Kuwait University

Immunological Analysis of Dental Pulp Inflammation

Submitted by:

Mohamed Maged Elsalhy

A Thesis submitted to the College of Graduate Studies in

Partial Fulfillment of Requirement for M.Sc

Degree in: Microbiology

Supervised by:

Prof. Raj Raghupathy

Dr. Kefah Barrieshi (Co-Supervisor)

Kuwait

Aug/2011

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©2011

All Rights Reserved

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Kuwait University

College of Graduate Studies

Signatory Page

(Thesis Examination Committee)

The undersigned certify that they have read, and recommend to the College of Graduate

Studies for acceptance, a Master's thesis entitled ''Immunological Analysis of Pulpal

Inflammation'' submitted by Mohamed Maged Elsalhy in partial fulfillment of the

requirements for M. Sc degree in Medical Microbiology, Faculty of Medicine.

Signatures of Committee Members Date

__________________________________________ ___________

Prof. Ziauddin Khan, Professor (Convener)

__________________________________________ ___________

Prof. Raj Raghupathy, Professor (Supervisor)

__________________________________________ ___________

Prof. Abu Salim Mustafa, Professor (Member)

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Abstract

Assessing the degree of inflammation in the dental pulp poses a diagnostic

dilemma as it influences the decision between conservative versus invasive dental

treatment. However, there are no objective, quantitative and clinically-practical methods

for evaluating pulpal inflammation. Cytokines have been suggested to be markers of

pulpal inflammation, but estimation of cytokine levels has been possible only after

extraction of inflamed teeth. We set out to develop a method to measure cytokines from

the dental pulp prior to decision making.

108 dental pulp blood samples were obtained with cotton pellets from pulp sites

exposed on pulpectomy. 25 samples were from normal teeth, 40 from asymptomatic

pulps with caries exposure and 43 from symptomatic pulps clinically diagnosed as

irreversible pulpitis. The levels of the inflammatory cytokines IL-2, IL-6, IL-8, TNF-α

and IFN-γ and the anti-inflammatory cytokine IL-10 were quantified using high-

sensitivity ELISA. Levels of cytokines and ratios of inflammatory cytokines to IL-10

were compared using Kruskal–Wallis and Mann–Whitney tests.

Significantly higher levels of IL-6, IL-8, IL-10, TNF-α and IFN-γ were detected

in caries-exposed and irreversible pulpitis as compared to normal teeth. IL-2 levels were

higher in caries-exposed as compared to normal teeth. IL-2 and IL-10 levels were higher

in caries-exposed pulps as compared to irreversible pulpitis, while IL-8 was higher in

irreversible pulpitis as compared to caries-exposed teeth. Most interestingly, IL-6/IL-10

and IL-8/IL-10 ratios were significantly higher in irreversible pulpitis compared to both

caries-exposed and normal teeth.

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IL-8 levels and the IL-8/IL-10 ratio promise to be good indicators of irreversible

pulpitis. An important and potentially very useful outcome of this study is the

demonstration that cytokine estimation in pulpal blood may help in the diagnosis of

pulpal inflammation.

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Table of Contents

Chapter No. Page

Title Page …………………………………………………………… i

Copyright Page …………………………………………………….. ii

Signatory Page ……………………………………………………... iii

Abstract …………………………………………………………….. iv

Table of Contents ………………………………………………….. vi

List of Figures ……………………………………………………… ix

Acknowledgements …………………………………………………..

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Chapter I Introduction ………………………………………………………….. 1

1.1. Anatomy of the tooth ……………………………………………. 1

1.2. Caries and its treatment options ………………………………… 1

1.2.1. Enamel caries …………………………………………. 1

1.2.2. Dentin caries not reaching the pulp …………………… 1

1.2.3. Dentin caries reaching the pulp ……………………….. 2

1.2.4. Extensively carious teeth ……………………………… 3

1.3. The biology and histology of dental pulp ……………………….. 3

1.4. Regulation of dental pulp repair by immune cells in carious teeth 7

1.5. Clinical classification of pulpal status …………………………... 9

1.5.1. Healthy pulp …………………………………………... 9

1.5.2. Reversible pulpitis …………………………………….. 9

1.5.3. Irreversible pulpitis …………………………………… 10

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1.5.4. Necrotic pulp …….…………………………………… 13

1.6. Cytokines and the dental pulp ………………………………….. 13

1.6.1. Interleukin-2 (IL-2) …………………………………… 14

1.6.2. Interleukin-6 (IL-6) …………………………………… 15

1.6.3. Interleukin-8 (IL-8) …………………………………… 17

1.6.4. Interleukin-10 (IL-10) ………………………………… 21

1.6.5. Tumor Necrosis Factor-α (TNF-α) ……………………. 22

1.6.6. Interferon-γ (IFN-γ) …………………………………… 24

Chapter II Aims and Objectives …………………………………………………. 26

Chapter III Materials and Methods ……………………………………………… 27

3.1. Patient selection …………………………………………. 27

3.2. Sample collection ……………………………………….. 28

3.3. Sample preparation ……………………………………… 28

3.4. Estimation of cytokine levels …………………………… 28

3.5. Total protein determination ……………………………... 30

3.6. Data Analysis …………………………………………… 30

Chapter IV Results ………………………………………………………………… 31

4.1. Testing of normality …………………………………………….. 31

4.2. Cytokine levels ………………………………………………….. 31

4.2.1. IL-2 ……………………………………………………. 31

4.2.2. IL-6 ……………………………………………………. 33

4.2.3. IL-8 ……………………………………………………. 34

4.2.4. IL-10 …………………………………………………... 35

4.2.5. TNF-α …………………………………………………. 36

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4.2.6. IFN-γ ………………………………………………….. 37

4.3. Ratio of inflammatory to anti-inflammatory cytokines …………. 39

4.3.1. IL-2/IL-10 ratio ……………………………………….. 39

4.3.2. IL-6/IL-10 ratio ……………………………………….. 40

4.3.3. IL-8/IL-10 ratio ……………………………………….. 41

4.3.4. TNF-α/IL-10 ratio …………………………………….. 42

4.3.5. IFN-γ/IL-10 ratio ……………………………………… 43

Chapter V Discussion …………………………………………………………….. 45

5.1. Cytokines and the dental pulp ………………………………….. 47

5.2. Ratios of inflammatory to anti-inflammatory cytokines ……….. 55

References ……………………………………………………………. 57

Curriculum Vitae ……………………………………………………. 68

Abstract in Arabic …………………………………………………… 70

Title Page in Arabic …………………………………………………. 71

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List of Figures

Figure No. Page

Figure 1 Mean IL-2 levels in different groups ……………………………… 32

Figure 2 Mean IL-6 levels in different groups ……………………………… 33

Figure 3 Mean IL-8 levels in different groups ……………………………… 34

Figure 4 Mean IL-10 levels in different groups …………………………….. 35

Figure 5 Mean TNF-α levels in different groups …………………………… 36

Figure 6 Mean IFN-γ levels in different groups ……………………………. 37

Figure 7 Summary of mean cytokines in different groups …………………. 38

Figure 8 Mean IL-2/IL-10 ratio in different groups ………………………... 39

Figure 9 Mean IL-6/IL-10 ratio in different groups ………………………... 40

Figure 10 Mean IL-8/IL-10 ratio in different groups ………………………... 41

Figure 11 Mean TNF-α/IL-10 ratio in different groups ……………………... 42

Figure 12 Mean IFN-γ/IL-10 ratio in different groups ………………………. 43

Figure 13 Summary of cytokine ratios in different groups ………………….. 44

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Acknowledgements

We gratefully acknowledge the financial support of College of Graduate Studies,

Kuwait University.

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Chapter 1

Introduction

1.1. Anatomy of the tooth

The tooth can be divided into the crown and the root. The crown is the portion

covered by enamel while the root is the portion covered by cementum and lies within

the alveolar bone. From inside out, the tooth has a pulp surrounded by dentin. Dentin is

covered by enamel in the crown and cementum in the root. Enamel, dentin and

cementum are mineralized tissue components of teeth (Berkovitz et al., 2009). Dentin is

a hard tissue with dentinal tubules penetrating throughout the entire thickness. The

dental pulp is a heterogeneous soft tissue located in the center of teeth, which contains a

variety of cell types and extracellular matrix (ECM) molecules. Both dentin and the

pulp are derived from neural crest cells (Zhang & Yelick, 2010).

1.2. Caries and its treatment options

Caries is defined as progressive destruction of tooth structure by acids produced

by bacteria. Treatment of caries depends on its extension into the tooth structures.

1.2.1. Enamel caries

When caries is only in the enamel, fluoride or fissure sealants can be applied to

prevent further progression of caries and it should be closely observed (Griffin et al.,

2008).

1.2.2. Dentin caries not reaching the pulp

If caries reaches the dentin, the defective and infected dentin has to be removed

and the tooth should be restored with a restorative material (Ricketts & Pitts, 2009).

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1.2.3. Dentin caries reaching the pulp

When the patient has no symptoms and caries is thought to extend close to, or

into the pulp, excavation of the pulpal caries can be stopped at stained but firm dentin.

A calcium hydroxide lining is applied over the pulpal dentin followed by glass ionomer

restoration. This is classically referred to as the indirect pulp cap. Later caries removal

is repeated after 6 to 12 months. The difficulty with this technique is knowing how

rapid the carious process has been, how much reactive dentin will be formed and

knowing exactly when to stop excavating to avoid pulp exposure. This technique has

been evolved to avoid pulp exposure and direct pulp capping (Ricketts, 2001).

A direct pulp cap usually involves the placement of a calcium hydroxide

preparation directly in contact with an exposed pulp (Ricketts, 2001). Teeth exposed

during caries removal will inevitably have some degree of inflammation although the

histological extent of this cannot be accurately predicted from a clinical examination.

Direct pulp capping with calcium hydroxide has questionable long term prognosis as the

failure rate is high (Barthel et al., 2000; Al-Hiyasat et al., 2006).

In the other hand, when the patient has severe symptoms of pulpal inflammation,

pulp is opened and pulpal tissue is removed. The tooth then undergoes root canal

treatment and may need an extensive restorative treatment (Torabinejad & Walton,

2009).

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1.2.4. Extensively carious teeth

Teeth with extensive caries that affect their restorability have poor prognosis and

extraction could be the treatment of choice (Torabinejad & Walton, 2009).

1.3. The Biology and Histology of Dental Pulp

In the healthy adult tooth, odontoblasts and cells of the sub-odontoblastic

(Höehl's) layer form a thin border located between the inner margin of the dentin and

the outer limit of the pulp. These post-mitotic polarized cells are responsible for the

production of dentine. Primary dentine is the dentin formed before tooth eruption while

secondary dentine is formed after eruption when the tooth becomes functional.

Although decreasing in number and activities, odontoblasts produce dentin extracellular

matrix molecules continuously as long the tooth is alive and a gradual thickening of

dentin takes place over time (Zhang & Yelick, 2010).

By contrast, in the dental pulp, fibroblasts are renewed constantly (Baume,

1980). In the fully mature and even in aging teeth, the pulp remains a soft tissue.

However, aging influences pulp mineralization, and in many cases pulp stones or areas

of diffuse mineralization develop gradually. The reduced mineralization potential of

dental pulp fibroblasts underlines the fact that they are distinctly different entities from

odontoblast/sub-odontoblastic cells, even if they share a common embryological origin

(Goldberg et al., 2008).

It is now well established that the normal dental pulp contains heterogeneous

cell populations including a majority of fibroblast-like cells, but also inflammatory and

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immune cells, and latent or dormant pulp stem cells (progenitors), mostly involved in

self-renewal. After damage of the tooth, the progenitors may contribute to pulp repair

and mineralization. Nerves, vascular and perivascular cells are also present within the

pulp (Goldberg & Smith, 2004).

Immune cells are present both in the normal and pathologic pulp. In the normal

pulp, they regulate cell density, and are crucial in the control of cell proliferation and

apoptosis (Vermelin et al., 1996; Nishikawa et al., 1999). In the exposed pulp, they may

contribute to resolve inflammatory processes. Several immune cell types have been

identified, specifically, lymphocytes, macrophages, and neutrophils (Trowbridge, 1990;

Stashenko, 1990). CD4+ T helper, CD8

+ T suppressor/cytotoxic cells and macrophages

were detected in normal pulps by Jontell et al (1998). These investigators extracted

human teeth with vital normal pulps under local anesthetia, sectioned the pulp tissue

and subjected them to indirect immunohistochemistry with monoclonal anti-CD4 and

anti-CD8 antibodies as well as to macrophage markers. T helper, T suppressor/cytotoxic

cells, macrophages and dendritic cells (DC) were observed; however, no B cells were

detected in any of the samples. These immune cells are motile. They migrate

independently in contrast to pulp fibroblasts that are closely associated through

junctional complexes, and therefore presumably translocate as a whole from the central

to the outer part of the pulp, as a syncytial structure (Jontell et al., 1987; Jontell et al.,

1998; Nishikawa et al., 1999).

Hahn et al. (1989) demonstrated an increase in the ratio of CD4+/CD8

+

lymphocytes in irreversibly inflamed pulps compared to normal pulps. CD8+ T

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lymphocytes were predominant in normal pulps, while the levels of CD4+ T

lymphocytes relative to CD8+ T lymphocytes were increased in the irreversible group.

Kettering and Torabinejad (1993) detected the presence of natural killer (NK) cells in

human chronic periapical lesions.

In response to a slow carious decay, pathological abrasion or to superficial tooth

preparation by a dentist, the odontoblasts that are still alive have the capacity to produce

a reactionary dentin, more or less similar to the physiological dentin (Lesot et al., 1993).

In case of an invasive carious lesion, the odontoblasts are destroyed by bacterial toxins

or altered by noxious molecules released by the restorative material, by necrotic cells or

by enzymes released by the degradation of the ECM. The cells located in the sub-

odontoblastic layer may in that case ultimately differentiate and replace the wounded

cells in producing a layer of reactionary/reparative dentin at the site (Goldberg et al.,

2008).

If the pulp is exposed, odontoblasts and Höehl cells can no longer perform repair

of the lesion and another process takes place. Stem cells or progenitors located within

the pulp get recruited. They proliferate and differentiate into osteoblast-like or

odontoblast-like cells (Six et al., 2004) and start to produce an ECM, which will

ultimately undergo mineralization. This cascade of events leads to the elaboration of a

reparative dentin (Goldberg & Smith, 2004), in the form of a thin dentinal bridge

occluding the exposure site, or a bone-like structure (osteo-dentin) filling the pulp

partially or totally (Six et al., 2004).

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Autoradiographic data obtained using tritiated thymidine after pulp capping with

calcium hydroxide in monkeys have shown that a first replication is observed in the

central part of the pulp, and later a second cell division occurs in the outer part of the

pulp, near the site of pulp exposure (Fitzgerald et al., 1990). The distribution of labeled

cells indicates that cells involved in the reparative process divide at least twice, and that

progenitors initially located in the central pulp migrate toward the exposure site. This

further suggests that a continuous influx of differentiating cells that contribute to the

formation of a dentinal bridge, excluding odontoblasts and sub-odontobastic cells in the

formation of reparative dentin. It seems that these reparative cells originate within the

pulp and only a subpopulation of the pulp cells are capable of being committed and to

be involved in the repair process. In addition, the identification of stem cells within the

dental pulp has led to the hypothesis that the reparative cell progenitors are stem cells,

but this has not been clearly demonstrated (Gronthos et al., 2002; Goldberg et al., 2008).

Progenitors or adult stem cells exist as dormant or latent cells, and are scarce in

the sound pulp (Gronthos et al., 2002). During the inflammatory process there is a

general increase in the number of pulp cells, but it is not known if this increase is

related to fibroblast proliferation, or represent a massive migration of inflammatory

cells, or to the proliferation of stem cells (Goldberg et al., 2008).

A link between inflammatory molecules and the regulation of pulp cell

population has been suggested previously by studies on the effects of essential fatty acid

deficiency (EFAD) (Vermelin et al., 1995). When rats were fed with an EFAD diet, the

cell density was increased two-fold and three-fold within the central and outer pulp

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respectively, a time-dependent effect related to the period of EFAD diet. In these

studies, the increased pulp cell population might result from a decrease or arrest of

apoptotic events that normally occur (Vermelin et al., 1996; Nishikawa et al., 1999). In

contrast, this diet did not modify either the number or shape of the odontoblasts. As the

essential fatty acids are transformed into prostaglandins and leukotrienes, it is possible

that these mediators play roles in the balance regulating cell death and cell renewal, as is

the case for other cells and tissues.

For years, inflammation in the tooth has been considered mostly as a negative

factor leading to pulp destruction by necrosis or apoptosis. However, some data on the

reaction to carious lesions and implantation of biomolecules suggest that the

inflammatory reaction might be a prerequisite for the burst of progenitors implicated in

repair of the pulp (Goldberg et al., 2008).

1.4. Regulation of dental pulp repair by immune cells in carious teeth

The role of odontoblasts and pulp fibroblasts in the regulation of the dental pulp

immune and inflammatory responses to cariogenic bacteria suggest that interactions

between immune/inflammatory cells and odontoblasts and their precursors influence the

process of pulp repair (Goldberg & Smith, 2004).

The progression of bacteria from the outermost enamel to the pulp–dentin

interface triggers inflammatory and immune events in the underlying dental pulp

through the diffusion of bacterial by-products into dentin tubules. These events may be

prevented when reactionary/reparative dentin is formed by odontoblasts at the pulp–

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dentin interface, eliminating the bacteria and blocking the route of infection. In the

absence of odontoblast reaction, or in case of odontoblast death, bacterial invasion leads

to irreversible pulpitis, pulp necrosis, infection of the root canal system and periapical

disease (Love & Jenkinson , 2002; Heyeraas & Mjör, 2001).

In the dental pulp, when dentin is being destroyed by caries, immature antigen-

presenting dendritic cells (APC) rapidly migrate to the odontoblast layer facing the

lesion in a strategic location near the foreign antigens (Yoshiba et al., 1996). Then, a

progressive and sequential accumulation of T-lymphocytes, macrophages, neutrophils

and B-lymphocytes occurs in the pulp, concomitant with the deepening of the dentin

caries lesion, the increase of the bacterial insult and the development of the pulp

inflammatory process (Hahn & Liewehr , 2007b; Jontell et al., 1998). So, a close

relationship exists between immune cells, especially DCs, that are involved in the initial

steps of the immune response in the pulp, and odontoblasts in order to regulate

odontoblast dentinogenic activities (Farges et al., 2003).

The rapid accumulation of DCs into the odontoblast layer during the early phase

of pulp repair suggests that odontoblast-derived chemotactic molecules might be

responsible for the recruitment of DCs that ensure immunosurveillance in the pulp

tissue and/or patrolling in pulp blood vessels (Farges et al., 2003). This hypothesis was

confirmed by in vitro data that demonstrated the role of odontoblasts in triggering

immune/inflammatory events in response to specific bacterial components (Durand et

al., 2006; Veerrayutthwilai et al., 2007; Staquet et al., 2008). In addition, odontoblasts

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were also found to be more potent attractants of dendritic cells than pulp fibroblasts

(Staquet et al., 2008).

1.5. Clinical classification of pulpal status

1.5.1. Healthy pulp

Healthy pulp is a vital pulp, without inflammation. It is asymptomatic and reacts

to vitality tests such as heat, ice and/or electric pulp tester (EPT). With increasing

amount of secondary dentin as the pulp ages, its reaction to thermal test might be

decreased (Torabinejad & Walton, 2009). However, a healthy pulp should predictably

react to EPT (Fulling & Andreasen, 1976).

1.5.2. Reversible pulpitis

A diagnosis of reversible pulpitis implies that the pulp is vital, but has some

local area/s of inflamed tissue that will heal after conservative vital pulp therapy; i.e.

removing caries and applying a restoration. Symptoms can be very misleading in this

diagnostic category, from none to very intense and sharp sensation associated with

thermal stimuli. It is well established that there is a poor correlation between clinical

symptomalogy and the histopathological state of the pulp (Seltzer et al., 1963; Lundy &

Stanley, 1969; Baume, 1970; Dummer et al., 1980). The history of symptoms will most

often reveal pain or sensation only on stimulation, such that the tooth will bother the

patient only when the tooth is exposed to a stimulus that is hot and/or cold (Asgeir,

2003; Torabinejad & Walton, 2009).

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According to the classification, reversible pulpitis should heal once the irritant is

removed or, in case of an exposed dentin surface, once the exposed dentin is adequately

sealed. However, there is a much higher risk of diagnosing a pulp with mild symptoms

as being reversibly inflamed, when in actuality the pulp is irreversibly inflamed. Thus,

mistakes in diagnosis of this pulpal category are common and understandable and it is

essential to recall and test all patients who have had treatment based on this diagnostic

category in order to confirm that the progression of pulpal reaction has gone according

to expectation, i.e. that the pulp has healed (Asgeir, 2003).

1.5.3. Irreversible pulpitis

In irreversible pulpitis, the pulp is still vital but is severely inflamed so that

healing is an unlikely outcome with conservative pulp therapy. Thus, pulp necrosis and

infection is the predicted outcome if vital pulp therapy is attempted. Apical periodontitis

will be the final outcome (Asgeir, 2003). In order to avoid pulp necrosis, the pulp is

aseptically removed and the entire space filled with a root canal filling material

(Torabinejad & Walton, 2009). As with reversible pulpitis, symptoms can be very

misleading. It has been well documented that in most cases a pulp that is irreversibly

inflamed is asymptomatic. It has been reported that dental pulps can progress from

vitality to necrosis without pain in 26–60% of all cases (Bender, 2000).

According to several studies, neither gender nor tooth type appears to matter in

case of asymptomatic pulpitis; however, the older the patient is, the less likely is there

pain associated with the pulpitis (Hasler & Mitchell, 1970; Michaelson & Holland,

2002).

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If the pulp is symptomatic it is most often very sensitive to thermal changes, and

the pain sensation has a tendency to linger as a dull ache after the stimulus has been

removed. This fact can be used with caution to predict if the pulp is likely to be

irreversibly inflamed or not. Therefore, the more dull, throbbing, poorly localized,

lingering pain is experienced after the stimulus has been removed, the more severe the

inflammation is likely to be and thus the more likely it is to be irreversible in nature

(Asgeir, 2003).

It has also been shown that the more severe the pain and the longer it has been

symptomatic, the more likely it is to be irreversibly inflamed (Bender, 2000). Perhaps

the clearest sign of irreversible inflamed pulp is the history of spontaneous pain, which

will ‘hit’ the patient without any thermal stimulation to the teeth, and even wake the

patient from sound sleep (Seltzer et al., 1963).

Also included in this category are teeth with carious exposures (Torabinejad &

Walton, 2009). Capping of caries-exposed teeth has showed long term failure (Baume

& Holz, 1981; Matsuo et al., 1996; Barthel et al., 2000; Al-Hiyasat et al., 2006) and this

why it is considered irreversible pulpitis. However, this was not very conclusive. The

reluctance to place a direct pulp cap on an exposure in a carious field is based on

unpredictable outcomes using traditional materials and treatment protocols. Changes in

the materials may change the outcomes.

Moreover, when bacterial byproducts induce pulpal inflammation, compromise

immune responses and impede cellular differentiation and recruitment, normal pulpal

12

repair mechanisms may not function properly. Success rates with direct pulp capping in

a carious field vary depending on the technique and materials. Most of the studies with

low success rates used calcium hydroxide as pulp-capping materials (Matsuo et al.,

1996; Barthel et al., 2000; Al-Hiyasat et al., 2006). Calcium hydroxide does not provide

close adaptation to dentin, does not promote consistent odontoblast differentiation and

has been shown to be cytotoxic in cell cultures. Also, the resultant reparative dentin is

characterized by tunnel defects. Tunnel defects within dentin bridges may provide a

pathway for the penetration of microorganisms to activate circulating immune cells,

induce pulpal irritation and produce subsequent dystrophic changes (Cox et al., 1996;

Andelin et al., 2003).

However, the introduction of mineral trioxide aggregate (MTA) as a capping

material is showing promising results. Over an observation period of nine years, Bogen

et al. (2008) followed MTA-capped teeth and found that 98 % had favorable outcomes

on the basis of radiographic appearance, subjective symptoms and cold testing. MTA

can be a reliable pulp-capping material on direct carious exposures in permanent teeth.

This would change carious-exposed teeth from the irreversible pulpitis category to

reversible pulpitis. However, a critical requirement for this is the development of

methods that can differentiate between the two categories before capping (Bogen et al.,

2008).

Clinically, pulp repair after capping occurs when bacterial contamination is

limited and inflammation is mild. However, information gained by examining the

13

patients before pulp capping is very difficult to assess and does not reflect the actual

pulpal status.

1.5.4. Necrotic pulp

The diagnostic category of necrotic pulp implies partial or total necrosis of

pulpal tissue. If the pulp is completely necrotic in a tooth with undeveloped root, it is

now possible in some cases to disinfect the canal space and stimulate the root to

continue formation (Iwaya et al., 2001). In case of a fully formed tooth, root canal

therapy is always indicated for both partially and fully necrotic pulp. If the pulpal space

is not already infected, it will in most cases become infected in time if left untreated.

Prevention of formation of periapical lesion has been shown to have a much more

reliable outcome than a treatment on a tooth with a periapical lesion (Sjogren et al.,

1990).

1.6. Cytokines and the dental pulp

Cytokines are polypeptides secreted by leukocytes and other cells that act

principally on hematopoietic cells, and whose effects include modulation of immune

and inflammatory responses. They are pleiotropic in their effects and can have multiple

target cells and consequently multiple actions. Structurally dissimilar cytokines can

have overlapping but typically non-identical actions, a given effect often being mediated

by several different cytokines. Another characteristic of cytokines is that a single

cytokine frequently induces or influences the action of other cytokines, and can function

synergistically. Cytokines can be classified according to their immune response into

Th1 and Th2 cytokines. Th1 cytokines are those involved in cell mediated immunity

14

like IFN-γ, IL-2 and TNF-α. TH2 cytokines are those involved in antibody mediated

immune response like IL-4, IL-5, IL-6 and IL-10. Another classification is by their

inflammatory/anti-inflammatory function. IL-2, IL-6, IL-8, IFN-γ and TNF-α are

inflammatory cytokines while IL-4, IL-10 and IL-13 are anti-inflammatory cytokines.

As the names indicate, inflammatory cytokines increase inflammation while anti-

inflammatory cytokines suppress inflammation. Levels of these cytokines can be used

as a reflection of the degree of inflammation.

1.6.1. Interleukin-2 (IL-2)

The best known action of IL-2 is to augment the proliferation of T lymphocytes

in response to antigenic stimulation, including the generation of both cytotoxic and

suppressor T cells (Dooms et al., 2004). Thus, IL-2 controls the 'amplification' phase of

the T-cell immune response. IL-2 exerts effects on cellular metabolism and glycolysis

that are necessary for long term survival of T cells. It is also important for the

differentiation of CD4+ T cells into Th1 and Th2 effector subsets (Gaffen & Liu, 2004)

and for the development of memory T cells (Williams et al., 2006).

In addition to its function as a T-cell growth and survival factor, IL-2 plays a

key role in Fas-mediated activation-induced cell death of CD4 + T cells in response to

antigen re-stimulation. This is critical in peripheral tolerance for the elimination of auto-

reactive T cells (Gaffen & Liu, 2004). IL-2 also promotes the growth and

differentiation of mitogen- or antigen-activated B cells in vitro (Mingari et al., 1984).

15

IL-2 also augments the cytolytic activity of natural killer cells, induces

lymphokine activated killer cell activity (Dooms et al., 2004) and increases the

proliferation of large granular lymphocytes (London et al., 1986). The actions of IL-2

on monocytes include the stimulation of cytotoxic activity against tumor targets and

induction of IL-l β and IL-6 mRNA (Saraya & Balkwill, 1993; Musso et al., 1995).

These activities emphasize the importance of IL-2 in the immune response

particularly in priming the immune response in pulpal inflammation. Significant

differences in detectable IL-2 levels in normal and inflamed pulps were reported by

Rauschenberger et al. (1997) in their study consisting of 40 human pulp samples; they

postulated that presence of elevated levels of IL-2 could be used as a marker to confirm

the presence of inflammation in human pulp tissue and be used in clinical diagnostic

procedures.

However, in another study by Anderson et al. (2002), no significant difference

between the concentrations of IL-2 was observed in any of the 80 pulp samples studied.

Thus significant discrepancy exists in the context of IL-2 and further investigation is

warranted to determine if a correlation exists between the concentration of IL-2 and the

degree of inflammation in the dental pulp.

1.6.2. Interleukin-6 (IL-6)

IL-6 is a multifunctional cytokine with biological activities including regulation

of immune response, inflammation, and hematopoiesis (Kishimoto, 2005). IL-6 is

responsible for multiple inflammatory manifestations (Kishimoto, 2005; Mima &

16

Nishimoto, 2009). In vivo treatment with IL-6 induces systemic inflammatory

symptoms such as fever, generalized fatigue and anorexia, as well as abnormalities in

laboratory test results, including increases in levels of acute-phase proteins, C-reactive

protein, serum amyloid A and fibrinogen and decreases in serum concentrations of

albumin (Nishimoto, 2010).

In inflammatory tissues, IL-6 induces local infiltration of immunocompetent

cells via up-regulation of adhesion molecules. It also induces angiogenesis by

augmenting the production of vascular endothelial growth factor. This growth factor

increases vascular permeability and leads to inflammatory edema. In the affected joints

of rheumatoid arthritis (RA) patients, vascular endothelial growth factor–mediated

angiogenesis is necessary for synovial pannus formation, and this causes destruction of

the joint. In bone metabolism, IL-6, in the presence of soluble IL-6 receptor, induces

osteoclast differentiation, resulting in the characteristic bone resorption and joint

destruction seen in RA. This function of IL-6 also explains why osteoporosis is

associated not only with chronic inflammation but also with postmenopausal status

(Nishimoto, 2010).

In addition, excessive IL-6 signaling induces the production of suppressors of

cytokine signaling molecules, intracellular negative feedback factors that inhibit the

Janus kinase–signal transducer and activator of the transcription signaling pathway.

Given that transduction of the erythropoietin receptor signal requires the same pathway,

the action of erythropoietin can be suppressed by suppressors of cytokine signaling.

Excessive production of IL-6 therefore contributes to the hypoferremic anemia of

17

chronic inflammation. IL-6 is a growth factor not only for malignant cells of multiple

myeloma and renal cell carcinoma but also for non-tumor cells, including the mesangial

cells of the kidney. This suggests that IL-6 overproduction may be involved in the

pathogenesis of mesangial proliferative glomerulonephritis (Nishimoto, 2010).

Due to its important role in immune responses and inflammation, IL-6 has been

investigated in the dental pulp by Barkhordar et al. (1999). Six inflamed human pulps

and six human periapical lesions were compared; pulp samples from impacted third

molars were used as controls. The inflamed dental pulp tissue and periapical lesions

demonstrated elevated levels of IL-6 compared with normal pulp values. This would

suggest that inflamed pulps and periapical tissues have elevated levels of IL-6 which is

produced and released locally in inflamed pulpal and periapical lesions, especially in

chronically inflamed sites of bone resorption (Barkhordar et al., 1999). This study is

consistent with a strong positive correlation of increased expression of IL-6 and IL-8 in

gingival fibroblasts isolated from periodontitis patients in vitro (Dongari-Bagtzoglou &

Ebersole, 1998).

On the other hand, Nakanishi et al. (1995) detected IL-6 in only 2 of the 18

blood samples from inflamed pulps, and even then were present in extremely small

quantities. It was not detected in the normal pulp group.

1.6.3. Interleukin-8 (IL-8)

IL-8 is made by a wide variety of cell types, including virtually all nucleated

cells within the body. However, among these cells, monocytes and macrophages

18

typically represent the principal cellular source. IL-8 is induced by multiple stimuli

including lipopolysaccharide (LPS), live bacteria, and other early proinflammatory

cytokines such as tumor necrosis factor (TNF-α) and IL-1 (Remick, 2005). IL-8 is

actively secreted into the extracellular space following stimulation of cells. IL-8 is

relatively unique as it may be produced early in the inflammatory response but will

persist for a prolonged period of time, even days and weeks. In this regard, it is in

contrast to most of the other inflammatory cytokines, which are typically made and

cleared during in vivo situations in a matter of a few hours (DeForge et al., 1993).

IL-8 is a chemokine and induces chemotaxis of inflammatory cells. IL-8 bears

principal responsibility for recruitment of neutrophils, the signature cell of the acute

inflammatory response (Webb et al., 1993). The cellular recruitment occurs through the

development of the chemotactic gradient so that the inflammatory cell moves towards

an area of increased chemokine concentration. In vivo, this gradient may be generated

by IL-8 binding to basement membrane proteins (Gimbrone et al., 1989). The

chemotactic gradient helps to both bring cells toward the local site of inflammation and

also to retain them once they have arrived. In addition to recruitment, IL-8 also serves to

stimulate the neutrophil to a higher state of activation (Remick, 2005).

IL-8 is important in the regulation of the acute inflammatory response. It is

rapidly synthesized at local sites of inflammation where it will fulfill its function to

recruit and activate acute inflammatory cells. IL-8 is a small protein that is resistant to

heat and proteolysis and relatively resistant to acidic environments. These biochemical

characteristics make it an ideal candidate molecule to function at a site of acute

19

inflammation, where it must withstand suboptimal conditions. An example of a harsh

environment is the collection of pus located within an abscess. Another important

component of IL-8 is its relative longevity at sites of acute inflammation. As indicated,

it may be found for several days during in vivo situations to continue the recruitment of

inflammatory cells to combat bacterial infections (Remick, 2005).

Interleukin-8 was induced in vitro when pulpal fibroblasts or human pulp stem

cells were challenged by endodontic pathogens, substance P, LPS or TNF (Nagaoka et

al., 1996; Yang et al., 2003). However, IL-8 in inflamed pulps is

immunohistochemically localized in odontoblasts, lymphocytes, macrophages and

endothelial cells, but not in fibroblasts. Hahn & Liewehr (2007a) suggested that IL-8

secreted by fibroblasts may not be relevant to pulpitis.

Huang et al. (1999) studied the expression of IL-8 in inflamed human dental

pulp using ELISA and immunohistochemical analysis. Fourteen pulp tissue samples

from teeth diagnosed with irreversible pulpitis compared with 15 freshly extracted

normal teeth for IL-8 by ELISA. They found that about half of the normal pulps had

barely detectable levels of IL-8; the majority of the inflamed pulps had statistically

significantly higher levels of IL-8. Immunohistochemical analysis showed that diseased

samples were either weakly or moderately stained, whereas normal samples were either

negative or weakly stained. In addition, not all diseased samples showed inflammatory

cell infiltrates.

20

Guo et al. (2000) investigated the level of IL-8 from normal and inflamed

human dental pulp tissues. Microliters of pulpal blood from 8 normal, 26 acute pulpitis

and 22 chronic pulpitis teeth were obtained using filter paper strips and IL-8 levels were

measured by ELISA. No IL-8 was detected in the samples from normal pulp, but

significant amounts of IL-8 were measured in inflamed pulp tissues, and the level of IL-

8 in exudates of acute stage of pulpitis was higher than that of chronic stage. This study

demonstrates that IL-8 is produced and accumulated in pulp inflammation and may play

a role in the occurrence and development of human pulpitis.

Silva et al. (2009) investigated the location, distribution and concentration of IL-

8 in healthy and inflamed dental pulps. Twenty pulps were studied, obtained from

healthy third molars and from pulpectomies by immunohistochemistry and ELISA.

Immunohistochemically, it was observed that inflamed pulps were strongly stained for

IL-8 in inflammatory cells, while healthy pulps were not. ELISA on tissue extracts

quantitatively confirmed higher levels of IL-8. It was concluded that inflamed pulps

have higher amounts of IL-8 than healthy pulps. Furthermore, pulp fibroblasts

stimulated with bacterial LPS produced higher levels of IL-8 than the control group.

An examination of steady-state mRNA expression of various cytokines in

symptomatic and clinically-healthy human dental pulps and produced evidence

supporting the notion that pulpitis is associated with the up-regulation of IL-6, IL-8 and

IL-18 (Zehnder et al., 2003).

21

1.6.4. Interleukin-10 (IL-10)

IL-10 is produced by a number of different cell types, especially inflammatory

cells such as T lymphocytes, and is an important inhibitor of cytokine synthesis and

macrophage activity. It also inhibits the production of proiflammatory cytokines and

extracellular matrix metalloproteinases (Anker & von Haehling, 2004).

IL-10 inhibits the production of several cytokines such as TNF-α, IL-1β and IL-

6 in various cell types, and is also self-modulated (Anker & von Haehling, 2004).

Furthermore, studies have shown that IL-10 production increases in inflammatory

processes such as anemia and rheumatoid arthritis exerting a predominantly

immunomodulatory role in such conditions (Yamaoka et al., 1999). It also has an

immunoregulatory role in pregnancy (Szereday et al., 1997).

IL-10 has also been described by its protective, i.e. anti-inflammatory, properties

in delaying the progression of disease, since high levels of IL-10 are associated with a

reduction in apoptosis (Mallat et al., 1999). Also, IL-10 has protective effects in

experimental endotoxemia and rescues mice from LPS-induced toxic shock (Marchant

et al., 1994).

Hahn et al. (2000) studied the expression of IL-10, IL-4 and IFN-γ mRNA

expression in dental pulps. The prevalence of IL-10 mRNA was significantly higher in

deep-caries than in shallow-caries.

22

1.6.5. Tumor Necrosis Factor-α (TNF-α)

TNF-α coordinates the early response to injury and thus represents an important

point of regulation in inflammatory diseases. TNF has systemic endotoxic activity

leading to fever, hypotension and shock (Malik & Balkwill, 1992). Detailed

investigations on its biological activities have confirmed that TNF-α is one of the most

prominent inflammatory mediators and absolutely central in starting off the

inflammatory reactions of the innate immune system including induction of cytokine

production, activation and expression of adhesion molecules and growth stimulation

(Clauss et al., 2001; Hehlgans & Pfeffer, 2005).

The blockade of TNF-α with specific antibodies is associated with a reduction in

the expression of other proinflammatory cytokines, such as IL-1 and IL-6, both in vitro

and in vivo (Pfeffer, 2003). TNF induces the expression of endothelial adhesion

molecules and chemokines that attract inflammatory leukocytes to sites of tissue injury

and stimulates leukocytes and parenchymal cells to release additional chemokines and

inflammatory cytokines such as IL-1, which facilitates further local accumulation and

subsequent activation of immunologic effector cells (Hehlgans & Pfeffer, 2005).

Consistent with these functions of TNF-α, the blockade of those cytokines in

rheumatoid arthritis patients reduced the TNF-dependent cytokine response and

diminished leukocyte recruitment to inflamed tissue (Charles et al., 1999). TNF also has

important immunoregulatory properties. It is essential for the development of secondary

lymphoid organ structures in lymph nodes, spleen and Peyer’s patches, and its

23

deficiency results in the absence of germinal centers and follicular dendritic cells

(Pfeffer, 2003).

Moreover, TNF-α induces apoptotic cell death in both leukocytes and

parenchymal cells. This is important for the immunoregulatory functions of TNF-α, but

also may contribute substantially to organ-specific damage, as reported in some acute

non-immune nephropathies (Van Herreweghe et al., 2010).

In a study by Nakanishi et al. (1995), TNF-α was detected in 10 of 20 pulpal

blood samples in the inflamed pulp group. In the normal pulp group, 2 of the 6 samples

contained a detectable amount of TNF- α. However, there was no significant difference

between the two groups.

In another study of normal and inflamed human dental pulps, Pezelj-Ribaric et

al. (2002) reported high levels of TNF-α in pulpal tissue from teeth clinically diagnosed

with irreversible pulpitis, but considerably lower expression of this cytokine in samples

from normal teeth.

Kokkas et al. (2007) found a significant increase of TNF-α gene expression

associated with irreversible inflammation compared with healthy controls. No such

difference was detected in reversibly inflamed pulp in comparison to healthy teeth.

These authors concluded that gene expression of TNF-α in inflamed human dental pulp

tissue is positively associated with the severity of clinical symptoms.

24

1.6.6. Interferon-γ (IFN-γ)

IFN-γ is the predominant physiologic macrophage activating factor. It plays a

crucial role in promoting non-specific host-defense mechanisms against a number of

pathogens. In vitro and in vivo studies have demonstrated that macrophages activated

by IFN-γ have the capacity to non-specifically kill a variety of intracellular and

extracellular parasites as well as neoplastic cells (Gattoni et al., 2006).

One of the major immunoregulatory roles of IFN-γ is its ability to promote

adaptive immune responses by influencing both the number of MHC molecules on the

cell surface, as well as the repertoire of peptides presented by MHC molecules. IFN-γ

plays an important role in the development of the Th1 response. In vitro, antibody

neutralization of IFN-γ greatly reduces the development of Th1 cells and augments the

development of Th2 cells (Hsieh et al., 1993).

IFN-γ plays a complex role in regulating humoral immunity. IFN-γ is

predominantly responsible for regulating three specialized B-cell functions: B-cell

development and proliferation, immunoglobulin (Ig) secretion and Ig-heavy chain

switching. Also, IFN-γ plays an important role in the immune-mediated rejection of

established tumors (Ikeda et al., 2002).

Hahn et al. (2000) found IFN-γ mRNA in pulp tissue in 67% of shallow-caries

teeth and it was about twofold more prevalent than IL-4 and IL- 10, supporting Th1

polarization of the response. Furthermore, in 43% of these samples IFN-γ mRNA was

the only cytokine message found. In the deep-caries group IFN-γ mRNA remained very

25

common, but IL-4 and IL-10 were found in similar frequencies and a polarization

toward Th1 was no longer apparent.

There are significant gaps in our current understanding of pulpal inflammation

and its relation to pulpal immune responses. The quantitative analyses of

immunologically-relevant cells and the patterns of production of inflammatory

cytokines and chemokines should help elucidate the immunopathologic mechanism of

pulpitis. The knowledge gained in these areas can then be applied to immunotherapy of

vital pulp in the future. In addition, the understanding of the biochemical and molecular

networks and pathways involved in early reversible and later irreversible pulpitis may

then be applied clinically to ensure that the dental pulp is vital and healthy.

26

Chapter 2

Aims and Objectives

1. To determine pulpal levels of IL-2, IL-6, IL-8, IL-10, TNF-α and IFN-γ in

irreversible pulpitis, asymptomatic caries exposure and normal pulps using pulpal

blood.

This will help in identifying potential markers to confirm the presence of inflammation

in human pulp tissue. Using this marker in clinical diagnosis would decrease the

number of caries exposure cases undergoing root canal treatment which would result in

reduction in visits to the clinic and treatment costs. It will help in predicting the

prognosis of pulp capping.

2. To compare ratios of inflammatory to anti-inflammatory cytokines in human dental

pulps in irreversible pulpitis, asymptomatic caries exposure cases and normal pulps.

This will contribute to our understanding of the pathogenesis of irreversible pulpitis.

27

Chapter 3

Materials and Methods

3.1. Patient selection

The study was approved by the Joint Committee for Protection of Human

Subjects in Research at Kuwait University and all subjects gave their written informed

consent. One hundred and eight subjects were involved (57 male and 51 female). The

medical histories of all patients in this study were non-contributory. The pulpal status in

these patients was categorized as normal, asymptomatic caries-exposed pulp and

symptomatic irreversible pulpitis.

The normal pulp group consisted of 25 subjects aged 18-35 years old (12 male

and 13 female). Clinical and radiographic examination of these teeth indicated that these

teeth had no caries, crown fractures or any restorations. These teeth were planned for

extraction for orthodontic reasons.

The asymptomatic caries-exposed pulp group consisted of 40 subjects aged 18-

54 years (21 male and 19 female). The teeth in this group had no history of pain to

thermal stimuli and were designated for pulpectomy and root-canal treatment due to

caries exposure. Clinical and radiographic examination indicated that these teeth had

caries and/or restorations and no pulpal necrosis. Pulp sensitivity test to cold yielded

sharp instant pain and not prolonged lingering pain.

The irreversible pulpitis group consisted of 43 subjects aged 18-60 years (24

male and 19 female). The teeth in this group had a history of prolonged pain to thermal

28

stimuli or spontaneous pain and were designated for pulpectomy and root-canal

treatment. Clinical and radiographic examination revealed the presence of caries and/or

restorations with or without pulp exposure. Pulp sensitivity test on cold elicited

immediate prolonged lingering pain reaction.

3.2. Sample collection

After the tooth was isolated with a rubber dam, caries was removed and the pulp

was carefully exposed with a spoon excavator and/or a low speed round bar. Blood from

the exposed surface of the pulp was collected with a cotton pellet. The pellet was held at

the exposed site to allow absorption of the pulpal blood. The pellets were then placed in

1 ml saline in tubes coated with heparin. Samples were stored at -20 °C until they were

tested.

3.3. Sample preparation

After thawing, the samples were centrifuged first at 10,000 rpm for 10 min

followed by centrifuging at 5,000 rpm for 5 min. The cotton pellet was then removed

and the samples were ready for testing.

3.4. Estimation of cytokine levels

IL-2, IL-6, IL-8, IL-10, TNF-α and IFN-γ levels in pulpal blood were

determined with commercially available ELISA kits. High sensitivity ELISA kits were

used for IL-2, IL-6, IL-10, TNF-α and IFN-γ (Bender Medsystems, Austria) with

sensitivities of 0.4, 0.03, 0.05, 0.13 and 0.06 pg/ml, respectively. Regular ELISA kits

29

were used for IL-8 with a sensitivity of 11.0 pg/ ml. All assays were performed in

accordance with manufacturer's instructions.

Generally in regular ELISA kits, antibody specific for the cytokine of interest is

bound to the walls of plastic microtiter wells. Samples are incubated in the wells, and

any cytokine in the sample gets bound to the antibody on the well walls. The wells are

then washed, and a second antibody (Horseradish peroxidase (HRP)- conjugated

antibody) is added to this one also specific for the cytokine, but recognizing epitopes

different from those bound by the first antibody. After incubation, the wells are washed

again, removing any unattached antibody. Attached to the second antibody is an

enzyme, which, when presented with its substrate (Tetramethylbenzidine (TMB),

produces a colored product, the intensity of the color produced being proportional to the

amount of bound cytokine. The intensity of the color is then read by a

spectrophotometer at 450 nm wave length.

In high sensitivity kits a biotin-conjugated antibody is added first followed by

strepatavidin-HRP antibody after washing. Then, two amplification reagents are added,

namely biotinyl-tyramide and strepatavidin-HRP, separated by a washing step. This is

followed by the TMB substrate solution. The intensity of the color is then read by a

spectrophotometer at 450 nm. Cytokine concentrations were expressed as picograms per

total protein which was measured using the Bio-Rad™ protein assay.

30

3.5. Total protein determination

The Bio-Rad protein assay was used to determine total protein concentration.

Dye reagent was prepared by diluting 1 part dye reagent concentrate with 4 parts de-

ionized distilled water. Then, the solution was filtered by filter paper to remove

particles. Samples were diluted 1: 40 in saline. Ten microliters of each diluted sample

solution was added into separate microliters plate wells. Two hundred microliters of

diluted dye reagents was added to each well and mixed thoroughly. Samples were

incubated at room temperature for 30 minutes. Color intensity was measured at 595 nm

wave length. Bovine serum albumin (BSA) was used as control. Five dilutions of BSA

were prepared with range between 0.05 mg/ml to 0.5 mg/ml. All solutions were assayed

in triplicate.

3.6. Data Analysis

Levels of cytokines were estimated and the ratios of cytokines to IL-10 ratio

were calculated. Data was tested for data normality by the Shapiro-Wilk test. All

concentrations calculated below the sensitivity level of the kits were recorded as the

lower sensitivity limit of the kit for the convenience of statistical analysis. Statistical

differences between the groups were determined by Kruskal–Wallis and Mann-Whitney

U tests for non-parametric comparisons. Differences were considered significant if the P

value was equal to or less than 0.05. Data was analyzed using SPSS 17.

31

Chapter 4

Results

4.1. Testing of normality

Data were tested for normality using the Shapiro-Wilk test. As the data were not

normally distributed, levels of cytokines and ratios of inflammatory cytokines to IL-10

were compared using the Kruskal–Wallis and Mann–Whitney tests.

4.2. Cytokine levels

4.2.1. IL-2

IL-2 was detected in 17 out of 25 samples (68 %) in the normal pulp group with

levels ranging from 0.005 to 1.373 pg/mg with a median of 0.054 pg/mg (mean +/-

standard deviation; 0.173 +/- 0.379 pg/mg). In the caries exposure group, IL-2 was

detected in 32 out of 40 samples (80 %) with levels varying from 0.087 to 18.886 pg/mg

with a median of 0.875 pg/mg (3.259 +/- 4.811 pg/mg). In the irreversible pulpitis

group, IL-2 was detected in 30 out of 43 samples (70 %) with levels from 0.057 to

12.138 pg/mg with a median of 0.261 pg/mg (1.205 +/- 2.109 pg/mg) (Figure 1).

There were significant differences between the groups (p<0.001). IL-2 was

significantly higher in the caries exposure group compared to both the normal group (p<

0.01) as well as the irreversible pulpitis group (p< 0.05). However, there was no

significant difference between the irreversible pulpitis group and the normal group

(p>0.05).

32

Normal

Caries Exposure

Irreversible

Pulpitis

0

0.5

1

1.5

2

2.5

3

3.5

4

IL-2

(p

g/m

g)

Figure 1. Mean IL-2 levels in different groups.

33

4.2.2. IL-6

IL-6 was not detectable in the normal pulp group. In the caries exposure group,

IL-6 was detected in 21 out of 40 samples (50 %) with levels from 0.019 to 1.760 pg/mg

with a median of 0.0244 pg/mg (0.233 +/- 0.421 pg/mg). In the irreversible pulpitis

group, IL-6 was detected in 30 out of 43 samples (70 %) with levels ranging from 0.086

to 2.309 pg/mg and a median of 0.159 pg/mg (0.459 +/- 0.578 pg/mg) (Figure 2).

There were significant differences between the groups (p<0.001). IL-6 was

significantly higher in both the caries exposure group as well as the irreversible pulpitis

group as compared to normal pulps (p< 0.001). However, there was no significant

difference between irreversible pulpitis and caries exposure groups (p>0.05).

Normal

Caries Exposure

Irreversible

Pulpitis

0

0.1

0.2

0.3

0.4

0.5

0.6

IL-6

(p

g/m

g)

Figure 2. Mean IL-6 levels in different groups.

34

4.2.3. IL-8

IL-8 was not detectable in the normal pulp group. In the caries exposure group,

IL-8 was detected in 16 out of 40 samples (40 %) with levels from 2.294 to 290.957

pg/mg (21.826 +/- 66.091 pg/mg). In the irreversible pulpitis group, IL-8 was detected

in 35 out of 43 samples (81 %) with a range from 4.267 to 1107.575 pg/mg and a

median of 42.08 pg/mg (154.866 +/- 270.838 pg/mg) (Figure 3).

Significant differences were observed between the groups (p<0.001). IL-8 was

significantly higher in both the caries exposure group and the irreversible pulpitis group

when compared to normal pulps (p< 0.001). In addition, IL-8 was significantly higher in

irreversible pulpitis than in caries exposure (p<0.001).

Normal

Caries Exposure

Irreversible

Pulpitis

0

20

40

60

80

100

120

140

160

180

IL-8

(p

g/m

g)

Figure 3. Mean IL-8 levels in different groups.

35

4.2.4. IL-10

IL-10 was detected in 17 out of 25 samples (68 %) in the normal pulp group

with levels ranging from 0.048 to 0.605 pg/mg with a median of 0.266 pg/mg (0.217 +/-

0.183 pg/mg). In the caries exposure group, IL-10 was detected in 39 out of 40 samples

(97.5 %) with amount varied from 0.279 to 17.921 pg/mg with a median of 2.303 pg/mg

(4.487 +/- 4.894 pg/mg). In the irreversible pulpitis group, IL-10 was detected in 35 out

of 43 samples (81 %) with detectable range 0.163 to 29.353 pg/mg with a median of

0.967 pg/mg (3.894 +/- 6.967 pg/mg) (Figure 4).

There were significant differences between the groups (p<0.001). IL-10 was

significantly higher in caries exposure and irreversible pulpitis groups than in the

normal group (p< 0.001). In addition, IL-10 was significantly higher in caries exposure

than in irreversible pulpitis (p<0.01).

Normal

Caries Exposure

Irreversible

Pulpitis

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

IL-1

0 (

pg

/mg

)

Figure 4. Mean IL-10 levels in different groups.

36

4.2.5. TNF-α

TNF-α was detected in 23 out of 25 samples (92 %) in the normal pulp group

with levels from 0.038 to 0.212 pg/mg and a median of 0.099 pg/mg (0.105 +/- 0.061

pg/mg). In the caries exposure group, TNF-α was detected in all 40 samples (100 %)

with levels from 0.089 to 39.447 pg/mg and a median of 1.314 pg/mg (5.802 +/- 9.815

pg/mg). In the irreversible pulpitis group, TNF-α was detected in 41 out of 43 samples

(95 %) with levels ranging from 0.065 to 8.811 pg/mg and a median of 1.929 pg/mg

(1.653 +/- 2.291 pg/mg) (Figure 5).

Significant differences between the groups were evident (p<0.001). TNF-α was

significantly higher in caries exposure (p<0.05) and irreversible pulpitis (p< 0.001) than

in normal pulps. However, no significant difference was observed between irreversible

pulpitis and caries exposure (p>0.05).

Normal

Caries Exposure

Irreversible

Pulpitis

0

0.5

1

1.5

2

2.5

TN

F-α

(p

g/m

g)

Figure 5. Mean TNF-α levels in different groups.

37

4.2.6. IFN-γ

IFN-γ was detectable in 8 out of 23 samples (35 %) in the normal pulp group

with amounts ranging from 0.001 to 0.019 pg/mg and a median of 0.004 pg/mg (0.006

+/- 0.007 pg/mg). In the caries exposure group, IFN-γ was detected in 19 out of 27

samples (70 %) with levels from 0.007 to 0.699 pg/mg with a median of 0.025 pg/mg

(0.141 +/- 0.227 pg/mg). In the irreversible pulpitis group, IFN-γ was detected in 22 out

of 28 samples (79 %); levels ranged from 0.01 to 0.621 pg/mg with a median of 0.028

pg/mg (0.131 +/- 0.197 pg/mg) (Figure 6).

There were significant differences between the groups (p<0.001). IFN-γ was

significantly higher in caries exposure (p<0.05) and irreversible pulpitis (p< 0.001) as

compared to normal. However, there was no significant difference between the

irreversible pulpitis and caries exposure groups (p>0.05).

Normal

Caries Exposure Irreversible

Pulpitis

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

IFN

-γ

(pg

/mg

)

Figure 6. Mean IFN-γ levels in different groups.

38

In summary, IL-2, IL-6, IL-8, IL-10, TNF-α, IFN-γ were significantly higher in

irreversible pulpitis and caries exposure compared to normal pulps. IL-2 and IL-10 were

significantly higher in caries exposure compared to irreversible pulpitis while IL-8 was

significantly higher in irreversible pulpitis compared to caries exposure (Figure 7).

Figure 7. Summary of mean cytokines in different groups.

39

4.3. Ratio of inflammatory to anti-inflammatory cytokines

The absolute levels of individual cytokines per se may not be as informative as

the ratios of cytokines, i.e. the levels of inflammatory cytokines relative to the level of

IL-10, an anti-inflammatory cytokine. Keeping this in mind, the ratios of inflammatory

cytokines to IL-10 were calculated.

4.3.1. IL-2/IL-10 ratio

There is no significant difference in IL-2/IL-10 ratio between all the groups (p>

0.05) (Figure 8).

Normal

Caries Exposure Irreversible

Pulpitis

0

1

2

3

4

5

6

7

IL-2

/IL

-10

Figure 8. Mean IL-2/IL-10 ratio in different groups.

40

4.3.2. IL-6/IL-10 ratio

The IL-6/IL-10 ratio is significantly higher in irreversible pulpitis as compared

to both caries exposure and normal pulps (p< 0.05). However, there is no significant

difference between normal pulps and caries exposure pulps (p> 0.05) (Figure 9).

Normal

Caries Exposure

Irreversible

Pulpitis

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

IL-6

/IL

-10

Figure 9. Mean IL-6/IL-10 ratio in different groups.

41

4.3.3. IL-8/IL-10 ratio

The IL-8/IL-10 ratio is significantly higher in irreversible pulpitis when

compared to both caries exposure pulps (p<0.001) and normal pulps (p< 0.01), but there

is no significant difference between normal and caries exposure pulps (p> 0.05) (Figure

10).

Normal

Caries Exposure

Irreversible

Pulpitis

0

20

40

60

80

100

120

140

160

IL-8

/IL

-10

Figure 10. Mean IL-8/IL-10 ratio in different groups.

42

4.3.4. TNF-α/IL-10 ratio

The TNF-α /IL-10 ratio is significantly higher in caries exposure group

compared to irreversible pulpitis (p<0.05). However, there is no significant difference

between normal and both irreversible pulpitis and caries exposure (p> 0.05) (Figure 11).

Normal

Caries ExposureIrreversible

Pulpitis

0

1

2

3

4

5

6

7

8

TN

F-α

/IL

-10

Figure 11. Mean TNF-α /IL-10 ratio in different groups.

43

4.3.5. IFN-γ/IL-10 ratio

The IFN-γ/IL-10 ratio is significantly higher in normal pulps compared to both

caries exposure and irreversible pulpitis (p<0.05). There is no significant difference

between irreversible pulpitis and caries exposure (p> 0.05) (Figure 12).

Normal

Caries Exposure

Irreversible

Pulpitis

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

IFN

-γ/I

L-1

0

Figure 12. Mean IFN-γ/IL-10 ratio in different groups.

44

In summary, IL-6/IL-10 and IL-8/IL-10 ratios were significantly higher in

irreversible pulpitis compared to both normal and caries exposure pulps (Figure 13).

Figure 13. Summary of cytokine ratios in different groups.

45

Chapter 5

Discussion

Pulpal diagnosis is indispensable to correct treatment planning. To date,

diagnosis and classification of pulpal diseases have been based on symptoms that are

not always consistent with histological findings of pulpal pathosis (Seltzer, 1972). In the

early stage of pulpal pathosis, the status of the pulp changes from reversible to

irreversible pulpitis (Torabinejad & Walton, 2009). Because the dental pulp is enclosed

in hard tissues, obtaining direct information on pulpal status is usually difficult unless

evaluated microscopically. To determine the histological status of pulp, it is necessary

to examine the pulp itself.

However, when the pulp is exposed, it is possible to obtain pulpal blood samples

that may contain inflammatory factors that reflect the inflammatory state of the pulp.

Analysis of the blood sample would provide valuable information regarding pulpal

status. This information could then be used to determine the appropriate treatment.

Due to the necessity of determining pulpal status, Prader (1949) reported

objective methods for the diagnosis of pulp disease using pulpal blood. However, he

analyzed the white blood cell differential count of the sampled pulpal blood after

extirpation or resection of the pulp and that was only qualitatively.

Nakanishi et al. (1995) attempted to take pulpal blood samples rather than the

entire pulps. They established a method for collecting pulpal blood samples from

exposed pulpal sites. Blood from the exposed surface of the pulp was collected with a

46

pre-weighed pellet of absorptive material. The pellet was held at the exposed site for 5

seconds to allow absorption of the pulpal blood. Then the quantity of sampled blood

was determined by measuring the increased weight of the pellets. Using this method,

they quantitatively analyzed the inflammatory factors in these blood samples. However,

weighing the pellets accurately is not easy and is difficult to implement in clinical

practice. Also, the amount of blood collected was very small to detect many cytokines

usually present in minute quantities.

In this study, we tried to make the method of Nakanishi et al. (1995) easier to

use clinically. We used cotton pellets which were directly placed in a heparin-coated

tube containing 1 ml saline. Later the levels of cytokines were expressed per total

amount of protein present in the sample. As the total amount of protein is proportional

to the total amount of blood, it will be an accurate reflection of the amount in the

sample. The method in this study can be used both for the clinical diagnosis of pulpitis

(reversible versus irreversible) and for the elucidation of pulpal pathogenesis.

All previous studies which measured cytokines in the dental pulp focused only

on two extremes. They compared normal pulps with irreversible pulpitis. They did not

consider the intermediate stage of reversible pulpitis where the pulp still has healing

capacity; this intermediate stage is very important in understanding pulpal pathogenesis

as well as diagnosis. We measured the levels of different cytokines in normal pulps,

asymptomatic caries-exposure pulps and irreversible pulpitis.

47

5.1. Cytokines and the dental pulp

IL-2, a cytokine secreted by CD4+ T cells, primarily acts to stimulate T cell

proliferation, activates natural killer cells and promotes B cell function through T helper

cell activation. Activated T helper cells assist B cells to recognize antigen, produce

antibody, and differentiate into plasma cells. These activities emphasize the importance

of IL-2 in the immune response. In addition, IL-2 activates transcription of

proinflammatory cytokines and increases the cytolytic activity of natural killer cells

(Gaffen & Liu, 2004). In the absence of IL-2, acute disease is reduced, and a chronic,

inflammatory disorder develops instead (Hoyar et al., 2008).

IL-2 was first measured in the dental pulp is by Rauschenberger et al. (1997).

Twenty healthy pulps obtained from soft tissue impacted third molars were compared

with twenty inflamed pulpal samples collected from patients diagnosed with irreversible

pulpitis. After extraction, teeth were sectioned and half of the pulpal tissue was

histologically evaluated while the second half was prepared for measurement of IL-2

levels by ELISA kits prepared in their own laboratory. They detected IL-2 in all vital

pulpal samples. Histologically uninflamed pulps exhibited low concentrations of IL-2

irrespective of their clinical classification (normal or irreversibly inflamed). The highest

concentrations of IL-2 were detected in mildly inflamed pulps followed by the moderate

and severely inflamed tissues. Additionally, they demonstrated that lL-2 concentrations

were significantly higher in irreversibly inflamed pulpal tissues as compared to

asymptomatic normal samples.

48

Anderson et al. (2002) tried to repeat the Rauschenberger et al. (1997) study

using commercial IL-2 assay kits. They detected IL-2 in only 20 of 72 samples with no

significant difference between the experimental groups. Thus, data on IL-2 in inflamed

pulps has been somewhat contradictory.

In our study, IL-2 was detected in 17 of 25 normal samples, 32 of 40 caries

exposure samples and 30 of 43 in the irreversible pulpitis group. IL-2 was significantly

higher in the caries exposure group compared to both the normal group (p< 0.01) as

well as the irreversible pulpitis group (p< 0.05).

Our data support the Rauschenberger et al. (1997) study. Both studies found that

IL-2 was significantly higher in carious teeth compared to normal. IL-2 was higher in

mildly inflamed pulps (i.e. caries-exposed in our study) compared to highly inflamed

(i.e. irreversible pulpitis in our study). The failure of Anderson et al. to detect IL-2 in

most samples may be explained by the low sensitivity of the assay used which was 7000

pg/ml compared to our study which has a sensitivity of 0.4 pg/ml.

Having higher levels of IL-2 in the caries-exposure group compared to both the

normal and irreversible pulpitis may suggest that the pulp is initiating an immunological

repair by stimulating the expansion of the helper T-cell population. As pulpal

inflammation progressed to irreversible stage, IL-2 concentration decreased. This

decrease in IL-2 represents a point where the tissue is in the late phases of irreversible

inflammation and is progressing toward total tissue necrosis. Further research

49

correlating the levels of IL-2 and both histological status and healing capacity is needed

to understand its role in pulpal inflammation.

IL-6 is an important cytokine in immune responses. It stimulates the

differentiation of mature B lymphocytes to antibody-producing plasma cells, activates

T-cells and enhances hematopoiesis. It also displays multiple biological effects and acts

as a major mediator of the host response following tissue injury and infection as well as

inflammation. It increases the levels of acute-phase proteins, C-reactive protein, serum

amyloid A and fibrinogen. IL-6 causes up-regulation of adhesion molecules and induces

angiogenesis leading to increase in vascular permeability and leads to inflammatory

edema. In addition, it induces osteoclast differentiation and bone resorption (Nishimoto,

2010). In general, pulpal symptoms were always explained by increase in the intra-

pulpal pressure due to edema (Bender, 2000). This makes IL-6 a cytokine worth

studying in the pulp. Levels of IL-6 can be correlated to the amount of inflammation

and edema in the pulp in addition to its role as a mediator of host response following

tissue injury and infection.

IL-6 has been studied by Barkhordar et al. (1999) in six inflamed human pulps

tissue and six human periapical lesions. The inflamed dental pulp tissue and the

periapical lesions demonstrated elevated IL-6 levels compared with the normal pulp.

Nakanishi et al. (1995) studied levels of IL-6 in pulpal blood. IL-6 was detected

in only 2 of the 18 blood samples from inflamed subjects and was present in extremely

small quantities. It was not detected in the normal pulp.

50

In our study, IL-6 was not detectable in the normal pulp. It was detected at high

incidence in irreversible pulpitis (70 %) than in asymptomatic caries exposure (53 %).

IL-6 was significantly higher in both the caries exposure group as well as the

irreversible pulpitis group as compared to normal pulps (p< 0.001). However, there was

no significant difference between the irreversible pulpitis and caries exposure groups.

Our data is consistent with results of Barkhordar et al. (2000) on pulpal tissue.

Although we and Nakanishi et al both used pulpal blood as samples, they could

not detect IL-6 in most of the samples. We may attribute our ability to detect IL-6 to the

higher sensitivity of ELISA in our study.

IL-8 is one of the most potent chemokines with strong chemoattractive activity

for neutrophils. It creates a chemotactic gradient toward the area of increased

chemokine concentration. This gradient helps to both bring cells toward the local site of

inflammation and also to retain them once they have arrived. In addition to recruitment,

IL-8 also serves to stimulate the neutrophil to a higher state of activation. It is rapidly

synthesized at local sites of inflammation where it fulfills its function of recruiting and

activating acute inflammatory cells (Remick, 2005).

For this reason, it has been studied in order to provide more information on its

action in the dental pulp. In diseased pulps, IL-8 was mainly produced by pulpal

inflammatory cells and endothelial cells in addition to odontoblasts. It has been

demonstrated that odontoblasts are capable of expressing IL-8 messenger RNA in

response to lipopolysaccharide stimulation (Levin et al., 1999).

51

Huang et al. (1999) studied IL-8 in dental pulp tissue by both ELISA and

immunohistochemistry. They detected 23-fold higher levels of IL-8 in inflamed pulps as

compared to normal pulps. In addition, stronger IL-8 histological staining was observed

in inflamed pulps than normal pulps. Guo et al. (2000) used pulpal blood as a sample.

They detected higher levels of IL-8 in inflamed pulps while it was undetectable in

normal pulps. They also found higher levels in patients with acute symptoms than

chronic pulpitis symptoms. In a study by Silva et al. (2008), inflamed pulps showed

higher levels of IL-8 by both ELISA and immunohistochemistry as compared to healthy

pulps. They also demonstrated that pulp fibroblasts stimulated by bacterial LPS produce

higher levels of IL-8 than the control group.

In our study, IL-8 was significantly higher in pulps with irreversible pulpitis and

caries exposure than normal pulps. It was not detectable in normal pulps. Irreversible

pulpitis pulps have 7-fold higher mean IL-8 levels than asymptomatic caries exposure

pulps.

TNF-α is one of the most prominent inflammatory mediators and absolutely

central in initiating the cascade of inflammatory reactions of the immune system

including induction of cytokine production, activation and expression of adhesion

molecules and growth stimulation. It coordinates the early response to injury and thus

represents an important point of regulation in inflammatory disease (Hehlgans &

Pfeffer, 2005).

52

Nakanishi et al. (1995) reported that TNF-α was detected in 10 out of 20 pulpal

blood samples in the inflamed pulp group. In the normal pulp group, 2 out of the 6

samples contained a detectable amount of TNF-α. However, there was no significant

difference between the two groups. Pezelj-Ribaric et al. (2002) studied the levels of

TNF-α in pulpal tissue from irreversible symptomatic pulpitis, asymptomatic

irreversible pulpitis and healthy pulps. They reported high levels of TNF-α in pulpal

tissue from teeth clinically diagnosed with irreversible pulpitis, but considerably lower

expression of this cytokine in samples from normal teeth. Also, they found significantly

higher levels of TNF-α in symptomatic pulpitis patients. Kokkas et al. (2007) found a

significant increase in TNF-α gene expression in pulp tissue associated with irreversible

inflammation as compared to healthy controls. No such difference was detected in

reversibly inflamed pulp in comparison to healthy teeth.

Our study focused on groups similar to the study by Pezelj-Ribaric et al.

However, we considered asymptomatic irreversible pulpitis cases as caries exposure

cases since irreversibility was still doubtful. Our data showed significantly higher levels

of TNF-α in pulpitis cases than in normal subjects with no significant difference

between caries exposure and irreversible pulpitis. We used pulpal blood while Pezelj-

Ribaric et al (2002) used pulpal tissue which makes comparison is difficult. However,

both studies showed that a higher level of TNF-α is present in pulps with inflammation.

The low levels in the Nakanishi et al (1995) study may be due to low sensitivity of the

assay used.

53

IL-10 is a very important anti-inflammatory cytokine. It is the main inhibitor of

cytokine synthesis and macrophage activity. It also inhibits the production of the pro-

inflammatory cytokines like TNF-α, IL-1β and IL-6 in various cell types (Anker & von

Haehling, 2004). IL-10 can further inhibit inflammation by increasing the release of IL-

1 receptor antagonist by macrophages (Akdis, 2001). It was shown that IL-10

production is increased in inflammatory processes exerting an immunomodulatory role

(Yamaoka et al., 1999, Markert, 2003). IL-10 inhibits the differentiation and maturation

of DCs (Mosser & Zhang, 2008).

Hahn et al. (2000) found that incidence of IL-10 mRNA was significantly higher

in deep-caries than shallow-caries. IL-10 has not been investigated in the dental pulps

or been related to pulpal inflammation.

In our study, significantly higher levels of IL-10 were detected in caries

exposure and irreversible pulpitis compared to normal pulps. Higher levels of IL-10

were detected in caries-exposed pulps compared to irreversible pulpitis. This suggests

that IL-10 may be associated with pulpal symptoms as high levels were present in

asymptomatic pulps. Also, having high levels may be an indication that the pulp is

attempting to suppress inflammation before it reaches an irreversible stage as in other

inflammatory diseases. For example, IL-10 has a critical role in suppressing intestinal

inflammation and colitis (Barbara et al., 2000). In addition, Sasaki et al. (2000) found

that, IL-10 is an important endogenous suppressor of infection-stimulated periapical

bone resorption in vivo.

54

IFN-γ is one of the predominant cytokines in immune responses. It plays a

crucial role in both innate and adaptive immunity as well as in inflammation. IFN-γ

activated macrophages produce a variety of toxic substances. It is the main trigger for

production and release of reactive oxygen species such as reactive oxygen intermediates

and reactive nitrogen intermediates. These powerful oxidants bestow macrophages with

cytostatic or cytotoxic activity against bacteria, viruses, fungi, protozoa, helminths and

tumor cells (MacMicking, 2009). Inflammatory actions of IFN- are closely involved in

both rheumatoid synovitis and atherosclerosis. Also, in concert with other pro-

inflammatory cytokines, IFN-is the most important trigger for the formation and

release of reactive oxygen species (Schroecksnadel et al., 2006).

Hahn et al (2000) found IFN-γ mRNA in pulp tissue of both shallow and deep

caries teeth. This is the only published study of IFN-γ in the pulp.

In our study, we found significantly higher levels of IFN-γ in caries exposure

and irreversible pulpitis compared to normal pulps. There was no significant difference

between caries exposure and irreversible pulpitis. In spite of its immunemodulatory

effects, IFN-γ may not reflect differences in the degree of inflammation between caries-

exposure and irreversible pulpitis.

55

5.2. Ratios of inflammatory to anti-inflammatory cytokines

Measuring inflammatory to anti-inflammatory cytokines ratios is a good

indicator of the cytokine balance in tissues. Also, ratios have no units which make

comparisons easy when different types of samples are used. We calculated ratios of

inflammatory cytokines to IL-10.

The IL-2/IL-10 ratio showed no significant difference between the different

groups. The TNF-α /IL-10 ratio was significantly higher in the caries-exposure group

compared to irreversible pulpitis. However, the TNF-α /IL-10 ratio was not significantly

different between normal and irreversible pulpitis and between normal and caries-

exposure and may not therefore be a good marker of pulpal inflammation.

The IFN-γ/IL-10 ratio was significantly higher in normal pulps compared to

both caries-exposure and irreversible pulpitis. However, there is no significant

difference between irreversible pulpitis and caries exposure.

The IL-6/IL-10 and IL-8/IL-10 ratios were significantly higher in the irreversible

pulpitis group compared to both caries exposure and normal pulps with no significant

difference between normal and caries exposure pulps. This makes these two ratios

potentially very good markers of irreversible pulpitis.

Pulpal blood is a potentially good sample for pulpal diagnosis in caries exposure

cases, as it is easily obtained in clinical setting. Choosing a marker of inflammation and

evaluating its levels with the success and failure of pulp capping is key to implement

56

these cytokine studies in clinical practice. This can help in predicting the long-term

prognosis of direct pulp capping. We suggest the use of the IL-8/IL-10 ratio because it

gives more information about overall cytokine balance in of the pulp. IL-8 and IL-8/IL-

10 are therefore recommended for studies on larger sample sizes, as it may contribute to

easy diagnosis of pulpal inflammation in caries-exposure.

57

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68

Mohamed Elsalhy

Curriculum Vitae

Personal Information

Full Name: Mohamed Maged M M Elsalhy

Birth Date: 21 December 1982

Birth Place: Kuwait

Gender: Male

Nationality: Egyption

Contact Information

Telephone Number: +965 66857788

E-mail Address: alsalhy1@hotmail.com

Undergraduate Education

High School: Abad bin Bishr School, (1996-2000). Scored 96.3% (Science).

Bachelor of Medical Sciences (B.Med.Sc.): Health Sciences Center – Kuwait

University (2000 – 2004) / GPA: 3.17

Bachelor of Dental Medicine (BDM): Faculty of Dentistry- Kuwait University (2004

- 2007) GPA: 3.19 – Ranked 4th in a 25 student batch

Postgraduate Education

Membership of the Faculty of Dentistry of the Royal College of Surgeons in Ireland

Diploma (MFDS RCSI)

Part I: passed April 2008

Part II: passed June 2010

Master of Science in Medical Microbiology- Faculty of Medicine- Kuwait University

(September 2008 – current).

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Work Experience

February 2007 - January 2008: Internship in Ministry of Health Dental Specialty

Centers - Kuwait .

Publications and Abstracts

ElSalhy M, Sayed Zahid I, Honkala E. EEffects of Xylitol Mouthrinse on Streptococcus

mutans: submitted to Journal of Dentistry; under review.

ElSalhy M. Short Term Effects of Polyols on Streptococcus mutans and Lactobacilli;

Presented as a poster in First African and Middle-East IADR Federation Conference

2005, Kuwait.

Current Research

Investigating mediators of Inflammation (IL-2, IL-6, IL-8, IL-10, TNF-α, and IFN-γ) in

Irreversible pulpitis and asymptomatic caries exposure cases. Elsalhy M. Raghupathy R.

Barrieshi-Nusair KM (Master Thesis).

Interests

Reading, Swimming, Traveling

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مهخصان

من المعضالت الحشخيصية في طب اسنبن ه بق جربذقش مربذاس ابلحيب ب فبي لب اسنبن هر مه جربذقش مربذاس

مقضببقةية كميببة ابلحيبب ب فببي الهبب ميببي تببذا فببي جرشقببش العببالذ الحألسضببي ال الدببزسق ل نببن هر ققتببذ طشقرببة

لحكقه مؤششات بلحي ب (Cytokines)سيحقكين ت ةمهية لحرذقش مرذاس ابلحي ب ححى اآلهر في الس بق جي مقحشاح ال

ل اسنن ه لكن جرذقش كميبة السبيحقكين ت كب ه فربك ممكنب بعبذ سهبن اسنبن ه المهحيذبةر فبي بزم الذسانبة قمنب بحنسيبز

طشقرة لري ط كمية السحقكين ت من اله بذ ه سهن اسنن ه قذل مجخ ر الرشاس.

بقانطة قطنبة غبريشم مبن مكب ه الهب المكشبقع سبال ةمهيبة مصالبة ل نني 101جي جدمين ةينة دم من

ةينبة مبن لب النبن ه س ليبة مبن اسةبشاا لكنيب مكشبقفة 40ةينة جي السز من النن ه نبهيم 25اله الدضئيةر

ةينة من النن ه مشخصة بإلحي ب اله غيبش البشد در جبي قيب ط كميبة السبحقكين ت المألسبضم 43اله بسذ الحسقط

-IFN منحشفيبش ه ت مب (TNF-α) ة مل نخبش البقسم اللسب 2 6 1 (Interleukin) إللحي ب ي منحشلقكينل

γ ر بقانببطة فألبب 10( السببيحقكن المضبب د لإللحيبب ب ببق منحشلببقكينELISA ةبب لي الذقببةر جببي جرببذقش مسببحق

–Kruskalطة السألقغ ت ابحصب ئية بقان 10السيحقكين ت نسذة السحقكينث المألسضم لإللحي ب مر بل منحشلقكين

Wallis) (Mann–Whitney.

منحشفيبش ه ت مب (TNF-α) ة مبل نخبش البقسم اللسب 10 1 6 تذن معبذ ت مشجسعبة مبن منحشلبقكين

IFN-γ) اسنن ه المكشقفة بقانطة الحسقط رات ابلحيب ب الهذبي غيبش البشد د مر سنبة ب سنبن ه السبهيمةر في ل

الةهبى 10 2الةهى في اسنن ه المكشقفة فربك مر سنبة ب لسبهيمةر كمب كب ه معبذ منحشلبقكين 2ك ه معذ منحشلقكين

الةهى في ملحي ب الهب 1 لكن ك ه منحشلقكين في اسنن ه المكشقفة ب لمر سنة ب سنن ه رات ملحي ب اله غيش الشد د

10/منحشلبقكين 6غيش الشد د مر سنة ب سنن ه المكشقفة بسذ الحسقطر من المثيبش لإل حمب م اله النسبذة منحشلبقكين

ك نبث مشجسعبة فبي ملحيب ب الهب غيبش البشد د مر سنبة بكبل مبن اسنبن ه المكشبقفة 10/منحشلبقكين 1النسذة منحشلقكين

قط اسنن ه السهيمة.بسذ الحس

قمكببن اله قكقنببقا 10/منحشلببقكين 1 النسببذة منحشلببقكين 1نسببحنحم مببن ببزم الذسانببة اله مسببحق منحشلببقكين

مشيشات مميضم بلحي ب اله غيش الشد در مه زم الذسانة جقضح اله معذ السبيحقكين ت المقتبقدم فبي لب اسنبن ه

ب ل اسنن ه.قمكن اله جكقه نيهة مسيذم في جشخي ملحي

71

جامعــــــة انكىيـــــث

األسنان انتحهيم انمناعي إلنتهاب نة

انمقذمة مه :

محمذ ماجذ انصانحي

أطروحة مقذمة نكهية انذراسات انعهيا إلستيفاء جزء مه متطهثات درجة انماجستير

في انعهىو انطثية )انميكروتيىنىجي(

تإشراف

راجيىتاثي .د. راج جىتال أ

د. كفاح محمىد تاريشي )مشرف مشارك(

انكىيث

2011أغسطس/