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Title: Comparison of decrease in interlukin-6 levels after using two
intracanal medicaments during root canal treatment: in-vivo study.
Article Type: Clinical Research
Keywords: Calcium hydroxide, Interlukin-6, Triple antibiotic paste
Corresponding Author: Dr. Munish Batra, M.D.S
Corresponding Author's Institution: Gian Sagar Dental College and
Hospital
First Author: Munish Batra, M.D.S
Order of Authors: Munish Batra, M.D.S; Rajveer Kaur; Jayati Batra
Manuscript Region of Origin: Middle East & India
Abstract: Objective:
Periapical inflammation as the result of pulpal infection is
characterized by the accumulation of inflammatory cells. Elevated
inflammatory markers (IL-6) are associated with chronic inflammatory
disease. Little literature is available to correlate levels of
inflammatory markers after placing intracanal medicament. Thus the aim of
the study was to compare the decrease in levels of interleukin-6 after
using two intracanal medicaments during root canal treatment.
Materials and method:
Among 50 patients, 25 were included in test group in which triple
antibiotic paste was used as intracanal medicament and 25 were included
in control group in which calcium hydroxide was placed as intracanal
medicament. Periapical fluid was collected with the help of paper points
before and after the placement of intracanal medicament. IL-6 levels were
measured using ELISA kit and ELISA reader.
Results:
In the control group there was a reduction in IL-6 levels from 3.761.42
pg/ml pre-peratively to 3.23 0.98 pg/ml post-operatively and in the
test group there was a reduction from 3.74 1.75 pg/ml pre-operatively
to 2.64 1.03 pg/ml post-operatively. The results showed that there was
a significant reduction in IL-6 levels in the test group compared to
control group.
Conclusion:
Triple antibiotic paste can be effectively used for sterilization of
canals and healing of periapical pathology by reducing inflammatory
response as compared to traditionally used calcium hydroxide paste as
intracanal medicament.

.Title Page (Only file with author names)

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Manuscript type: Original Research


Title: Comparison of decrease in interlukin-6 levels after using two intracanal
medicaments during root canal treatment: in-vivo study.
Author names and affiliations
1. Dr. Munish Batra
Senior Lecturer
Department of Conservative Dentistry and Endodontics
Gian Sagar Dental college and Hospital, Patiala
Punjab, India
Email: munish7667@yahoo.in
2. Dr. Rajveer Kaur
Senior Lecturer
Department of Periodontology and Oral Implantology
Luxmi Bai dental College and Hospital, Patiala
Punja, India
Email: rajveerkr07@gmail.com
3. Dr. Jayati Batra
MD student
Department of Dermatology, Venerology and Leprology
Guru Gobind Singh Medical College and Hospital, Faridkot
Punjab, India
Corresponding Author:
Dr. Munish Batra
Email: munish7667@yahoo.in
Address: H.no. 37-A, dashmesh Nagar, street 7, Patiala, Punjab, India. PIN 147001
Phone: +91-9988950233
Acknowledgement:
The authors deny any conflicts of interest. I affirm that I/We have no financial affiliation (e.g.,
employment, direct payment, stock holdings, retainers, consultantships, patent licensing arrangements
or honoraria), or involvement with any commercial organization with direct financial interest in the
subject or materials discussed in this manuscript, nor have any such arrangements existed in the past
three years. Any other potential conflict of interest is disclosed."

*Statement of Clinical Relevance (max 40 words)

Statement of clinical relevanve


Periapical inflammation as the result of pulpal infection is characterized by the
accumulation of inflammatory cells leading to elevated levels of inflammatory markers.
Thus the aim of the study was to compare the decrease in levels of interleukin-6 after
using two intracanal medicaments during root canal treatment.

*Manuscript
Click here to view linked References

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ABSTRACT
Objective: Periapical inflammation as the result of pulpal infection is characterized by
the accumulation of inflammatory cells. Elevated inflammatory markers (IL-6) are
associated with chronic inflammatory disease. Little literature is available to correlate
levels of inflammatory markers after placing intracanal medicament. Thus the aim of the
study was to compare the decrease in levels of interleukin-6 after using two intracanal
medicaments during root canal treatment.
Materials and method: Among 50 patients, 25 were included in test group in which triple
antibiotic paste was used as intracanal medicament and 25 were included in control
group in which calcium hydroxide was placed as intracanal medicament. Periapical fluid
was collected with the help of paper points before and after the placement of intracanal
medicament. IL-6 levels were measured using ELISA kit and ELISA reader.
Results: In the control group there was a reduction in IL-6 levels from 3.761.42 pg/ml
pre-peratively to 3.23 0.98 pg/ml post-operatively and in the test group there was a
reduction from 3.74 1.75 pg/ml pre-operatively to 2.64 1.03 pg/ml post-operatively.
The results showed that there was a significant reduction in IL-6 levels in the test group
compared to control group.
Conclusion: Triple antibiotic paste can be effectively used for sterilization of canals and
healing of periapical pathology by reducing inflammatory response as compared to
traditionally used calcium hydroxide paste as intracanal medicament.
Keywords: Calcium hydroxide, Interlukin-6, Triple antibiotic paste

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INTRODUCTION
Periapical lesions are inflammatory diseases that develop as a result of root canal
bacterial infections and may result in periapical bone destruction because of host
defensivemicrobial disturbances.1, 2 Periapical inflammation as the result of root canal
infection is characterized by the accumulation of inflammatory cells and bone
resorption.3 Cytokines are low-weight messenger molecules between the host cells
secreted by different immune cells and believed to have an important role in
pathogenesis of many inflammatory diseases such as periradicular lesions.4, 5
Interleukins are a group of cytokines (secreted proteins and signal molecules)
that were first seen to be expressed by white blood cells (leukocytes).6 Among various
interleukins, IL-6 is a pro-inflammatory cytokine involved in osteoclast formation and
plasma cell proliferation (American Academy of Periodontology 1999).

Interleukin- 6

(IL-6) has been traditionally considered to be a pro-inflammatory cytokine that may have
a part in inflammatory process of periapical lesions.7 IL-6 may also release locally in
inflamed pulp and periradicular lesions, especially of chronic types.8
Elicitation of the immune inflammatory response against bacteria from the
infected root canal is known to play a pivotal role in chronic apical periodontitis,
involving phagocytosis, activation of humoral and cellular responses and production of
inflammatory mediators, including cytokines, such as interleukin (IL)-1, interleukin (IL)-6
and tumor necrosis factor (TNF)- and matrix metalloproteinases.9 As consequence, the
breakdown of the extracellular matrix from the periodontal tissues leads to the
development and progression of an apical lesions of endodontic origin.10

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Calcium hydroxide paste has been used as an intracanal medicament for many years.
Recently, Triple antibiotic paste (TAP) containing metronidazole, ciprofloxacin and
minocycline has been reported to be a successful regimen as intracanal medicament.
So the aim of this study was to compare the decrease in interlukin-6 levels after using
Calcium Hydroxide and TAP as intracanal medicaments during root canal treatment.

MATERIALS AND METHOD


50 patients in the age group 20 to 40, with chronic apical periodontitis were selected for
the study. Teeth included were maxillary anteriors and mandibular premolars with
evident radiolucent periapical area. Individuals with positive history of acute infection in
last 3 months, with systemic chronic inflammatory or immunological conditions such as
arthritis, gastrointestinal disorders, skin conditions, bronchitis etc, pregnant or lactating
females and individuals who were diabetic and smoker were excluded. All individuals
were informed verbally and in writing about the purpose of the study and an informed
consent document was obtained from them prior to participation. After exclusion 50
patients were divided into two groups:
1. Test group: 25 patients with chronic periapical lesion in which Triple antibiotic paste
was given as intracanal medicament.
2. Control group: 25 patients with chronic periapical lesion in which calcium hydroxide
paste was given as intracanal medicament.

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Procedure:
Files and instruments used in this study were sterilized by autoclaving. After
administering local anesthesia the involved tooth was isolated with a rubber dam.
Access to the pulp cavity was made with sterile high speed burs and pulpectomy was
done using hyflex CMTM no. 25 rotary file (Coltene/Whaledent) with 4% taper. Irrigation
was done with 5% sodium hypochlorite and EDTA. Commercially available calcium
hydroxide paste (Ultradent, USA) was used for the study. Triple antibiotic Paste was
prepared by mixing Ciprofloxacin, Metronidazole and Doxycycline in the ratio 1:1:1 and
propylene glycol was used as a vehicle to carry the mix.

Collection of Periapical fluid


In both groups, periapical fluid sample was collected by absorbent (sterile and
apyrogenic) paper points # 15. The paper points were inserted into the root canal and
extended beyond the apical foramen for the collection of periapical fluid.
Then the intracanal medication was placed by means of lentulo spiral and packed with a
cotton pellet at the level of canal entrance. A provisional restoration (CavitTM G) was
placed and after 14 days, the provisional restoration was removed and the periapical
fluid sample was recollected in both groups i.e. test group and control group.
For completion of endodontic treatment, all root canals were filled with gutta-percha
points and AH plus cement (Dentsply De Trey) by using the active lateral
condensation technique. The teeth were restored with permanent restoration.

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Interleukin Detection Assay:


The wetted length of each paper point was measured immediately upon removal. Each
paper point was placed in a centrifuge tube containing 150 l of 10 mM phosphatebuffered saline. After centrifugation, the supernatant was collected and stored at -20C
until the measurement of IL-6 concentrations was done.
The levels of IL-6 were determined using an ELISA kit (Ray Biotech, Inc.) as shown in
figure 1 and readings were calculated by ELISA reader (Erba Lisascan II, ERBA
Mannheim, Germany) as shown in figure 2, 3. The minimum detectable dose of IL-6 by
this kit was less than 3pg/mL.

Statistical analysis:
The data so collected was tabulated and put to statistical analysis and the results were
compiled. Paired t test were used for obtaining the p value. The p < 0.05 was
considered to be significant and p < 0.001 was considered highly significant. On the
basis of above analysis, conclusion was drawn.
RESULTS
Results showed that decrease in levels of IL-6 was observed in test group after 7 days
which was found to be statistically significant (p = 0.002) as shown in table 1 and figure
4 as compared to control group in which calcium hydroxide paste was used as
intracanal medicament.

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DISCUSSION:
The present study has shown that interleukin levels were significantly decreased after
root canal treatment by using triple antibiotic paste as root canal medicament than
calcium hydroxide. Endodontic therapy is aimed at elimination of bacteria from the
infected root canal11 and hence reduction of inflammatory mediators like IL-6 due to
improve immune response to eliminated micro-organisms.
Among various inflammatory cytokines, IL-6 is a cytokine that has multiple functions
such as differentiation or activation of macrophages and lymphocyte-T, development
and differentiation of lymphocyte B, stimulation of hematopoietic process, stimulation of
collagen synthesis and glycosaminoglycan, production of fibroblasts and proliferation of
epithelial cells.12 IL-6 was detected in neutrophils from peripheral blood and
inflammatory periradicular tissues.12,13 Prabhu et al(1996) in their study showed that IL-6
mRNA was significantly higher in diseased periodontal tissues compared to healthy
controls. They also had shown the correlation between extent of tissue damage and
bone destruction.14 Ivana Brekalo Pro et al(2012) evaluated the presence of the
cytokines tumor necrosis factor-alpha (TNF-) and interleukin-6 (IL-6) in human
periapical lesions and showed that the IL-6 levels in symptomatic group were
significantly in higher concentration as compared to control group (uninflammed
periapical tissues).15
In present study periapical fluid was collected to evaluate the levels of interleukin-6.
One outstanding feature of analyzing periapical fluid was that it enabled researchers to
study the inflammatory response to a microbial infection in a closed environment.16
Periapical fluid could be especially helpful to monitor early signs of healing and

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inflammatory response in a two-visit treatment approach.17,

18

In the present study

patients were recalled 14 days after the placement of intracanal medicament since the
minimum inter-appointment time interval should be no less than 14 days as
inflammation takes at least 10-14 days to subside or heal.
The traditional approach for treating cases of a necrotic permanent tooth was
apexification with calcium hydroxide.14 It is difficult to get an appropriate apical seal in
teeth with open apices by using the conventional endodontic treatment methods. Longterm use of calcium hydroxide has several disadvantages such as multiple treatment
appointments, probable recontamination of the root canal system during treatment
period and increased brittleness of root dentin which increases the risk of future cervical
root fractures.19, 20
Present study showed comparison of levels of interleukin-6 after using Triple antibiotic
paste as intracanal medicament. Bacteria may be present within areas of the root canal
system that are inaccessible to irrigants and to the mechanical cleaning processes
within the canal. Hence, an antibiotic contained within an intracanal medicament must
be able to diffuse into these areas to reduce the number of viable bacteria. If such a
reduction is achieved then decreased levels of inflammatory cytokines, (IL-6) due to
reduced inflammatory response, would be expected. The advantage of Triple Antibiotic
paste as intracanal medicament was that its local application in the root canal had been
suggested to overcome the potential risk of adverse systemic effects of antibiotics and
as an effective mode for drug delivery in teeth lacking blood supply due to necrotic
pulps or pulp-less status.21 Because root canal infections were polymicrobial consisting
of both aerobic and anaerobic bacterial species, single antibiotic might not be effective

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in canal disinfection. Therefore, combination of antibiotics, mainly consisting of


ciprofloxacin, metronidazole, and minocycline, referred to as triple antibiotic (TA) paste
had been suggested for root canal disinfection.22, 23 Triple antibiotic paste was proved to
be biocompatible. Tetracycline inhibits inflammatory cytokine, collagenases and matrix
metalloproteinases, is not cytotoxic and increases the level of interleukin-10, an antiinflammatory cytokine.19
Maria Tanumihardja et al in their study concluded that triantibiotic paste showed
additional antimicrobial effect over commonly used calcium hydroxide, and may prevent
the growth of bacteria. It had been proved that if the lesion was effectively evacuated of
the inflammatory exudates and the microbial load was reduced with an effective
intracanal medicament, it was possible to stimulate the immunological system to induce
repair.24
Present study showed significant improvement in IL-6 levels after root canal treatment
by using triple antibiotic paste whereas Maryam Ehsani found no significant change in
IL-6 after use of Ibuprofen.25

Conclusion:
Micro-organisms at periapical region may cause re-infection and failure and
inflammatory response to these micro-organisms may cause additional destruction of
periapical tissues. Thus complete sterilization of canal may be not possible alone by
root canal treatment. Triple antibiotic paste can be effectively used for sterilization of
canals and healing of periapical pathology by reducing inflammatory response as
compare to traditionally used calcium hydroxide paste as intracanal medicament. The

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present study showed decreased levels of IL-6 at periapical region by using Triple
Antibiotic paste as compared to calcium hydroxide paste.

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24. Maria Tanumihardja ,Wahyuni S, Pattelongi IJ , Samad R , Wahid S. Antimicrobial


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FIGURE LEGENDS
Figure 1- Elisa kit for Interlukin-6
Figure 2- Elisa reader
Figure 3- Micropipettes used
Figure 4- Graph showing comparison of levels of IL-6 in test group pre & postoperatively (intergroup) and control group pre & post-operatively (intergroup).

Table 1: Showing comparison of levels of IL-6 (pg/ml) pre & post-operatively in test
group and control group.
Control
group
(preoperative)

Control
group
(postoperative)

Test group
(preoperative)

Test group
(post-operative)

3.76 1.42

Mean Standard deviation


3.25 0.98 3.74 1.75

2.64 1.03

p1

p2

0.141NS

0.002*

** p <0.001: Highly Significant; * p < 0.05: Significant; NS: p > 0.05: Not Significant
p1 p value on comparison between pre-operatively and post-operatively mean value in
control group
p2 - p value on comparison between pre-operatively and post-operatively mean value in test
group

Elisa kit for Interlukin-6


Click here to download high resolution image

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Elisa Reader
Click here to download high resolution image

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Micropipettes used
Click here to download high resolution image

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Graph showing comparison of levels of IL-6


Click here to download high resolution image

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