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Photodynamic

therapy- A
promising
alternate
Dr. Saji George
antimicrobial
Research Fellow
Faculty of Dentistry
strategy National University of
Singapore
Republic of Singapore.
Bacterial growth modes that defies current
antimicrobials

Biofilm

Community of immobilized
bacteria in embedded in
self made matrix

Intracellular pathogens

Bacteria residing inside host


cells
Root canal infection- eg of biofilm
mediated infection
Biofilm mode of bacterial
growth
(Nair 1987, Gulabivala et al 2005)

RCT with NaOCl and


Chlorhexidine- reduction but not
elimination
(Sequeira et al )-PCR based

Resistance to endodontic
disinfectants (Peter et al (2002),
Sjögren et al 1997, Sequeira et al
2007, Sathorn et al 2007)

Site of bacterial growth


Anatomically inaccessible regions Endodontics 3rd Edition: Edt Stock et al, 2004.
(Nair et al 2005)
35-53% of root canal surface
uninstrumented (Peter et al
Search for newer magic bullets

Biofilm and intracellular bacterial


growth-
Additional barriers for older magic
bullets
Photodynamic Therapy (PDT) as an
antimicrobial agent
Photosensitizer +
light

=
Bacterial Killing
Sensitized bacterial cell Damaged cell

Photoreactions LIGHT
Excited photosensitizer Singlet oxygen
Type 1- Direct PS**
O**

interaction of PS with
other molecules

Type 2- Mediated via PS PS O2


Damaged
Biomolecules
singlet oxygen
(Photodynamic effect)
Mechanism of antimicrobial
action Water

MIX

Cell wall damage due to PDT


Mark Con L+ WL- M L- WL+ M L+
Marker Con WL- ML- WL+ ML+

Outer membrane profile


DNA damage caused by PDT
before and after PDT
S. George and Kishen A. Photochemistry and Photobiology (in press)
Is PDT a magic
bullets?
Bacteria associated with
fibroblast
y= 95.939e-
0.0764x

The rate of bacterial killing -


faster than loss of mammalian y= 137.48e-
cells.
1.0088x

(George and Kishen 2007)

Bacteria internalized by
THP1

100% elimination of
internalized bacteria at the
cost of 70% THP1 cells with
damaged cell membrane!!!
(unpublished data)
Confidentia
Is PDT a magic
bullets…? A

Mitochondrial (MTT
assay) and cell membrane
damage (Trypan blue
exclusion assay) on THP1
cells induced by PDT
1
B 2

DNA damage (comet


3 4
assay) in THP1 cells
induced by PDT
1. Control 5

2. H2O2
3. Irradiation
4. Sensitization
5. PDT

Confidentia
Modified PDT in comparison with
conventional RCT and conventional PDT

A. 1 week old biofilm A B

B. 4 weeks old biofilm


The structure and
composition of root canal
(in vitro) biofilm changes
along with the stage of
maturation 12

1 wee k
10
4 wee ks
Antimicrobial
log numebr of bacteria
8
treatment in four
6
weeks old root
canal biofilm is 4

less effective than 2

1 week old biofilm 0


Modified
Control Conventional ANILAD RCT
PDT
PDT
Lim et al. J Oral Rehabil . Communicated.
Confidentia
The ultrastructure of matured root canal
biofilm –limited accessibility to the biofilm
core
Confocal microscopy

Biofilm

Dentine

Fluid phase
Photosensitiz
er

Bacteria
mediated
dentine
dissolution

Dentine
surface

A. Kishen, S. George and R. Kumar (2006). J Biomed Mat Res A. 77(2):406-415.


Hypothesis

Incorporation of matrix disrupting and


oxygenating agents in
photosensitization formulation can
improve the biofilm eradication
potential of photodynamic therapy
Materials and
methods
Light Source- Diode Laser (30mW)
Photosensitizer- Methylene Blue
Photosensitizing formulation
PF1, PF2, PF3 and PF4
Bacteria-Enterococcus faecalis (ATCC
29212) NATA oxidation
(50µM MB with 10µM NATA)
Fluence rate- 100mw/cm2
Photochemical
Singlet oxygen yield
Photosensitizer (DPBF bleaching assay)
in different (50µM MB with 100µM
formulations DPBF)
Fluence rate- 100mw/cm2
Photobiological Antibiofilm potential
Biofilm in multiwell plate-
31.84J/cm2- CLSM
observation
Biofilm in dentine- 127J/cm2
Materials and
methods
17
%
1 2 3 ED

Na 5%
l
OC
(n=30) TA

5.2
Control specimen
Incubation 10 weeks PDT (Dose-127J/cm2) RCT

Splitting the root canal open and


collecting the dentine shavings using burr

Incubating the dentin shavings in fresh medium

Incubated for 6 hours to enrich bacterial number

Culturing on agar plates to


enumerate colony forming units
Result: Photooxidation
potential
A B

Fig 1. (A) The oxidation of NATA caused by oxygen


based free-radicals measured as the reduction of
NATA concentration.

(B) Compared to any other formulation, PF4 showed


a significant increase in the rate of oxidation of
DPBF, showing the increased rate of singlet-oxygen
production.

Confidentia
Result: Biofilm
disruption A B

The Laser Confocal


Scanning Microscopy of
biofilm subjected to
PDT. C D

PDT using PF4


resulted in E F

eradication of
biofilm

A- biofilm receiving no treatment, B- biofilm subjected to


irradiation alone, C- biofilm subjected to sensitization with
100μM MB, D- biofilm subjected to sensitization with MB
followed by irradiation, E- biofilm subjected to PF4, F-
biofilm subjected to PF4 and irradiation.

Confidentia
Result: Antibiofilm
property
Treatment groups log10 after 4 Number of tooth
hours of specimens positive for
enrichment bacteria after 24 hours
of enrichment
Control 7.147 (±0.601)5/5

Conventional-PDT 5.639 5/5


(±0.066)
Conventional RCT 0.0 3/5
PDT using PF4 0.0 0/5
Conventional 0.0 0/5
Conventional
RCT+PDT PDT failed to disinfect the root-canal
using PF4

Conventional root-canal treatment (RCT) showed re-


growth in
66% samples after 24-hour enrichment

PDT using PF4 alone or in combination with RCT


-complete
elimination of bacteria
Confidentia
Discussion

 PDT can break down the matrix components of biofilm


(Wainwright et al., 2002).

 Bacterial killing during PDT - mostly confined to the


outer surface of biofilm (Zanin et al., 2005)

 PDT using PF4 could breakdown the biofilm matrix and


inactivate bacteria.

 Due to the complementing function of matrix disrupting


and enhanced molecular oxygen availability

Confidentia
Discussion

 60% of the root-canal shavings from RCT


group confirmed bacterial growth after 24
hours of incubation.

 PDT using PF4 alone or in combination with


conventional disinfection technique showed
the absence of bacteria even after 24 hours
of incubation

Confidentia
Conclusion

‘Matured’ bacterial biofilm, that is


generally resistant to antimicrobial
agents, can be eradicated by PDT
using PF4
Thank You