Development of wound healing agent
from bacterial exopolysaccharides and
understanding their mechanism of action
Sahana T G
Junior Research Fellow
Yenepoya Research Centre
1
Funding agency applied for:
Indian Council of Medical Research (ICMR)
2
Principle Investigator:
Dr. Rekha PD
Deputy Director
Yenepoya Research Centre
Yenepoya University
Co- investigator:
Sahana T G
Junior Research Fellow
Yenepoya Research Centre
3
Introduction
• Wound is a growing challenge affecting all age groups
• Healing involves
– Hemostasis
– Inflammation
– Proliferation
– Re-modeling
• Annually 4% of total health care costs are spent on wound
care products (Smith et al., 2016)
4
Introduction cont’d
Source: http://i0.wp.com/blog.mediligence.com/2013/06/wound-pie-2013
5
Introduction cont’d
• Passive dressings act as physical barrier, hence active
dressings are recommended
• Biopolymer based material are the good alternatives
• Exopolysaccharides are the biopolymers secreted by
bacteria
• In this study, bacterial exopolysaccharides will be used
to study wound healing properties and the mechanism
of action
6
Rationale of the study
• Current wound management techniques are clinical
based
– Debridement
– Irrigation
– Suturing
– Negative pressure therapy
– Grafting
– Growth factor supplementation
7
Rationale of the study
• In-appropriate management leads to
– Fibrosis
– Chronic wounds
– Loss of tissue functioning
• Hence alternative approaches are necessary
• Potential applications of microbial exopolysaccharides
are not explored
8
Aim of the study
To develop wound healing material from bacterial
exopolysaccharides and to understand the
mechanism of action
9
Objectives
• To study the wound healing potential of EPS of bacterial
origin by in vitro approaches.
• To understand the mechanisms involved in the wound
healing using in vivo experimental animal models.
• To improve material properties to enhance the bioactivity
based on the molecular and structural features of the
EPS and to develop an end product for treating wounds.
10
Methodology
Isolation of bacterial EPS
Purification of EPS by dialysis and column chromatography
Structural characterization of EPS by FTIR and NMR
In vitro wound healing activity by cell migration assay on Human
Dermal Fibroblasts and Keratinocytes
11
Methodology cont’d
Isolation of protein and RNA
RT-PCR to check
expression profile
ELISA, western blot for
VEGF, TGF-β, TNF-α
12
Methodology cont’d
In vivo experiments using excision, incision and burn wound
models in Albino Wistar rats
Histopathology using H and E stainning
and
Isolation of protein and RNA for RT-PCR and western blot
for VEGF, TNF- α, TGF-β
13
Methodology cont’d
Enhancing the property by functionalization
Developing an end product for healing the wounds
14
Work plan
Time period
Feb 29th -June 29th 2016 (0-4
Work to be completed
Course work
months)
1st July -31st Dec 2016 (5th -10th
Extraction of EPS and purification
month)
process
Enhancing material properties
1st Jan-31st Dec 2017
In vitro experiments
1st Jan-31st Dec 2018
In vivo experiments
1st Jan 2019
Thesis writing
15
Expected outcome
• Development of biopolymer based material for treating
the wounds
• Adds to the current wound healing management
techniques
• Understanding the molecular mechanism in wound
healing with respect to the exopolysaccharides
16
Estimated budget
Staff
1st
2nd
3rd
year
year
year
Total
1,92,000/- 1,92,000/- 2,16,000/- 6,00,000/-
Recurring
Mice, culture media,
ZM
media,
disposable pipettes,
ELISA
kits,
antibodies
for
western
blot,
primers for RT-PCR,
Cell lines (HDF)
2,00,000/- 2,00,000/- 1,00,000/- 5,00,000/-
17
Estimated budget
Non recurring
1,00,000/- 50,000
50,000
2,00,000/-
Micropipette tips,
glass wares, plastic
wares
Contingencies
Travel
Overhead charges
Total
5000/-
5000/-
5000/-
15,000/-
-
-
-
-
20,000/-
15,000/-
15,000/-
50,000/-
5,17,000/- 4,62,000/- 3,86,000/- 13,65,000
18
Animal ethical clearance
Ethical clearance from the Institutional Animal
Ethics Committee for involving experimentation
on Albino Wistar rats
19
References
•
•
•
•
•
•
•
•
Martin P. Wound healing--aiming for perfect skin regeneration. Science. 1997 Apr
4;276(5309):75-81.
Singer AJ, Clark RA. Cutaneous wound healing. New England journal of medicine.
1999 Sep 2;341(10):738-46.
Broughton 2nd G, Janis JE, Attinger CE. The basic science of wound healing. Plastic
and reconstructive surgery. 2006 Jun;117(7 Suppl):12S-34S.
Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and regeneration.
Nature. 2008 May 15;453(7193):314-21.
Werner S, Grose R. Regulation of wound healing by growth factors and cytokines.
Physiological reviews. 2003 Jul 1;83(3):835-70.
Diegelmann RF, Evans MC. Wound healing: an overview of acute, fibrotic and
delayed healing. Front Biosci. 2004 Jan 1;9(1):283-9.
Clark R, editor. The molecular and cellular biology of wound repair. Springer Science
& Business Media; 2013 Nov 11.
Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic‐Canic M. Growth factors
and cytokines in wound healing. Wound Repair and Regeneration. 2008 Sep
1;16(5):585-601.
20