EVALUATION OF THE LEVELS OF INTERLEUKIN 8 AND ...

I

EVALUATION OF THE LEVELS OF INTERLEUKIN 8 AND

INTERLEUKIN 4 AROUND MINI-IMPLANTS DURING

ORTHODONTIC TOOTH MOVEMENT - A CLINICAL STUDY

By

Dr. BIANCA B. RODRIGUES

Dissertation Submitted to the

Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore

In partial fulfillment of the requirements for the degree of

MASTER OF DENTAL SURGERY

in the speciality of

ORTHODONTICS AND DENTOFACIAL ORTHOPAEDICS

Under the guidance of

Prof. (Dr.) SANJU SOMAIAH M.K.

Professor

DEPARTMENT OF ORTHODONTICS AND DENTOFACIAL

ORTHOPAEDICS

COORG INSTITUTE OF DENTAL SCIENCES

VIRAJPET, COORG, KARNATAKA-571218.

2016– 2019

VI

ACKNOWLEDGEMENT

As this dissertation draws to an end I would like to take this opportunity to express

my gratitude to several people who have been instrumental in the completion of this

study.

I thank god almighty for showering his immense blessings and providing strength

for completing this study successfully.

I express my deepest sense of gratitude to my teacher and guide,

Dr. Sanju Somaiah M.K., Professor, Department of Orthodontics and Dentofacial

Orthopaedics, Coorg Institute of Dental Sciences, for the proficient guidance, vigilant

supervision and incessant encouragement given to me throughout the study.

I am immensely obliged to Dr. Goutham B., Professor and Head, Department Of

Orthodontics and Dentofacial Orthopedics, Coorg Institute Of Dental Science for his

pricely support and constant encouragement. I have been amazingly fortunate to have

an advisor who gave me the freedom to explore on my own.

I would also like to thank Dr.Sunil Muddaiah, Dean, Professor, Department Of

Orthodontics And Dentofacial Orthopedics, for his valuable suggestions and

overwhelming support.

I take this opportunity to express my gratitude and heartfelt thanks to

Dr.Balakrishna Shetty, Professor, Department Of Orthodontics And Dentofacial

Orthopedics for timely help and encouragement.

VII

I also thank Dr. Roopa S., Dr.Vikram Susil Faculty, Department Of Orthodontics

And Dentofacial Orthopedics, Coorg Institute Of Dental Sciences for all the help,

guidance and encouragement rendered throughout the study.

I am also thankful to Dr. Bhakti Sadhu, Department of Public Health Dentistry,

Coorg Institute Of Dental Sciences for helping me with the statistical analysis of the

data for my study.

I thank Dr. K. C. Ponnapa, Principal, Coorg Institute of Dental Sciences, for his co-

operation and guidance throughout my post-graduation course.

I also thank my co post-graduates, Dr. Dixit T.P., Dr. Athul Tom, Dr. Justin Jolly,

Dr. Muhasin and Dr. Nikhil for standing by me and lending me helping hands

throughout these past years of postgraduate training. Their support and friendship were

constant during this tenure; I appreciate it from the bottom of my heart.

I take immense pleasure to thank my seniors, Dr. Jeffy, Dr. Shafeeq,

Dr. Shanahaz, Dr. Abin, Dr. Saran, Dr. Shafad for their constant advice and support.

It would be unjust on my part if I don’t extent my gratitude to my juniors

Dr. Basil, Dr. Ann, Dr. Razeen, Dr. Roni, Dr. Tripthi, Dr. Manu, Dr. Amric,

Dr. Elbin, Dr. Eric, Dr. Anna, Dr. John, Dr. Noufal for their constant help and

encouraging me out at various levels of work.

A Special thanks to my friends Dr.Manju, Dr.Anusha, Dr.Dhanya and

Dr.Nithya for their constant encouragement and support during my thesis.

I would also like to extend my gratitude to all the nonteaching staff of my

department for rendering timely support.

IX

LIST OF ABBREVIATIONS

ELISA ENZYME LINKED IMMUNOSORBANT ASSAY

GCF GINGIVAL CREVICULAR FLUID

IL- 8 INTERLUEKIN 8

IL- 4 INTERLUEKIN 4

mm MILLIMETER

MSI MINISCREW IMPLANT

OTM ORTHODONTIC TOOTH MOVEMENT

pg/μl PICOGRAMS PER MICROLITER

PMICF PERI-MINISCREW IMPLANT CREVICULAR FLUID

TAD TEMPORARY ANCHORAGE DEVICE

X

LIST OF TABLES

Table.

No.

Title Page No.

1 Comparison of Interleukin 8 levels at different intervals

among study subjects around miniscrew implants using

paired t test.

37

2 Comparison of Interleukin 8 levels at different intervals

among study subjects around miniscrew implants between

maxilla and mandible using independent t test.

38

3 Comparison of Interleukin 4 level among study subjects

around miniscrew implants using paired t test.

40

4 Comparison of Interleukin 4 levels among study subjects

around miniscrew implants between maxilla and mandible

using independent t test.

41

XI

LIST OF FIGURES

Figure

No.

Title Page No.

1 Comparison of mean Interleukin 8 levels at T1, T2, and T3. 36

2 Comparison of mean Interleukin 4 levels at T1, T2, and T3. 39

3 Eppendorf tube and microcappilary tube used for sample

collection.

64

4 Peri-miniscrew implant crevicular fluid sample collection. 64

5 Samples stored in Eppendorf tubes. 64

6 ELISA kits of IL-8 and IL-4. 65

7 Contents of ELISA kits IL-8 and IL-4. 65

8 Performing the ELISA test. 65

9 Yellow colour change after adding stop solution. 66

10 Elisa spectrometer for reading colour change 66

11 Dontrix gauge 66

XIII




Background and objectives:

The evaluation of cytokines in the peri-miniscrew implant crevicular fluid (PMICF)

has been proposed as a non-invasive means of monitoring the healthy or diseased

status of peri-implant tissues. The objective of this study was to evaluate the levels of

interleukin 8 (IL-8) and interleukin 4 (IL-4) around miniscrew implants during

orthodontic tooth movement and to draw clinical inferences from the same.

Methodology:

The present study was done around 22 healthy miniscrew implants in subjects

within an age range between 16-25 years without any signs of peri-implantitis. The

selected subjects were in retraction stage using titanium miniscrew implants with a

force of 150g. The samples were collected over a month time period after force

application on miniscrew implants according to the following schedule: T1, T2 and

T3 at 1 hour, 7th

day and 21st day respectively. The PMICF samples were put through

ELISA test for determining the concentration of IL-8 and IL-4.

Result:

It was observed that in healthy miniscrew implants the IL- 8 levels increased from

one hour to 7th

day due to perimucositis and slightly decreased or remained the same

from 7th

to 21st day indicating that the progress of inflammation has regressed.

XIV

The IL-4 levels decreased from one hour to 7th

day due to perimucositis. It

decreased from 7th

to 21st day. This could be attributed to the persistence of

inflammation through 21st day.

Conclusion:

These findings demonstrated that the levels of IL-8 and IL- 4 in PMICF could be

considered in detecting the stability of miniscrew implants.

Keywords: Miniscrew implant, Peri-miniscrew implant crevicular fluid,

Interleukin 8, Interleukin 4.

Introduction

1




INTRODUCTION

Anchorage is a critical issue during orthodontic treatment and if inadequate can be

the most limiting factor, no matter which technique or philosophy the clinician

follows especially when treating adults. Orthodontic tooth movement has always been

limited to action-reaction reciprocal force mechanics in anchorage control.

Although extraoral anchorage can be used to supplement tooth-borne anchorage

and deliver force in directions not possible with an intraoral anchorage, extraoral

anchorage has such limitations that it requires excellent patient cooperation. Over the

past 10 to 15 years, several techniques at a skeletally based anchorage have been

attempted clinically which include fixation wires, bone plates, fixation screws,

miniaturized dental implants in the palate and retromolar areas and palatal onplants.

The control of anchorage is one of the most crucial factors in orthodontic treatment

planning. Achieving absolute anchorage has been one of the main objectives for an

orthodontist and mini implants have become the most effective and powerful tool for

achieving this. Miniscrew implants that have increasingly been used for anchorage

control in orthodontic treatment for non-compliant patients have many advantages,

such as ease of placement and removal, small size, and low cost.1

Introduction

2

Background of the study:

Clinicians and researchers have tried to use endosseous implants as orthodontic

anchorage for over half a century. The first successful attempt to move teeth against a

stationary screw was published as early as in 1969 by Linkow, although routine

application of dental implants for orthodontic purposes in humans began 20 years

later.2

The disadvantages of dental implants became apparent during this period. The

requirement of specific surgical procedures during insertion and removal, the demand

for complicated clinical and laboratory procedures to accurately transfer the implant

position to cast models and to facilitate safe and precise implant insertion, delayed

loading resulting from the waiting period necessary for osseointegration increasing

total treatment time, possible location limited only to edentulous or retromolar areas

of the maxilla and mandible and large dimensions apparently increases the risk of

damage to the adjacent tissues or root injuries.

To overcome these problems, Kanomi in 1997 and Costa et al. in 1998 introduced

miniscrew implants as temporary anchorage devices (TADs). Their decreased

diameter facilitates insertion in almost all sites of the jaws, with no flap surgery

required. Osseointegration does not usually occur; therefore, they provide only

temporary stationary anchorage; consequently, there is no 6-month waiting period.

Immediate loading of TAD apparently decreases total treatment time.2

From this point onwards, the single most important implant used during

orthodontic treatment for an anchorage is the miniscrew implant. The clinical use of

miniscrew implants as temporary anchorage devices has been widespread. This is due

to the possibility of absolute orthodontic anchorage and the ease of installation and

Introduction

3

removal of these devices. Miniscrew implants are routinely used to anchor retraction

of the anterior segment, mesiodistal movement of the posterior teeth, asymmetrical

tooth movement, intrusive mechanics and orthopaedic corrections.

Miniscrew implants (MSI), are customarily placed without a flap or after a punch

incision under local infiltration only. It is important to realize that the potential

complications are relatively insignificant relative to major orthognathic surgical

procedures; however, tooth root damage, bony and soft tissue infection, and implant

failure are possible.

Need and significance of the study:

It was reported in a review article that the miniscrew implants have an average

survival rate of 84% (range 57–95.3%) which is largely dependent on factors

governing primary and secondary stability. The primary stability pertains to the

mechanical holding of MSI in the bone, while the secondary stability relates to

biological retention. Broadly, the factors affecting the stability can be grouped into a

host, miniscrew implant and technique-related factors. Contemporary research has

focused on the biological seal between the implant and host tissue, more so on its

surrounding peri-implant oral epithelium.3

The occurrence of screw failures has required ongoing investigation of potential

risk factors. Since peri-implantitis turned out to be the main cause of screw loosening,

assessment of factors involved in such inflammation has become crucial to increase

TAD stability in orthodontics.2 An early and reliable detection of any adverse peri-

miniscrew tissue reaction is essential for patients being treated with miniscrew.

Introduction

4

As in periodontitis and other infections, pathogens and their virulence factors

stimulate the release of several immunoinflammatory biomarkers by peri-implant

cells. Among the numerous biomarkers, proinflammatory cytokines [e.g., tumor

necrosis factor (TNF)-a, interferon-c, interleukin(IL)-1b, IL-6, IL-12, IL-17,IL-8 and

RANKL] and anti-inflammatory cytokines(e.g. IL-4, IL-10 and IL-1 receptor

antagonist) have been suggested to be important mediators of inflammation and

immunity in the pathogenesis of peri-implantitis.

Peri-implantitis accounts for about 30% of miniscrew failures.4, 5

Peri-implantitis

is a progressive peri-implant bone loss in conjunction with soft tissue inflammatory

lesion. Initially, peri-mucositis (a reversible inflammation of the soft tissues

surrounding the miniscrew) occurs, which if left untreated may progress to peri-

implantitis.4, 6

Peri-implantitis is clinically characterized by increased probing depth,

pain, and/or radiographic bone loss, which may cause implant failure. An early and

reliable detection of any adverse peri-miniscrew tissue reaction is essential for

patients being treated with miniscrew.4, 7

Orthodontic forces cause acute inflammatory reactions, vascular changes, and

migration of leucocytes. Recent studies show that in periodontal and peri miniscrew

tissues cytokines, tumour necrosis factor and transforming growth factor have

important roles in regulating and amplifying inflammatory response.4, 7, 8, 9

Cytokines

are involved in initiating, amplifying, perpetuating and resolving inflammatory

responses in periodontal and peri-implant tissues.1

Cytokines are classified as pro-inflammatory and anti-inflammatory. Pro-

inflammatory cytokines include TNF, IL-1, IL-2, IL-6 and IL-8. These are ‘alarm’

cytokines that induce vascular dilation through increased permeability and enhanced

Introduction

5

inflammatory response, whereas IL-4, IL-10, IL-13, IFN-alpha and transforming

growth factor-beta are recognized as anti-inflammatory cytokines and are involved in

the reduction of inflammatory reactions.1

Many studies on dental implants have

reported that an increase in proinflammatory cytokine levels in peri-implant crevicular

fluid causes peri-implantitis.1, 10, 11, 12

IL-8 plays a key role in recruiting and activating neutrophils during inflammation.

It is important for regulating alveolar bone resorption during tooth movement by

acting at an early stage in the inflammatory response. IL-8 is secreted mainly by

monocytes, macrophage, epithelial cells and endothelial cells and is essential in early

inflammatory response with a role in neutrophil recruitment and degranulation during

inflammation. Evidence from research studies also supports high IL-8 levels in

periodontitis and at peri-implant inflammation sites.3

The various activities of IL-8 indicate that this cytokine plays a major role in

mediating inflammatory responses. In a study conducted to analyse the levels of

interleukin-8 during tooth movement, it was found that orthodontic forces evoke

changes in IL-8 levels.13

In another study it was found that IL-8 levels were

significantly higher in patients with failing implants than in those with healthy

implants.1, 14

Interleukin-4 (IL-4) is a pleiotropic cytokine secreted by activated T lymphocytes,

mast cells, eosinophils, and basophils. It is a key regulator in humoral and adaptive

immunity that regulates the function of lymphocytes and macrophages.15

In a recent

study it was found that IL-4, secreted by T helper type 2 (TH2) cells, is a potent

inhibitor of the RANKL induced osteoclastogenic process.16

The effect of IL-4 in

osteoclastogenesis is supported by the finding from studies which revealed that

Introduction

6

over-expression of IL-4 in vivo prevents bone erosion in animal models of

inflammatory arthritis.17, 18, 19

In periodontitis sites where IL-4 levels were found to be too low, there was

persistent accumulation of activated macrophages, leading to increased destruction of

the periodontium. It has been hypothesized by researchers that localized absence of

IL-4 at the site of gingival inflammation plays a fundamental role in the progression

of gingivitis to periodontitis. In a study done in India an inverse relationship has been

reported in between gingival crevicular fluid IL-4 levels and periodontal status; IL-4

was higher in the periodontally healthy group, but very low in the periodontally

diseased group.20

It was found that the anti-inflammatory effect of IL-4 results from

its efficient inhibition of the production of proinflammatory cytokines such as tumor

necrosis factor-a (TNF-a), IL-1a, IL-1b, IL-6 and IL-8 by monocytes/ macrophages.

In a recent article it was stated that IL-4 plays an important role in the regulation of

the immunoinflammatory response.17, 20

Additionally, it was reported in two studies

that IL-4 inhibited TNF-α-mediated osteoclast formation in vitro 17, 21

and in vivo via

both TNF-α- activated stromal cells and TNF-α-activated osteoclast precursors.17, 22

Peri-miniscrew crevicular fluid (PMCF) is an osmotically mediated inflammatory

exudate originating from the vessels of the gingival plexus. Its composition is similar

to that of the gingival crevicular fluid (GCF) containing host-derived enzymes and

their inhibitors, inflammatory mediators, host response modifiers and tissue

breakdown products.23

Analysis of PMCF offers a non-invasive means of studying

host response in peri-miniscrew disease and may provide an early indication of

patients at risk for active disease.24

Introduction

7

The possible correlation of the immune response and prime instability of cortical

TADs may help to improve control of risk factors responsible for the premature loss

of orthodontic miniscrew implants essential in the sophisticated and modern treatment

of malocclusions. Therefore, the evaluation of such cytokines in the peri-miniscrew

implant crevicular fluid (PMICF) has been proposed as a noninvasive means of

monitoring the healthy or diseased status of peri-implant tissues as well as the local

response of peri-implant treatments.

Despite investigative efforts to identify the levels of several cytokines in the

PMICF, the efficacy of these parameters to predict or to contribute to the diagnosis of

peri-implantitis is still undetermined. Hence, the investigator is interested to evaluate

the levels of interleukin 8 and interleukin 4 around mini-implants during orthodontic

tooth movement and to draw clinical inferences from the same.

Aims & Objectives

8

AIM AND OBJECTIVES

The present study was conducted with the following aim and objectives:

Aim

The aim of the study was to determine whether the levels of interleukin 8 and

interleukin 4 could be considered in detecting the stability of miniscrew implants.

Objectives

1. To assess the levels of interleukin IL-8 and IL-4 in peri-miniscrew implant

crevicular fluid around miniscrew implants.

2. To analyse the levels of interleukin IL-8 and IL-4 at different intervals in

peri-miniscrew implant crevicular fluid around miniscrew implants.

3. To draw clinical inferences from the differences in the IL-8 and IL-4 at

different intervals in peri-miniscrew implant crevicular fluid around miniscrew

implants.

Review of literature

9

REVIEW OF LITERATURE

The review of literature for the present study has been organised under the

following headings.

Miniscrew implants as an effective anchorage system.

Role of cytokines as inflammatory markers.

Different cytokines around mini-implants.

Interleukin 8, a biomarker.

Interleukin 4, a biomarker.

Miniscrew implants as an effective anchorage system:

A review was done about mini-implants used in orthodontics as a temporary

anchorage device for orthodontic tooth movement. The review analysed all the

different aspects of mini implants such as classification, size and shapes, site and

method of insertion, advantages, diagnosis and treatment planning and mechanics.

Clinical implications of mini implants are closure of extraction spaces (retraction),

single tooth intrusion, and correction of canted occlusal plane, molar intrusion,

mesializaion and distalization. It was concluded that implants could be used in

minimal patient cooperation cases with absolute anchorage needs.25

The authors in a study illustrated three cases. One was treated with maxillary

microscrew implants, another with mandibular microscrew implants, and the third

with both maxillary and mandibular microscrew implants. With the maxillary

microscrew implants, the maxillary anterior teeth were retracted bodily with a slight

intrusion and all the premolar extraction space was closed without loss of anchorage.

Furthermore, the maxillary posterior teeth showed distal movement. The mandibular


10

microscrew implants controlled the vertical position of the mandibular posterior teeth

and played an important role in improving the facial profile. Sliding mechanics with

maxillary microscrew implants provide anchorage for bodily retraction with a slight

intrusion by making the force pass near the centre of resistance. The maxillary

posterior teeth and anterior teeth can both be retracted without anchorage loss. The

mandibular microscrew implants control the vertical mandibular molar position and

contribute to improvement of the facial profile.26

Role of cytokines as inflammatory markers:

A short review was conducted to evaluate the importance of substances as valid

biomarkers of periodontal health during orthodontic movements. The conclusion is

that GCF is a powerful vehicle for clinical diagnostics, since it contains different

biochemical and cellular arrays in relation to different clinical situations indicative of

the state of periodontal health during orthodontic treatment.The main gingival

crevicular fluid (GCF) biomarkers related to orthodontic movements and classified

them into four main groups: biomarkers of inflammation, bone metabolism, cell death

and bone deposition and mineralization. Biomarkers of inflammation were

Interleukins (IL-1𝛽, IL-6, IL-8), Tumour Necrosis factors (TNF-𝛼), Colony-

stimulating factors (M-CSF, G-CSF, GM-CSF), Prostaglandins (PGE), Vascular

endothelial growth factors (VEGF), Calcitonin gene related peptide (CGRP),

Substance P. Increased levels of these proinflammatory cytokines are demonstrated in

GCF during orthodontic tooth movement.27

The authors performed an in situ hybridization to measure the messenger RNA

expression of IL-1β, IL-6, and TNF- α at 3, 7, and 10 days after the application of

orthodontic force on the maxillary first molars of 12 rats. The contralateral side and 3


11

untreated rats served as controls. Measurements of the messenger RNA expression

were selected as the means to investigate the role of orthodontic force in de novo

synthesis of proinflammatory cytokines. After the application of force, the induction

of IL-1β and IL-6 was observed to reach a maximum on day 3 and to decline

thereafter. No messenger RNA induction of either cytokine was measured in the

control teeth. The messenger RNA expression of TNF-α was not detected at any time

point of this study in the experimental or contralateral sides or in the control animals.

The data support the hypothesis that these proinflammatory cytokines may play

important roles in bone resorption after the application of orthodontic force.28

The authors studied the pattern of expression of mRNA encoding several pro- and

anti-inflammatory cytokines in relation to several extracellular matrix and bone

remodeling markers, in tension (T) and compression (C) sides of the PDL of human

teeth subjected to rapid maxillary expansion, by means of real-time polymerase chain

reaction (PCR). The PDL of normal teeth was used as a control. The results showed

that both T and C sides exhibited significantly higher expression of all targets when

compared with controls, except for type I collagen (COL-I) and tissue inhibitor of

metalloproteinase-1 (TIMP-1) on the C side. Comparing C and T sides, the C side

exhibited higher expression of tumor necrosis factor-α (TNF-α), receptor activator of

nuclear factor-κB ligand (RANKL), and matrix metalloproteinase-1 (MMP-1),

whereas the T side presented higher expression of interleukin-10 (IL-10), TIMP-1,

COL-I, osteoprotegerin (OPG), and osteocalcin (OCN). The expression of

transforming growth factor-β (TGF-β) was similar in both C and T sides. The data

demonstrate a differential expression of pro- and anti-inflammatory cytokines in

compressed and stretched PDL during orthodontic tooth movement.29


12

The authors concluded through a review that the interaction between bacteria and

host cells are important both for the induction of responsiveness to infectious agents

and for the induction of non-responsiveness to commensal bacteria. These interactions

can result in the production of cytokines. The lipopolysaccharides (LPS) are the key

bacterial component inducing pro-inflammatory cytokine (IL-1,IL-6,IL-8 & TNF)

gene expression. In this study the authors also classified cytokines based on their

receptors: immunoglobin receptor (IL-1,IL6), haemopoietin receptor(IL-3,IL4), nerve

growth factor receptor(TNF.R I &II) , G-protein coupled receptor(IL-8), tyrosine

kinase –coupled receptor(EGF.R,IGF.R).30

Different cytokines around mini-implants:

The authors did a literature review and found six relevant studies: two on IL-1β;

one on IL-2, IL-6 and IL-8; one on TNF-α; one on chondroitin sulphate (CS); and one

on receptor activator of nuclear factor kappa-B ligand

(RANKL)/osteoprotegerin(OPG) ratio. One study showed an increase in IL-1β levels

upon miniscrew implant (MSI) loading, peak in 24 hours (h), followed by a decrease

in 21 days to reach baseline in 300 days. A 6.87% decrease in IL-2 levels was seen

before loading and a 5.97% increase post-loading. IL-8 showed a 6.31% increase after

loading and IL-6 increased by 3.08% before MSI loading and 15.06% after loading.

RANKL/OPG ratio increased in loaded compared to unloaded MSIs. It was concluded

that cytokines (mainly ILs and TNF-α) and RANKL/OPG ratio showed alteration in

PMICF levels upon loading of MSIs as direct or indirect anchorage.3

The aim of this review was to conduct an assessment of the immunological

response following application of cortical temporary anchorage devices, titanium

miniscrew implants in orthodontic patients. They reviewed studies evaluating the


13

immune response subsequent to insertion of dental implants. It was concluded that the

following factors are associated with dental implant rejection: (1) correlation of the

volume of gingival and peri-implant crevicular fluid and the amount of interleukin

(IL)-1b with mucosal inflammation, thus serving as a peri-implantitis evaluation

index; (2) significantly more frequent marginal bone loss around implants in patients

with IL-1B-511 2/2 genotype; (3) humoral response to Staphylococcus aureus.2

A study was conducted to describe levels of selected pro-inflammatory cytokines

in clinically healthy peri-implant and periodontal sites, and to examine whether

cytokine levels may be related to specific bacterial/viral pathogens. Eleven subjects

(mean age 56.2) participated in the study. Subgingival microbial samples were

cultured for periodontopathic bacteria. Gingival crevicular fluid samples were

analyzed by nested polymerase chain reaction for Cytomegalovirus (HCMV) and

were tested for the quantification of Interleukin (IL)-8, IL-1b, IL-6, IL-10,Tumor

Necrosis Factor (TNF)-a, and IL-12p70 using flow cytometry (FACS). Findings for

microbiota composition and cytokine levels were compared between implants and

teeth (chi square, Kruskall–Wallis, Mann–Whitney; p 2 .05). Both the frequency (%)

and levels (%) of periodontopathic bacteria were higher around teeth than implants.

The concentration (picogram per milliliter) of cytokines was more prominent around

implants than teeth, reaching nearly twofold differences in some instances. Cytokine

levels were higher when the sites analyzed were positive for any bacteriatested.

HCMV was not detected. Pro-inflammatory cytokine production was unrelated to

heavy bacterial challenge and when periodontopathic bacteria were detected by

culture, cytokine levels were increased around both implants and teeth.14

A study was done to analyse IL-1β in peri-miniscrew crevicular fluid obtained

from crevices around the miniscrews inserted in 11 patients (eight females and three


14

males, mean age 17.3 ± 4.64 years) with all first premolar extraction and maximum

anchorage requirement using miniscrew-supported anchorage. Miniscrews were

loaded at 3 weeks after placement by 200g nitinol closed coil springs of 9-mm length

for en masse retraction. Peri-miniscrew crevicular fluid was collected at miniscrew

placement (T1), at 3 weeks (T2/baseline) and on loading at 0 (T3) and 1 day (T4), 21

(T5), 72 (T6), 120 (T7), 180 (T8) and 300 (T9) days. IL-1β levels were estimated by

enzyme-linked immunosorbent assay (ELISA). Peri-miniscrew tissue was examined

for signs of inflammation, and also, miniscrew mobility was assessed with Periotest

and handles of two mouth mirrors. It was concluded that the changing levels of IL-1β

levels in PMCF over duration of 300 days are suggestive of the underlying

inflammatory process. IL-1β levels in PMCF show a significant rise during miniscrew

insertion and on immediate loading. The trend of gradually reducing IL-1β levels

around the miniscrew over the period after loading towards baseline is suggestive of

adaptive bone response to stimulus.4

The authors did a study to determine the peri-miniscrew implant crevicular fluid

receptor activator of nuclear factor-kB ligand (RANKL) and osteoprotegerin (OPG)

levels around loaded and unloaded miniscrew implants at different time intervals.

Twenty loaded and 16 unloaded miniscrew implants were included in this study. All

miniscrew implants were placed bilaterally between the maxillary second premolars

and first molars as anchorage units for canine distalization. Peri-miniscrew implant

crevicular fluid was taken from the mesiobuccal aspects of the loaded and unloaded

miniscrew implants before loading; at 24, 48, and 168 hours; and on day 30 after

force application. ELISA kits were used to determine RANKL and OPG levels in the

peri-miniscrew implant crevicular fluid samples. Although the total amount of OPG

was not different between the groups, the total amount of RANKL was significantly


15

elevated in the loaded miniscrew implant group (P<0.05) at all time periods.

Peri-miniscrew implant crevicular fluid volume was the highest at 48 hours in the

loaded group. Also, the OPG/RANKL ratio in the peri-miniscrew implant crevicular

fluid was significantly decreased in the loaded miniscrew implant group. They

concluded that OPG and RANKL levels vary around loaded and unloaded miniscrew

implants as a result of force application.31

The authors did a study to measure Tumor Necrosis Factor-α (TNF-α) levels

around miniscrews used for anchorage during a 3-month period of canine

distalization. Sixteen patients whose upper first premolars were extracted for

orthodontic treatment were included in this study. Miniscrews were used as an

anchorage unit in canine distalization. Thirty-two miniscrew implants were placed

bilaterally in the alveolar bone between the maxillary second premolars and first

molars. The treatment, miniscrew, and control groups comprised upper canines,

miniscrew implants, and upper first premolars, respectively. Peri-miniscrew implant

crevicular fluid and gingival crevicular fluid were obtained before applying force and

at 1, 24, and 48 hours, and at 7 and 21 days, and 3 months after applying force. In

conclusion during the 3-month period, the TNF-α level increased significantly at 24

hours only in the treatment group. In the miniscrew and control groups, there were no

statistically significant changes. No significant differences was observed between

groups.32

The authors did a study to monitor changes in chondroitin sulphate (CS; WF6

epitope) levels in peri-miniscrew implant crevicular fluid (PMICF) during orthodontic

loading. Ten patients (seven males and three females; aged 22.0 ± 3.4 years), who

required orthodontic treatment with extraction of all four premolar teeth, participated

in the study. Twenty miniscrew implants (used as orthodontic anchorage) were


16

placed, two in each patient, buccally and bilaterally in the alveolar bone between the

roots of the maxillary posterior teeth. Sentalloy closed-coil springs (50 g) were used

to load the miniscrew implants and to move the maxillary canines distally. During the

unloaded period, PMICF samples were collected on days 1, 3, 5, and 7 after

miniscrew implant placement and on days 14,21, 28, and 35 during the loaded period.

Clinical mobility assessments of the miniscrew implants were recorded at each visit.

The competitive enzyme-linked immunosorbent assay with monoclonal antibody

WF6 was used to detect CS (WF6 epitope) levels in the PMICF samples. The

differences between the CS (WF6 epitope) levels during the unloaded and loaded

periods were determined by a Mann – Whitney U -test. They concluded that CS (WF6

epitope) levels in PMICF can be detected and may be used as biomarkers for

assessing alveolar bone remodelling around miniscrew implants during orthodontic

loading.33

A study was conducted to determine whether interleukin 1(IL-1) levels are

elevated around microscrew implants that are used as anchorage for tooth movement.

Ten young adults, aged 16.3 +/- 2.5 years and with all four premolars extracted ,

comprised the study group. Twenty maxillary microscrew implants were placed

bilaterally in the alveolar bone between the maxillary second premolars and first

molars as anchorage units for distal movement of the maxillary canines. The

maxillary canines served as the treatment group, and the microscrew implants were

designated as the implant group. The mandibular canines were used as controls. Peri-

microscrew implant crevicular fluid (MICF) and gingival crevicular fluid (GCF) were

collected at the beginning of tooth movement (2 weeks after implant placement); at

24, 48, and 168 hours later; and on days 14 and 21. An automated enzyme

immunoassay was used to measure 1L-1 in the MICF and the GCF. It was found that


17

the mean 1L-1 level in the treatment group was significantly elevated at 24 hours ,

whereas the levels in the control and implant groups did not change significantly

during the experimental period. Also, the mean 1L-1level of the treatment group was

significantly higher than in both the control and implant groups at 24 and 48 hours.

The conclusion of the above study was that the microscrew implants did not

demonstrate increased 1L-1levels during tooth movement.9

Interleukin 8, a biomarker:

A study was conducted to identify the levels of interleukin IL-2, IL-6 and IL-8

around miniscrews used for anchorage during canine distalization. Sixteen patients

(eight males and eight females; mean age, 16.6 ± 2.4 years) who were treated with

bilateral upper first premolar extractions were included in the study. Thirty-two

maxillary miniscrew implants were placed bilaterally in the alveolar bone between the

maxillary second premolars and first molars as anchorage units for maxillary canine

distalization. Three groups were constructed. The treatment, miniscrew, and control

groups consisted of upper canines, miniscrew implants, and upper second premolars,

respectively. Peri-miniscrew implant crevicular fluid and gingival crevicular fluid

(GCF) were obtained at baseline (T1) and at 1 (T2), 24 (T3), and 48 (T4) hours, 7

(T5) and 21 (T6) days, and 3 months (T7) after force application. Paired sample t-tests

were used to determine within-group changes and Dunnett’s t and Tukey’s honestly

significant difference tests for between-group multiple comparisons. During the 3

month period, IL-2 levels significantly increased (P < 0.01) but only in the treatment

group after 24 hours. IL-6 levels were unchanged at all times points in the three

groups. IL-8 levels increased significantly at 1 (P < 0.05), 24 (P < 0.01), and 48 (P <

0.01) hours in the treatment group and at 24 (P < 0.05) and 48 (P < 0.01) hours in the


18

miniscrew group. It appears that miniscrews can be used for anchorage in

orthodontics when correct physiological forces are applied.1

A study was conducted to evaluate the levels of IL-8 during mechanical forces on

periodontal tissues at different stages of orthodontic therapy. Ten canine teeth of

patients having different Angle classifications were selected for the study. After the

premolars were extracted, the maxillary/mandibular canines were tipped distally.

Gingival crevicular fluid was sampled from mesial and distal gingival crevices of

each canine separately at baseline and one hour, 24 hours, six days, 10 days, and 30

days after the application of the force. An enzyme-linked immunosorbent assay for

quantitative detection of IL-8 was used. Although there was an increase in the

concentration of IL-8 at tension (mesial) sites after one hour, 24 hours, six days, and

10 days, a decrease was observed at 30 days. Pressure (distal) sites did not

demonstrate such an increase at any period except at 10 days. However, the

concentration of IL-8 at both sites showed a similar decrease and approached each

other at day 30. They concluded that local host response toward the orthodontic forces

might lead to an increase in IL-8 and neutrophil accumulation, and this may be one of

the triggers for bone remodelling processes.13

The authors measured intercellular messenger and cytokines that are regulatory for

osteoblast and osteoclast function. Production of osteocalcin, a marker for osteoblast

maturation was also estimated. Human osteoblastlike cells from osteosarcoma cell

line MG 63 were grown in wells in the presence of titanium (Ti), titanium alloy

(Ti6A14V) and stainless steel implant materials incubated at 370C. Interleukin- 1α

(IL-1α), IL-6, IL-8, IL-11 and osteocalcin were quantitated using standard enzyme

linked immunosorbant assay (ELISA) kits from the growth media extracted at specific

intervals over the critical ten day period. In all dishes, cells were seen adhering to the


19

base after 24 hours and to confluence at 96 hours. Both IL-1α and IL-11 were not

produced in sufficient quantities to be measured in the assay (<pg/ml).Interleukin-6

production was significantly higher for stainless steel than for titanium and the alloy.

There was a progressive rise in osteocalcin production for titanium contrasted to a

basal rate for stainless steel and alloy. Interleukin-8 levels for all metals and controls

increased markedly after two days implicating inherent cellular characteristics. A

relatively high constant range for macrophage colony stimulating factor from the first

day was seen for all metals, including the controls. In conclusion, it appears that

titanium implants activate osteocalcin production while stainless steel activates IL-6.10

A study was done to determine the distribution of IL-8 and IL-8R in gingival

tissues and cultured human gingival keratinocytes in vitro. Standard

immunohistochemical and immunocytochemical techniques were utilized in order to

localize IL-8 and its receptors CXCR-1 and CXCR-2 in archival gingival specimens

(eight periodontitis and four non-inflamed controls) and in cultured gingival

keratinocytes. It was demonstrated that, in vivo, IL-8 and IL-8R were present in

gingival epithelium, MVEC and leukocytes. It was concluded that IL-8 and IL-8

receptors are expressed in gingival epithelium both in vivo and in vitro.34

A study was done with the aim to determine levels of interleukins 2, 6, and 8

during tooth movement, and test whether they differ from each other with levelling

and distalization forces used in various treatment stages of standard orthodontic

therapy. Fifteen patients (9 female, 6 male; ages, 15-19 years; mean age, 16.7+/-2.3

year participated in this study. Each underwent a session of professional oral hygiene

and received oral hygiene instructions. Two months later, a fixed orthodontic

appliance was placed. The patients were seen at baseline, at days 7 and 21, and as the

teeth were leveled. Records of the baseline scores for the distalization forces were


20

taken at the sixth month. Scores of days 7 and 21 after 6 months of the distalization

treatment were also recorded. It was found that the increases were seen in the volume

of gingival crevicular fluid and the concentrations of interleukins 2, 6, and 8. From

this study it was concluded that levelling and distalization of the teeth evoke increases

in interleukins 2, 6, and 8 levels in the periodontal tissues that can be detected in

gingival crevicular fluid. Distalization forces increased IL-8 levels at the 7th day and

decreased them the 21st day.35

In an in vitro study, understand the contribution of stromal cells, such as

granulation tissue fibroblasts, to peri-implantitis with regard to (1) the secretion of

constitutive factors promoting migration/survival of infiltrates into osseointegrated

sites; and (2) the effect of exogenous infiltrate cytokines on the cells' secretion.

Fibroblasts were cultured from eight peri-implantitis sites. Multiplexed enzyme-

linked immunosorbent assay was used to quantify factors secreted by the cells either

unstimulated or stimulated with gamma interferon (lFNγ), interleukin 4 (1L4), or

tumor necrosis factor alpha (TNFα). Controls consisted of fibroblasts cultured from

healthy gingival and chronic periodontitis granulation tissues. Peri-implantitis

fibroblasts differed significantly from periodontitis fibroblasts in their reduced

secretion of the collagen inducer transforming growth factor beta-1 (TGFβ1) and

tissue inhibitor of metalloproteinase-1. The cells exhibited enhanced secretion of

angiogenic factor vascular endothelial growth factor (VEGF) and collagenolytic

matrix metalloproteinase 1 (MMP1) compared to both healthy and periodontitis

fibroblasts. Fibroblasts from both periodontitis and peri-implantitis sites exhibited a

pronounced proinflammatory profile compared to normal gingival fibroblasts with

respect to secretion of chemokines 1L6, 1L8, and monocyte chemoattractant protein 1

(MCP1). Fibroblasts stimulated with TNFα showed increased levels of 1L6, 1L8,


21

MCP1; neutrophil chemokine growth-related oncogene alpha stimulation with IFNγ

increased MCP1; and stimulation with 1L4 increased VEGF. The results indicate that

peri-implantitis fibroblasts represent a distinct stromal population. The cells might

participate in the pathogenesis of peri-implantitis by up-regulating both vascularity

and matrix breakdown, thus promoting migration/maintenance of infiltrates into the

site. Cytokines produced by infiltrates could enhance the inflammatory nature of the

cells in a self-feeding loop. It was concluded that the fibroblasts of the patients with

peri-implantitis and periodontitis synthesized more IL-6 and IL-8, and thus presented

a more accentuated proinflammatory profile.36

A study was done to evaluate the expression of IL-1b, IL-4, and IL-8 in the

gingival crevicular fluid (GCF) of children, adolescents, and young adults with and

without fixed orthodontic appliances. Eighty systemically healthy children and

adolescents participated in the study: 56 aged between 8 and 16 years without any

orthodontic appliance (Group A) and 24 aged between 10 and 20 years having worn

fixed orthodontic appliances for at least 12 months (Group B). Clinical examination

included presence or absence of plaque, probing depth, bleeding on probing, and

gingival overgrowth. GCF was collected by means of Durapore strips from four

randomly selected sites per subject. The contents of interleukin-1 beta (IL-1b),

interleukin-4 (IL-4), and interleukin-8 (IL-8) were detected by ELISA, measured as

total amounts (pg/30s) and expressed in log scale. Statistically significant differences

were noted for the mean log IL-1b, IL-4, and IL-8 between the two groups: Group B

showed significantly higher mean levels in log IL-1b and log IL-8 compared to Group

A. Mean levels of log IL-4 were lower in Group B, although they did not reach

statistical significance. Furthermore, mean levels of log IL-1b and log IL-8 were

associated with bleeding sites (pB0.001) and gingival overgrowth, while mean level


22

of log IL-4 was associated with non-bleeding sites and no gingival overgrowth

(pB0.001). It was concluded that fixed orthodontic appliances result in an increase in

the expression of IL-1b and IL-8. This may reflect biologic activity in the

periodontium during orthodontic tooth movement.41

Interleukin 4, a biomarker:

The authors did a study to investigate the effect of IL-4 on tooth movement and its

associated root resorption in a mouse model. The maxillary first molars of four male

mice for each experimental group were subjected to mesial force by a nickel titanium

coil spring for 12 days. Control mice were not given appliances and injections.

Varying doses of IL-4 were injected locally, adjacent to the first molar. Two sets of

experiments were designed. The first set was composed of three groups: the control,

treatment with phosphate-buffered saline (PBS), or 1.5 μg/day of IL-4. The second set

was composed of five groups: the control, treatment with 0 (PBS only), 0.015, 0.15,

or 1.5 μg/day of IL-4.The distance of orthodontic tooth movement (OTM) was

measured and tartrate-resistant acid phosphatase positive cells along the loaded

alveolar bone and root surface were identified. The root resorption associated with

OTM was evaluated by a scanning electron microscope. The authors found the

amount of OTM and the number of osteoclasts was significantly decreased in the IL-

4-treated mice. Moreover, IL-4 significantly suppressed force-induced odontoclasts

and root resorption. It was concluded that IL-4 inhibits tooth movement and prevents

root resorption in the mouse model. These results suggest that IL-4 could be used as a

useful adjunct to regulate the extent of OTM and also to control root resorption.17

The authors investigated the effect of IL-4 on TNF-α-mediated osteoclast

formation in vivo. TNF-α was administered with and without IL-4 into the


23

supracalvariae of mice. The number of osteoclasts and the levels of mRNA for

cathepsin K and tartrate-resistant acid phosphate, both osteoclast markers, in mice

administered TNF-α and IL-4 were lower than those in mice administered TNF-α

alone. The level of tartrate-resistant acid phosphatase form 5b (TRACP5b) as a

marker of bone resorption in mice administered both TNF-α and IL-4 was also lower.

It showed that IL-4 inhibited TNF-α-mediated osteoclast formation in osteoclast

precursors in vitro. It was concluded that IL-4 inhibited TNF-α-mediated osteoclast

formation by inhibiting expression of RANKL in TNF-α-activated stromal cells, and

directly inhibited TNF-α-activated osteoclast precursors in vivo via a T cell-

independent mechanism.22

A study was done to assess the relation between clinical parameters and

concentrations of IL-4 within gingival crevicular fluid from inflamed gingiva and

periodontitis sites and, subsequently, after treatment of the periodontitis sites. A total

of 60 subjects were divided into three groups based on gingival index (GI), pocket

probing depth and clinical attachment loss (CAL): healthy (group 1), gingivitis (group

2) and chronic periodontitis (group 3). A fourth group (group 4) consisted of 20

subjects from group 3, 6–8 weeks after treatment (i.e. scaling and root planing).

Gingival crevicular fluid samples collected from each patient were quantified for IL-4

using the enzymatic immunometric assay. It was concluded that the mean

concentration of IL-4 decreased from periodontal health to disease. Thus, they

suggested that type 2 helper T cell cytokine, as represented by IL-4, was associated

with the remission or improvement of periodontal disease.20

A study was done to define the molecular mechanism(s) by which interleukin(IL)-

4 reversibly inhibits formation of osteoclasts (OCs) from bone marrow macrophages

(BMMs), The authors examined the capacity of this T cell-derived cytokine to impact


24

signals known to modulate osteoclastogenesis, which include those initiated by

macrophage colony stimulating factor (M-CSF), receptor for activation of NF-kB

ligand (RANKL), tumor necrosis factor (TNF) and IL-1. They concluded that IL-4

reversibly arrests osteoclastogenesis in a STAT6-dependent manner by 1) preventing

IB phosphorylation and thus NF-B activation, and 2) blockade of the JNK, p38, and

ERK mitogen-activated protein kinase pathways.16

In another article the authors discussed about IL-4 receptor (IL -4R), its signalling

mechanisms and biologic functions. They gave emphasis on different pathways of IL-

4R signalling, its modulation. It was concluded that the signalling pathways that are

activated by IL-4R engagement, such as the IRS-1/2 and Jak-Stat pathways, mirror

those activated by a number of other cytokines. The activation of these pathways

results in a unique array of cellular responses to IL-4. The specific cellular responses

to IL-4 may also result from the unique character of the IL-4R.15

The study was done to determine the levels of interleukin (IL)-1b, IL-4, IL-6 and

IL-8 in gingival crevicular fluid of periodontaly healthy and diseased individuals and

to study their association to smoking, stress and clinical periodontal parameters. A

total of 80 patients were included in the study: 20 patients with early onset or

aggressive periodontitis (EOP), 20 with chronic adult periodontitis (AP), 20 with

gingivitis (G) and 20 patients with healthy periodontium (H). GCF was collected by

means of Durapore strips, from four sites per patient, randomly selected in each

quadrant. The contents of IL- 1b, IL-4, IL-6 and IL-8 were measured in 320 samples

by use of commercially available sandwich enzyme-linked immunoadsorbent assays.

In periodontally diseased subjects the total amounts of IL-1b, IL-6 and IL-8 were

significantly elevated as compared to healthy subjects, whereas IL-4 showed an

inverse relationship to periodontal status and higher amounts were found in the


25

healthy group. The amounts of all four cytokines were positively correlated with

probing depths. IL-4, IL-6 and IL-8 were significantly correlated to smoking while

stress was associated with IL-1b, IL-6 and IL-8 level. This study suggested that

crevicular IL-1b, IL-6 and IL-8 reflect the activity of periodontal destruction, whereas

IL-4 shows an inverse correlation to it. They also stated that the enhanced production

of inflammatory cytokines in the presence stress of smoking and stress may have

clinical consequences.37

A review was conducted in USA with the hypothesis that, in the case of Adult

periodontitis, a localized lack of the regulatory cytokine interleukin-4 (IL-4) in the

gingival tissues predisposes susceptible individuals to progress from gingivitis to

periodontitis. The authors found an analogous situation may exist in the case of

rheumatoid arthritis (RA). There are several similarities in the pathogenesis of tissue

damage in RA and periodontal disease, suggesting RA may provide a model for host

response mediated tissue destruction. The studies using the RA model in animals have

found that lL-4 injected directly into the lesion prevents the tissue destruction

observed in control sites. These results suggest several experimental treatment

protocols for periodontitis. IL-4 is biologically active in very low concentrations (3

U/ml, 29). Therefore, susceptible patients may benefit from local delivery of IL4 in a

sustained release vehicle. Such a vehicle may provide a localized source of IL-4 at the

site of inflammation. This may reduce the deleterious effects of the bacteria-host

interaction in a site specific manner, thereby reducing or preventing local periodontal

tissue destruction.38

A study was conducted to assess whether IL-1 ra and IL-4 play an important role

in the natural homeostatic mechanisms by regulating the effect of IL-1 on

inflammatory periodontal disease and whether they are present in GCF or not. GCF


26

samples were obtained from eight adult patients (mean age 46 years) with

periodontitis radiographic-evideoce of generalized alveolar bone loss and deep

pockets. As a negative control, GCF samples were obtained from healthy volunteers

without periodontal disease. The following clinical parameters were evaluated at the

time of sampling; gingiva) index (Gl), pocket depth, pus discharge, spontaneous pain,

and alveolar bone loss calculated from radiographs. lnterleukin-1 receptor antagonist

(IL-lra) and interleukin-4 (IL-4) were detected in gingival crevicular fluid (GCF) by

an immunochemical method. But they could not detect IL-4 in GCF from severe

inflammation sites. The cell types expressing CD 68 were identified as

monocytes/macrophages and stained positively for IL-lra. The helper T cells

identified by immunostaining for CD 4 stained positively for IL-4. These results

suggest that IL-4 is one of the mediators regulating the degree of local inflammation

in periodontal disease.39

A study was done to examine the impact of local IL-4 on bone erosion in the knee

joint of mice with collagen - induced arthritis (CIA), using gene transfer with an IL-

4–expressing adenoviral vector. They also investigated the effects of IL-4 on the

degradation and formation of collagen type I in bone samples from patients with

arthritis. It was reported that local overexpression of IL-4, introduced by a

recombinant human type 5 adenovirus vector (Ad5E1mIL-4) prevents joint damage

and bone erosion in the knees of mice with collagen arthritis (CIA). No difference

was noted in the course of CIA in the injected knee joints between Ad5E1mIL-4 and

the control vector, but radiographic analysis revealed impressive reduction of joint

erosion and more compact bone structure in the Ad5E1mIL-4 group. Although severe

inflammation persisted in treated mice, Ad5E1mIL-4 prevented bone erosion and

diminished tartrate-resistant acid phosphatase (TRAP) activity, indicating that local


27

IL-4 inhibits the formation of osteoclast-like cells. Messenger RNA levels of IL-17,

IL-12, and cathepsin K in the synovial tissue were suppressed, as were IL-6 and IL-12

protein production. Osteoprotegerin ligand (OPGL) expression was markedly

suppressed by local IL-4, but no loss of OPG expression was noted with Ad5E1mIL-4

treatment. Finally, in in vitro studies, bone samples of patients with arthritis revealed

consistent suppression by IL-4 of type I collagen breakdown. IL-4 also enhanced

synthesis of type I procollagen, suggesting that it promoted tissue repair.40

Materials and Methods

28

MATERIALS AND METHODS

Source of data

The study subjects were patients seeking fixed orthodontic treatment from the

Department of Orthodontics and Dentofacial Orthopaedics, Coorg Institute of Dental

Sciences, Virajpet, Karnataka. The nature and purpose of the study and the treatment

protocol was explained to the subjects included in the study and a written consent

from all patients / parents of patients under the age 18 was obtained before

commencing the study.

Inclusion criteria

Patients seeking fixed orthodontic treatment with miniscrew implants

In the age group 16 to 25.

With periodontal probing depth upto 3mm.

With no radiographic evidence of periodontal bone loss after a full–mouth

radiographic peri-apical examination.

Exclusion criteria

Patients seeking fixed orthodontic treatment with miniscrew implants

With periodontal problems.

With gingival inflammation.

With any systemic diseases.

Who have taken anti-inflammatory and antibiotic medications during the past

3 months.


29

Armamentarium:

PMICF collection-

1. Graduated capillary pipettes

2. Eppendorf tubes

Elisa analysis-

1. ELISA reader

2. Microplate

3. Micropipette

4. Disposable micropipette tips

5. ELISA kit

Procedure:

The study sample comprised of 22 miniscrew implants (12 in maxilla and 10 in

mandible) from 7 patients who are undergoing orthodontic treatment with miniscrew

implants. The selected subjects were in retraction stage using mini-implants. The

miniscrew implants were titanium implants (B K Surgicals) of the size 1.5mm

diameter, 8mm long and was placed by the same orthodontist in all the cases. A force

of 150g (measured using dontrix gauge) was applied on each miniscrew implant for

retraction.

Before commencement of study, the patients were instructed oral hygiene methods

and also were systematically checked for periodontal problems. An oral prophylaxis

was performed 1 week prior to study.


30

Sample collection:

Peri-miniscrew implant crevicular fluid (PMICF) was collected by isolating the

area with cotton rolls, drying the adjacent marginal gingiva and teeth with air and

using graduated micro capillary tubes from the mesial aspect of the miniscrew

implants.9 The samples were collected over a month time period according to the

following schedule: T1, T2 and T3 at one hour, 7th

day and 21st day after force

application on miniscrew implants respectively. The samples were stored in sterile

eppendrof tubes at -200 Celsius until the start of the experiment in Coorg Institute of

Dental Sciences.

Laboratory analysis:

Quantitative analysis of IL-8 and IL-4 in the PMICF samples was assessed using a

commercially available ELISA test (Elabscience® human interleukin 8 and

interleukin 4). A calibration curve was plotted by regression analysis and the optical

density of each sample was used to estimate the concentration of IL-8 and IL-4

(pg/μL). This was corrected for the original volume of PMICF and the result was

recorded as the concentration of interleukin-8/4 in the samples, expressed in pg/ml.

Test principle:

This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided

in this kit has been pre-coated with an antibody specific to Human IL-8/IL-4.

Standards or samples are added to the micro ELISA plate wells and combined with

the specific antibody. Then a biotinylated detection antibody specific for Human IL-

8/IL -4 and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively

to each micro plate well and incubated. Free components are washed away. The


31

substrate solution is added to each well. Only those wells that contain Human IL-8/

IL-4, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in

color. The enzyme-substrate reaction is terminated by the addition of stop solution

and the color turns yellow. The optical density (OD) is measured

spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is

proportional to the concentration of Human IL-8/IL-4. The amounts of IL-8/IL-4 in

each sample were compared with standard curves for these interleukins.

Reagent preparation:

1. Bring all reagents to room temperature (18~25℃) before use. Follow the

Microplate reader manual for set-up and preheat it for 15 min before OD

measurement.

2. Wash Buffer: Dilute 30 mL of Concentrated Wash Buffer with 720 mL of

deionized or distilled water to prepare 750 mL of Wash Buffer. Note: if crystals have

formed in the concentrate, warm it in a 40℃ water bath and mix it gently until the

crystals have completely dissolved

3. Standard working solution: Centrifuge the standard at 10,000×g for 1 min. Add 1.0

mL of Reference Standard & Sample Diluent, let it stand for 10 min and invert it

gently several times. After it dissolves fully, mix it thoroughly with a pipette. This

reconstitution produces a working solution of 4000 pg/mL. Then make serial dilutions

as needed. The recommended dilution gradient is as follows: 4000, 2000, 1000, 500,

250, 125, 62.50, 0 pg/mL.

Dilution method: Take 7 EP tubes, add 500uL of Reference Standard & Sample

Diluent to each tube. Pipette 500uL of the 4000 pg/mL working solution to the first


32

tube and mix up to produce a 2000 pg/mL working solution. Pipette 500uL of the

solution from the former tube into the latter one according to these steps.

4. Biotinylated Detection Ab working solution: Calculate the required amount before

the experiment (100 μL/well).

In preparation, slightly more than calculated should be prepared. Centrifuge the stock

tube before use, dilute the 100× Concentrated Biotinylated Detection Ab to

1×working solution with Biotinylated Detection Ab Diluent.

5. Concentrated HRP Conjugate working solution: Calculate the required amount

before the experiment (100μL/well). In preparation, slightly more than calculated

should be prepared. Dilute the 100× Concentrated HRP Conjugate to 1× working

solution with Concentrated HRP Conjugate Diluent.

Assay procedure:

1. Add the Standard working solution to the first two columns: Each concentration of

the solution is added in duplicate, to one well each, side by side (100 uL for each

well). Add the samples to the other wells (100 uL for each well). Cover the plate with

the sealer provided in the kit. Incubate for 90 min at 37℃. Note: solutions should be

added to the bottom of the micro ELISA plate well, avoid touching the inside wall and

causing foaming as much as possible.

2. Remove the liquid out of each well, do not wash. Immediately add 100 μL of

Biotinylated Detection Ab working solution to each well. Cover with the Plate sealer.

Gently mix up. Incubate for 1 hour at 37°C.


33

3. Aspirate or decant the solution from each well, add 350 uL of wash buffer to each

well. Soak for 1~2 min and aspirate or decant the solution from each well and pat it

dry against clean absorbent paper. Repeat this wash step 3 times.

4. Add 100 μL of HRP Conjugate working solution to each well. Cover with the Plate

sealer. Incubate for 30 min at 37°C.

5. Aspirate or decant the solution from each well, repeat the wash process for five

times as conducted in step 3.

6. Add 90 μL of Substrate Reagent to each well. Cover with a new plate sealer.

Incubate for about 15 min at 37°C. Protect the plate from light. Note: the reaction

time can be shortened or extended according to the actual color change, but not more

than 30min.

7. Add 50 μL of Stop Solution to each well. Note: Adding the stop solution should be

done in the same order as the substrate solution.

8. Determine the optical density (OD value) of each well at once with a micro-plate

reader set to 450 nm.

Statistical Analysis :

The data were collected, coded and fed in the SPSS (IBM version 23) and are

analyzed using descriptive and inferential statistics. The inferential statistics included

paired t test and independent t test.

Sample Size Estimation

34

SAMPLE SIZE ESTIMATION

Sample size was determined using nMASTER software.

Single Mean - Paired t-test was chosen.

The following parameters were chosen from a similar study by Hamamcı N et al 1 ;

Pre-test mean = 1292.30

Post-test mean = 1845.02

Standard deviation in Pre-test= 345.80

Standard deviation in Post-test = 1425.90

Effect size = 0.6239431054919

Power (%) = 80

Alpha Error (%) = 5

sided = 2

Required sample size = 22

The sample size for the study was determined as 22.

Results

35

RESULTS

The present study was carried out to evaluate the levels of interleukin 8 and

interleukin 4 in peri-miniscrew implant crevicular fluid (PMICF) around miniscrew

implants during orthodontic tooth movement.

In this study PMICF was collected by isolating the area with cotton rolls, drying the

adjacent marginal gingiva with air and using graduated micro capillary tubes from the

mesial aspect of the miniscrew implant.

The samples were collected over a month time period according to the following

schedule: T1, T2 and T3 at one hour, 7th

day and 21st day respectively after force

application on miniscrew implants. The collected PMICF was then tested for

Interleukin 8 and Interleukin 4 using ELISA test.

The data were collected, coded and fed in the SPSS (IBM version 23) and are

analyzed using descriptive and inferential statistics. The inferential statistics included

paired t test and independent t test.

The results of the present study were evaluated as follows:

The analysis is done under two sections.

Section 1 - Levels of Interleukin 8.

Section 2 - Levels of Interleukin 4.

Results

36

Section 1. Levels of Interleukin 8

The mean of IL-8 levels at one hour, 7th

day and 21st day is presented as bar diagram.

Figure 1. Comparison of mean Interleukin 8 levels at T1, T2, and T3.

Figure 1 shows that there is marked increase in level of IL-8 from 1 hour to 7th

day.

And there is a slight decrease in interleukin 8 level from 7th

day to 21st day.

7.88818

8.18773 8.17455

7.7

7.75

7.8

7.85

7.9

7.95

8

8.05

8.1

8.15

8.2

8.25

T1 T2 T3

LEV

ELS

OF

IL-8

TIME OF STUDY

MEAN INTERLEUKIN 8

Results

37

Table 1

Comparison of Interleukin 8 levels at different intervals among study subjects

around miniscrew implants using paired t test.

Mean

Standard

Deviation

t Sig.

Interleukin 8

T1 7.88818 .841505

-3.630 0.002**(H.S)

T2 8.18773 .930099

T1 7.88818 .841505

-2.508 0.020 *(S)

T3 8.17455 .992806

T2 8.18773 .930099

0.169 0.867 (N.S)

T3 8.17455 .992806

**p<0.01 *p<0.05

Table 1 shows that the mean of IL-8 level on 7th

day (T2) is higher than that of it at

1st hour (T1) after implantation and the difference is found to be statistically highly

significant (p<0.01). Table 1 also demonstrates that the mean of IL-8 levels on 21st

day is slightly less than that on 7th

day but the difference is statistically not significant

(p>0.05). It also shows that the difference in the mean of IL-8 levels after 1st hour and

21st day is statistically significant (p<0.05).

Results

38

Table 2

Comparison of Interleukin 8 levels at different intervals among study subjects

around miniscrew implants between maxilla and mandible using independent t

test.

Interleukin 8 Mean

Standard

deviation

t Sig.

T1

Maxilla 7.8950 .88252

-0.041

0.635

(N.S) Mandible 7.8800 .83681

T2

Maxilla 8.1325 .89329

-0.298

0.768

(N.S) Mandible 8.2540 1.01700

T3

Maxilla 8.0433 1.01679

-0.670

0.510

(N.S) Mandible 8.3320 .99286

Table 2 demonstrates that there is no statistically significant difference between the

Interleukin 8 levels of maxilla and mandible after one hour, 7th

day or 21st day of

miniscrew implant placement.

Results

39

Section 2. Levels of Interleukin 4

The mean of IL-4 at one hour, 7th

day and 21st day is presented as bar diagram.

Figure 2. Comparison of mean Interleukin 4 levels at T1, T2, and T3.

Figure 2 shows that there is a decrease in IL-4 level from 1 hour to 7th

day. And also

there is a decrease in interleukin 4 levels from 7th

day to 21st day.

1.2

1.22

1.24

1.26

1.28

1.3

1.32

1.34

1.36

T1 T2 T3

1.35545

1.30136

1.25818

LEV

ELS

OF

IL-4

TIME OF STUDY

MEAN INTERLEUKIN 4

Results

40

Table 3

Comparison of Interleukin 4 level among study subjects around miniscrew

implants using paired t test.

Mean

Standard

Deviation

t Sig.

Interleukin 4

T1 1.35545 .239399

3.545 0.002** (H.S)

T2 1.30136 .211306

T1 1.35545 .239399

6.445 0.000*** (H.S)

T3 1.25818 .250174

T2 1.30136 .211306

2.304 0.032* (S)

T3 1.25818 .250174

***p<0.001 **p<0.01 *p<0.05

Table 3 shows that the mean of IL-4 level on 7th

day (T2) is lower than that of it at

1st hour (T1) after implantation and the difference is found to be statistically highly

significant (p<0.01). Table 1 also demonstrates that the mean of IL-4 on 21st

day is

less than that on 7th

day and the difference is statistically significant (p>0.05). It also

shows that the difference of the means of IL-4 after 1st hour and 21

st day is

statistically highly significant (p<0.001).

Results

41

Table 4

Comparison of Interleukin 4 levels among study subjects around miniscrew

implants between maxilla and mandible using independent t test.

Interleukin 4 Mean

Standard

deviation

t Sig.

T1

Maxilla 1.32750 .224180

-0.591

0.561

(N.S) Mandible 1.38900 .264594

T2

Maxilla 1.28250 .205255

-0.450

0.410

(N.S) Mandible 1.32400 .227264

T3

Maxilla 1.24083 .214580

-0.349

0.451

(N.S) Mandible 1.27900 .298122

Table 4 shows that there is no significant difference between the Interleukin 4 levels

of maxilla and mandible after at one hour, 7th

day or 21st day of miniscrew implant

placement.

Results

42

The results obtained from the study were;

Section1. Levels of Interleukin 8.

Interleukin 8 level increased from 1 hour (7.8882 +/-0.8415) to 7th

day

(8.1877+/-0.9301) which is statistically highly significant with p value < 0.01.

There is a slight decrease in interleukin 8 levels from 7th

day (8.1877+/-

0.9301) to 21st day (8.1745+/-0.9928) which is statistically not significant with

p value > 0.050.

The difference between 1 hour and 21st day is significant with p value < 0.05.

No statistically significant difference (p value >0.05) were observed in levels

of interleukin 8 between maxilla and mandible at one hour, 7th

day or 21st day

of miniscrew implant placement.

From the above findings, it is concluded that in healthy miniscrew implants the

Interleukin 8 levels significantly increases from one hour to 7th

day. Eventhough there

is a slight decrease in levels of IL-8 from 7th

day to 21st day, it was not statistically

significant.

Section 2. Levels of Interleukin 4.

Interleukin 4 level decreased from 1 hour (1.3554+/-0.2394) to 7th

day

(1.3014+/-0.2113) which is statistically highly significant with p value < 0.01.

There is a decrease in interleukin 4 levels from 7th

day (1.3014+/-0.2113) to

21st day (1.2582+/-0.2502) which is statistically significant with

p value < 0.05.

The difference between IL-4 levels at 1st hour and 21

st day is highly significant

with p value < 0.01.

Results

43

No statistically significant difference (p value > 0.05) were observed in levels

of IL-4 between maxilla and mandible at one hour, 7th

day or 21st day of

miniscrew implant placement.

From the above findings, it is concluded that in healthy miniscrew implants the

Interleukin 4 levels decreases from one hour to 7th

day and from 7th

to 21st day.

Discussion

44

DISCUSSION

An early and reliable detection of any adverse peri-miniscrew implant tissue

reaction is essential for patients being treated with miniscrew implants. The

evaluation of cytokines in the peri-miniscrew implant crevicular fluid (PMICF) has

been proposed as a noninvasive means of monitoring the healthy or diseased status of

peri-implant tissues as well as the local response of peri-implant treatments. In this

scenario, the present study was done to evaluate the levels of interleukin 8 and

interleukin 4 in PMICF around healthy mini-implants.

The present study was conducted in 22 healthy miniscrew implants from 7 patients

(6 females and 1 male) within an age range between 16-25 years with 12 miniscrew

implants in the maxilla and 10 in the mandible without any signs of peri-implantitis.

The selected subjects were in retraction stage using miniscrew implants. The

miniscrew implants were titanium implants (B K Surgicals) of the size 1.5mm

diameter, 8mm long and was inserted by the same orthodontist in all the cases. A

force of 150g (measured using dontrix gauge) was applied on each miniscrew implant

for retraction.

The samples were collected over a month time period according to the following

schedule: T1, T2 and T3 at 1 hour, 7th

day and 21st day after force application on

miniscrew implants respectively. The samples were stored in sterile eppendorf tubes

at -200 Celsius until the start of the experiment. The PMICF samples were put through

Elisa test (Elabscience® human interleukin 8 and interleukin 4) for determining the

concentration of IL-8 and IL-4. This was corrected for the original volume of PMICF

and the results were recorded as the concentration of interleukin-8/4 in the samples,

expressed in pg/ml.

Discussion

45

In the present study, Interleukin 8 level was used to assess the peri-miniscrew

implant tissue inflammation because the various activities of IL-8 indicate that this

cytokine plays a major role in mediating inflammatory responses.1 Sfakianakis et al

reported the precise localization of IL-8 receptors in periodontitis and noninflamed

human gingivae and suggested that IL-8 plays a multifunctional role in the

pathogenesis of periodontal disease.34

In a previous study, it was demonstrated that

orthodontic forces evoked changes in the levels of IL-8.13

In the present study, Interleukin 4 level was used to assess the peri miniscrew

implant tissue inflammation because Interleukin-4 plays an important role in the

regulation of the immunoinflammatory response.20

The anti-inflammatory effect of

IL-4 results from its efficient inhibition of the production of proinflammatory

cytokines such as tumor necrosis factor-a (TNF-a), IL-1a, IL-1b, IL-6 and IL-8 by

monocytes/ macrophages.17

In the present study the IL-8 levels in healthy implants increases from one hour to

7th

day. It could be related to the perimucositis due to the injury during insertion of

miniscrew implant.

These findings agree with the study by Shaama where the author measured

intercellular messenger and cytokines that are regulatory for osteoblast and osteoclast

function. Human osteoblast like cells from osteosarcoma cell line MG 63 were grown

in wells in the presence of titanium (Ti), titanium alloy (Ti6A14V) and stainless steel

implant materials incubated at 370C. Interleukin- 1α (IL-1α), IL-6, IL-8, IL-11 and

osteocalcin were quantitated using standard enzyme linked immunosorbant assay

(ELISA) kits from the growth media extracted at specific intervals over the critical ten

Discussion

46

day period. It was reported that IL-8 levels in implant crevicular fluid increased from

day 1 but decreased by day 10.10

In the present study, there was a slight decrease in interleukin 8 levels from 7th

day to 21st day. But it was statistically not significant with p value 0.867.

The present study is also supported by Hamamcı N et al who aimed to identify the

levels of interleukin IL-2, IL-6 and IL-8 around miniscrews used for anchorage during

canine distalization. Peri-miniscrew implant crevicular fluid and gingival crevicular

fluid (GCF) were obtained at baseline (T1) and at 1 (T2), 24 (T3), and 48 (T4) hours,

7 (T5) and 21 (T6) days, and 3 months (T7) after force application. IL-8 levels

increased significantly in both the miniscrew and the canine groups at 24 and 48

hours. No statistically significant difference in the levels of IL-8 was noted on 7th

and

21st day from baseline in this study.

1

The present study finding is also supported by a study done with the aim to

determine levels of interleukins 2, 6, and 8 during tooth movement and test whether

they differ from each other with levelling and distalization forces used in various

treatment stages of standard orthodontic therapy. Fifteen patients (9 female, 6 male;

ages, 15-19 years; mean age, 16.7+/-2.3 year participated in this study. The patients

were seen at baseline, at days 7 and 21, and as the teeth were levelled. Records of the

baseline scores for the distalization forces were taken at the sixth month. Scores of

days 7 and 21 after 6 months of the distalization treatment were also recorded. From

this study it was concluded that levelling and distalization of the teeth evoke increases

in IL-8 levels in the periodontal tissues that can be detected in gingival crevicular

fluid and distalization forces increased IL-8 levels at the 7th

day and decreased on the

21st day.

35

Discussion

47

In the present study, the IL-8 level decrease from 7th

to 21st day but it was not

statistically significant (p value>0.05). It indicates that the progress of inflammation is

slightly reduced or regressed. This can be attributed to fact that there is no progression

towards peri-implantitis.

This could be stated in accordance with an in vitro study by Bordin et al. The

authors evaluated the fibroblasts of patients with peri-implantitis, patients with

periodontitis, and healthy subjects. They found that the fibroblasts of the patients with

peri-implantitis and periodontitis synthesized more IL-8 and thus presented a more

accentuated proinflammatory profile.36

In the present study, it was concluded that in healthy implants the IL-4 levels

decreases from one hour to 7th

day. From this it can be inferred that there is some

amount of perimucositis around the implant due to the tissue injury during insertion of

the miniscrew implants. In the present study, the decrease in IL-4 from 7th to 21st day

could be attributed to the persistence of inflammation through 21st day.

The present study findings is consistent with a study conducted in India aimed to

assess the relation between clinical parameters and concentrations of IL-4 within

gingival crevicular fluid from inflamed gingiva and periodontitis sites and,

subsequently, after treatment of the periodontitis sites. A total of 60 subjects were

divided into three groups based on gingival index (GI), pocket probing depth and

clinical attachment loss (CAL): healthy (group 1), gingivitis (group 2) and chronic

periodontitis (group 3). A fourth group (group 4) consisted of 20 subjects from group

3, 6–8 weeks after treatment (i.e. scaling and root planing). Gingival crevicular fluid

samples collected from each patient were quantified for IL-4 using the enzymatic

immunometric assay. It was concluded that the mean concentration of IL-4 decreased

Discussion

48

from periodontal health to disease. Thus, they suggested that type 2 helper T cell

cytokine, as represented by IL-4 was associated with the remission or improvement of

periodontal disease.20

The findings of the present study is congruent with the findings of another study

conducted to determine the levels of interleukin (IL)-1b, IL-4, IL-6 and IL-8 in

gingival crevicular fluid of periodontally healthy and diseased individuals and to

study their association to smoking, stress and clinical periodontal parameters. A total

of 80 patients were included in the study: 20 patients with early onset or aggressive

periodontitis (EOP), 20 with chronic adult periodontitis (AP), 20 with gingivitis (G)

and 20 patients with healthy periodontium (H). In periodontally diseased subjects the

total amounts of IL-8 were significantly elevated as compared to healthy subjects,

whereas IL-4 showed an inverse relationship to periodontal status and higher amounts

were found in the healthy group.37

This study was also supported by the previous

studies by Shapira et al and Kabashima et al who reported lack of IL-4 in gingival

crevicular fluid from severe inflammatory sites.38, 39

In conclusion, the present findings demonstrate that the levels of IL-8 and IL-4

could be considered in detecting the stability of miniscrew implants.

LIMITATION:

The study is done in a smaller sample size.

SCOPE FOR FUTURE STUDY:

1. A study can also be done on a larger scale with a higher sample size to get

more accurate and authentic results.

Discussion

49

2. A longitudinal study can be conducted to assess the levels of IL-8 and IL- 4

with clinical correlation of implants.

3. Further studies are required to elaborate on the levels of interleukins in

different condition such as in health, in progression to peri-implantitis and

during peri-implantitis.

4. A study can be conducted to compare the levels of other cytokines like IL-2,

IL-6 as these also haven been associated with peri-implantitis or bone

turnover.

Conclusion

50

CONCLUSION

Within the limitations of the present study, it was observed that:

In the present study the IL-8 levels in healthy implants increases from one hour to

7th

day. It could be related to the perimucositis due to the injury during insertion of

miniscrew implant. The IL-8 level decreases or remains the same from 7th

to 21st day

indicating that the progress of inflammation is reduced or regressed. This can

attributed to fact that there is no progression towards peri-implantitis.

In the present study, the IL-4 levels in healthy implants decreases from one hour to

7th

day. From this it can be inferred that there is some amount of perimucositis around

the implant due to the tissue injury during insertion of the miniscrew implants. In the

present study, IL-4 levels the decreases from 7th

to 21st day. This could be attributed

to the persistence of inflammation through 21st day.

These findings demonstrate that the levels of Interleukin 8 and Interleukin 4 could

be considered in detecting the stability of miniscrew implants.

Summary

51

SUMMARY

The study was carried out in the Department of Orthodontics and Dentofacial

Orthopaedics, Coorg Institutes of Dental Sciences, Virajpet, Karnataka.

The purpose of this study was to evaluate the levels of interleukin 8 and interleukin

4 around mini-implants during orthodontic tooth movement and to draw clinical

inferences from the same.

The study was done around 22 healthy miniscrew implants from 7 patients (6

females and 1 male) within an age range between 16-25 years with 12 mini-implants

in the maxilla and 10 in the mandible without any signs of peri-implantitis were taken

into consideration. The selected subjects were in retraction stage using miniscrew

implants. The miniscrew implants were titanium implants (B K Surgicals) of the size

1.5mm diameter, 8mm long and was inserted by the same orthodontist in all the cases.

A force of 150g (measured using dontrix gauge) was applied on each miniscrew

implant for retraction.

The samples were collected over a month time period after force application on

mini-implants according to the following schedule: T1, T2 and T3 at 1 hour, 7th

day

and 21st day respectively, after force application on miniscrew implant. The samples

were stored in sterile eppendorf tubes at -200 Celsius until the start of the experiment.

The GCF samples were put through Elisa test (Elabscience® human interleukin 8

and interleukin 4) for determining the concentration of IL-8 and IL-4. The results

were analysed using descriptive and inferential statistics.

Within the limitations of the study, it was observed that in healthy miniscrew

implants the Interleukin 8 levels increased from one hour to 7th

day. It could be related

Summary

52

to the perimucositis due to the injury during insertion of miniscrew implant. The IL-8

level slightly decreased or remained the same from 7th

to 21st day indicating that the

progress of inflammation is reduced or regressed. This can attributed to fact that there

is no progression towards peri-implantitis.

In this study, the IL-4 levels in healthy implants decreased from one hour to 7th

day. From this it can be inferred that there is some amount of perimucositis around the

implant due to the tissue injury during insertion of the miniscrew implants. The IL-4

levels the decreased from 7th

to 21st day .This could be attributed to the persistence of

inflammation through 21st day.

These findings demonstrated that the levels of Interleukin 8 and Interleukin 4 in

PMICF around could be considered in detecting the stability of miniscrew implants.

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53

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collagen arthritis suppresses synovial IL-17 and osteoprotegerin ligand and

prevents bone erosion. The Journal of clinical investigation. 2000 Jun

15;105(12):1697-710.

41) Giannopoulou C, Mombelli A, Tsinidou K, Vasdekis V, Kamma J. Detection of

gingival crevicular fluid cytokines in children and adolescents with and without

fixed orthodontic appliances. Acta Odontologica Scandinavica. 2008 Jan

1;66(3):169-73.

Consent form

60

CONSENT FORM

This Informed Consent form is for the Participant.

TITLE OF RESEARCH:

EVALUATION OF THE LEVELS OF INTERLEUKIN 8 AND INTERLEUKIN 4

AROUND MINI-IMPLANTS DURING ORTHODONTIC TOOTH MOVEMENT - A

CLINICAL STUDY.

I am Dr. Bianca B. Rodrigues, Post Graduate Student, Coorg Institute of Dental Sciences,

Virajpet. Under the guidance of Dr. Sanju Somaiah, Professor, Department of Orthodontics

and Dentofacial Orthopaedics, I am doing a “EVALUATION OF THE LEVELS OF

INTERLEUKIN 8 AND INTERLEUKIN 4 AROUND MINI-IMPLANTS DURING

ORTHODONTIC TOOTH MOVEMENT - A CLINICAL STUDY”. This research

includes an oral examination and collection of peri mini-implant crevicular fluid. I am going

to give you information and invite you to be a part of this research. Before you decide, you

can talk to anyone if you feel comfortable with, about the research. There may be some words

that you do not understand. Please ask me to stop as we go through this information sheet and

I will take time to explain. If you have questions later, you can ask them to me.

Your participation in this research is entirely voluntary. It is your choice whether to

participate or not. There may not be any benefits for you at this stage of the research, but your

participation is likely to help us find the answer to the research question. By participating in

this research you will not be at any risk. The information that we collect from this research

project will be kept confidential. You are entitled to withdraw from the study at any point of

time. The procedure will be of free of cost.

If you have any questions you may ask them now or later, even after the study has started.

If you wish to ask questions later, you may contact me as follows:

Investigator:

DR.BIANCA B. RODRIGUES

POST GRADUATE STUDENT

Contact no. – 9481665642

Email id: [email protected]

Guided by:

DR. SANJU SOMAIAH

PROFESSOR

DEPARTMENT OF ORTHODONTICS

COORG INSTITUTE OF DENTAL SCIENCES, VIRAJPET

Contact no. 9945527999

Email id:[email protected]

This proposal has been reviewed and approved by Institutional Review Board of Coorg

Institute of Dental Sciences, Virajpet, which is a committee whose task is to make sure that

research participants are protected from harm. By signing on the consent form you will be

waving off all legal liabilities against the institution and staff.

Consent form

61

CERTIFICATE OF CONSENT

I have read the foregoing information or it has been read to me. I have had the opportunity

to ask questions about it and any questions that I have asked have been answered to my

satisfaction.

I consent voluntarily to participate as a participant in this research. I understand that I have

the right to withdraw from the study at any time before the publication of the data. I have

been assured that the data will be kept confidential and anonymous. I also give consent for

my data to be published or presented if the above conditions are met.

Name of Participant

Signature of Participant

Date

If illiterate

I have witnessed the accurate reading of the consent form to the potential participant and

the individual has had the opportunity to ask questions. I confirm that the individual has

given consent freely.

Name of witness Thumb print of participant

Signature of witness

Date

Statement by the researcher/person taking consent

I have accurately read out the information sheet to the potential participant, and to the best

of my ability and made sure that the participant understands that the following will be done.

1. Oral examination.

2. Collection of peri mini-implant crevicular fluid

I confirm that the participant was given an opportunity to ask questions about the study,

and all questions asked by the participant have been answered correctly and to the best of my

ability.

I confirm that the individual has not been forced into giving consent and the consent has

been given freely and voluntarily. A copy of informed consent form has been provided to the

participant.

Name of Researcher/person taking consent

Signature of Researcher/person taking consent

Date

Consent form

62

INFORMED CONSENT FROM PARENT/GUARDIAN

I, Dr. BIANCA B RODRIGUES, post graduate student, Department of Orthodontics and

Dentofacial Orthopedics, Coorg institute of Dental Sciences, Virajpet certify that all the

elements including the nature, purpose risks of the above study and described in this consent

document have been fully explained to the subjects. In my judgment the guardian/parents

possess the legal capacity to give informed consent to participate in this research and are

voluntarily and knowingly giving informed consent to participate.

Name of investigator: Dr. Bianca B Rodrigues

Signature of investigator:

CONSENT

I……………………the undersigned, parent/ guardian of …………….. agree to give the

voluntary consent for the participation of my ward in the study. I …………have read the

information in this form (or it has been read to me). I was free to ask any questions and they

have been answered.

1. I have read and understood this consent form and the information provided to me.

2. I have had the consent document explained to me

3. I have been explained about the nature of the study.

4. My ward’s rights and responsibilities have been explained to me by the investigator.

5. I have been advised about the risks associated with my ward’s participation in the study.

6. I agree to cooperate with the investigator and I will inform him/ her immediately if any

ward’s suffer unusual symptoms.

7. My ward has not participated in any research study within the past months.

8. I am aware of the fact that I can opt of the study at any time without having to

give any reason and this will not affect my future treatment in the dental hospital.

9. I am also aware that the investigator may terminate my participation in the study at any

time, for any reason, without my consent.

10. If, despite following the instructions, my ward is physically harmed because of any

substance or any procedure as stipulated in the study plan.

11. I have had my questions answered to my satisfaction.

Date: Signature of the Parent/guardian

Ethical Committee Clearance Certificate

59

ETHICAL COMMITTEE CLEARANCE CERTIFICATE

Proforma Data

63

PROFORMA DATA

Patient name:

Age/Sex:

Deleterious Habits:

Dental history:

Medical history:

1. Systemic Diseases:

2. Medication:

3. Allergy:

4. Others:

Clinical examination:

1. Gingiva:

a. Inflammation

b. Bleeding on probing

2. Periodontal pockets:

Radiographic examination:

1. Periodontal bone loss:

2. Other findings:

IMPLANT NO

IMPLANT

SITE

UPPER RIGHT

UR

UPPER LEFT

UL

LOWER RIGHT

LR

LOWER LEFT

LL

Peri-implantitis/

implant

loosening

T1 T2 T3 T1 T2 T3 T1 T2 T3 T1 T2 T3

Figures

64

FIGURES

Figure 3. Eppendorf tube and microcappilary tube used for sample collection.

Figure 4. Peri-miniscrew implant crevicular fluid sample collection.

Figure 5. Samples stored in Eppendorf tubes.

Figures

65

Figure 6. ELISA kits of IL-8 and IL-4.

Figure 7. Contents of ELISA kits IL-8 and IL-4.

Figure 8. Performing the ELISA test.

Figures

66

Figure 9. Yellow color change after adding stop solution.

Figure 10. Elisa spectrometer for reading colour change.

Figure 11. Dontrix gauge

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