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Application Form

2017 AOSpine East Asia Research Grant

Applications must be completed on this form in Arial Size 10 font, adhering to the
specified page limits. Boxes may be expanded as required.

Part 1: GENERAL INFORMATION

Research Project Title:

The role of Doxycyline as an MMP inhibitor in rabbit model of tuberculosis of the spine

Project Duration (from YYYY/MM/DD to YYYY/MM/DD):

January 2017 until November 2017

Investigators
Title First Name Surname Institution Membership No.*
Department of
Orthopaedic and
Traumatology, Faculty
dr Otman Siregar 16826
of Medicine, University
of North Sumatera,
Medan, Indonesia
Department of
Orthopaedic and
Traumatology, Faculty
dr Benny Benny
of Medicine, University
of North Sumatera,
Medan, Indonesia
*At least 1 investigator must be a subscribing member of AOSpine

Part 2: PERSONAL INFORMATION


Lead Investigator:
(Surname, First Siregar, Otman
Name)

Position and Title: Head of Spine Division

Department of Orthopaedic and Traumatology, Faculty of Medicine,


Institution:
University of North Sumatera, Medan, Indonesia

Address: Jl. Bungalau no.17, Medan, Indonesia

Contact Number: 62-61-8367300 / 62-817751755 / 62-85262369908

Email Address: otmansiregar114@yahoo.com

AOSpine Asia Pacific


Unit 1310-11 Tower 1, Millennium City 1, 388 Kwun Tong Road, Kowloon, Hong Kong
Phone: +852 2581 1727, Fax: +852 2581 1772
researchAOSAP@aospine.org I www.aospine.org
Part 3: DETAILS OF PROPOSAL

Aims and Objectives:

[maximum ½ page]

Aims
To investigate the role of MMP inhibitor, Doxycyline, to decrease tissue destruction as
shown by the decrease in the serum level of MMP-9

Objectives
 To investigate the relationship between MMP -9, TNF-α and (IL)-1 β and
tuberculosis infection of the spine in rabbit model
 To investigate the relationship between the administration of oral doxycyline in
multiple doses and the serum level of MMP-9, TNF-α and (IL)-1 β in tuberculosis
of the spine in rabbit model
 To investigate the relationship between the serum level of MMP -9 and the
expression of MMP-9 in the tissue

Background:
[maximum 1 page]

Tuberculosis kills more than 1.5 million people per year, and standard treatment has
remained unchanged for more than 30 years, and multidrug - and C drug-resistant strains
are progressively emerging. Mortality rates remain high among patients even after they
have commenced TB treatment.

Approximately 10% of TB infection are cases with extrapulmonary tuberculosis. About 10 -


35% cases of extrapulmonary tuberculosis involve the bones and joints. Spinal
tuberculosis accounts for about 50% cases of skeletal tuberculosis.

A characteristic hallmark of spondylitis TB is tissue destruction, causing morbidity,


mortality, and transmission of infection.

Both pro-inflammatory and anti-inflammatory cytokines play a central role in progression


and control of infection of Mycobacterium tuberculo sis. It has also been shown that the
cytokine profil differs in different stages of the disease. Serum and CSF levels of pro -
inflammatory cytokines TNF-α, IL-1-β, IFN-γ, IL-6 and IL-8; anti-inflammatory cytokines
IL-10 and MMP 2 and 9 has been shown signif icantly higher in patients with spinal
tuberculosis compared to controls, and poor outcome is associated with higher pro
inflammatory serum TNF-α and IFN-γ, and CSF MMP-9 levels, and lower anti-
inflammatory serum IL-10 level.

The only pharmacological agent licensed as an MMP inhibitor in the United States is
doxycycline, a tetracycline antibiotic, which is used in periodontal disease at
subantimicrobial doses to suppress collagenase activity, and has been proposed as an
agent to limit pathology in other infectious and inflammatory disease such as
atherosclerosis, arthritis, neurosistercosis and gram positive infection.

Doxycyclin suppressed multiple MMPs in cellular models of TB infection. Consequently,


doxycyclin may reduce pathology in human TB by effect s on both MMP gene transcription
and activity. Doxycyclin also suppressed TNF -α secretion, a key cytokine in the immune
response to TB. TNF-α drives stromal cell MMP secretion via intercellular networks and
may also drive cachexia ini TB, so doxycycline ma y have multiple immunomodulatory
effects.

AOSpine Asia Pacific


Unit 1310-11 Tower 1, Millennium City 1, 388 Kwun Tong Road, Kowloon, Hong Kong
Phone: +852 2581 1727, Fax: +852 2581 1772
researchAOSAP@aospine.org I www.aospine.org
In this study, we first investigated the hypothesis that the MMP inhibitor doxycycline will
reduce tissue destruction in rabbit model of tuberlucosis of the spin e as shown by
decreased in the level of serum MMP-9 and decreased in the expression of MMP-9 in
tissue

Methods:

In this experimental study, we will purchase 40 New Zealand white rabbits (aged 3 -4
weeks; body weights of 3.25±0.5 kg) and Mycobacterium tuberculosis strain H37Rv from
the institute of biologic product.

Single cell suspensions will be prepared by trituration. A total of 3 -4 Mycobacterium


tuberculosis colonies on agar slant culture medium will be transferred to a homogenizer
with a bamboo stick. An appropriate amount of saline will be ad ded into the homogenizer
and the bacteria will be homogenized. A bacterial suspension will be collected and diluted
with saline to prepare suspensions of 0.5 mg/ml.

All animal experimental protocols will be processed by the Ethics Committee in Indonesia.

After numbering and weighing, the 40 rabbits will be randomly divided into 2 groups. A.
rabbits will be treated with M. tuberculosis (infection group; n = 30); and B. rabbits will be
treated with sterile normal saline (control group; n = 10). All rabbits will be examined
preoperatively by X-ray to exclude any with vertebral anomalies.

Preoperatively, peripheral blood samples will be collected from infection group and
control group. Sera will be separated by centrifugation at 1,000 g for 30 minutes and will
immediately stored at -80 °C. Measurement of TNF-α, IL-1β and MMP-9 will be performed
by ELISA as per manufacture’s instructions.

Under general anesthesia, a longitudinal incision will be made down from the 6 th lumbar
spinous process of the rabbits. At the upper part of the 6 th lumbar vertebra, an electric
drill will be used to bore a hole (0,3 mm diameter). This hole will be filled with medical
gelatin sponges. The, the gelatin sponges will be impregnated slowl y using a syringe with
0.2 ml of a TB suspension at a consentration of 0.5 mg/ml for the infection group or 0.2 ml
sterile normal saline for the control group. Rabbits then will be held individually in cages
for 8 weeks.

At 8 weeks postoperatively, X-ray, Ct-scan and polymerase chain reaction (PCR)


examination will be performed for all rabbits to establish spinal tuberculosis infection, as
well as measurement of serum TNF-α, IL-1β and MMP-9 performed by ELISA as per
manufacture’s instructions.

Then, the infection group then will be divided into three groups. The first group will
receive no treatment, the second group will receive doxycylin dosage 1, the third group
will receive dosage 2..

After 4 weeks of follow-up, serum TNF-α, IL-1β and MMP-9 will be measured by ELISA as
per manufacture’s instructions. Then, each rabbit will euthanized and
immunohistochemical examination will be revealed from the pathological vertebra. The
parameters measured objectively are serum TNF -α, IL-1β and MMP-9

AOSpine Asia Pacific


Unit 1310-11 Tower 1, Millennium City 1, 388 Kwun Tong Road, Kowloon, Hong Kong
Phone: +852 2581 1727, Fax: +852 2581 1772
researchAOSAP@aospine.org I www.aospine.org
Expected Outcomes & Significance:

[maximum ½ page]
Administration of doxycyline in tuberculosis of the spine in rabbit model is expected to
decrease tissue destruction as shown by the decrease in the level of serum MMP -9 and
the decrease in the expression of MMP-9 in pathological tissue by immunohistochem istry
examination.

Research Plan (timeline and milestones):


[maximum ½ page]

January-February 2017 Literature review


March 2017 Reseach proposal
April 2017 Ethical approval
May-August 2017 Animal study
September 2017 Data Management
October 2017 Data analysis
November 2017 Manuscript preparation

Budget:
[maximum ½ page]

New Zealand white rabbits (@ 50 USD ) x 40 = USD 2,000


Institute Pertanian Bogor Animal Hospital Expenses USD 25,000
Mycobacterium tuberculosis strain H37Rv USD 1,000
Xray USD 400
CT Scan USD 3,000
PCR USD 6,000
ELISA kit USD 6,000
Doxycycline USD 50
Stationary USD 200
Stastician USD 1,000
Allowance USD 1,000
Unexpected Expenses USD 4,564

TOTAL USD 50,215

AOSpine Asia Pacific


Unit 1310-11 Tower 1, Millennium City 1, 388 Kwun Tong Road, Kowloon, Hong Kong
Phone: +852 2581 1727, Fax: +852 2581 1772
researchAOSAP@aospine.org I www.aospine.org
Please return this application to researchAOSAP@aospine.org by 18:00 Hong Kong time
on 25 th November 2016.

Thank you

AOSpine Asia Pacific


Unit 1310-11 Tower 1, Millennium City 1, 388 Kwun Tong Road, Kowloon, Hong Kong
Phone: +852 2581 1727, Fax: +852 2581 1772
researchAOSAP@aospine.org I www.aospine.org