Photodynamic therapy-

A promising alternate
antimicrobial strategy

Dr. Saji George

Research Fellow
Faculty of Dentistry
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
(Nair et al 2005)
35-53% of root canal surface
uninstrumented (Peter et al 2001)
Endodontics 3rd Edition: Edt Stock et al, 2004.
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
O**
Type 1- Direct interaction of PS**

PS with other molecules

Type 2- Mediated via singlet Damaged

oxygen (Photodynamic effect) PS PS O2 Biomolecules
 Mechanism of antimicrobial action
10
Water
9 Without Light

Ratio of intact to dam aged cells

8 With Light

7
6

5
4 MIX
3
2

1
0
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 before

DNA damage caused by PDT
and after PDT
S. George and Kishen A. Photochemistry and Photobiology (in press)
 Is PDT a magic bullets?

120
E. faecalis
Bacteria associated with fibroblast 100 Fibroblast

% cell surv iv a l
80

The rate of bacterial killing - faster 60

y= 95.939e-
0.0764x

than loss of mammalian cells. 40

(George and Kishen 2007) 20 y= 137.48e-

1.0088x
0
0 Min 1 Min 5 Min 10 Min 20 Min
Duration of irradiation
6 90

E. faecalis
80
THP1
Bacteria internalized by THP1 5
70

% of THP1 with damaged membran

log number of bacteria
4 60

100% elimination of internalized 3

50

40

bacteria at the cost of 70% THP1 2 30

cells with damaged cell membrane!!! 1

20

10

(unpublished data) 0 0
0 min 5 mins 10 mins 20 mins
Duration of sensitization

Confidential
 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 week
10
log numebr of bacteria 4 weeks
Antimicrobial treatment in 8
four weeks old root 6
canal biofilm is less
4
effective than 1 week old
2
biofilm
0
Control Conventional ANILAD
Modified RCT
PDT PDT

Lim et al. J Oral Rehabil . Communicated.

Confidential
 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 in (DPBF bleaching assay)
different formulations (50μM MB with 100μM 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
%

Na 5 %
1 2 3 ED

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
300 2
PF1

A
PF1
PF2 1.8 B PF2
250 PF3 PF3
1.6
fluorescence intensity(AU) @ 360nm PF4 PF4
1.4

Absorbance @ 420nm
200
1.2

150 1

0.8
100
0.6

0.4
50
0.2

0 0
0 min 1 min 2 min 3 min 4 min 5 min 0 min 1 min 2 min 3 min 4 min 5 min

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.

Confidential
Result: Biofilm disruption
A B

The Laser Confocal Scanning

Microscopy of biofilm
subjected to PDT.
C D

PDT using PF4 resulted in

eradication of biofilm
E F

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.

Confidential
Result: Antibiofilm property
Treatment groups log10 after 4 Number of tooth specimens
hours of positive for bacteria after 24
enrichment hours of enrichment
Control 7.147 (±0.601) 5/5
Conventional-PDT 5.639 (±0.066) 5/5
Conventional RCT 0.0 3/5
PDT using PF4 0.0 0/5
Conventional 0.0 0/5
RCT+PDT using PF4

¾Conventional PDT failed to disinfect the root-canal

¾Conventional root-canal treatment (RCT) showed re-growth in

66% samples after 24-hour enrichment

¾PDT using PF4 alone or in combination with RCT -complete

elimination of bacteria

Confidential
 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

Confidential
 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

Confidential
 Conclusion

‘Matured’ bacterial biofilm, that is generally

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