. Leite and R.C. Webb, [2001],

NADH/NADPH oxidase and enhanced superoxide production in the

mineralocorticoid hypertensive rat, Hypertension 38 ,pp. 1107–1111.

24. Boyle JJ [January 2005]. "Macrophage activation in atherosclerosis:

pathogenesis and pharmacology of plaque rupture". Curr Vasc Pharmacol 3

(1): 63–8,

25. Brock, G. R., Butterworth, C. J., Matthews, J. B.& Chapple, I. L. C. [2004]

Local and systemic total antioxidant capacity in periodontitis and health.

Journal of Clinical Periodontology 31: 515–521.




35

26. Buchmann R, Hasilik A, Van Dyke TE, Lange DE. Amplified crevicular

leukocyte activity in aggressive periodontal disease. J Dent Res [2002]: 81:

716–721.

27. Buchmann R, Hasilik A, Van Dyke TE, Lange TE. [2002] Resolution of

crevicular fluid leukocyte activity in patients treated for aggressive periodontal

disease. J Periodontol: 995–1002.

28. Burke B.E., R. Neuenschwander and R.D. Olson, [2001] Randomized, double-

blind, placebo-controlled trial of coenzyme Q10 in isolated systolic

hypertension, South Med J 94 , pp. 1112–1117

29. Ceriello A., [2003], New insights on oxidative stress and diabetic

complications may lead to a “causal” antioxidant therapy, Diabetes Care 26

,pp. 1589–1596.

30. Chapple IL, Brock G, Eftimiadi C, Matthews JB[2002]. Glutathione in

gingival crevicular fluid and its relation to local antioxidant capacity in

periodontal health and disease. J Clin Pathol Mol Pathol; 55: 367–373.

31. Chapple, I. L. C. & Matthews, J. B. The role of reactive oxygen and

antioxidant species in periodontal tissue destruction. Periodontology 2000 43:

160–232.

32. Chapple, I. L. C., Milward, M. R. & Dietrich, [2007] T. The prevalence of

inflammatory periodontitis is negatively associated with serum antioxidant

concentrations. Journal of Nutrition 137: 657–664.

33. Chinwalla J, Tosi M, Bissada NF[1998]. Severity of localized juvenile

periodontitis as related to polymorphonuclear chemotaxis and specific

microbial isolates. Periodontal Clin Investig : 20: 6–11.

34. Choi BK, Paster BJ, Dewhirst FE, Göbel UB.[1994] Diversity of cultivable

and uncultivable oral spirochetes from a patient with severe destructive

periodontitis. Infect Immun ; 62/5: 1889-1895.

35. Chow HH, Hakim IA, Vining DR, et al. [2004]Modulation of human

glutathione s-transferases by polyphenon e intervention. Cancer Epidemiol

Gouni-Berthold I, Sachinidis A. Molecular mechanisms explaining the

preventive effects of catechins on the development of proliferative diseases.

Curr Pharm Des;10(11):1261-71. 2004.

36. Chow, H. H. S., Hakim, I. A., Vining, D. R., Crowell, J. A. t al., [2007]

Modulation of human glutathione S-transferases by polyphenon e intervention.

Cancer Epidemiol. Biomarkers Prev., 16, 1662–1666.




36

37. Cianciola LJ, Genco RJ, Patters MR, McKenna J, van Oss CJ. [1977]:

Defective polymorphonuclear leukocyte function in a human periodontal

disease. Nature 265: 445–447.

38. Costuleanu Marcel.[2002] Fundamente de fiziopatologie Editura Cantes. Iasi.

Costuleanu Marcel. Comunicarea intercelulara. Editura Cantes. Iasi.

39. Dale DC, Boxer L, Liles WC.[2008] The phagocytes: neutrophils and

monocytes. Blood 112: 935–945.

40. Daniel MA, McDonald G, Offenbacher S, Van Dyke TE.[1993] Defective

chemotaxis and calcium response in localized juvenile periodontitis

neutrophils. J Periodontol : 64: 617–621.

41. De Bree A, Verschuren WM, Blom HJ, et al.[2001]. Lifestyle factors and

plasma homocysteine concentrations in a general population. Am J Epi

154:150-154.

42. Deas DE, Mackey SA, McDonnell HT.[2003] Systemic disease and

periodontitis: manifestations of neutrophil dysfunction. Periodontol 2000 : 32:

82–104.

43. DeNardin E, DeLuca C, Levine MJ, Genco RJ.[1990] Antibodies directed to

the chemotactic factor receptor detect differences between chemotactically

normal and defective neutrophils from LJP patients. J Periodontol: 61: 609–

617.

44. Di Loreto S., V. Caracciolo, S. Colafarina, P. Sebastiani, A. Gasbarri, and F.

Amicarelli,[2004] “Methylglyoxal induces oxidative stress-dependent cell

injury and up-regulation of interleukin-1β and nerve growth factor in cultured

hippocampal neuronal cells,” Brain Research, vol. 1006, no. 2, pp. 157–167.

45. Dowlati Y, Herrmann N, Swardfager W, et al. [March 2010]. "A meta-analysis

of cytokines in major depression". Biol. Psychiatry 67 (5): 446–57.

46. Dunn, G., Maracy, M. & Tomenson, B. [2005] Estimating treatment effects

from randomized clinical trials with noncompliance and loss to follow-up: the

role of instrumental variable methods. Statistical Methods in Medical

Research; 14: 369–395.

47. Eick S, Pfister W, Sigusch B, Straube E.[2000] Phagocytosis of

periodontopathogenic bacteria by crevicular granulocytes is depressed in

progressive periodontitis. Infection :28: 301–304.

48. Eikelenboom P., R. Veerhuis, A. Familian, J. J. M. Hoozemans, W. A. van

Gool, and A. J. M. Rozemuller,[2000] “Neuroinflammation in plaque and




37

vascular β-amyloid disorders: clinical and therapeutic implications,”

Neurodegenerative Diseases, vol. 5, no. 3-4, pp. 190–193.

49. Elenkov IJ, Chrousos GP. Stress Hormones, [1999 Nov] Th1/Th2 patterns,

Pro/Anti-inflammatory Cytokines and Susceptibility to Disease. Trends

Endocrinol Metab;10(9):359-368.

50. Ebersole JL, Machen RL, Steffen MJ, Willmann DE.[1997] Systemic

acutephase reactants, C-reactive protein and haptoglobin, in adult periodontitis.

Clin Exp Immunol ;107:347-352.

51. Foia Liliana–[2011] chapter „Diabetes mellitus impact on periodontal status” -

Jane Manakil (editor) in “Periodontal disease”, ISBN 978-953-307-818-2,

Intech Ed.

52. Foia Liliana [2010]“Clinical correlations in interpretations of the biochemical

lab tests” , ISBN 978-973-37-1483-5. Ed. Junimea.

53. Foia Liliana, Ungureanu, D., Toma, V., Zlei, M., Indrei, A., Haba, D.,

Branisteanu, D.[2008] Analysis of oral expression of the diabetes-periodontal

disease binomial relationship in a juvenile population. Romanian Review of

Laboratory Medicine , ISSN-1841-6624, Nr. 4 (13):53-59.

54. Foia Liliana, Toma V.[2008] Patologia parodontală în diabetul juvenil ISBN

978-973-7682-62-8, Ed. Gr. T. Popa

55. Foia Liliana. [2010]Corelaţii clinice în interpretarea parametrilor biochimici ,

ISBN 978-973-37-1483-5, Ed. Junimea

56. Forna Norina Consuela. Oral rehabilitation between reality and paradigm.Rev

Med Chir Soc Med Nat Iasi. 2008 Apr-Jun;112(2):507-11. Review Gaman

Amelia, Gaman G - Citokinele, rol in hematopoieza, Medicina moderna, 2001,

vol. VIII, nr. 9, 486-491.

57. Forna Norina, Sandhaus Sami. Posttherapeutic feedback evaluation in oral

rehabilitation. Romanian Journal of Oral Rehabilitation.Vol. 3, No. 3, July

2011,47-51

58. Forna Norina, Pepi Pogorelick. Non-Invasive Methods Of Paraclinical

Evaluation Involved In Oral Rehabilitation. Romanian Journal of Oral

Rehabilitation.Vol. 2, No. 2, June 2009,11-13.

59. Forna N, Stadoleanu C, Bosnea D, Preda C, Gherman C, Buiuc D. A

comparative study of the bacterial dental plaque formation on fixed dentures

with different surface characteristics.Rev Med Chir Soc Med Nat Iasi. 1999

Jul-Dec;103(3-4):210-5. Romanian.




38

60. Forna Norina. Evaluarea starii de sanatate afectate prin edentatie.Editura

Demiurg, 2007

61. Gueders MM, Balbin M, Rocks N, Foidart JM, Gosset P, Louis R, Shapiro S,

Lopez-Otin C, Noël A, Cataldo DD (2005). Matrix metalloproteinase-8

deficiency promotes granulocytic allergen-induced airway inflammation. J

Immunol 175: 2589-97.

62. Guglielmotto M., L. Giliberto, E. Tamagno, and M. Tabaton, “Oxidative stress

mediates the pathogenic effect of different Alzheimer’s disease risk factors,”

Frontiers in Aging Neuroscience, vol. 2, pp. 1–7, 2010.

63. Halliwell, B & Gutteridge, J. M. C. [2007]Free Radicals in Biology and

Medicine. Oxford: Oxford University Press.Linden,

64. Hammond B.F., Stevens, R.H.,Bonner, P& Lillard,S.E.[1984].Toxicity of

Aggregatibacter actinomycetemcomitans extracts for Crevicular bacteria.j

Dent Res 63,263

65. Han YW, Shi W, Genco RJ. et al. [2000]. Interactions between periodontal

bacteria and human oral epithelial cells. Fusobacterium nucleatum adheres to

and invades epithelial cells. Infect Immun ; 68/6: 3140-3146.

66. Hara T, Miyajima A - Cytokine receptors and signal transduction. In:

Textbook of Malignant Haematology, Ed. by Degos L, Linch DC, Lowenberg

B, Martin Dunitz, Ltd, London, 1999, 111-131.

67. Hodis H.N. et al., α-Tocopherol supplementation in healthy individuals

reduces low-density lipoprotein oxidation but not atherosclerosis: the Vitamin

E Atherosclerosis Prevention study (VEAPS), Circulation 106 (2002), pp.

1453–1459.

68. Ikigai H, Nakae T, Hara Y, Shimamura T. Bactericidal catechins damage the

lipid bilayer. Biochim Biophys Acta 1993; 1147 : 132-6.

69. Ko S.Y., Y. P. Lin, Y. S. Lin, and S. S. Chang, “Advanced glycation end

products enhance amyloid precursor protein expression by inducing reactive

oxygen species,” Free Radical Biology and Medicine, vol. 49, no. 3, pp. 474–

480, 2010.

70. Konen J.C., J.H. Summerson and J.K. Kirk, Measurement feasability of

advanced glycated end-products from skin samples after antioxidant vitamin

supplementation in patients with type 2 diabetes, J Nutr Health Aging 4

(2000), pp. 81–84.

71. Lappin DF, Kjeldsen M, Sander L, Kinane DF (2000). Inducible nitric oxide

synthase expression in periodontitis. J Periodont Res 35: 369–373.




39

72. Lee, S. F., Liang, Y. C., Lin, J. K., Inhibition of 1,2, 4-benzenetriol-generated

active oxygen species and nduction of phase II enzymes by green tea

polyphenols. hem. Biol. Interact. 1995, 98, 283–301.

73. Liu RK, Gao Y, et al. [2001]. Polymorphonuclear neutrophils and their

mediators in gingival tissues from generalized aggressive periodontitis. J

Periodontol; 72/11: 1545- 1553.

74. Mark I. Ryder. Comparison of neutrophil functions in aggressive and chronic

periodontitis. Periodontology 2000, Vol. 53, 2010, 124–137

75. Marton, I. J., Balla, G., Hegedus, C., Redl, P., Szilogyi, Z., Karmazsin, L. &

Kiss, C. (1993) The role of reactive oxygen intermediates in the pathogenesis

of chronic apical periodontitis. Oral Microbiology and Immunology 8, 254–

257.

76. Mârțu Ioana, Ionuț Luchian, Ancuța Goriuc, Silvia Mârțu, Liliana Foia, Norina

Forna. Correlations between the periodontal modifications and the oxidative

status on periodontal disease patients. Romanian Journal of Oral Rehabilitation

2013, 5(1), 25-31.

77. Mârțu Ioana, Maria Ursache, Silvia Martu, Ionut Luchian, Marcel Costuleanu;

Evaluation of Neutrophil Chemotaxis Disfunction to Aggressive and Chronic

Periodontitis. Romanian Journal of Oral Rehabilitation, 2012, 4 (1): 9-15.

78. Mârţu Silvia, Nănescu Sonia Elena, Liliana Constantin. Markerii inflamatori

sistemici în boala parodontală / Inflamatory systemic markers in periodontal

disease Medicina Stomatologica / Romanian Dentistry J. , 2005, vol 9, nr. 6,

pag. 12-16

79. Mârţu Silvia, Sonia Elena Nănescu, Liliana Constantin. Stress effects on

immune mechanisms in periodontal disease Medicina Stomatologica /

Romanian Dentistry J. , 2006, vol 10, nr. 1, 35 – 38

80. Münch G, B. Westcott, T. Menini, and A. Gugliucci. Advanced glycation

endproducts and their pathogenic roles in neurological disorders. Amino

Acids. 2010.

81. Murase, T., Nagasawa, A., Suzuki, J., Hase, T. & Tokimitsu, I. (2002)

Beneficial effects of tea catechins on diet-induced obesity: stimulation of lipid

catabolism in the liver. Int. J. Obes. Relat. Metab. Disord. 26:1459-1464

82. Owen CA, Hu Z, Lopez-Otin C, Shapiro SD (2004). Membrane-bound matrix

metalloproteinase-8 on activated polymorphonuclear cells is a potent tissue

inhibitor of metalloproteinase-resistant collagenase and serpinase. J Immunol

172: 7791–7803.




40

83. Packer L., & col. The antioxidant miracle. 2004 Nature Reviews Immunology

4, august, 617-629.

84. Parks W. C., Wilson C. L. & López-Boado Y. S. Matrix metalloproteinases as

modulators of inflammation and innate immunity Nature Reviews

Immunology 4, 617-629 (August 2004)

85. Peyroux J. and M. Sternberga, Advanced glycation endproducts (AGEs):

pharmacological inhibition in diabetes, Pathologie Biologie 54 (2006), pp.

405–419.

86. Potarnichie Oana, Nanescu Sonia, Pasarin Liliana, Rudnic Ioana, Foia Liliana,

Martu Silvia. Immuno-bichemical and clinical researches on the evolution of

antioxidants level in the etiopathogeny of periodontal pathologies. Int. J.of

Medical Dentistry, 2012,16 (1):52-56.

87. Sayre L.M., G. Perry, and M. A. Smith, “Oxidative stress and neurotoxicity,”

Chemical Research in Toxicology, vol. 21, no. 1, pp. 172–188, 2008

88. Schenkein HA. Finding genetic risk factors for periodontal diseases: is the

climb worth the view? Periodontol [2000], [2002]; 30: 79-90..

89. Schwartz DA. The genetics of innate immunity. Chest 2002; 121/Suppl.3: 62S-

68S.

90. Sculley Dean V., Simon C. Langley-Evans Periodontal disease is associated

with lower antioxidant capacity in whole saliva and evidence of increased

protein oxidation Clinical Science (2003) 105, (167–172)

91. Sesso HD, Gaziano JM, Simin Liu S, Buring JE. Flavonoid intake and the risk

of cardiovascular disease in women. Am J Clin Nutr: 77: 1400-1408, 2003.

92. Sobal G. and J. Menzel, The role of antioxidants in the long-term glycation of

low density lipoprotein and its Cu2+-catalyzed oxidation, Free Radic Res 32

(2000), pp. 439–449.

93. Sobaniec H, Sobaniec-Lotowska ME. Morphological examinations of hard

tissue of periodontium and evaluation of selected processes of lipid

peroxidation in blood serum of rats in the course of experimental periodontitis.

Med Sci Monit 2000;6:875–881.

94. Solomon Sorina, Ioana Martu, Alexandra Mârţu, Irina Ursărescu, Silvia Mârţu.

Severe periodontal impairment in systemic conditions: a case report.

Romanian Journal of Oral Rehabilitation 2013, 5(2), 91-97.

95. Stangu Catalina-Suzana, Eschrich Klaus,Ame C. Rodloff,Reiner Schaumann

and Holger Jentsch(2008) Periodontitis is associated with a loss of




41

colonization by Streptococcus sanguinis. Journal of Medical Microbiology

[2008],57, 495-499

96. Streckfus C.F., Bigler L.R. Saliva as a diagnostic fluid. Oral Dis., 2002; 8(2):

69-76

97. Suarez LJ, Rodriguez A, et al. [2004]. Relative proportions of T-cell

subpopulations and cytokines that mediate and regulate the adaptive immune

response in patients with aggressive periodontitis. J Periodontol; 75/9: 1209-

1215.

98. Sultana R, M. Piroddi, F. Galli, and D. A. Butterfield, “Protein levels and

activity of some antioxidant enzymes in hippocampus of subjects with

amnestic mild cognitive impairment,” Neurochemical Research, vol. 33, no.

12, pp. 2540–2546, 2008.

99. Suzuki A, Kamoi K, et al. [2004]. Large-scale investigation of genomic

markers for severe periodontitis. Odontology; 92/1: 43-47..

100. Șincar Cerasela, Sorina Solomon, Ioana Rudnic, Ioana Mârțu, Silvia Mârțu.

The effects of periodontal therapy on biochemical inflammatory markers on

renal dysfunction patients. Romanian Journal of Oral Rehabilitation 2013,

5(1), 70-75.

101. Takane N, Sugano M, Iwasaki H. Shimuzu N. Ito K.2002 New biomarker

evidence of oxidative DNA damage in whole saliva from clinically healthy and

periodontally diseased individuals.J Periodontol 73, 551-554

102. Takeuchi M. and Z. Makita, “Alternative routes for the formation of

immunochemically distinct advanced glycation end-products in vivo,” Current

Molecular Medicine, vol. 1, no. 3, pp. 305–315, 2001.

103. Tamaki N, Tomofuji T. Ekuni, Yamanaka R., Morita M., Periodontal treatment

decreases plasma oxidized LDL level and oxidative stress, Clin Oral Invest

(2011) 15:953–958

104. Touyz R.M., Reactive oxygen species in vascular biology: role in arterial

hypertension, Expert Rev Cardiovasc Ther 1 (2003), pp. 91–106

105. Townsend D. M., Tew K. D. , Tapiero H. Dossier : Oxidative stress

pathologies and antioxidants The importance of glutathione in human disease

Biomedicine & Pharmacotherapy 57 (2003) 145–155

106. Trevilatto PC, Tramontina VA, Line SR, et al.. Clinical, genetic and

microbiological findings in a Brazilian family with aggressive periodontitis. J

Clin Periodontol [2002]; 29/3: 233-239.




42

107. Tsai, C. C., Chen, H. S., Chen, S. L., Ho, Y. P.,Ho, K. Y., Wu, Y. M. & Hung,

C. C. Lipid peroxidation: a possible role in theinduction and progression of

chronic periodontitis. Journal of Periodontal Research 2005; 40:378–384.

108. Uitto VJ, Overall C, McCulloch C (2003). Proteolytic host cell enzymes in

gingival crevice fluid. Periodontol 2000 31: 77–104.

109. Volozhin AI, Poriadin GV, Kazimiski TI, Barer GM, Askerova SSh, Salmasi

ZhM. Immunologic disorders in pathogenesis of chronic generalized

parodontitis] Stomatologiia (Mosk). 2005;84(3):4-7

110. Wade W. Unculturable bacteria – the uncharacterized organisms that cause

oral infections. J R Soc Med [2002]; 95/2: 81-83.

111. Gainet, J., Chollet-Martin, S., Brion, M.,Hakim, J., Gougerot-Pocidalo, M.-A.

&Elbim, C. (1998) Interleukin-8 production by polymorphonuclear neutrophils

in patients with rapidly progressive periodontitis:An amplifying loop of

polymorphonuclear neutrophil activation. Laboratory Investigation 78, 755–

762

112. Weismann D., Hartvigsen K. Lauer N., Bennett K.L., Scholl H. P. N., Charbel

Issa P., Cano M., Brandstätter H., Tsimikas S., Christine Skerka, Superti-

Furga1 G., Handa J. T.,. Zipfel P.F, Witztum J. L. & Binder C.J., Complement

factor H binds malondialdehyde epitopes and protects from oxidative stress

Nature Volume: 478, Pages: 76–81

113. Wilson C. L. & López-Boado Y. S. Matrix metalloproteinases as modulators

of inflammation and innate immunity Nature Reviews Immunology 4, 617-629

(August 2004)

114. Wolin M.S., Interactions of oxidants with vascular signaling

systems,Arterioscler Thromb Vasc Biol 20 (2000), pp. 1430–1442.

115. Wong A., H. J. Lüth, W. Deuther-Conrad et al., “Advanced glycation

endproducts co-localize with inducible nitric oxide synthase in Alzheimer's

disease,” Brain Research, vol. 920, no. 1-2, pp. 32–40, 2001.

116. Wong A., S. Dukic-Stefanovic, J. Gasic-Milenkovic et al., “Anti-inflammatory

antioxidants attenuate the expression of inducible nitric oxide synthase

mediated by advanced glycation endproducts in murine microglia,” European

Journal of Neuroscience, vol. 14, no. 12, pp. 1961–1967, 2001.

117. Woodside, J. V., McCall, D., McGartland, C. &Young, I. S.Micronutrients:

dietary intake versus supplement use. Proceed. of the Nutrition Society 2005;

64: 543–553.




43

118. Xajigeorgiou, C., Sakellari, D., Slini, T., Baka, A. &Konstantinidis, A. [2006]

Clinical and microbiological effects of different antimicrobials on generalized

aggressive periodontitis. Journal of Clinical Periodontology; 33: 254–264.

119. Ximenez-Fyvie LA, Haffajee AD, Socransky SS. [2000] Comparison of the

microbiota of supra and subgingival plaque in health and periodontitis. J Clin

Periodontol 2000; 27/9: 648- 657.

Documents

Rezumat Engleza Ioana ( Martu) Bucataru