Effect of the examination stress on periodontal health status and salivary IL-1β among Iraqi dental...

Effect of the examination stress on

periodontal health status and salivary

IL-1β among Iraqi dental students

A thesis submitted to the council of the College of Dentistry at

the University of Baghdad, in partial fulfillment of the

requirements for the degree of Master of Science in

Periodontics

By

Athraa Ali Mahmood

B.D.S.

Supervised by

Prof. Dr. Leka'a M. Ibrahim

B.D.S., M. Sc.

October/ 2013 Dhu al-Hijjah / 1434

Approved by the council of the college of Dentistry/ University of Baghdad.

Prof. Dr. Nabeel abdul-Fatah

B.D.S., M.Sc. (Prosthodontics) UK

Dean of the college of Dentistry

University of Baghdad

Committee Certification

We, the members of the examining committee, certify that we

have read this thesis and examined the graduate student “Athraa Ali

Mahmood“ in its contents and in our opinion, it meets the standard of thesis

for the degree of Master of Science in Periodontics.

Assist. Prof. Dr. Maha Abdul Aziz Ahmed

B.D.S., M.Sc.

(The Chairman)

Assist. Prof. Dr. Batool Hassan Al-Ghurabei

B.S.C., M.Sc., Ph.D.

Member

B.D.S M.Sc.

Member

Assist. Prof. Dr. Kadhim Jawad Hanau

B.D.S., M.Sc.

Member

To my precious mother

& father

To my lovely Hashim

Dedication

Athraa…

To my sweet daughters

Retaj & Fatima

To my dear sisters &

brothers

I

Acknowledgement

First of all, I would like to thank almighty “ALLAH” for inspiring me the

energy, patience and strength to accomplish this work. A special peace to our

messenger Mohammed (peace be upon him).

My sincere appreciation to Prof. Dr. Nabeel abdul-Fatah, Dean of the

College of Dentistry, University of Baghdad, for continuously supporting the

postgraduate students.

My deepest thanks and admiration goes to Prof. Dr. Leka'a M. Ibrahim,

Head of Department of Periodontology and my supervisor, for her guidance, kindness,

high ethics, scientific support and continuous helpful advices throughout my study.

I would like to express my deep thanks and respect to the members of the

Department of Periodontology, Baghdad University; Dr. Maha Shukri, Dr. Basima

Ghafori, Dr. Maha Abdul Aziz, Dr. Saif Siham, Dr. Alaa Umran for their help and

encouragement through the study period.

I am highly indebted to the members of Department of Periodontology,

Almustansiriyah University; Dr. Kadhim Jawad, Dr. Ra'ad Aziz, Dr. Hussein Owaid,

Dr. Enas Razzoqi, for their valuable advices, support and continuous assistance.

Much thanks to Assistant Prof. Dr. Batool Hassan in Department of basic

science, Baghdad University, for her generous helpful throughout my study.

My mellifluous appreciation and gratitude to all medical staff of Department

of Immunology in Teaching Laboratories of Baghdad Medical City, especially Dr.

Nahla and Mr. Amjad, for their help in facilitating the performing of this work.

My thanks and appreciation to all the postgraduate colleagues in the

Department of Periodontology, for their cooperation throughout my study.

I gratefully acknowledge to the cooperation and participation of dental

students in this study.

Finally, I am unable to express my sincere gratitude, appreciations and love

to my dear husband (Dr. Hashim) and mother for their unlimited support and love.

II

Abstract

Background: Periodontal diseases are common chronic inflammatory diseases caused

by pathogenic microorganisms colonizing the gingival area and inducing local and

systemic elevations of pro-inflammatory cytokines resulting in tissue destruction by a

destructive inflammatory process affecting tooth-supporting tissues. Many local or

systemic risk factors contribute to development of periodontal disease, stress was

considered as one of the important factors that causes many inflammatory diseases

including periodontal disease. When the stress increase for long period, it may

influence inflammatory process leading to systemic or local diseases such as

periodontal disease.

Interleukin-1beta is a highly pro-inflammatory cytokine strongly associated

with periodontal disease. In addition, interleukin-1beta is a critical mediator of adaptive

stress responses as well as stress-associated neuropathology and psychopathology.

Aims of the study: To determine and compare clinical periodontal parameters (plaque

index, gingival index and bleeding on probing), stress level and salivary interleukin-

1beta level among dental students before, during and after mid-year exam periods. In

addition, to find the correlation among stress, salivary interleukin-1beta and clinical

periodontal parameters.

Materials and methods: The sample was consisted of twenty-four dental students;

twelve male and twelve female aged (21-23) years, they were examined in this follow

up study at three main periods:

First period at least one month before mid-year exam (Period I).

Second period during mid-year exam (Period II).

Third period at least one month after mid-year exam (Period III).

Depression Anxiety Stress Scale (DASS-21) was used to measure stress level

in all periods. Saliva samples were collected to determine the salivary interleukin-1beta

level by enzyme-linked immunosorbent assay (ELISA). Clinical periodontal

III

parameters were recorded at four sites per tooth including plaque index, gingival index

and bleeding on probing.

Results: The results showed that the means of all clinical periodontal parameters were

higher in the period II than in the periods I and III with highly significant differences

at (P ≤ 0.01). As well as, the means concentrations of salivary interleukin-1beta were

higher in the period II than in the periods I and III with highly significant differences

at (P ≤ 0.01). Also, by using Pearson's Correlation Coefficient, stress shows highly

significant strong positive correlation with the immunological parameter (salivary

interleukin-1beta) and clinical periodontal parameters at (P ≤ 0.01).

Conclusions: The results of this study provided strong evidence of association between

examination stress and periodontal disease, where dental students during mid-year

exam have higher levels of stress, clinical periodontal parameters and salivary

interleukin-1beta as compared with before and after mid-year exam periods. Also, there

were strong correlation among stress, salivary interleukin-1β and clinical periodontal

parameters.

IV

List of Contents

Page

No.

Subject

I Acknowledgement

II Abstract

IV List of Contents

VII List of Tables

VIII List of Figures

IX List of Abbreviations

1 Introduction

3 Aims of the study

Chapter One-Review of Literature

4 1.1. Periodontal diseases

4 1.1.1. Definition

5 1.1.2. Epidemiology

5 1.1.3. Pathogenesis of periodontitis

5 1.1.3.1. Bacterial factors

7 1.1.3.2. Host responses factors

8 I- Innate immune response

9 II-Adaptive immune response

10 1.1.3.3. Viral factors

10 1.1.3.4. Other risk factors

12 1.2. Stress

14 1.2.1. Stress scales

18 1.2.2. Molecular and endocrine mechanisms of the stress response

22 1.2.3. Behavioral changes of stress response

23 1.2.4. Stress and microbiology of periodontal disease

24 1.2.5. Role of stress on gingivitis

25 1.2.6. Role of stress on periodontitis

26 1.2.7. Role of stress on periodontal treatment

27 1.3. Saliva

28 1.3.1. Functions of saliva

29 1.3.2. Salivary composition

30 1.3.3. Saliva and diagnosis

V

31 1.3.4. Saliva and stress

32 1.4. Cytokines

34 1.4.1. Interleukin-1β

35 1.4.1.1. Interleukin-1β regulation of the periodontal ligament

36 1.4.1.2. Interleukin-1β and its role in periodontal disease

37 1.4.1.3. Stress-induced interleukin-1β production

Chapter Two-Materials and Methods

41 2.1. Materials

41 2.1.1. Instruments and Equipment

42 2.1.2. Test kit

42 2.2. Methods

42 2.2.1. Sample description

43 2.2.2. Exclusion criteria

43 2.2.3. Study design

44 2.2.4. Depression anxiety and stress scale

45 2.3. Clinical periodontal parameters examination

45 2.3.1. Plaque index

46 2.3.2. Gingival index

46 2.3.3. Bleeding on probing

46 2.4. Saliva sample collection and preparation

47 2.5. Detection of interleukin-1β

47 2.5.1. Test principle

48 2.5.2. Reagent preparation

49 2.5.3. Procedure

50 2.5.4. Calculations of interleukin-1β

50 2.6. Calibration

51 2.7. Statistical analysis

Chapter Three-Results

56 3.1. Descriptive statistical analysis of demographic data

56 3.2. Clinical periodontal parameters analysis




62 3.3. Stress analysis

63 3.4. Salivary interleukin-1β statistical analysis

VI

65 3.5.Correlations between clinical periodontal parameters with

immunological marker (salivary interleukin-1β) and stress

Chapter Four-Discussion

67 4.1. Demographic data

68 4.2. Periodontal health status




70 4.3. Stress data

71 4.4. Salivary interleukin-1β level

73 4.5. Correlations between stress, interleukin-1β and clinical periodontal

parameters

Chapter Five-Conclusions and Suggestions

74 5.1. Conclusions

75 5.2. Suggestions for further studies

References

76 References

Appendices

106 Appendices

الخالصة

VII

List of Tables

Page

No.

Table title Table

No.

15 DASS severity-rating index 1-1

15 DASS 42-item questionnaire version 1-2

17 DASS 21-item questionnaire version 1-3

29 Functions of saliva components 1-4

33 Effects attributed to cytokines identified in periodontal disease 1-5

45 Stress questionnaires of DASS 21-item version 2-1

45 DASS severity-rating index 2-2

56 Descriptive statistical results of students' ages 3-1

56 Descriptive statistical results of plaque index for each period 3-2

57 ANOVA test for plaque index 3-3

58 LSD test to compare the means of plaque index between each two

periods

3-4

58 Descriptive statistical results of gingival index for each period 3-5

59 ANOVA test for gingival index 3-6

60 LSD test to compare the means of gingival index between each

two periods

3-7

61 Percentages and numbers of scores of bleeding on probing for

each period and comparison among periods

3-8

61 Inter-period comparisons for bleeding on probing among periods 3-9

62 Percentages and numbers of stress range and comparison among

periods

3-10

63 Descriptive statistical results of interleukin-1β for each period 3-11

64 ANOVA test of interleukin-1β 3-12

64 LSD to compare the means of interleukin-1β between each two

periods

3-13

65 Person correlation coefficient of salivary interleukin-1β with

plaque index, gingival index, bleeding on probing score (1 and 0)

and stress levels among periods

3-14

66 Person correlation coefficient of stress with salivary interleukin-

1β and plaque index, gingival index, bleeding on probing score (1

and 0) among periods

3-15

VIII

List of Figures

Page

No.

Title Figure

No.

7 Schematic illustration of the pathogenesis of periodontitis 1-1

21 Effects of stress on hypothalamic pituitary adrenal axis 1-2

21 Physiological model of effects of stress on periodontal disease 1-3

23 Psychosocial stress and its effect on behavior as manifested

by alterations in periodontal disease

1-4

40 Interleukin 1 mediates stress-induced activation of the

hypothalamic pituitary adrenal axis. Immunological and

psychological stressors increase the levels of interleukin 1

1-5

52 Salimetrics salivary interleukin-1β kit 2-1

52 A. Adjustable micropipettes and pipettes yellow tips

B. Adjustable micro multichannel pipettes

2-2

52 Centrifuge (Kokusan Corporation –Japan) 2-3

53 Auto vortex mixer (Frost instrument LTD, Great Britain) 2-4

53 A. Rotatest shaker (R 100 Luck ham, England)

B. Microtitre plate of interleukin-1β kit

2-5

53 Microplate ELISA washer device (Human, Germany) 2-6

54 Microtitre plate of interleukin-1β kit after adding stop solution 2-7

54 Microplate ELISA reader device (Human, Germany) 2-8

55 Quadratic parameter curve fit of software program 2-9

57 Bar chart graph for means of plaque index for each period 3-1

59 Bar chart graph for means of gingival index for each period 3-2

61 Bar chart graph for percentages of scores of bleeding on

probing for each period

3-3

63 Bar chart graph for means of interleukin-1β for each period 3-4

65 Bar chart graph for correlations of interleukin-1β with (plaque

index, gingival index, bleeding on probing score (1)) and

stress

3-5

66 Bar chart graph for correlations of stress with (plaque index,

gingival index, bleeding on probing score (1)) and interleukin-

1β

3-6

IX

List of Abbreviations

Aggregatibacter actinomycetemcomitans A. actinomycetemcomitans

Adrenocorticotropic hormone ACTH

Analysis of variance ANOVA

Acute necrotizing ulcerative gingivitis ANUG

Bleeding on probing BOP

Degree of centigrade ºc

Cluster of differentiation CD

Central nervous system CNS

Chronic periodontitis CP

Corticotropin releasing hormone CRH

Depression anxiety stress scale DASS

example e.g.

Enzyme-linked immunosorbent assay ELISA

Glucocorticoids GC

Gingival crevicular fluid GCF

Gingival index GI

Interleukine-1 receptor antagonist IL-1ra

Human cytomegalovirus HCMV

Hypothalamic pituitary adrenal HPA

Horseradish peroxidase HRP

Highly significant HS

Interferon IFN

Immunoglobulin Ig

Interleukin-1 IL-1

Interleukin-1beta IL-1β

Interleukin-10 IL-10

Lipopolysaccharide LPS

Least significant differences LSD

Maximum Max.

Membrane Cluster of differentiation mCD

Minimum Min.

Milliliter ml

Matrix metalloproteinases MMPs

X

Natural killer cell NK

Nanometer nm

Number No.

Non-significant NS

Optical density OD

Pathogen associated molecular pattern PAMPs

Periodontal diseases PD

Periodontal ligament PDL

Prostaglandin E2 PgE2

Porphyromonas gingivalis P. gingivalis

Prevotella intermedia P. intermedia

Picogram per milliliter pg/ml

Plaque index PLI

Polymorphonuclear leukocyte PMN

Picomole per liter pmol/L

Probability value P-value

Person's correlation coefficient R

Revolution per minute rpm

Significant S

Soluble cluster of differentiation sCD

Suprachiasmatic nuclei SCN

Standard deviation SD

Standard error SE

Significance Sig.

Substance P SP

Statistical package for social science SPSS

Transforming growth factor beta TGF-β

T helper Th

T helper type 1 Th1

T helper type 2 Th2

Tetramethylbenzidine TMB

Tumor necrosis factor TNF

Microliter μL

Introduction

&

Aims of the study

1

Introduction

Periodontal diseases are one of the most wide spread diseases of humankind,

no nation and no region of the world being free from them (Buatongsri et al., 2002).

The extent, severity and course of periodontal diseases are affected by several factors

such as personal oral hygiene, diet, genetics, public preventive services as well as

personal dental preventive, diagnostic and therapeutic services (Brown et al., 2002).

Such diseases may be found in every age group, but is more commonly found in adult

population regardless of the sex, race, education, residence, or socioeconomic status

(El-Qadri and Taani, 2004).

Periodontal diseases are multifactorial infection characterized by destructive

inflammatory process affecting tooth-supporting tissues caused by pathogenic

microorganisms, which induce elevations of pro-inflammatory cytokines resulting in

tissue destruction. Evolution of periodontal diseases is influenced by many local or

systemic risk factors (Cazalis et al., 2009; Malathi and Sabale, 2013). Dental plaque,

which harbours specific periodontal pathogens, its primary etiologic factor. Where host

tissue damage in periodontal disease is mainly due to the action of oral microbes and

associated host immune-inflammatory responses (Van Dyke and Serhan 2003; Van

Dyke, 2007). In addition, several risks and susceptibilities have been associated with

periodontal disease, like systemic diseases, socioeconomic or educational status,

smoking and psychological stress. Several clinical studies have investigated the

possible relationship between psychological stress and periodontal disease and have

suggested that stress may play a role in development of periodontal disease (Elter et

al., 1999; Hamissi et al., 2010; Vered et al., 2011; Akcali et al., 2013).

Stress is defined as the reactions of the body to forces of a deleterious nature,

infections and various abnormal states that tend to disturb its normal physiological

equilibrium (Lathrop and Thomas, 2008). It's nevertheless a confirmed and important

factor in the etiology and maintenance of many inflammatory diseases, including

periodontal disease (Keshava and Sangeeta, 2013). Stress results in delayed healing

2

of the connective tissues and bone, apical migration of the junctional epithelium and

formation of periodontal pocket (Chandna and Bathla, 2010). It's said to influence

the host defenses, exerting an immunosuppressive effect, increasing one's vulnerability

to disease (Ishisaka et al., 2008; Goyal et al., 2011). Cytokines and other humoral

mediators of inflammation are potent activators of the central stress response, and the

glucocorticoids released via this mechanism might regulate the recruitment of immune

cells into inflamed tissues, in order to cope with the psychological stress and depression

(Breivik and Thrane, 2001). When the inflammatory action is sufficiently long and

profound, the systemic manifestations of the disease may become evident, as could

happen with periodontal disease.

Many physiopathological processes are involved in periodontal destruction

in terms of the inflammatory and immune host response, especially proinflammatory

cytokines or matrix metalloproteinase (Dahan et al., 2001; Kiecolt-Glaser et al.,

2003; Van Dyke and Kornman, 2008). Interleukin-1β is a highly pro-inflammatory

cytokine strongly associated with periodontal breakdown (Dayan et al., 2004). In

addition, interleukin-1, produced following exposure to immunological and

psychological challenges, plays an important role in the neuroendocrine and

neurobehavioral stress responses (Goshen and Yirmiya, 2009; Debnath et al., 2011).

Saliva is a mirror to the general health condition that reflects various

systemic changes in the body (Nagler et al., 2002; Chiappelli et al., 2006; Nagler,

2008). So its' interest as a diagnostic fluid has grown exponentially in recent years.

Where the composition of saliva immediately reflects the sympathetic and

parasympathetic nervous systems, hypothalamic-pituitary-adrenal axis and immune

system response to stress (Khaustova et al., 2010). In addition, salivary levels of

various biochemical parameters have been measured in infectious diseases,

autoimmune diseases and psychiatric disorders (Streckfus and Bigler, 2002).

3

Aims of the study

1. To determine and compare the periodontal parameters (plaque index, gingival index

and bleeding on probing) among dental students before, during and after mid-year

exam period by clinical examination.

2. To determine and compare the stress level among dental students before, during and

after mid-year exam period by using DASS-21.

3. To estimate and compare the level of salivary interleukin-1β among dental students

before, during and after mid-year exam period by using ELISA.

4. To correlate the stress with the immunological parameter (salivary interleukin-1β)

and clinical periodontal parameters (plaque index, gingival index and bleeding on

probing).

Chapter One

Review of Literature

Review of Literature Chapter One

4

Review of Literatures

1.1. Periodontal diseases

1.1.1. Definition

Periodontal diseases (PD) are group of inflammatory diseases caused by

pathogenic microflora organized in biofilms surrounding the teeth and divided into two

main forms, gingivitis is a superficial and reversible affection of gingiva without

destruction of alveolar bone and periodontitis corresponding to profound disease

associated with destruction of teeth-supporting tissues that can lead to tooth loss

(Pihlstrom et al., 2005).

Periodontal diseases begins when bacteria in plaque causes the gingiva to

become inflamed (American Academy of Periodontology, 2004). As a rule, PD

develops through gingivitis, an inflammation of the marginal periodontium. However,

not every gingivitis develops further into periodontitis; both the amount and virulence

of the microorganisms and the resistance factors of the host (risk factors and immune

status) are crucial for the progression of the periodontal destruction (Saini et al., 2009).

There is equilibrium that exists between microbial challenge and host's

immune response; any alteration to that with the addition of other modifying factors is

responsible for clinical manifestation of PD. The oral cavity works as a continuous

source of infectious agents, and its condition often reflects progression of systemic

pathologies (Saini et al., 2009; Soory, 2010).

Studies showed the essential role of bacteria in periodontitis but bacteria

alone seem to be insufficient to explain occurrence or progression of the disease

(Leininger et al., 2010). Age, tobacco use, systemic diseases and psychological stress

have been identified as important risk factors for periodontitis (Peruzzo et al., 2007;

Cronin et al., 2008).

Several studies have demonstrated a relationship between psychological

stress and inflammatory diseases such as rheumatoid arthritis and periodontitis


5

(Walker et al., 1999; Hilgert et al., 2006; Saini et al., 2010; Goyal et al., 2011;

Reddy et al., 2012; Rivera et al., 2012; Refulio et al., 2013).

1.1.2. Epidemiology

The PD are highly prevalent and can affect in the initial and acute phases up

to 90% of the population worldwide. The disease affects a relatively high percentage

of the adult population in developed and developing countries (Zadik et al., 2008).

Periodontal disease is widely regarded as the second most common disease

worldwide, after dental decay, and in the United States has a prevalence of 30-50% of

the population, but only about 10% have sever forms. Like other conditions that are

intimately related to access to hygiene and basic medical monitoring and care,

periodontitis tends to be more common in economically disadvantaged populations or

regions (Pihlstrom et al., 2005). Generally, in Yemenite, North-African, South Asian,

or Mediterranean origin have higher prevalence of PD than individuals from European

descent individuals living in East Asia (e.g. Japan, South Korea and Taiwan) have the

lowest incident of PD in the world. This could be attributed to genetic predisposition

as well as environmental and behavioral differences (e.g. smoking, oral hygiene, stress

and access to dental treatment) between populations (Zadik et al., 2008).

1.1.3. Pathogenesis of periodontitis

There are a number (No.) of models by which pathogenesis of periodontitis

can be explained. One suggests continuous progression of disease where the loss of

attachment occurs slowly over time and then another suggests progression of

attachment loss occurs rapidly over short period of time, or in an episodic burst manner.

It appears that, depending on the patient and the sites, both these models could occur

together (Jepsen, 1996).

1.1.3.1. Bacterial factors

Periodontitis is a common, chronic and complex inflammatory disease

caused by bacterial biofilms that accumulate on the tooth surface and gingival sulcus,


6

and it is characterized by progressive destruction of the structures that support teeth

(Van Dyke, 2007; Kornman, 2008).

Microbial plaque accumulation on teeth surfaces adjacent to the gingival

tissues brings the oral sulcular and junctional epithelial cells into contact with the waste

product, enzymes and surface components of colonizing bacteria. As the bacterial load

increases, so does the irritation of the host tissues by these substances. The microbial

substances that can direct its action against vasculature and leukocytes, causing

vasodilation, increased gingival crevicular fluid (GCF) flow and migration of

neutrophils. In addition, these components interact with host systems involved in

inflammatory responses that lead to produce proinflammatory cytokines and other

chemical mediators of inflammation. These mediators begin an inflammatory response

within the tissues, which follows the classical inflammatory response. The tissues

become swollen as fluid accumulates and cell infiltrate is recruited into the lesion that

secretes proinflammatory mediators, including prostaglandin E2 (PGE2), interleukin

1(IL-1), IL-6 and tumor necrosis factor alpha (TNF-α), and clinical gingivitis develops

(Ranney, 1991; Ebersole and Cappelli, 2000). Many physiopathological processes

are involved in periodontal destruction in terms of the inflammatory and immune host

response, especially proinflammatory cytokines or matrix metalloproteinase (MMP)

(Dahan et al., 2001; Kiecolt-Glaser et al., 2003; Van Dyke et al., 2008). MMPs

participate in the destruction of the extracellular matrix (Teba et al., 2005).

Etiology of PD is highly related to periodontal bacteria such as

Porphyromonas gingivalis (P. gingivalis), Prevotella intermedia (P. intermedia) or

Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) (Castillo et al.,

2011). These bacteria induce the destruction of periodontal tissues with their numerous

virulence factors such as fimbriae and lipopolysaccharide (LPS) that cause activation

of monocytes and subsequent production of tissue destruction can lead to connective

tissue attachment loss and bone loss. Cytokines can cause tissue destruction by

allowing the secretion of tissue MMPs, thus further contributing to the pathogenesis of

PD (Curtis et al., 2005) (Fig. 1-1).


7

Fig. 1-1: Schematic illustration of the pathogenesis of periodontitis (Carranza et al., 2006).

In healthy periodontium, neutrophils can form a wall and fend back bacteria,

keeping their virulent factors away from the tissues. The neutrophils get the assistance

of opsonizing antibodies from reservoirs of the GCF. In the diseased state, GCF

is an inflammatory exudate that flows through the junctional epithelium into the

gingival crevice. It contains many molecules such as locally produced antibodies, host

cell derived products, endotoxin, amines, enzymes, bacterial collagenase and immune

response product such as cytokines (Embery and Waddington, 1994; Delima and

Van Dyke, 2003). The antibodies in the GCF allow neutrophils to recognize, ingest,

and degrade bacteria. When bacteria have invaded the periodontal tissues, host-

mediated destruction occurs and host cells release cytokines into the environment,

which provide the signals to other cells to begin affecting their function (Liu et al.,

1996; Rawlinson et al., 2000).

1.1.3.2. Host responses factors

Although bacteria initiate PD, the host response is believed to play an

essential role in the breakdown of connective tissue and bone. Microbial antigens and


8

virulence factors elicit an inflammatory and immune reaction, in which both innate and

adaptive immune responses are involved (Van Dyke and Kormman, 2008). The

response varies among individuals, depending on potential variations in cytokine and

other antimicrobial responses, environmental factors and the subjects' genetics

(Gemmell et al., 2007; Kinane et al., 2007).

I. Innate immune response:

The first line of defense in infectious diseases, the innate immunity, is

challenged to detect pathogens and mount a rapid defensive response, which requires

no prior immune learning or experience. It is responsible for the defense during the

initial hours and days of the infection (Kirkwood et al., 2006; Kinane et al., 2007).

It acts through the recruitment of immune cells, activation of complement system,

identification and removal of foreign substances and activation of the adaptive immune

system (Vandyke and Kormman, 2008).

Periodontal disease may in most cases be considered a localized infection.

However, it is speculated that the inflamed and ulcerated subgingival pocket epithelium

could form an easy port of entry for dental plaque bacteria to disperse systemically

(Hujoel et al., 2001; Moutsopoulos and Madianos, 2006). Neutrophils

(polymorphonuclear leukocytes (PMNs)) are the first cells to encounter pathogens,

soon reinforced by the recruitment of monocytes/macrophages (Janeway et al., 2005).

Neutrophil infiltration takes place in the periodontal tissue (Dixon et al., 2004).

Inflammatory cell infiltrate in gingival tissue and GCF is predominantly formed by

neutrophils, B-cells and plasma cells are also present (Ebersole, 2003).

The complement system is a part of innate response, composed of 30 proteins

that participate in tissue destruction and in inflammatory processes, it can be activated

by the alternative pathway (LPS or other bacterial products) or by the classical pathway

(detection of antigen antibody complexes), giving rise to bacterial opsonisation

(Bascones and Gonzalez-Moles, 2003).

The innate immune system recognizes common pathogen associated

molecular patterns (PAMPs) that are expressed on microorganisms, but not on host


9

cells, by extra- and intracellular receptors like cluster of differentiation 14 (CD14). The

CD 14 proteins found in two distinct forms: membrane CD14 (mCD14), expressed

primarily on the surface of monocytes/ macrophages and neutrophils, and a soluble

form (sCD14) (Le Van et al., 2001; Bas et al., 2004). Activation of the CD14 receptor

activates monocytes and endothelia cells for secretion of proinflammatory molecules

such as IL-1β, TNF-α, PGE2 and IL-6 (Dixon et al., 2004; Martinez et al., 2009).

II. Adaptive immune response:

The innate immunity system is closely linked to the adaptive immune

response; it acts the second defense line that helps clear the infection and builds specific

immunity with a memory component. Activation of the adaptive response occurs

through cytokine secretion, antigenic processing and presentation and differentiation

of effector cells (Hornef et al., 2002).

The primary cells involved in the adaptive immune response are

lymphocytes, B-cells and T-cells. B-cells are mainly antibody producers, while T-cells

are functionally divided into two main classes. One class differentiates on activation

into cytotoxic T-cells, which kill cells infected with viruses and express CD8 molecule

on their cell surfaces. T helper (Th) cells, on the other hand, are marked by the

expression of the cell surface molecule CD4, and play an important role in the initiation

of immune responses by providing help to other cells (Janeway et al., 2005).

T-cells are considered to have a central role in controlling the progression of

PD, and different types of T cell clones have been demonstrated to play destructive

roles (Taubman and Kawai, 2001). The early periodontal lesion (gingivitis)

characterized by increased Nos. of T-cells, but there is only limited consensus

regarding the nature of Th cells that predominate in inflamed gingival tissue. Most of

the studies support the notion that T helper type 1 (Th1) cells and their cytokines are

associated with early /stable periodontal lesions, while T helper type 2 (Th2) response

in periodontium predispose to susceptibility to disease progression (Gemmell et al.,

2007). Although T-cells act as the main regulatory group of cells in periodontitis

lesions, B-cells have several critical roles. After encounter with antigen, B-cells


10

differentiate into antibody secreting plasma cells, the production of antibodies involves

the response to foreign antigens as well as to self-antigens, plasma cells that have the

property of specifically binding to the antigen (Bascones and Gonzalez-Moles, 2003;

Berglundh et al., 2007).

Periodontal pathogens give rise to a marked humeral immune response such

as production of immunoglobulin (Ig) that can be measured locally in saliva or GCF or

systemically in serum (Korn et al., 2007; Pussinen et al., 2007; Gaffen and

Hajishengallis, 2008).

1.1.3.3. Viral factors

In addition to bacteria and host response factors, viruses also play a role in

the pathogenesis of periodontitis (Slots and Contreras 2000; Slots, 2005).

Herpes virus infections can induce pro-inflammatory cytokine release as a

response from the immune system. Cytokines normally play a positive role in the

immune system by exerting antiviral activity to fight off infection or invasion by

pathogens (Alcami and Koszinowski, 2000). However, herpes viruses can interfere

with these functions and cause paradoxical effects leading to tissue damage. In

particular, the viruses can change the cytokine production and prevent antiviral

responses (Tortorella et al., 2000).

The over production of pro-inflammatory cytokines such as IL-1β, 1L-6, IL-

12 and TNF-α are often evident in human cytomegalovirus (HCMV) infections and

can lead to detrimental effects (Mogensen et al., 2004). A productive herpes virus

infection can exacerbate periodontal tissue destruction through a virus-induced

impairment of the periodontal immune defense, leading to a more virulent population

of resident bacteria (Slots and Contreras, 2000; Mogensen et al., 2004; Slots, 2005).

1.1.3.4. Other risk factors

Although oral microorganism seem to play a predominant role in etiology of

chronic periodontitis (CP), other risk indicators have been identified. Risk factors are

important in periodontopathy as they affect the severity and prevalence of periodontal

diseases among population. The assessment of risk factor is essential and necessary in


11

order to limit and control the disease and treatment for subjects with increased risk

(Tanner et al., 1997). CP has been considered more common in people aged more than

30, having systemic disease, inadequate oral hygiene and /or high levels of stress and

low socioeconomic status (Akhter et al., 2005).

Oral hygiene, a considerable No. of studies have suggested an important role

of oral hygiene in periodontal health status such as (Hirschfeld and Wasserman in

1978; Konig et al., in 2001), during their classical investigations with optimal

individual hygiene procedures, resulted in improvement of periodontitis and stable

periodontal health status (under highly standardized conditions).

Smoking is one of the most significant risk factor for the initiation and

progression of PD and associated with increased prevalence and severity of destruction

of PD in term of periodontal pocketing and attachment loss. Additionally, smoking can

lower the chances for successful treatment (AL-Ghamdi and Anil, 2007).

Genetic factors influence susceptibility to the different forms of early onset

periodontitis. However, it is unlikely that a specific gene will be identified as causing

enhanced disease susceptibility. It is more likely that the genetic influences are as

multifactorial as the diseases themselves, and a complex interplay between genetically

determined host responses and environmental challenges may determine whether

disease is present (Jindal, 2009).

Alcohol consumption as well as alcohol sensitivity may be a risk factor for

periodontitis progression (Shimazaki et al., 2005; Nishida et al., 2010).

Psychological factors, many studies have demonstrated that individuals

under psychological stress are more likely to develop clinical attachment loss, loss of

alveolar bone and increased the periodontitis (Mawhorter and Lauer, 2001; Pistorius

et al., 2002; Wimmer et al., 2002).


12

1.2. Stress

Stress originates from a Latin word: 'stringere', which means 'tight', 'strained'.

In 1935, Cannon described stress as the result of the homeostasis and showed the

influence of the sympathetic system. In 1950, Selye published a book known as (Relief

from stress) which had over 1000 pages and more than 5000 references. He elaborated

stress as a response state of the organism to forces acting simultaneously on the body

which if excessive that is straining the capacity of adaptive process beyond their limits

leads to disease of adaptation and eventually disease of exhaustion and death. He

defined forces that have the potential to challenge the adaptive capacity of the organism

as stressor (LeResche and Dworkin, 2002; Sateesh et al., 2010).

The stressors are all situations that can constitute aggressions or feel like that.

Various origins can take place such as physical or psychological (mental) (Sateesh et

al., 2010). When any type of stressor exceeds the threshold, the stress system mediates

the generalized stress response. Selye recognized that stressor acting to produce

changes in the body could be positive (e.g. exciting, pleasurable), leading to a response

state known as eustress, or stressor could be negative, threatening homeostasis with

pain, discomfort and physical pathology which is known as distress (LeResche and

Dworkin, 2002).

A subject exhibits stress response or not depends upon the factors, including

coping behaviors, genetic predisposition, concomitant stressors, level of social support

and their lifestyle factors. Potential effects of stress response that may be observed or

even measured, includes anxiety, depression, impaired cognition and altered self-

esteem (Boyapati and Wang, 2007). The coping is the effort to try to reduce, control

or tolerate the state of stress. It needs adjustment, adaptation and confrontation

strategies (Sateesh et al., 2010).

Reners and Breex in 2007 defined stress as physiological and metabolic

perturbations caused by various aggressive agents and psychophysiological response

of an organism facing the perception of a challenge or a threat.


13

Different kinds of stress have been defined, such as work related, negative

life experiences and socioeconomic status (Genco et al., 1998; Mead et al., 2001;

Soares et al., 2007).Stress is often classified as acute and chronic. Acute stress lasts

for a period of minutes to hours, whereas chronic stress persists for several hours, a

day, weeks or even months. In case of acute stress, stress response may prepare the

immune system for challenges such as infection that may be imposed by the stressor

(Dhabbar, 2002). When stress becomes chronic, it may influence inflammatory

processes leading to development of systemic or local diseases such as rheumatoid

arthritis (Culshaw et al., 2011), diabetes (Chida and Hamer, 2008), cardiovascular

diseases (Backe et al., 2012) or PD (Stabholz et al., 2010). So stress can be mediates

disease and illness, LeResche distinguished between disease and illness in that the

maladaptation of organs or organ systems manifests itself as the pathological states that

we call disease, such as inflammatory disease, malignancy and genetic malformations.

While people have an illness, which is typically based on the self-attribution or belief

that a disease is present. Illness behaviors may become maladaptive, such as excessive

worry about poor outcomes, depression, anxiety, overzealous health-care seeking and

interruption of normal behaviors such as work, eating and social activities (LeResche

and Dworkin, 2002).The magnitude of stress is measure according to the peak levels

of stress hormones, neurotransmitters and other physiological changes, including

increased levels in heart rate and blood pressure and by the duration of time during

which these changes linger during and after exposure to a stressor. Therefore, the

combination of intensity and duration comprise the magnitude of stress (Dhabbar,

2002).

An academic exam can be considered as a psychological stressor, consisting

of a period of preparation, anticipation and then the exam itself. Furthermore, an

academic exam plays an important role in evaluating student learning outcomes and

their mastery of a subject. Recent studies have reported high levels of anxiety among

dental and medical students (Omigbodun et al., 2006; Smith et al., 2007). Students

who participated in a major exam had significantly more dental plaque and more


14

gingival inflammation compared with students who didn't participate in any exam

(Deinzer et al., 2005). Academic stress appears to affect periodontal health status

(Deinzer, 2001; Johannsen et al., 2010). In addition, some studies have shown that

the levels of pro-inflammatory cytokines, IL-1β, IL-6 and IL10 in serum and GCF, are

increase in patients with depression and academic stress (Deinzer, 1998; Maes et al.,

1998; Paik et al., 2000; Waschul et al., 2003; Johannsen et al., 2006; Johannsen et

al., 2007; Von Kanel et al., 2007; Johannsen et al., 2010).

1.2.1. Stress scales

There are many types of stress scales such as holme rahe stress scale (HRSS),

college undergraduate stress scale (CUSS), traditional college student stress scale

(TCSS) and two versions of depression anxiety stress scale (DASS); DASS versions

are a 42-item questionnaire version and a 21-item questionnaire version, Table (1-2)

and (1-3), which includes three self-report scales designed to measure the negative

emotional states of depression, anxiety and stress. Each of the three scales contains 14

items in a 42-item questionnaire version and 7 items in a 21-item questionnaire version.

The depression scale assesses dysphoria, hopelessness, devaluation of life, self-

deprecation, lack of interest, anhedonia and inertia. The anxiety scale assesses

autonomic arousal, skeletal muscle effects, situational anxiety and subjective

experience of anxious affect. The stress scale is sensitive to levels of chronic non-

specific arousal. It assesses difficulty relaxing, nervous arousal and being easily

agitated, over-reactive and impatient. Respondents are asked to use 4-point severity

scales to rate the extent to which they have experienced each state over the past week.

Scores of depression, anxiety and stress calculated by summing the scores for the

relevant items. The depression scale items are 3, 5, 10, 13, 16, 17, 21, 24, 26, 31, 34,

37, 38 and 42 in a 42-item questionnaire version and in a 21-item questionnaire version

include 3,5,10, 13, 16,17and 21. The anxiety scale items are 2, 4, 7, 9, 15, 19, 20, 23,

25, 28, 30, 36, 40 and 41 in a 42-item questionnaire version and in a 21-item

questionnaire version include 2, 4,7,9,15,19 and 20. The stress scale items are 1, 6, 8,


15

11, 12, 14, 18, 22, 27, 29, 32, 33, 35 and 39 in a 42-item questionnaire version and in

a 21-item questionnaire version include 1, 6, 8,11,12,14 and 18. The score for each of

the respondents over each of the sub-scales then evaluated as per the severity-rating

index, Table (1-1) (Lovibond and Lovibond, 1995).

Table (1-1): DASS severity-rating index (Lovibond and Lovibond, 1995).

Table (1-2): DASS 42-item questionnaire version (Lovibond and Lovibond, 1995).

1 I found myself getting upset by quite trivial things 0 1 2 3

2 I was aware of dryness of my mouth 0 1 2 3

3 I couldn't seem to experience any positive feeling at all 0 1 2 3

4 I experienced breathing difficulty (e.g. excessively rapid breathing,

breathlessness in the absence of physical exertion)

0 1 2 3

5 I just couldn't seem to get going 0 1 2 3

6 I tended to over-react to situations 0 1 2 3

7 I had a feeling of shakiness (e.g. legs going to give way) 0 1 2 3

8 I found it difficult to relax 0 1 2 3

9 I found myself in situations that made me so anxious I was most

relieved when they ended

0 1 2 3

10 I felt that I had nothing to look forward to 0 1 2 3


16

11 I found myself getting upset rather easily 0 1 2 3

12 I felt that I was using a lot of nervous energy 0 1 2 3

13 I felt sad and depressed 0 1 2 3

14 I found myself getting impatient when I was delayed in any way (e.g.

lifts, traffic lights, being kept waiting)

0 1 2 3

15 I had a feeling of faintness 0 1 2 3

16 I felt that I had lost interest in just about everything 0 1 2 3

17 I felt I wasn't worth much as a person 0 1 2 3

18 I felt that I was rather touchy 0 1 2 3

19 I perspired noticeably (e.g. hands sweaty) in the absence of high

temperatures or physical exertion

0 1 2 3

20 I felt scared without any good reason 0 1 2 3

21 I felt that life wasn't worthwhile 0 1 2 3

22 I found it hard to wind down 0 1 2 3

23 I had difficulty in swallowing 0 1 2 3

24 I couldn't seem to get any enjoyment out of the things I did 0 1 2 3

25 I was aware of the action of my heart in the absence of physical

exertion (e.g. sense of heart rate increase, heart missing a beat)

0 1 2 3

26 I felt down-hearted and blue 0 1 2 3

27 I found that I was very irritable 0 1 2 3

28 I felt I was close to panic 0 1 2 3

29 I found it hard to calm down after something upset me 0 1 2 3

30 I feared that I would be "thrown" by some trivial but unfamiliar task 0 1 2 3

31 I was unable to become enthusiastic about anything 0 1 2 3

32 I found it difficult to tolerate interruptions to what I was doing 0 1 2 3

33 I was in a state of nervous tension 0 1 2 3

34 I felt I was pretty worthless 0 1 2 3


17

35 I was intolerant of anything that kept me from getting on with what

I was doing

0 1 2 3

36 I felt terrified 0 1 2 3

37 I could see nothing in the future to be hopeful about 0 1 2 3

38 I felt that life was meaningless 0 1 2 3

39 I found myself getting agitated 0 1 2 3

40 I was worried about situations in which I might panic and make fool

of myself

0 1 2 3

41 I experienced trembling (e.g. in the hands) 0 1 2 3

42 I found it difficult to work up the initiative to do things 0 1 2 3

Table (1-3): DASS 21-item questionnaire version (Lovibond and Lovibond, 1995).

1 I found it hard to wind down 0 1 2 3

2 I was aware of dryness of my mouth 0 1 2 3

3 I couldn't seem to experience any positive feeling at all 0 1 2 3

4 I experienced breathing difficulty (e.g. excessively rapid breathing) 0 1 2 3

5 I found it difficult to work up the initiative to do things 0 1 2 3

6 I tended to over-react to situations 0 1 2 3

7 I experienced trembling (e.g. in the hands) 0 1 2 3

8 I felt that I was using a lot of nervous energy 0 1 2 3

9 I was worried about situations in which I might panic and make

a fool of myself

0 1 2 3

10 I felt that I had nothing to look forward to 0 1 2 3

11 I found myself getting agitated 0 1 2 3

12 I found it difficult to relax 0 1 2 3

13 I felt down-hearted and blue 0 1 2 3

14 I was intolerant of anything that kept me from getting on with

what I was doing

0 1 2 3


18

15 I felt I was close to panic 0 1 2 3

16 I was unable to become enthusiastic about anything 0 1 2 3

17 I felt I wasn't worth much as a person 0 1 2 3

18 I felt that I was rather touchy 0 1 2 3

19 I was aware of the action of my heart in the absence of physical

exertion (e.g. sense of heart rate increase, heart missing a beat)

0 1 2 3

20 I felt scared without any good reason 0 1 2 3

21 I felt that life was meaningless 0 1 2 3

1.2.2. Molecular and endocrine mechanisms of the stress response

The immune response doesn't operate autonomously but in close cooperation

with the neuroendocrine systems. When the body is in stress, there is an increase of

stress markers and immune cells in the plasma mobilized from lymphoid organs. A

negative feedback, with the activation of the immune system that is associated with the

increase of circulating cytokines, increases the activity of the corticotropin releasing

hormone (CRH) and activates the hypothalamic pituitary adrenal (HPA), causing an

elevation of the levels of cortisol (Fig. 1-2). When the inflammatory action is

sufficiently long and profound, systemic manifestations of the disease might become

evident, as could happen with periodontitis (LeResche and Dworkin, 2002).

Where stress can result in the degeneration of the immune system, mediated

primarily through HPA axis and sympathetic adrenal medullary axis (Fig. 1-3). Stress

perceived by the brain stimulates the hypothalamus to produce CRH, which is release

into the hypophyseal portal system, activating the pituitary gland to release

adrenocorticotropic hormone (ACTH), which in turn induces release of glucocorticoids

(GC) from the adrenal cortex (Boyapati and Wang, 2007). Cortisol, known more

formally as hydrocortisone, is a steroid hormone, more specifically is the main adrenal

GC hormone, and it is released in response to stress and a low level of blood GC

(Marieb et al., 2013).


19

Glucocorticoids, including cortisol, exert major suppressive effects through

highly specific mechanisms at multiple levels. For example, in vivo GC reduce the No.

of circulating lymphocytes, monocytes and eosinophil. They also inhibit the

accumulation of eosinophil, macrophages and neutrophils at inflammatory sites. At the

molecular level, GC profoundly inhibit important functions of inflammatory cells

including macrophages, neutrophils, eosinophil and mast cells in functions such as

chemotaxis, secretion and degranulation. GC also inhibit the cascade of the immune

response by inhibiting macrophage-antigen presentation, lymphocyte proliferation and

lymphocyte differentiation to effector cell types such as helper lymphocytes, cytotoxic

lymphocytes, natural killer cells and antibody-forming B cells. Corticosteroids also

inhibit production of cytokines including IL-1, IL-2, IL-3, IL-6, TNF, interferon-

gamma (IFN-γ), granulocyte and monocyte colony stimulating factors. GC inhibit

arachidonic acid-derived proinflammatory mediators such as PG. Hence, the stress-

related stimulation of the HPA axis with the production of GC such as cortisol has

major suppressive actions on immune and inflammatory responses. This represents the

major effector arm of the CNS-hormonal axis. There is also an afferent or feedback

arm consisting of stimulation of the HPA axis by cytokines (Kunz-Ebrecht et al.,

2003; Boyapati and Wang, 2007; Groer et al., 2010; Papacosta and Nassis, 2011).

Glucocorticosteroids, including cortisol, then depresses immunity including

secretory IgA, IgG and neutrophil functions, all of which may be important in

protection against infection by periodontal organisms. Secretory IgA antibodies may

protect by reducing initial colonization of periodontal pathogens. IgG antibodies may

exert protection by opsonizing periodontal organisms for phagocytosis and killing by

neutrophils. This then gives rise to increased susceptibility, which leads to the

establishment of periodontal infection, which, in turn, results in destructive

periodontitis. Periodontitis is brought about by tissue destroying factors such as IL-1

and MMPs activated by the periodontal pathogens, as well as by the direct effects of

pathogenic bacteria (Grossi et al., 1998).


20

The second major pathway to be activated is the sympathetic nervous system

called ‘flight or fight’ response to potentially harmful stimuli. Stress activates the nerve

fibers of the autonomic nervous system, which innervate the tissues of the immune

system, results in the release of catecholamine from the adrenal medulla that lead to

the development of hyperglycemia by directly stimulating glucose production and

interfering with the tissue disposal of glucose. Catecholamines are alter the blood flow.

Peripheral vasoconstriction may affect important oxygen–dependent healing

mechanisms, such as angiogenesis, collagen synthesis and epitheliazation (Boyapati

and Wang, 2007). In addition, catecholamines are the most important molecules to

relay information from the CNS to the immune system (Marketon and Glaser, 2008).

The release of catecholamine results in hormonal secretion of

norepinephrine/ epinephrine (noradrenaline/adrenaline) by adrenal medulla and

sensory nerve fibers, which results in a range of immune functions including cell

proliferation (Sanders and Straub, 2002; Hamaguchi et al., 2008), inhibition of pro-

inflammatory cytokines such as IFN-γ, IL-2, IL-6, IL-12 and TNF-α (Hansel et al.,

2010), suppression of lymphocyte proliferation, Natural killer (NK) cell activity (Ben-

Eliyahu et al., 2000), antibody production and cytolysis activity (Padgett and Glaser,

2003).

Increased sympathetic stimulation can also act to decrease salivary

secretions typically experienced as anxiety induced dry mouth. Stress that is associated

with immune challenge has been called immune stress or inflammatory stress

(Boyapati and Wang, 2007).

Autonomous nervous system can also moderate the HPA axis by stimulating

CNS and sensory nerve fibers leading to secretion of neuropeptides such as substance

P (SP) (Rosenkranz, 2007). Neuropeptides are generated primarily in the CNS and

play important roles in neurogenic inflammation, including vasodilatation, plasma

extravasation and recruitment of immune cells (Kabashima et al., 2002; Lundy and

Linden, 2004). SP is important in initiating and sustaining inflammation, increasing


21

proinflammatory cytokine production and by limiting the production of transforming

growth factor beta (TGF-β) and IFN-γ activated macrophages (Pradeep et al., 2009).

Fig. 1-2: Effects of stress on hypothalamic pituitary adrenal axis (Genco et al., 1998).

Fig. 1-3: Physiological model of effects of stress on periodontal disease (Boyapati and Wang,

2007).


22

1.2.3. Behavioral changes of stress response

It is hypothesized that the main effects of stress occur through behavioral

changes which affect at risk health behaviours such as smoking, poor oral hygiene and

poor compliance with dental care. There is also a possibility that stress leads to other

behavioral changes such as overeating, especially a high-fat diet, which then can lead

to immunosuppression through increased cortisol production (Fig. 1-4) (Grossi et al.,

1998).

There are certainly much other possible behavior that could be affected by

stress, inadequate coping and distress, such as depression, which would have

significant effects on PD. Croucher et al., in 1997 studied the relationship between

life events and PD and found that both negative life events leading to oral health risk

behaviours such as poor oral hygiene and smoking as important determinants of PD.

Stress, distress and inadequate coping may influence a multitude of

behaviors, for example depression, thus considerably affecting PD. Health risk

behaviors must be evaluated to reveal the extent to which they contribute to the

interaction between stress and PD (Genco et al., 1998).

Klages et al., in 2005 discovered that mental stress could influence life-style

and dental hygiene habits. This influence wasn't only decreases the frequency as well

as the quality of the dental hygiene but also it increases tobacco use and alcohol

consumption, changes in food habits leading to a diminution of the general health. This

was in agreement with the study conducted by Suchday et al., in 2006.

Deinzer et al., in 2005 have discussed that plaque is a valid indicator of oral

hygiene behavior even under academic stress conditions, there seems to be good

evidence to add oral hygiene behavior to the list of health behavior's which gets

adversely affected by stress. These results confirm the findings of Deinzer et al., in

2001 on stress associated alterations in oral health behavior. It further extends by

demonstrating an increase of gingivitis in exam going students as compared with

controls 4 weeks after the exams. Gingivitis rates of posterior sextants of exam going

students nearly doubled those of control participants.


23

Fig. 1-4: Psychosocial stress and its effect on behavior as manifested by alterations in

periodontal disease (Grossi et al., 1998).

1.2.4. Stress and microbiology of periodontal disease

Periodontitis is mainly related to changes in the composition of oral biofilms

especially colonizing species such as P. gingivalis, P. intermedia or A.

actinomycetemcomitans (Socransky and Haffajee, 2005). Stress induced by

psychosocial factors could influence periodontal ecology. Where several

microorganisms have the ability to recognize the hormones that are found within the

host and use them effectively to adapt to their environment and to promote bacterial

growth and infectious diseases. This supports the idea bacterial infections may develop

in response to stress. In an in vitro study done by Roberts et al., in 2002 to determine

whether noradrenaline and adrenaline, which are, released during human stress

responses, signals to alter the growth of 43 microorganisms found within subgingival

microbial complexes. The researchers found that 20 species within the subgingival

biofilm significantly grew from inoculation with noradrenaline and 27 species

significantly grew when adrenaline was introduced. There was also marked difference


24

in the growth response within bacterial species and within or between microbial

complexes. They concluded that this variation might influence in the vivo composition

of the subgingival biofilm in response to stress-induced changes in local catecholamine

levels and thus play a vital role in the etiology and pathogenesis of PD.

Shortly after this research published, another research also discovered that

chronic psychological stress has a marked impact on the localized immune response to

P. gingivalis (Houri-Haddad et al., 2003).

These observations indicate that stress-induced hormones may have specific

effects depending on species of bacteria. Periodontal destruction is the result of an

imbalance between bacterial aggression and host response. Stress-related hormones are

likely to favor the infection by increasing bacterial growth, thereby inducing a

breakdown in oral biofilms. Specific mechanisms underlying these effects on

periodontal microbiota remain unknown, and further studies are required to evaluate

possible effects of these hormones, especially on triggering of virulence factors

(Roberts et al., 2005).

1.2.5. Role of stress on gingivitis

Stress has shown to reduce the saliva flow and it increases the formation of

plaque. A study has shown that responses to emotional or psychological stress may

influence immune activities directly via nerve messenger substances

(neurotransmitters and neuropeptides) and/or indirectly via neuroendocrine (hormone)

substances and may modulate the immune response to bacteria, thus be expected to

influence the progression and course of gingivitis and periodontitis (Breivik et al.,

1996). Another study has shown that emotional stress modifies pH and its IgA secretion

(Reners and Breex, 2007). A series of studies done by Deinzer et al., examine the role

of academic stress during their examination period on periodontal health status.

Academic stress as shown to be a risk factor for gingival inflammation with increasing

crevicular IL-1β levels and a decrease in the quality of the oral hygiene (Deinzer et

al., 1998; Deinzer et al., 1999; Deinzer et al., 2000; Deinzer et al., 2001).

http://www.ncbi.nlm.nih.gov/pubmed?term=Breivik%20T%5BAuthor%5D&cauthor=true&cauthor_uid=8930578


25

In a pilot study done by Axtelius et al., in 1998 showed the presence of

cortisol in GCF. A study conducted by Johannsen et al., in 2006 also confirmed that

persons with depressive signs show an elevation in cortisol levels in GCF.

Over the past decade, it has become more apparent that stress can negatively

influence the oral health status, which can lead to increased amounts of dental plaque

and gingival inflammation (Klages et al., 2005; Johannsen et al., 2007). In addition,

Deinzer et al., in 2005 and Johannsen et al., in 2010, who found increased dental

plaque and gingival inflammation in students who experienced academic stress.

Academic stress appears to affect periodontal health status, shown by more

plaque accumulation, gingival inflammation and increased amounts of IL-6, IL-10 in

GCF and cortisol in saliva. Therefore, the clinical implication should be to inform

individuals about stress as a possible risk factor for gingivitis and periodontitis and to

introduce additional preventive strategies in these individuals (Johannsen et al.,

2010).

1.2.6. Role of stress on periodontitis

Psychosocial factors are predisposing factors for the development of necrotic

PD. The first studies showing this influence were done by Giddon et al., in 1963;

Giddon et al., in 1964 (more necrotic PD in college during examination period). Many

of the main risk factors for necrotic PD such as past episode of necrotic PD, bad oral

hygiene, unusual emotional stress, bad sleep, tobacco, alcohol and recent illness are

often related to stress (Sateesh et al., 2010).

In addition, Stress linked with acute necrotizing ulcerative gingivitis

(ANUG) since early study evaluated the association between PD and psychosocial

stress (Murayama et al., 1994). Compared to other times, college students experience

an increase in the incidence of ANUG during examinations (Genco et al., 1998).

One study by Monteiro da Silva et al., in 1996 showed that people with

aggressive periodontitis were more depressed and socially isolated people than people


26

with CP or the control group. These studies show the interconnection that exists

between aggressive periodontitis and psychosocial stress.

Several clinical studies have investigated the possible relationship between

psychological stress and periodontitis; have suggested that stress may play an

important role in the development of PD and increase severity of PD (Hilgert et al.,

2006; Saini et al., 2010; Doyle and Bartold, 2012). Where, in a longitudinal study by

Linden et al., in 1996 predicted the future attachment loss depending on occupational

stress. In addition, the subjects who felt stress were more prone to develop PD than

subjects without stress (Akhter, 2005).

In contrary, Castro et al., in 2006 couldn't show any association between

life events, anxiety and depression with periodontitis. The association between

psychosocial factors and periodontitis is derived mainly from cross-sectional studies

(Linden et al., 1996; Genco et al., 1999; Johannsen et al., 2005; Klages et al., 2005).

1.2.7. Role of stress on periodontal treatment

A study conducted by Axtelius et al., in 1998 showed that patients with

psychosocial strain and passive dependent traits didn't respond to treatment when

compared to patients with less stressful psychosocial situation and a rigid personality.

Another study done by Kamma and Baehni showed that supportive

periodontal care was more effective in less stressful patients with aggressive

periodontitis (Kamma and Baehni, 2003).

Wimmer et al., in 2005; Reners and Brecx in 2007 explained the influence

of coping with stress on periodontal therapy and concluded that patients who had

maladaptive coping strategies have more advanced disease and those patients showed

a poor response to non-surgical periodontal treatment. In addition, Gamboa et al., in

2005 showed the influence of emotional intelligence which was used as a measure of

the coping mechanism in patients with CP on the initial responses to periodontal

treatment. The results of this study showed a decrease in plaque formation and

reduction in bleeding on probing (BOP) in patients with active coping strategy.


27

1.3. Saliva

Saliva is a unique complex, important body fluid and contains a No. of

systems, which serves a wide spectrum of physiological needs to protect the oral

mucosa and the whole body from infection (Harris and Godoy, 2004; Nanci, 2003;

Peter, 2004). It's produced from three-paired major salivary glands (parotid,

submandibular and sublingual), plus that from 200-400 minor salivary glands which

scattered throughout the oral cavity (Nanci, 2003; Costanzo, 2010). When the fluids

from all major and minor glands mix with each other, this secretion become known as

whole saliva or mixed saliva. Whole saliva is further altered by the presence of particles

of food, tissue fluid, lysed bacteria, sloughed epithelial cells and inclusion of living

cells and their metabolic products (Lawrence, 2002; Harris and Godoy, 2004).

Unstimulated (resting) saliva is a mixture of secretions, which enter the

mouth in the absence of exogenous stimuli (Ship et al., 1991; Thylstrup and

Fejerskov, 1994). Several factors affecting unstimulated salivary flow rate as the

degree of hydration, body position, exposure to light, previous stimulation, circadian

rhythms (peak during late afternoon), circannual rhythms (peak during winter) and

medications (Leone and Oppenheim, 2001; Thie et al., 2002; Guggenheimer, 2003).

Stimulated saliva is secreted in response to either masticatory or gustatory

stimulation and to lesser extent by activation of the vomiting center (Ghezzi et al.,

2000; Stooky, 2008). Stimulated salivary flow rate is affected by nature of the

stimulus, vomiting, smoking, gland size, gag reflex, olfaction and food intake

(Sreebny, 2000; Chausau et al., 2002; Inoue et al., 2009).

A loss or reduction of saliva results in significant problems such as dental

caries, PD, difficulties with eating, talking, altered taste sensation, as well as higher

risks of candidiasis and mucositis, which result in an overall reduction in the quality of

life (Buhlink et al., 2002; Hujoel et al., 2002; Thomson and Spencer, 2002).


28

1.3.1. Functions of saliva

De Almeida et al., in 2008; Greabu et al., in 2009 stated the functions of

saliva as follow:

1. Taste: The hypotonicity of saliva (low levels of glucose, sodium, chloride, and urea)

and its capacity to provide the dissolution of substances allows the gustatory buds

to perceive different flavors. Gustin, a salivary protein appears to be necessary for

the growth and maturation of these buds.

2. Buffer Capacity: Saliva behaves as a buffer system to protect the mouth as follows:

a) It prevents colonization by potentially pathogenic microorganisms by denying

them optimization of environmental conditions.

b) Saliva buffers (neutralizes) and cleans the acids produced by acidogenic

microorganisms, thus, prevent enamel demineralization.

3. Protection and Lubrication: Saliva forms a seromucosal covering that lubricates and

protects the oral tissues against irritating agents.

4. Dilution and cleaning: In addition to diluting substances, its fluid consistency

provides mechanical cleansing of the residues present in the mouth such as

nonadherent bacteria, cellular and food debris.

5. Integrity of tooth enamel: Saliva plays a fundamental role in maintaining the

physical-chemical integrity of tooth enamel by modulating remineralization and

demineralization.

6. Digestion: Saliva is responsible for the initial digestion of food, favoring the

formation of the food bolus, this action occurs mainly by the presence of the

digestive enzyme in the composition of saliva.

7. Tissue repair: A tissue repair function attributed to saliva since clinically the

bleeding time of oral tissues appears to be shorter than other tissues.

8. Defense such as spiting and oxidative stress.

9. Excretion, speaking, water balance, drug testing and denture retention.

10. Antibacterial, antifungal and antiviral: Saliva contains spectrum proteins with

antibacterial properties.


29

1.3.2. Salivary composition

Salivary gland secretion is mainly under autonomic nervous control, but

various hormones may also modulate salivary composition (Turner and Sugiya,

2002). Saliva consists of two component that are secreted by independent mechanisms

first component includes ions (inorganic), which is produced mainly by

parasympathetic stimulation and second component include protein (organic), which

is released mainly in response to the sympathetic stimulation (Scully, 2003).

Saliva possesses a multiplicity of immunological and nonimmunological

defense systems against toxins, fungi, viruses and bacteria (Dean and Simon, 2003;

Surdacka et al., 2007).

Saliva is principally a mixture of water and electrolytes such as sodium,

potassium, chloride and bicarbonate ions (Guyton and Hall, 2000; Johnson, 2003).

Saliva also contains organic compounds. These organic products are mostly proteins

or peptides, including enzymes, mucins, lactoferrin, lysozyme, cystatins and histatins

(Nieuw et al., 2004). The organic and inorganic components of saliva serve a wide

range of functions. Some of the more important of these are summarized in the Table

(1- 4) (Nieuw, 2007).

Table (1-4): Functions of saliva components (Nieuw, 2007).

Mucins Lubricate food

Protect teeth against acid

Help protect against bacteria, viruses, fungi

Digestive enzymes α-Amylase – digests starches

Lipase – digests fats

Protease – digests proteins

Lysozyme

Peroxidases

Lactoferrin

Histatins

Anti-bacterial agents


30

Cystatins

Secretory IgA

Histatins

Cystatins

Anti-fungal, anti-viral agents

Bicarbonate ions

Phosphate ions

Proteins

Help protect teeth and soft tissues against

acidic conditions

Calcium ions

Phosphate ions

Proline-rich proteins

Help maintain mineral content of tooth enamel

Concentrations of some components in whole saliva can be altered because

of differing flow rates from the principal glands. While in the unstimulated rest state,

the parotid glands contribute only a relatively small proportion of the total mix, and the

viscous, mucin-rich saliva from the minor, sublingual and submandibular glands

predominates. When stimulated, however, the parotid glands disproportionately

increase their output of watery saliva, effectively lowering the concentration of mucins

in the mixed saliva (Nieuw, 2007).

1.3.3. Saliva and diagnosis

Local and systemic disorders may disturb and interrupt the complex balanced

functions of saliva, which can lead to mucosal and tooth damages. In other cases,

systemic disorders induce salivary changes without any significant local effects. Many

such changes are of high diagnostic interest because they can be rather specific to the

causing conditions and can be used for screening and early diagnosis of several local

and systemic disorders (Fabian et al., 2007).

Interest in saliva as a diagnostic fluid has grown exponentially in recent

years. Saliva is a unique diagnostic fluid, the composition of which immediately

reflects the sympathetic nervous system, parasympathetic nervous system, HPA axis


31

and immune system response to stress (Khaustova et al., 2010). In addition, salivary

levels of various biochemical parameters have been measured in infectious diseases,

autoimmune diseases, cancers and psychiatric disorders (Edgar, 1992; Boyle et al.,

1994; Streckfus and Bigler, 2002).

Saliva collection is rapid, painless, non-invasive, economically, by

individuals with limited training, no special equipment is needed for the collection of

saliva and yields findings that are reproducible (Akcali et al., 2013).

1.3.4. Saliva and stress

Saliva can be used to monitor the systemic as well as the oral health status

(Buduneli et al., 2011). Furthermore, stress is also a factor that can be followed by

analysis of saliva, especially by determining the levels of stress-related markers (Pani

et al., 2011). These markers have biological properties that influence genesis and

development of PD. There are numerous stress-related molecules involved in different

aspects of stress response (Cortisol, Catecholamines, Chromogranin A, α-amylase,

Neuropeptides) (Akcali et al., 2013).

Physical and psychological stress have been shown to affect the secretion of

salivary proteins (Bosch et al., 2001; Allgrove et al., 2008). Recent investigations

have explored the use of salivary α-amylase as a biomarker of stress and there has been

much interest in its ability to serve as a convenient and non-invasive measure of

sympathetic nervous activity (Van Stegeren et al., 2006; Granger et al., 2007; Davis

and Granger, 2009; Nater and Rohleder, 2009; Spinrad et al., 2009; Fisher et al.,

2010; Strahler et al., 2010; Rudolph et al., 2011). However, due to the role that the

parasympathetic nervous system also plays in the control of protein secretion, a recent

study has questioned the ability of salivary α-amylase to serve exclusively as a

sympathetic marker (Bosch et al., 2011).


32

1.4. Cytokines

Cytokines are soluble proteins, secreted by cells in both the innate and

adaptive host response, act as messenger molecules that transmit signals to other cells,

cytokines have many overlapping functions, they interlinked to form an active network,

which controls the host response, and the responses caused by these substances are

varied and interrelated. In general, cytokines control growth, mobility and

differentiation of lymphocytes, but they also exert a similar effect on other leukocytes

and some non-immune cells (Greenwold et al., 2007).

Between the important members of the cytokine group is interleukins

involved in communication between leukocytes and other cells, such as epithelial cells

and fibroblasts engaged in the inflammatory process, they play role in cell-mediated

immune responses (Lindhe et al., 2008).

In other words cytokines are biologically an active nucleus released by

specific cells that elicit a particular response from other cells on which they act,

cytokines are effective in very low concentrations, are produced transiently, act locally

in the tissue where they are produced (Page et at, 1997). It also has systemic effects

(Okada and Murakami, 1998). They are often self-regulatory and able to induce their

own expression in an autocrine (affecting the behavior of the cell that releases the

cytokine) or paracrine fashion (affecting the behavior of adjacent or even distant cells).

Cytokines act on their target cells by binding to specific receptors and

initiating intracellular second messengers resulting in phenotypic changes (physical

characteristics) in the cell via altered gene regulation (Taylor et al., 2004). There is a

significant overlap and redundancy between the function of individual cytokines. They

don't act in isolation, but rather as a complex network, bringing together element of

both innate and adaptive immunity (Banyer et al., 2000).

Cytokines play an important role in a No. of different processes, but if

expressed inappropriately, they also induce pathology. An inflammatory cytokine is

defined as a cytokine, which is induced during the course of an inflammatory response

and is closely associated with its onset and/or progression. IL1α, IL-1 ß, IL-6, IL-8 and


33

TNF-α are generally classified as pro-inflammatory cytokines (Okada and

Murakami, 1998). Pro-inflammatory cytokines enhance the bactericidal capacity of

phagocytes, recruit additional innate cell populations to sites of infection, induce

dendritic cell maturation and direct the ensuing specific immune response to the

invading microbes (Hornef et al., 2002). Anti-inflammatory cytokines block this

process or at least suppress the intensity of the cascade. Cytokines such as IL4, IL-10,

IL-13 and TGF-β suppress the production of IL1, TNF, chemokine such as IL8, and

vascular adhesion molecules (Dinarello, 2000).

Under pathologic conditions such as those that occur in PD, the balance

between pro- and anti-inflammation is directed towards proinflammatory activity

(Graves and Cochran, 2003). In periodontal tissue destruction three proinflammatory

cytokines, IL-1, IL-6 and TNF-α, appear to have a central role (Nikolopoulos et al.,

2008). Some of the effects attributed to cytokines identified in PD are listed in Table

(1-5) (Ranney, 1991).

Table (1-5): Effects attributed to cytokines identified in periodontal disease (Ranney, 1991).

Function induced or facilitated Cytokine

Antigen presentation IL-1

Attachment of PMN and monocytes to epithelium IL-1, TNF

T-cell growth and proliferation IL-2, IL-6, IL-7

TNF production IL-2

B-cell maturation to plasma cell IL-6

Cytoxicity TNF

Metalloproteinase synthesis IL-2, INF-γ

Bone resorption IL-1, IL-6, TNF, INF-γ


34

1.4.1. Interleukin-1β

Interleukin-1 is a key mediator of the host response to various infectious,

inflammatory and immunologic challenges two distinct polypeptides of IL-1 are

found (IL-lα and IL-lβ), which mediate IL-1 biological activities and bind to the same

cell surface receptor (Dinarello, 2000).

The primary sources of IL-1 are blood monocytes and tissue macrophages,

other specialized cells such as T and B-lymphocytes, various epithelial such as mucosal

epithelial cells ,dendritic cells, endothelial, keratinocytes and fibroblasts cells also

produce IL-1, IL-1β represent the major form described in plasma and other biological

fluids (Maruyama et al., 2005; Nikolopoulos et al., 2008). IL-1β is also present in

the saliva of both healthy and diseased individuals (Perrier et al., 2002). IL-1β also

found to be synthesize and release from acinar and ductal cells in mouse salivary

glands, which may be present as components of whole saliva (Tanda et al., 1998; Seil

et al., 2012). Human tear fluid and GCF also contain IL-1β (Solomon et al. 2001;

Miller et al., 2010).

The relationship between salivary and circulating levels of IL-1β is not clear.

Studies involving antigenic challenge by injection of LPS in mice produced increases

in salivary levels of IL-1β similar to that seen in the circulation in response to LPS

challenge (Yao et al., 2005; Yao et al., 2006; Reinhardt et al., 2012). Another study

that examined both plasma and salivary cytokine responses to LPS injections in pigs

found that plasma levels of TNF-α, but not IL-1β, were increased following injection

of a low dose of LPS. Levels of both cytokines found to be elevated in the pigs’ saliva,

which were higher than those found in plasma (Kakizaki et al., 1999). IL-1β levels are

generally higher in saliva than in plasma or serum, and serum/plasma levels are often

below the limit of detection (Llamas Moya et al., 2006; Brailo et al., 2012; Aliefendic

et al., 2013). One study has reported that the correlation between human passive drool

saliva samples and plasma for IL-1β isn't statistically significant (Williamson et al.,

2012).


35

Interleukin-1 has essential role in T-cell activation, providing one of the

necessary signals for IL-2 production, it's the main mediator of inflammatory processes

by acting on the nervous system (fever, sleep, anorexia), bone marrow derived cells

(chemotaxis and or activation of neutrophils, monocyte and lymphocyte) and on

various tissues (fibroblast proliferation, resorption of cartilage and bone matrices and

stimulation of endothelial cell) (Gourin, 1997; Dinarello, 2009). Most of these

activities are directly attributable to IL-β, but others are mediate in collaboration with

other cytokines such as IL6, IFN and TNF. IL-1 stimulates the production or acts

synergistically with these cytokines and the final biological activity in thus the result

of network of interaction between these various mediators and the biological properties

of IL-1 (Aboyoussef et al., 1998).

1.4.1.1. Interleukin-1β regulation of the periodontal ligament

In periodontal tissues, IL-1 known to stimulate the proliferation of

keratinocytes, fibroblasts and endothelial cells to enhance fibroblast synthesis of type

I procollagen, collagenase, hyaluronate, fibronectin and PGE2. IL-1 is therefore a

critical competent in the homeostasis of periodontal tissues, and its unrestricted

production may lead to tissue damage (Okada and Murakami, 1998), and it's also a

potent stimulator of bone resorption (Shirodaria et al., 2000).

Agarwal et al., in 1998 examined how IL-1β regulates periodontal ligament

(PDL) cell functions that are maintain the attachment between the tooth and alveolar

bone and are constantly challenged during infections by bacterial by-products. In

addition, he demonstrated that IL-1β down-regulates or inhibits the osteoblast-like

characteristics of PDL cells (that can grow new bone in periodontal defects under

certain conditions) and become unable to produce TGF-β1 and proteins needed for

bone mineralization, such as alkaline phosphatase and osteocalcin. In other words, IL-

1β changes the phenotype of the PDL cells, so that they are now able to respond to LPS

and subsequently produce proinflammatory cytokines.

Agarwal et al., in 1998 proposed that proinflammatory cytokines regulate

homeostasis in PDL, a role that may be central to maintaining the stability of both the


36

PDL and the host immune response during inflammation. The potent

immunostimulatory agent, LPS and proinflammatory cytokines are the most significant

factors regulating tissue destruction and remodeling.

1.4.1.2. Interleukin-1β and its role in periodontal disease

Interleukin-1β is a proinflammatory cytokine that is consistently associated

with patients of PD. It is assists inflammatory cells at the site of infection, promotes

bone resorption and stimulates the release of MMPs then degrades the extracellular

matrix (Dinarello, 1997; Aboyoussef et al., 1998; Barksby et al., 2007).

Interleukin-1β is considered the most potent osteoclast-activating factor

within the mammalian organism (Nguyen et al., 1991). In addition, it is the most

important single agent that responsible for periodontal bone destruction at sites of

periodontal inflammation (Cavanaugh et al., 1998; Rasmussen et al., 2000). Not only

within the periodontium but also within the whole organism, the margin between IL-1

induced clinical benefit and IL-1 induced clinical destruction and toxicity seems to be

extremely small (Dinarello, 1998). Other destructive inflammatory diseases like

cardiovascular disease, arthritis, colitis and multiple sclerosis have been associated

with exceeding IL-1 concentrations. Even septic shock itself seems to be a consequence

of an endotoxin induced IL-1 overflow and the cascade of bodily responses there by

set off (Quan et al., 2001; Apostolakis et al., 2008; Bhaskar et al., 2011).

Rawlinson et al., in 2000 investigated cytokine IL-1β and its receptor

antagonist (IL-1ra) in GCF, in patients with adult periodontitis. The cytokine levels

were highest for bleeding periodontitis sites, lower for non-bleeding periodontitis sites

and lowest for periodontal healthy sites. IL-1ra levels were highest in the periodontal

healthy sites, lower for non-bleeding periodontitis sites and lowest for bleeding

periodontitis sites. Rawlinson et al. concluded that a significant relationship exists

between the severity of adult periodontitis and the higher GCF levels of IL and lower

levels of IL antagonist.

In addition, patients suffering from periodontitis who are under stressful

conditions have increased levels of IL-6 and IL-1β in GCF (Giannopoulou et al.,


37

2003; Kamma et al., 2004; Johannsen et al., 2007); similarly, patients with

aggressive forms of periodontitis have elevated levels of IL-6 and IL-1β in serum. On

contrary, another study failed to find any correlation between IL-6, IL-1β and cortisol

levels in peripheral blood of aggressive periodontitis patients (Mengel et al., 2002).

Serum level of pro-inflammatory cytokine IL-lβ was significantly higher in

patients with CP as compared with healthy control (Bodet et al., 2006; Guzeldemir et

al., 2011). Moreover, levels of IL-1β in saliva and GCF have been studied in relation

to gingival and PD and significant correlation to the presence of PD has been found

(Miller et al., 2006; Fitzsimmons et al., 2010; Han and Reynolds, 2012; Lee et al.,

2012). In addition, several reports have been demonstrated that a significant correlation

exists between IL-1β levels in GCF and periodontal parameters such as probing pocket

depth and attachment level (Figueredo et al., 1999; Engebretson et al., 2002; Orozco

et al., 2006). In other clinical study conducted by Guzeldemir et al., in 2011 noticed

that increased serum levels of IL-1β in CP patients have been associated with

inflammation in periodontitis and levels decrease after therapy.

Correspondingly, Gorska et al., in 2003 studied twenty-five patients with

CP in Poland, and found that the concentrations of IL-1β were significantly higher in

serum and gingival tissue biopsies samples in those patients as compared to healthy

control. In contrast, other reports mentioned that there were no differences in the

concentrations of IL-1β between CP patients and healthy control (Queiroz et al., 2008;

Yucel et al., 2008; Elkhouli, 2011).

1.4.1.3. Stress-induced interleukin-1β production

Interleukin-1 is a main mediator of the acute phase inflammatory responses

characterized by alteration in metabolic, endocrinlogic and immunologic functions

(Maruyama et al., 2005). IL-1β levels in the circulation change in response to stress.

(Glaser and Kiecolt-Glaser, 2005; Calcagni and Elenkov, 2006). IL-1β is a critical

mediator of adaptive stress responses as well as stress-associated neuropathology and

psychopathology (Goshen and Yirmiya, 2009; Debnath et al., 2011). IL-1β and its

associated signaling systems may play a role in mediating the response of the circadian


38

timing system to immune challenge as well as the basal functioning of the SCN

(suprachiasmatic nuclei of the anterior hypothalamus of the brain) (Beynon and

Coogan, 2010).

Where psychological and immunological stressors activate the IL-1 system

(brain and peripheral) and elevation in circulation by activated cells of the innate

immune system such as monocytes, macrophages and brainmicroglia then IL-1

activates the HPA axis not only during sickness, but also in response to various

psychological stressors. Interestingly, the physiological and behavioral outcomes of

both immune activation and exposure to other stressors are very similar, including

sympathetic and HPA axis activation, fever and a variety of sickness behavior like

symptoms, such as increased sleep and reduced exploratory, social and sexual

behavior. The interaction between IL-1 and the HPA axis is bi-directional. On the one

hand, IL-1 activates the HPA axis and on the other hand, GCs suppress the production

of IL-1 then brain IL-1 is also involved in the feedback regulation of the HPA axis (Fig.

1-5). This regulation is considered to involve inhibitory actions of GCs via GC

receptors (Goshen and Yirmiya, 2009).

The Levels of IL-1β in saliva and GCF have been observed to change in

response to various types of physical and psychological stressors, similar to the

response seen in the circulation (Ilardo et al., 2001; Deinzer et al., 2004; Zefferino

et al., 2006). Where it's accepted that stress and psychological tension can negatively

influence the immune system and function. The manner in which the host immune

system responds to periodontal pathogenic microorganisms appears to play a

significant role in PD. Of late, IL-1ß has become an important focal point for

periodontal researchers such as Deinzer et al., in 2000 found that supragingival plaque

stimulated the secretion of crevicular IL-1ß, and stress further increased the level of

IL-1ß. IL-1ß is consider the most powerful osteoclast-activating factor in humans. It

increases bone resorption and inhibits bone formation. Increased levels of IL-1β have

been noted in areas experiencing periodontal inflammation. Not only do studies show

that it is associated with future loss of alveolar bone, but the IL-1ß levels decrease


39

following periodontal treatment. Thus, it postulated that IL-1ß has a significant

function in the deterioration of periodontal tissues.

The first study to prove the effects of stress on the levels of IL-1β secretion

in GCF was done by Deinzer et al., in 1999. They conducted clinical oral examinations

during and after a major stressful academic examination on students. Experimentally

induced gingivitis was begun 7 days before the last exam. Increased levels of IL-1β

due to stress were noted at gingivitis sites with no oral hygiene for 21 days as well as

the perfect oral hygiene sites. Deinzer et al., in 2000 published another study, with a

modified experimental design to advance their understanding of the relationship

between stress and immune system relative to periodontal research. In this study, the

period of experimental gingivitis began immediately after stress was terminated, rather

than during the stress period as in 1999 study. Thus, the subjects were exposed to stress

before gingivitis and plaque accumulation were experimentally induced. Stress and

gingivitis weren't concurrently present. After termination of the stress, they wanted to

determine if the after-effects of stress on the plaque-induced IL-1ß secretion would still

be measurable. Eighteen medical students took a major academic examination prior to

the inception of the study, and thirteen students who were controls didn't take any

academic examinations 4 weeks before or after the study. No students in this study

smoked. The results revealed that pre-exposure to academic stress did increase levels

of IL-1β secreted at sites where plaque was allow accumulating in the study group, but

it didn't increase IL-1β levels at sites where perfect oral hygiene was maintained in the

control group. The levels of IL-1β secretion in GCF were most obvious 2 weeks after

stressful examinations. By this time, IL levels in control subjects had already achieved

their maximum (Max.), but the levels in the study subjects continued to rise.

In the first study in 1999, they weren't certain if the after-effects of stress

were cause by the exposure to plaque and stress that were both present at the same

time. Their second study confirmed that plaque and stress needn't be present

concomitantly to induce stress aftereffects. Pre-exposure to a protracted period of

academic stress was sufficient in itself to alter the consequent crevicular IL-1β response


40

to microbial plaque. However, the results indicate that a concomitant exposure to stress

and plaque leads to secretion of a larger level of crevicular IL-1β than successive

exposure to stress and plaque. They also established that after termination of stress, the

effects of stress on IL-1β could persist for at least two weeks (Deinzer et al., 2000).

Furthermore, academic stress has been associated with increased levels of

IL-1β, IL-6 and IL-10 in serum and GCF (Maes et al., 1998; Paik et al., 2000;

Waschul et al., 2003), but the data regarding IL-1β alterations have been inconsistent

with (Dugue et al., 1993; Lacey et al., 2000), Marques-Deak et al., in 2007 and

Johannsen et al., in 2010 reported similarities between IL-1β and IL-6 in both stressed

and non-stressed students.

Fig. 1-5: Interleukin 1 mediates stress-induced activation of the hypothalamic pituitary adrenal

axis. Immunological and psychological stressors increase the levels of interleukin 1 (Goshen and

Yirmiya, 2009).

Chapter Two

Materials and Methods

Materials and Methods Chapter Two

41

Materials and Methods

2.1. Materials

2.1.1. Instruments and Equipment

1. Dental mirrors.

2. Periodontal probes (the University of Michigan O probe, with William’s

markings at (1, 2, 3, 5, 7, 8, 9 and 10mm).

3. Dental tweezers

4. Cotton wool.

5. Gloves and masks.

6. Kidney dish.

7. Pan for sterilized instruments and sterilizer.

8. Disposable test tubes.

9. Case sheets form.

10. Disposable plastic cups.

11. Cooling box and freezer.

12. Marker pen.

13. Adjustable micro pipettes (Fig. 2-2).

14. Pipettes yellow tips (Fig. 2-2).

15. Adjustable micro multichannel pipettes (Fig. 2-2).

16. Water bath (Grant Instruments LTD, Barrington, England).

17. Centrifuge (Kokusan Corporation –Japan) (Fig. 2-3).

18. Filter papers.

19. Test tube racks.

20. Can tubes (Fig. 2-2).

21. Auto vortex mixer (Frost instrument LTD, Great Britain) (Fig. 2-4).

22. Rotatest shaker (R 100 Luck ham, England) (Fig. 2-5).

23. Microplate ELISA washer device (Human, Germany) (Fig. 2-6).


42

24. Microplate ELISA reader device (Human, Germany) (Fig. 2-8).

25. Digital timer.

2.1.2. Test Kit

Test kit, salivary IL-1β ELISA kit for research use only; for quantitative

determination of IL-1β in saliva from Salimetrics, USA. Catalog No. 1-3902, 96-well

kit order No. 1302502. Expiry date: 12.Oct.2013 (Fig. 2-1).

Materials Supplied with IL-1β kit are:

1. IL-1β microtitre plate with 96 mouse IL-1β antibodies coated wells.

2. IL-1β standard in a saliva-like matrix 200 pg/ml.

3. IL-1β controls high and low.

4. Wash buffer concentrate.

5. IL-1β assay diluent ready to use.

6. IL-1β sample diluent ready to use.

7. IL-1β antibody conjugate: biotin conjugated to antibody to human IL-1β.

8. Streptavidin conjugated to horseradish peroxidase (HRP).

9. Tetramethylbenzidine (TMB) substrate solution (non-toxic).

10. Three molar stop solution contains sulfuric acid (caustic).

2.2. Methods

2.2.1. Sample description

The original sample was consisted of fifty-four dental students, twenty-three

male and thirty-one female aged (21-23) years were randomly recruited in this follow

up study. They were fifth class students from the collage dentistry in University of

Baghdad and Almustansiriyah. Eleven male and nineteen female were excluded from

this study in the second and third periods because they were not fit for the criteria of

study. Therefore, the final sample was twenty-four students, twelve male and twelve

female continue to follow in this study at three main periods.

The students enrolled voluntarily in the study after a well explanation of

purpose of the investigation and consented to its protocol in period from November

2012 to March 2013.


43

All students in this study were systemically healthy, males and females in

the fifth class, cooperative, nonsmoker and not taking any antibiotics during the last

three months (Johannsen et al., 2010).

2.2.2. Exclusion criteria

Pregnant and in menstrual cycle women.

Any student had history of chronic systemic diseases with known associations

with PD (e.g. cardiovascular diseases and diabetes mellitus).

Any student taking psychotropic medication (e.g. prednisone).

Any student with retentive factor of plaque (e.g. orthodontic appliance and

partial denture).

2.2.3. Study design

In this follow up study, clinical periodontal parameters were measured and

stress questionnaire were recorded at three main periods:

1. The first period at least one month before mid-year exam: In this period, the

students were in the normal range of stress scale of DASS criteria and all students

given motivation and instructions about brushing technique and the use of dental

aids to reduce accumulation of dental plaque and gingival inflammation. This

period represents period I, which used as a base line for this study.

2. The second period during mid-year exam: In this period, the students subjected

to stress, also all students given motivation and instructions about brushing

technique and the use of dental aids to reduce accumulation of dental plaque and

gingival inflammation. This period represents period II.

3. The third period at least one month after mid-year exam: In this period, students

were in the normal range of stress scale of DASS criteria. This period represents

period III.

The students were subjected to questionnaire about name, age, full medical

history, periodontal health status and clinical examination of periodontal parameters in

each period by measuring plaque index (PI), gingival index (GI) and BOP for all teeth

except third molars. Clinical examinations were performed after recording the stress


44

questionnaires that include 7-items of DASS 21-item questionnaire version (a copy of

case sheet and DASS stress questionnaires are provided in appendices).

Two milliliter (ml) of saliva sample was collected from students one month

before mid-year exam period, during mid-year exam and one month after mid-year

exam period and stored at (-70ºc) for later centrifuged and analysis by ELISA for

determination of IL-1β. The laboratory test done in the Teaching Laboratories of

Baghdad Medical City.

2.2.4. Depression Anxiety and Stress Scale

The DASS 21 version was used in this study, which included three self-

report scales designed to measure the negative emotional states of depression, anxiety

and stress. Each of the three scales contains 7 items. Students were answered about

stress questionnaires during the three periods before the examination of clinical

periodontal parameters; Table (2-1). The items of stress scale are sensitive to levels of

chronic non-specific arousal. It assesses difficulty relaxing, nervous arousal, being

easily agitated, over-reactive and impatient.

Respondents were asked to use 4-points of severity scales to rate the extent

to which they have experienced each state over the past week. Scores of stress

calculated by summing the scores for the relevant items (1, 6, 8, 11, 12, 14 and 18).

Then, the sum for stress scores evaluated by the severity-rating index, Table (2-2)

(Lovibond and Lovibond, 1995).

The DASS case sheet include name of subject, date of questionnaire then

instruct the subject to understand each statement and refer a suitable No. either 0, 1, 2

or 3, which indicates how much the statement applied to his or her over the past week.

There are no right or wrong answers and don't spend too much time on any statement.

The rating scale is as follows:

0 Didn't apply to me at all.

1 Applied to me to some degree, or some of the time.

2 Applied to me to a considerable degree, or a good part of time.

3 Applied to me very much, or most of the time.


45

Table (2-1): Stress questionnaires of DASS 21-item version (Lovibond and Lovibond, 1995).

1. I found it hard to wind down 0 1 2 3

2. I tended to over-react to situations 0 1 2 3

3. I felt that I was using a lot of nervous energy 0 1 2 3

4. I found myself getting agitated 0 1 2 3

5. I found it difficult to relax 0 1 2 3

6. I was intolerant of anything that kept me from

getting on with what I was doing

0

1

2

3

7. I felt that I was rather touchy 0 1 2 3

Table (2-2): DASS severity-rating index (Lovibond and Lovibond, 1995).

2.3. Clinical periodontal parameters examination

Clinical Periodontal examination was performed by using the University of

Michigan O probe according to the following criteria:

2.3.1. Plaque index

The four surfaces of each tooth except 3rd molar were examined and scored

according to PI by (Silness and Loe, 1964).

The scores and criteria for this system as proposed by authors:

Score 0: No plaque in the gingival area.


46

Score 1: A film of plaque adhering to the free gingival margin and adjacent area

of the tooth surface, the plaque may be recognized only by running a probe

across the tooth surface.

Score 2: Moderate accumulation of soft deposits within the gingival pocket, on

the gingival margin and or adjacent tooth surface, which can be seen by naked

eye.

Score 3: Abundance of soft matter within the gingival pocket and or on the

gingival margin and adjacent tooth surface.

2.3.2. Gingival index

The gingival inflammation at the four surfaces of each tooth except 3rd molar

was assessed using the criteria of the GI (Loe and Silness, 1963). The criteria of this

index then modified by Loe in 1967 as the following:

Score 0: Absence of inflammation/normal gingival.

Score 1: Mild inflammation, slight change in color, slight edema, no BOP.

Score 2: Moderate inflammation, moderate redness, glazing and edema, BOP.

Score 3: Sever inflammation, marked redness and ulceration, tendency to

spontaneous bleeding.

2.3.3. Bleeding on probing

A periodontal probe was inserted to the bottom of the periodontal pocket and

was moved gently along the tooth (root) surface. If bleeding occurs within 30 seconds

after probing, the site was given as positive score (1) and a negative score (0) for the

non-bleeding site (Carranza et al., 2006).

2.4. Saliva sample collection and preparation

The students were instructed not eat or drink (except water) for at least 60

minutes before collection of the samples. Acidic or high sugar foods can compromise

assay performance by lowering saliva PH and influencing bacterial growth. To

minimize the effect of these factors, the student was rinsed his/her mouth thoroughly

with water then waited 2 minutes for water clearance before the sample was collected.


47

The subject was sat in a relax position then the whole unstimulated mixed

saliva was collected by tilting the head forward, allowing the saliva to pool on the floor

of the mouth, and then drop his/her saliva into the polyethylene tube until 2ml was

collected, a standardized method according to (Thylstrup and Fejerskov, 1994).

The tube was labeled with the No. of the subject that was previously written

on the case sheet and record the time and date of sample collection. Samples visibly

contaminated with blood were discarded (Schwartz and Granger, 2003; Kivlighan

et al., 2004). Saliva was collected between 8-12 a.m. and before clinical periodontal

examination.

After sample collection, it was put in a small cooling box to keep sample

cold within 30 minutes and freezing at (-70ºC) within 4 hours of collection until

assayed, in order to prevent bacterial growth and minimize loss of IL-1β in the sample.

Freezing saliva samples were precipitate mucins. On day of assay, thaw

completely by putting in the water bath at 37ºC then centrifuge at 3000 rpm for 15

minutes. Centrifuging removes mucins and other particulate matter, which may

interfere with antibody binding, leading to false elevated results, the clear supernatant

was separated by micropipette into can tubes then all samples were diluted 15 times in

IL-1β sample diluent before adding to assay plate.

2.5. Detection of IL-1β

2.5.1. Test principle

The ELISA test kit provides quantitative in vitro assay for the determination

of IL-1β in saliva. The test kit contains a microtitre plate that was coated with mouse

antibodies to IL-1β. IL-1β in standards and unknowns attach to the antibody binding

sites. After incubation, unbound components were washed away. Biotin conjugated to

goat antibodies to human IL-1β were added and attached to the bound IL-1β. After

incubation, unbound components were washed away. Streptavidin conjugated to HRP

was added and binds to the biotin conjugated to the goat antibodies. Bound

streptavidin-HRP was measured by the reaction of the HRP enzyme on the substrate

TMB. This reaction produces a blue color. A yellow color was formed after stopping


48

the reaction with 3-molar sulfuric acid (Fig. 2-7). Optical density (OD) was read on a

standard plate reader at 450 nm. The amount of streptavidin-HRP detected is

proportional to the amount of IL-1β present (Chard, 1990).

2.5.2. Reagent preparation

All reagents and microtitre plate were bring in sealed foil pouch to room

temperature. A minimum of 1.5 hours is for the 14ml of IL-1β assay diluent

used in steps 7 and 10 to come to room temperature. It was important to keep

the foil pouch with the plate strips closed until warmed to room temperature (as

humidity may have an effect on the coated wells).

All reagents were mixed before use.

Each control vial was reconstituted with 1.0ml of deionized water then let it to

sit 20 minutes at room temperature before using. It was mixed well immediately

before use.

IL-1β standard was reconstitute with deionized water according to the volume

on the standard vial label then let it to sit 20 minutes at room temperature before

using. It was mixed well immediately before use.

Prepared serial dilutions of the IL-1β standard as follows:

1. Labeled 6 can tubes 2 through 7.

2. 300μL of IL-1β assay diluent was pipetted into tubes 2 through 7.

3. Serially diluted the IL-1β standard by adding 300μL of the 200pg/ml standard

(tube 1) to tube 2 and mixed well. After changing pipette tips, removed 300μL

from tube 2 to tube 3 and mixed well then continue for tubes 4, 5, 6 and 7.

The final concentrations of standards for tubes 1 through 7, respectively, were

200pg/ml, 100pg/ml, 50pg/ml, 25pg/ml, 12.5pg/ml, 6.25pg/ml and

3.13pg/ml. Standard concentrations in pmol/L were 11.8, 6.0, 3.0, 1.5, 0.7,

0.4 and 0.2.

Prepared the wash buffer solution by diluting wash buffer concentrate 10-fold

with room-temperature deionized water (100ml of wash buffer concentrate to

900ml of deionized water).


49

2.5.3. Procedure

Step 1: The plate layout was determined.

Step 2: 14ml of IL-1β assay diluent was pipette into each of two different disposable

tubes and cap. They were set aside for 7 and 10 steps.

Step 3: Saliva was diluted 15 times in IL-1β sample diluent by using 20μL of saliva to

280μL of IL-1β sample diluent.

Step 4:

• 100μL of each standard, control and unknown (diluted) sample was pipette into

appropriate wells. Standards, controls and unknown samples were assayed in duplicate.

• 100μL of IL-1β assay diluent was pipetted into the duplicate zero wells.

Step 5: The plate was covered with adhesive cover provided then it was incubated at

room temperature for 1 hour on a horizontal shaker set at 500 rpm.

Step 6: The plate was washed 4 times with wash buffer solution; washing was done by

pipetting 300μL of wash buffer into each well and then decanting the liquid into a sink.

After each wash, the plate was blotted on paper towels but didn't let the wells dry out.

Step 7: The antibody conjugate was diluted 1:500 by adding 28μL of the antibody

conjugate to the 14ml of IL-1β assay diluent prepared in step 2 (antibody conjugate

tube was centrifuged for a few minutes to bring the liquid down to the tube bottom).

Immediately mixed the diluted antibody solution and added 100μL to each well by

using a multichannel pipette.

Step 8: The plate was covered with adhesive cover provided then it was incubated at

room temperature for 2 hour on a horizontal shaker set at 500 rpm.

Step 9: Repeated the washing procedure at step 6.

Step 10: The streptavidin-HRP was diluted 1:200 by adding 70μL of the streptavidin-

HRP to the 14ml of IL-1β assay diluent prepared in step 2 (Streptavidin-HRP tube was

centrifuged for a few minutes to bring the liquid down to the tube bottom). Immediately

mixed the diluted solution and added 100μL to each well using a multichannel pipette.

Step 11: The microtiter plate was incubated at room temperature for 20 minutes and

mixed constantly at 500 rpm.


50

Step 12: Repeated wash procedure from step 6.

Step 13: Added 100μL of TMB solution to each well with a multichannel pipette.

Step 14: The plate was incubated in the dark at room temperature for 20 minutes on a

horizontal shaker set at 500 rpm and avoided direct sunlight.

Step 15: Added 50μL of stop solution with a multichannel pipette into each well.

Step 16:

• The plate was mixed at room temperature for 3minutes on a horizontal shaker that set

at 500 rpm. All wells were turned yellow.

• The absorbencies were read in a plate reader at 450nm within 10 minutes of adding

stop solution. (It was corrected at 630nm).

2.5.4. Calculations of IL-1β

1. The average OD was computed for all duplicate wells.

2. The concentrations of the controls and unknowns were determined by using a

quadratic parameter curve fit of software program (Fig. 2-9).

3. All results for unknown samples were multiplied by the dilution factor of 15.

2.6. Calibration

The purpose of calibration, training and testing the design of the study,

calibration was carried out about three weeks before the conduction of the actual work,

for accurate calibration of the examiner for the clinical periodontal parameters was

assessed by:

(1) Inter examiner calibration: Four sites of all teeth except third molars were examined

by the examiner and supervisor for five subjects.

(2) Intra examiner calibration: Four sites of all teeth except third molars were examined

twice by the same examiner with interval of one week between the two examinations.

Measurements obtained by the inter- and intra-examiner calibrations were

computed and the results were with non-significant (NS) differences (P ≥ 0.05), when

student t-test was applied.


51

2.7. Statistical analysis

Data were processed and analyzed using (IBM® SPSS® Statistics version 21,

2012). Both descriptive and inferential analysis were used in order to analyze and

assess the results of the study.

1. Descriptive statistics:

a) Tables (Frequencies and percentages).

b) Ratio

c) Arithmetic mean (Mean).

d) Standard deviation (SD).

e) Standard error (SE).

f) Two extreme values (Min. and Max.).

g) Graphical presentation by Bar charts.

2. Inferential statistics:

These used to accept or reject the statistical hypotheses, which included:

a) Chi-square test.

b) student t-test

c) Analysis of variance (ANOVA) with least significant differences (LSD).

d) Pearson's correlations coefficient (R) was used for testing the correlation

between the two independent variables: the clinical and immunological

parameters.

The level of NS was P ≥ 0.05, the level of significant (S*) was at P ≤ 0.05,

the level of highly significant (HS**) when P ≤ 0.01.


52

Fig. 2-1: Salimetrics salivary interleukin-1β kit.

Fig. 2-2: A. Adjustable micropipettes and pipettes yellow tips.

B. Adjustable micro multichannel pipettes.

Fig. 2-3: Centrifuge (Kokusan Corporation–Japan).


53

Fig. 2-4: Auto vortex mixer (Frost instrument LTD, Great Britain).

Fig. 2-5: A. Rotatest shaker (R 100 Luck ham, England).

B. Microtitre plate of interleukin-1β kit.

Fig. 2-6: Microplate ELISA washer device (Human, Germany).


54

Fig. 2-7: Microtitre plate of interleukin-1β kit after adding stopper solution.

Fig. 2-8: Microplate ELISA reader device (Human, Germany).


55

Fig. 2-9: Quadratic parameter curve fit of software program.

Chapter Three

Results

Results Chapter Three

56

Results

3.1. Descriptive statistical analysis of demographic data

During this study, twenty-four dental students were involved; they were

continuous to followed up during three main periods (1st period/before examination

was called period I, 2nd period/during examination was called period II, 3rd period/after

examination was called period III). The ages of students ranged between (21-23) years

with a mean age of 22 and SD of 0.624 as shown in table (3-1). Furthermore, 50% of

students were female and 50% of students were male. Female to male ratio in this study

was 1:1.

Table (3-1): Descriptive statistical results of students' ages.

Number Min. Max. Mean SD

Age 24 21 23 22 0.62409

3.2. Clinical periodontal parameters analysis

3.2.1. Plaque index

The descriptive statistical results of PLI for each period were shown in table

(3-2); it was shown that the mean of PLI was elevated in period II in comparison with

I and III periods as shown in figure (3-1). Mean ± SD of PLI were 1.0283 ± 0.24701

in period II, while mean ± SD of PLI were 0.6375 ± 0.25674 and 0.2300 ± 0.22733

respectively in periods I and III.

Table (3-2): Descriptive statistical results of plaque index for each period.

Periods No. Min. Max. Mean SD SE

Period I 24 0.23 1.14 0.6375 0.25674 0.05241

Period II 24 0.56 1.40 1.0283 0.24701 0.05042

Period III 24 0.01 0.73 0.2300 0.22733 0.04640


57

Fig. 3-1: Bar chart graph for means of plaque index for each period.

For comparisons among periods, ANOVA test was used in this study; the

results showed that there were HS difference at P-value ≤ 0.01 among and within

periods, Table (3-3).

Least significant difference test (LSD) was performed for multiple

comparisons between each two periods; the results showed that there were HS

differences at P-value ≤ 0.01, Table (3-4).

Table (3-3): ANOVA test for plaque index.

ANOVA Sum of Squares

(SS)

Degree of

freedom

(df)

Mean Square

(MS)

F-test P-value Sig.

Among periods 7.649 2 3.825

64.240

0.000

**

Within periods 4.108 69 0.060

Total 11.757 71

P ≤ 0.01 High significant (HS) **


58

Table (3-4): LSD test to compare the means of plaque index between each two periods.



The descriptive statistical results of GI for each period were shown in table

(3-5); it was shown that the mean of GI was elevated in the period II in comparison

with the periods I and III as shown in figure (3-2). Where mean ± SD of GI were 0.6004

± 0.27085 in the period II, while mean ± SD of GI were 0.2029 ± 0.16843 and 0.1054±

0.08444 respectively in the periods I and III.

Table (3-5): Descriptive statistical results of gingival index for each period.

Periods Mean Difference (MD) SE P- value Sig.

Period I

Period II -0.39083 0.07044 0.000 **

Period III 0.40750 0.07044 0.000 **

Period II Period III 0.79833 0.07044 0.000 **

SE SD Mean Max. Min. No. Periods

0.03438 0.16843 0.2029 0.56 0.03 24 Period I

0.05529 0.27085 0.6004 1.17 0.20 24 Period II

0.01724 0.08444 0.1054 0.31 0.02 24 Period III


59

Fig. 3-2: Bar chart graph for means of gingival index for each period.

For comparison among periods, ANOVA test was used in this study; the

results showed that there were HS difference at P-value ≤ 0.01 among and within


For multiple comparisons between each two periods, LSD test was

performed. The results showed that there were HS differences at P-value ≤ 0.01

between periods I and II; II and III, while there was NS difference at P-value ≥ 0.05

between periods I and III as shown in Table (3-7).

Table (3-6): ANOVA test for gingival index.

ANOVA SS df MS F-test P-value Sig.

Among periods 3.300 2 1.650

45.475

0.000

** Within periods 2.504 69 0.036

Total 5.804 71



60

Table (3-7): LSD test to compare the means of gingival index between each two periods.

P ≥ 0.05 Non-significant (NS)



Descriptive statistical results for BOP were described in table (3-8); it was

clearly shown that the No. and percentage of bleeding sites in the period II was higher

than the periods I and III.

Where in the period II the No. and percentage of healthy sites were 2508

(93.3 %), whereas the bleeding sites were 180 (6.7 %). While in the period I the No.

and percentage of healthy sites were 2624 (97.6 %), whereas the bleeding sites were

64 (2.4 %). While in the period III the No. and percentage of healthy sites were 2657

(98.8 %), whereas the bleeding sites were 31 (1.2 %) as shown in figure (3-3).

For comparison of all periods, chi-square test was used; the results showed

that there was HS difference at P-value ≤ 0.01 among periods as shown in table (3-8).

Inter-period comparisons for BOP was shown in table (3-9); the results showed that

there were HS differences at P-value ≤ 0.01 of the comparison between each two

periods.

Periods MD SE P-value Sig.

Period I

Period II -0.39750 0.05499 0.000 **

Period III 0.09750 0.05499 0.081 NS



61

Table (3-8): Percentages and numbers of scores of bleeding on probing for each period and

comparison among periods.

Scores of BOP

Period I Period II Period III

No. % No. % No. % Chi-

square

df P-value Sig.

0 (no bleeding) 2624 97.6% 2508 93.3% 2657 98.8%

138.339

2

0.000

**

1(bleeding sites) 64 2.4% 180 6.7% 31 1.2%


Table (3-9): Inter-period comparisons for bleeding on probing between each two periods.

Sig. P-value df Chi-square Periods

** 0.000 1 57.770 Between period I and II

** 0.001 1 11.669 Between period I and III

** 0.000 1 109.516 Between period II and III


Fig. 3-3: Bar chart graph for percentages of scores of bleeding on probing for each period.


62

3.3. Stress analysis

Descriptive statistical results for stress were shown in table (3-10); it was

clearly appeared that all students in the period II were within mild to severe range of

stress, while in the period I and III were within normal range of stress.

In the period II, the Nos. and percentages of students in the mild, moderate

and sever ranges were 4 students (16.7 %), 15 students (62.5 %) and 5 students (20.8

%) respectively. Whereas in the periods I and III, Nos. and percentages of students in

normal range were 24 students (100 %) while in the other ranges were zero (0.0 %).

For comparison among periods, chi-square test was used; the results showed

that there was HS difference at P-value ≤ 0.01 among periods as shown in table (3-10).

Table (3-10): Percentage and number of stress range and comparison among periods.


Scores of

Stress

Period I Period II Period III

No % No % No % Chi-square df P-value Sig.

Normal

(0-14)

24 100.0% 0 0.0% 24 100.0%

72.000

6

0.000

**

Mild

(15-18)

0 0.0% 4 16.7% 0 0.0%

Moderate

(19-25)

0 0.0% 15 62.5% 0 0.0%

Sever

(26-33)

0 0.0% 5 20.8% 0 0.0%

Extremely

sever (34)

0 0.0% 0 0.0% 0 0.0%


63

3.4. Salivary interleukin-1β statistical analysis

The descriptive statistical results of IL-1β for each period were shown in

table (3-11); it was shown that the mean of IL1β was elevated in the period II in

comparison with the periods I and III as shown in figure (3-4). Where mean ± SD of

IL-1β were 552.5669 ± 275.62299 pg/ml in the period II, while mean ± SD of IL-1β

were 269.9206 ± 158.79005 pg/ml and 114.0194 ± 72.51898 pg/ml respectively in the

periods I and III.

Table (3-11): Descriptive statistical results of interleukin-1β for each period.

Fig. 3-4: Bar chart graph for means of interleukin-1β for each period.

SE SD Mean Max. Min. No. Periods

32.41288 158.79005 269.9206 674.85 60.39 24 Period I

56.26131 275.62299 552.5669 1228.07 122.04 24 Period II

14.80287 72.51898 114.0194 263.96 39.72 24 Period III


64

For comparison among periods, ANOVA test was used in this study; the

results were showed that there was HS difference at P-value ≤ 0.01 among and within


Least significant difference test (LSD) was performed for multiple

comparisons between each two periods; the results showed that there were HS

differences at P-value ≤ 0.01, Table (3-13).

Table (3-12): ANOVA test for interleukin-1β level.

ANOVA SS df MS F P-value Sig.

Among periods 2372144.097 2 1186072.049

33.429

0.000

** Within periods 2448150.213 69 35480.438

Total 4820294.310 71


Table (3-13): LSD to compare the means of interleukin-1β between each two periods.

Periods

MD SE P- value Sig.

Period I

Period II -282.64625 54.37557 0.000 **

Period III 155.90125 54.37557 0.005 **




65

3.5 Correlations between clinical periodontal parameters with

immunological marker (salivary interleukin-1β) and stress

Pearson's correlation coefficient (R) of immunological marker (salivary IL-

1β) with clinical periodontal parameters (PLI, GI and BOP) and stress were strong

(positive) correlations and HS differences at P-value ≤ 0.01 in all parameters, except

BOP score (0) the correlation was inverse (negative) correlation and NS difference at

P-value ≥ 0.05 as shown in table (3-14) and figure (3-5).

Table (3-14): Pearson's correlation coefficient of salivary interleukin-1β with plaque index,

gingival index, bleeding on probing score (1 and 0) and stress level among periods.

Salivary IL-1β

R P-value Sig.

PLI 0.543 0.000 **

GI 0.504 0.000 **

BOP score (1) 0.411 0.000 **

BOP score (0) -0.090 0.450 NS

Stress 0.661 0.000 **

P ≥ 0.05 Non-significant (NS) P ≤ 0.01 High significant (HS) **

Fig. 3-5: Bar chart graph for correlations of interleukin-1β with (plaque index, gingival

index, bleeding on probing score (1)) and stress.


66

Pearson's correlation coefficient of stress with immunological parameter

(salivary IL-1β) and clinical periodontal parameters (PLI, GI and BOP) were strong

(positive) correlations and HS differences at P-value ≤0.01 in all parameters, except

BOP score (0) the correlation was inverse (negative) correlation and NS difference at

P-value ≥ 0.05 as shown in table (3-15) and figure (3-6).

Table (3-15): Pearson's correlation coefficient of stress with salivary interleukin-1β and

plaque index, gingival index, bleeding on probing score (1 and 0) among periods.

P ≥ 0.05 Non-significant (NS) P ≤ 0.01 High significant (HS) **

Fig. 3-6: Bar chart graph for correlations of stress with (plaque index, gingival index,

bleeding on probing score (1)) and interleukin-1β.

Stress

R P-value Sig.

PLI 0.715 0.000 **

GI 0.684 0.000 **

BOP score (1) 0.545 0.000 **

BOP score (0) -0.016 0.450 NS

Salivary IL-1β 0.661 0.000 **

Chapter Four

Discussion

Discussion Chapter Four

67

Discussion

Periodontal disease is a multifactorial disease and research promoting the

identification of risk factors are gaining importance for treatment and prevention. It's

suggested that stress, depression and ineffective coping may contribute to development

of PD (Rosania et al., 2009). Stress is term refers to physiological and psychological

reactions that mobilize an organism’s defense against external or internal threats to

stressors. Different kinds of stress have been defined, such as work related, negative

life experiences and socioeconomic status (Genco et al., 1999; Mead et al., 2001;

Soares et al., 2007). Chronic stress may tend to have a negative effect on

immunological response of body, which represents an important example of mind-

body interaction that leading to an imbalance between host and parasites and further

resulting in periodontal breakdown (Reners and Brecx, 2007; Johannsen et al.,

2010).

Interleukin-1 is one of the most critical molecules involved in

neuroendocrine and neurobehavioral stress responses (Goshen and Yirmiya,

2009). In addition, IL-1 is important pro-inflammatory cytokine in pathogenesis of PD

(Dayan et al., 2004). Therefore, the current study is the first of its kind in Iraq that

reflect the association between examination stress, salivary IL-1β and periodontal

health status.

4.1. Demographic data

The results of this study involving dental students because recent studies

have reported high levels of stress among dental and medical students (Omigbodun et

al., 2006; Smith et al., 2007; Johannsen et al., 2010), aged 21-23 years of the fifth

class, we selected this class because final class represent the more stress class, they

follow up in three main periods:

1. The first period (period I) before mid-year exam: It was used as a base line for this

study to assess periodontal health status and salivary IL-1β at least one month before


68

mid-year exam to avoid the effect of examination stress on students because an

academic exam can be considered a psychological stressor, consisting of a period

of preparation, anticipation and then the exam itself (Deinzer et al., 1999;

Johannsen et al., 2010).

2. The second period (period II) during mid-year exam: in this period the students

during the stress at the third weeks of examination because IL-1β reach to the peak

level with gingival inflammation at this time (Deinzer et al., 1999). Students who

participated in a major exam had significantly more dental plaque and more gingival

inflammation compared with students who didn't participate in any exam (Deinzer

et al., 2005; Johannsen et al., 2010). Furthermore, academic stress has been

associated with increased levels of IL-1β, IL-6 and IL-10 in serum and GCF (Maes

et al., 1998; Paik et al., 2000; Waschul et al., 2003).

3. The third period (period III) at least one month after mid-year exam to avoid any

after-effects of stress on periodontal health status and IL-1β (Deinzer et al., 2000;

Johannsen et al., 2010).

4.2. Periodontal health status

4.2.1. Plaque index

The mean value of PLI of period II was significantly higher than that of

periods I and III. This result could be related to the effect of stress to achieve good oral

hygiene among the second period. The changes occurring in behavioral of stressful

students, as inattention leads to improper oral hygiene mechanism, have been

considered as a reason for association between stress and gingivitis.

Over the past decade, it has become more apparent that stress can negatively

influence oral health status, which can lead to increased amounts of dental plaque,

gingival inflammation and more severe periodontitis (Klages et al., 2005; Johannsen

et al., 2007). In the present study, students were found to have more plaque

accumulation and gingival inflammation during an exam period, suggesting that

examination stress might influence periodontal health status. This result is in


69

conformity with Deinzer et al., in 2001; Deinzer et al., in 2005 and Johannsen et al.,

in 2010, who found increased dental plaque and gingival inflammation in students who

experienced examination stress. The increased levels of dental plaque and gingival

inflammation in the present study may be explained by behavioral changes in the

stressed students, for example, oral hygiene might be less effective and ⁄ or reduced in

frequency during this time of stress. After the exam period, a reduced amount of dental

plaque was found and this may partly be explained by the Hawthorne effect (Adair,

1984), meaning that panelists involved in clinical trials might be affected because of

attention and interest.


There is a significant difference in the mean values of GI among periods.

This elevation of GI reflects a higher inflammation in the period II than periods I and

III. This result is agrees with Deinzer et al., in 1999; Deinzer et al., in 2005 and

Johannsen et al., in 2010.

Regarding gingival inflammation during examination stress, two possible

explanations may be considered, either by the direct influence of stress on immune

system (biologic model), through release of stress hormones or by an influence of

increase plaque accumulation during the exam period leading to gingival inflammation,

as the plaque is the causative factor of gingival inflammation (behavioral model), both

resulting in increased susceptibility to PD (Genco et al., 1998; Boyapati and Wang,

2007).


The percentage of sites with BOP was significantly higher in period II than

periods I and III. The potential altered abilities of period II to perform effective oral

hygiene could result in an increased BOP that exacerbates the risk for enhanced tissue

destruction in PD. Moreover, interesting observations regarding the complexity of the

oral and systemic challenge provide unique mechanisms by which dysregulation of

host responses could occur due to immunologic and behavioral changes, related to

examination stress may be lead to PD. Where examination stress appears to affect


70

periodontal health status, shown by more plaque accumulation, gingival inflammation

and increased amounts of proinflammatory cytokine salivary IL-1β therefore

examination stress appears as a possible risk factor for gingivitis and periodontitis. This

result is in agreement with Deinzer et al., in 1999; Deinzer et al., in 2005 and

Johannsen et al., in 2010.

4.3. Stress data

The students registered their perceived stress on DASS-21. The DASS is a

simple-to-administer, reliable and a valid measurement tool for evaluating stress

(Lovibond and Lovibond, 1995). The DASS summarized magnitude of psychological

derangement by summing the scores obtained from stress domain only from this scale,

that ranged from no stress at all, to the extremely sever stress imagined. Where students

were asked how stressed they felt on a 4-points scale before, during and after mid-year

exam to compare between them. According to the results of this study, the percentage

of stress was significantly higher in period II than period I and III. Where the DASS

scores were significantly higher during the exam period compared with before and after

the mid-year exam.

This result is agrees with Johannsen et al., in 2010, which used a visual

analogue scale (VAS) to register the perceived stress of students during the exam

period compared with after the exams, Michael et al., in 2011, which used DASS to

psychological distress in diurnal variations, Singh et al., in 2012, which used DASS in

measuring stress and it's effect on cortisol level in medical student and Premkumar et

al., in 2013, which used DASS in measuring changes in mood and assessed the

correlation with cortisol. The proper explanation for this result was dental students who

participated in the exam had significantly more stress compared with students who

didn't participate in any exam.


71

4.4. Salivary interleukin-1β level

Salivary IL-1β was used in this study instead of serum IL-1β because saliva

has been used as a diagnostic biofluid to measure host responses to a variety of

triggering factors in systemic and oral diseases (Forde et al., 2006). Saliva analyses

have advantages of quick and easy sample collection not requiring specialized

equipment or personnel. Moreover, its sampling is painless and noninvasive, therefore,

saliva sampling doesn’t cause stress to students. It's one of the most promising

mediums for its diagnostic potential for various diseases including stress-related

diseases, and it's readily available any time and for repeated samplings (Wolff et al.,

2004). If the relationship between stress and PD can be verified in saliva samples,

diagnosing stress by means of saliva analysis can be helpful for differential diagnosis

from the evidence-based point of view in various medical fields (Akcali et al., 2013).

In addition, IL-1β levels are generally higher in saliva than in plasma or serum, and

serum/plasma levels are often below the limit of detection (Llamas Moya et al., 2006;

Brailo et al., 2012; Aliefendic et al., 2013).

Interleukin-1 is important pro-inflammatory cytokine in the pathogenesis

of PD (Dayan et al., 2004). It induces widespread gene expression of

cyclooxygenase-2, inducible nitric oxide synthesis and MMP, which results in

activation of osteoclasts, bone resorption and downregulation of type I collagen

expression in bone (Okada and Murakami, 1998; Graves and Cochran, 2003).

Although both isoforms of IL-1 (1L-1α and IL-1β) have similar biological activities

and appear to be contributory, but IL-lβ is more potent in stimulating bone

resorption and is the form more frequently occurring in periodontitis (Stashenko et

al., 1987). In addition IL-1β is a critical mediator of adaptive stress responses as well

as stress-associated neuropathology and psychopathology (Goshen and Yirmiya,

2009; Debnath et al., 2011). For all these reasons, salivary IL-1β was used in this

study.


72

In the present study, salivary level of IL-1β inflammatory related cytokine

was assessed; the mean value of IL-1β of period II was significantly higher than that

of the period I and III. Where IL-1β stimulates the HPA axis activity and associated

with immune system and inflammation response during stress (Goshen and Yirmiya,

2009).

This result is agrees with Deinzer et al., in 1999 and Deinzer et al., in 2000,

who found higher amounts of IL-1β in GCF during academic examination stress and

Brydon et al., in 2005, who found higher amounts of IL-1β in human mononuclear

cells during psychological stress and disagrees with Marques-Deak et al., in 2007,

who reported similarities of IL-1β level in both stressed and non-stressed individuals

and Johannsen et al., in 2010, who reported non-significant difference of IL-1β level

between during and after exam. One problem in stress studies in general could be the

difficulty to know when the influence on the biomarkers by a stress period is over. An

explanation to why Marques-Deak et al., in 2007 and Johannsen et al., in 2010 didn't

find high IL-1β levels in GCF, despite a high degree of inflammation could be that

hormonal stress inhibits IL-1β response to stress (feedback regulation of IL-1β)

(Williams and Yarwood, 1990; Chrousos, 1995; Genco et al., 1998; Nguyen et al.,

2000). In addition, some studies have shown that the levels of pro-inflammatory

cytokine, IL-1β, are increased in patients with depression (Johannsen et al., 2006;

Johannsen et al., 2007; Von Kanel et al., 2007); however, contradictory results have

also been described (Rothermundt et al., 2001).

Interleukin-1β level is a sensitive and reliable marker of chronic

inflammatory disease activity and IL-1β elevation may demonstrate tissue destruction

(Lacopino et al., 1997; Keles et al., 2005). Thus, in this study the detection of elevated

levels of IL-1β in the saliva of subjects with stress was consistent with the cytokine's

role in inflammation and suggests that salivary lL-1β may be a good marker of

periodontal inflammation.


73

4.5. Correlations between stress, interleukin-1β and clinical periodontal

parameters

There was a strong positive correlations between stress, pro-inflammatory

cytokine(IL-1β) and clinical periodontal parameters, when stress increase pro-

inflammatory cytokine IL-1β level, plaque accumulation, gingival inflammation and

bleeding sites will be increase. These results of present study were in agreement with

Maes et al., in 1998; Paik et al., in 2000; Waschul et al., in 2003 where stress alters

immune function and affects different immune cells, hence increase production of pro-

inflammatory cytokine (humoral immunity) and decrease cellular immunity that lead

to periodontal inflammation. In addition, stress was related to PD because

psychological stress can directly affect periodontal health status by various biological

(physiological) mechanisms; also, it can have indirect effects through the behavioral

(psychological) changes in lifestyle such as ignoring oral-hygiene measures that lead

to increased levels of dental plaque, smoking more heavily and consuming more fat

and sugar in diet (LeResche and Dworkin, 2000).

Periodontal disease is develop during stressful condition by tissue destroying

factors such as IL-1β and MMPs activated by periodontal pathogens, as well as by the

direct effects of pathogenic bacteria in dental plaque (Grossi et al., 1998). Where

etiology of PD is highly related to periodontal pathogenic bacteria such as P. gingivalis,

P. intermedia or A. actinomycetemcomitans (Castillo et al., 2011). These bacteria

induce the destruction of periodontal tissues by their numerous virulence factors such

as LPS, fimbriae etc. (Curtis et al., 2005). In addition, many physiopathological

processes are involved in periodontal destruction in terms of the inflammatory and

immune host response, especially proinflammatory cytokines or MMPs (Dahan et al.,

2001; Kiecolt-Glaser et al., 2003; Van Dyke and Kornman, 2008).

All these factors lead to strong correlation among salivary IL-1β, dental

plaque, gingival inflammation, BOP and stress during examination period. This result

is in agreement with Deinzer et al., in 1999 and Deinzer et al., in 2000.

Chapter Five

Conclusions and

Suggestions

Conclusions and Suggestions Chapter Five

74

Conclusions and Suggestions

5.1. Conclusions

1. Highly significant differences in the immunological parameter (salivary

interleukin-1β) level and clinical periodontal parameters (plaque index, gingival

index and bleeding on probing) in different periods at P-values ≤ 0.01.

2. Psychological examination stress affected on periodontal health status by more

plaque accumulation, gingival inflammation and increased amounts of interleukin-

1β in saliva. Thus, examination stress appears as a possible risk factor for gingivitis.

3. Strong positive correlations between salivary interleukin-1β level, clinical

periodontal parameters and examination stress at P-value ≤ 0.01.

4. Salivary interleukin-1β can be used as indicator for examination stress that lead to

periodontal diseases.

Conclusions and Suggestions Chapter Five

75

5.2. Suggestions for further studies

1. Further studies in larger populations are required to explore the relationship

between stress and periodontal diseases.

2. Use another stress questionnaires such as Perceived Stress Scale (PSS) with another

stress markers such as salivary and serum cortisol, C-reactive protein, tumor

necrosis factor-alpha, and matrix metalloproteinase.

3. Further studies may include stress managements for the stressful patients such as

coping styles or medications and their relation with periodontal diseases.

4. Determine the relationship between periodontal diseases and other types of

psychological stress such as occupational stress (athletes); involuntary stress

(soldiers) and voluntary stress (musicians).

5. Compare the effects of any type of stress differences with the periodontal diseases

between males and females.

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Appendices

106

Appendices

Appendix I: DASS-21 case sheet

Name: Date:

Please read each statement and circle a number 0, 1, 2 or 3, which indicates how much

the statement applied to you over the past week.

There is no right or wrong answers. Do not spend too much time on any statement.

The rating scale is as follows:

0 Did not apply to me at all.

1 Applied to me to some degree, or some of the time.

2 Applied to me to a considerable degree, or a good part of time.

3 Applied to me very much, or most of the time.

1. I found it hard to wind down 0 1 2 3

2. I tended to over-react to situations 0 1 2 3

3. I felt that I was using a lot of nervous energy 0 1 2 3

4. I found myself getting agitated 0 1 2 3

5. I found it difficult to relax 0 1 2 3

6. I was intolerant of anything that kept me from

getting on with What I was doing

0

1

2

3

7. I felt that I was rather touchy 0 1 2 3

107

Appendix II: Case sheet

Patient name: No: Age:

Telephone No: Date: Time:

Previous medical history and medication:

Periodontal health status:

Smoking:

Plaque index:

Mean PLI =

Gingival index:

Mean GI =

Bleeding on probing:

Percent BOP =

الخالصة

عمر تست تسببها كائنات حية دقيقه ممرضه شائعه مزمنة هلتهابيإ أمراض هي ما حول األسنان أمراض الخلفية:

تدمير إلى يؤدي مما لاللتهاباتؤدية الم المدورات الخلوية في عامةو وضعيةم رتفاعاتإ على تحفزو منطقه اللثة

ةلخطرا عواملال من العديدهنالك لإلسنان.الداعمة األنسجة على تؤثر التيالمدمر لتهاباأل عملية بواسطة األنسجة

همةالم العوامل أهم من واحدك اإلجهاد يعتبرو األسنان،ما حول أمراض تطوير في تساهم لعامة التيا أو ةموضعيال

،ه طويلهلفتر اإلجهادزداد ي ما عندف .ما حول األسنان أمراض ذلك في بما هلتهابياأل األمراض من لعديدا التي تسبب

.ما حول األسنانأمراض مثلوضعية الم أو عامةال األمراض إلى ؤديي مما لتهاباأل عملية على ؤثري قد فإنه

إلى باإلضافة .ما حول األسنانيرتبط بقوة مع أمراض مؤدي لاللتهاب مدور خلويبيتا هو 1البين ابيضاضي

مراضلأل المصاحب اإلجهاد وكذلك األجهاد المتكيف ستجاباتحرج في إ وسيطهو بيتا 1ابيضاضي البين ذلك،

.النفسيةو لعصبيةا

ما حول األسنان )مؤشر الصفيحة الجرثومية، مؤشر إلتهاب اللثة، ؤشراتم لتحديد ومقارنة أهداف الدراسة:

اللعابي بين طالب طب األسنان قبل، بيتا 1ابيضاضي البينمستوى األجهاد ومستوى ، مؤشر النزف عند الجس(

بيتا 1ابيضاضي البين ،اإلجهاد العالقة بينيجاد إل ذلك، إلى وباإلضافة. خالل وبعد فترة إمتحان نصف السنة

حول األسنان. لما السريرية المؤشراتواللعابي

راوحتت أنثى عشر اثنإوذكرا عشر اثنإ ; األسنان طبل الباط عشرينو أربعة من تكونت عينةال المواد والطرق:

:رئيسية فترات ثالث فيالمتتابعه في هذه الدراسة فحصهم تمالذين ،عاما( 12-11) بين أعمارهم

فترةال) نصف السنة متحانإ قبلاألقل على واحد بشهر األولى الفترة (I.

نصف السنة متحانالثانية خالل إ الفترة (فترةال II).

نصف السنة بعد إمتحان األقل على واحد بشهر الثالثة الفترة (فترةال III).

عينات جمع تم .المجموعات كل في اإلجهاد مستوى لقياس( DASS-21) اإلجهادو والقلق اإلكتئاب مقياس استخدم

تم .بواسطة نظام مقايسة األنزيم المرتبط الممتز المناعي اللعابي بيتا 1ابيضاضي البين مستوى لتحديد اللعاب من

مؤشر الصفيحة الجرثومية، مؤشر إلتهاب سن تضمنت لكل واضعم أربعة فيتسجيل مؤشرات ما حول األسنان

.اللثة، مؤشر النزف عند الجس

فترةال يف أعلى تكان السريرية ؤشرات ما حول األسنانم جميعل المتوسطات الحسابية أن ائجالنت أظهرت النتائج:

وكذلك، .(P ≤ 0.01) عالية عند معنوية ختالفاتإ وجود مع والثالثة األولى فتراتال في عليه كانت مما الثانية

اتفترال في عليه كانت مما الثانية فترةال في أعلى تكان اللعابي بيتا 1ابيضاضي البين تركيزل االوساط الحسابي

يظهر بيرسون، رتباطإ معامل ستخدامإب ،كذلك .(P ≤ 0.01) عالية عند إختالفات معنوية وجود مع والثالثة األولى

ؤشرات لما حول والم( اللعابي بيتا 1ابيضاضي البين) المناعي مؤشرال مع قوية عالية عالقة معنوية وجود اإلجهاد

.(P ≤ 0.01) عند األسنان السريرية

يثح األسنان، وأمراض ما حول إجهاد اإلمتحان بين رتباطاإل على قويا دليال الدراسة هذه نتائج قدمت ستنتاج:اإل

و مؤشرات ما حول األسنانو جهادلإل يات أعلىمستو إمتلكوا إمتحان نصف السنة خالل طب األسنان طالبأن

قوي اطرتبإ هناك كان ،كذلك .نصف السنة متحانإ وبعد قبلفترات مع بالمقارنة اللعابي بيتا 1ابيضاضي البين

.والمؤشرات لما حول األسنان السريرية اللعابي بيتا 1ابيضاضي البين اإلجهاد، بين

لما صحيةال حالةال على متحاناإل إجهاد تأثير

بين اللعابيبيتا 1بيضاضيإالبين و حول األسنان

ينالعراق األسنان طب طالب

من كجزءجامعة بغداد / األسنان طب كلية مجلس الى مقدمة رسالة

أمراض وجراحة ما حول األسنان في الماجستير درجة نيل متطلبات

من قبل

عذراء علي محمود سنانواأل الفم وجراحة طب لوريوسابك

بأشراف

هيمرالق اء محمود إب أ. الدكتورة

مراض وجراحة ما حول األسنانماجستير أ

م2013 ين األولرتش / ه1434 الحجةذو /

Effect of the examination stress on periodontal health status and salivary IL-1β among Iraqi dental...

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