Photodynamic Photodynamic therapy- A therapy- A promising promising alternate alternate antimicrobial antimicrobial strategystrategy

Dr. Saji GeorgeDr. Saji GeorgeResearch FellowResearch FellowFaculty of DentistryFaculty of DentistryNational University of National University of SingaporeSingaporeRepublic of Singapore.Republic of Singapore.

Bacterial growth modes that defies current Bacterial growth modes that defies current antimicrobialsantimicrobials

Biofilm

Community of immobilized bacteria in embedded in self made matrix

Intracellular pathogens

Bacteria residing inside host cells

Biofilm mode of bacterial Biofilm mode of bacterial growthgrowth(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 growthSite 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.

Root canal infection- eg of biofilm Root canal infection- eg of biofilm mediated infectionmediated infection

Search for newer magic bulletsSearch for newer magic bullets

Biofilm and intracellular bacterial Biofilm and intracellular bacterial growth-growth-

Additional barriers for older magic Additional barriers for older magic bulletsbullets

Damaged Biomolecules O2

O**

PS

PS**

LIGHT

PS

PhotoreactionsPhotoreactions

Type 1- Type 1- Direct Direct interaction of PS with interaction of PS with other moleculesother molecules

Type 2- Type 2- Mediated via Mediated via singlet oxygen singlet oxygen (Photodynamic effect)(Photodynamic effect)

Singlet oxygen Excited photosensitizer

Sensitized bacterial cellSensitized bacterial cell Damaged cellDamaged cell

Photodynamic Therapy (PDT) as an Photodynamic Therapy (PDT) as an antimicrobial agentantimicrobial agent

Photosensitizer + light

=Bacterial Killing

Cell wall damage due to PDT

Water

MIX

DNA damage caused by PDTOuter membrane profile before and after PDT

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

Mechanism of antimicrobial Mechanism of antimicrobial actionaction

S. George and Kishen A. Photochemistry and Photobiology (in press)

Bacteria associated with fibroblast

The rate of bacterial killing - faster than loss of mammalian cells.(George and Kishen 2007)

Is PDT a magic Is PDT a magic bullets? bullets?

y= 95.939e-

0.0764x

y= 137.48e-

1.0088x

Bacteria internalized by

THP1

100% elimination of

internalized bacteria at the

cost of 70% THP1 cells with

damaged cell membrane!!!

(unpublished data)ConfidentiaConfidentiall

Mitochondrial (MTT

assay) and cell membrane

damage (Trypan blue

exclusion assay) on THP1

cells induced by PDT

DNA damage (comet

assay) in THP1 cells

induced by PDT

A

1. Control

2. H2O2

3. Irradiation

4. Sensitization

5. PDT

1 2

3 4

5

B

Is PDT a magic Is PDT a magic bullets…?bullets…?

ConfidentiaConfidentiall

Modified PDT in comparison with Modified PDT in comparison with conventional RCT and conventional PDTconventional RCT and conventional PDT

A. 1 week old biofilm

B. 4 weeks old biofilm

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

Antimicrobial treatment in four weeks old root canal biofilm is less effective than 1 week old biofilm

Lim et al. J Oral Rehabil . Communicated.

0

2

4

6

8

10

12

Control ConventionalPDT

ANILAD RCT

log

num

ebr

of b

acte

ria

1 week

4 weeks

A B

Modified PDT

ConfidentiaConfidentiall

The ultrastructure of matured root canal The ultrastructure of matured root canal biofilm –limited accessibility to the biofilm biofilm –limited accessibility to the biofilm

corecore

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

Photosensitizer

Fluid phase

Dentine surface

Bacteria mediated dentine dissolution

Dentine

Biofilm

Confocal microscopy

HypothesisHypothesis

Incorporation of matrix disrupting and Incorporation of matrix disrupting and

oxygenating agents in oxygenating agents in

photosensitization formulationphotosensitization formulation can can

improve the biofilm eradication improve the biofilm eradication

potential of photodynamic therapypotential of photodynamic therapy

Materials and Materials and methodsmethods

Light Source- Diode Laser (30mW)

Photosensitizer- Methylene Blue

Photosensitizing formulation PF1, PF2, PF3 and PF4

Bacteria-Enterococcus faecalis (ATCC 29212)

Photochemical

NATA oxidation(50µM MB with 10µM NATA)Fluence rate- 100mw/cm2

Singlet oxygen yield (DPBF bleaching assay)(50µM MB with 100µM DPBF)Fluence rate- 100mw/cm2

Photosensitizer in different formulations

Photobiological Antibiofilm potentialBiofilm in multiwell plate-31.84J/cm2- CLSM observation

Biofilm in dentine- 127J/cm2

Incubation 10 weeksControl specimen

Splitting the root canal open and collecting the dentine shavings using burr

Incubating the dentin shavings in fresh medium

Culturing on agar plates to enumerate colony forming units

(n=30)

Incubated for 6 hours to enrich bacterial number

PDT (Dose-127J/cm2) RCT

1 2 3

17% EDTA 5.

25%

N

aOC

l

Materials and Materials and methodsmethods

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.

A B

Result: Photooxidation Result: Photooxidation potentialpotential

ConfidentiaConfidentiall

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.

A

E

C D

B

F

The Laser Confocal Scanning Microscopy of biofilm subjected to PDT.

PDT using PF4 resulted in eradication of biofilm

Result: Biofilm Result: Biofilm disruptiondisruption

ConfidentiaConfidentiall

Treatment groups

log10 after 4 hours of enrichment

Number of tooth specimens positive for bacteria after 24 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 RCT+PDT using PF4

0.0 0/5Conventional PDT failed to disinfect the root-canal

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

Result: Antibiofilm Result: Antibiofilm propertyproperty

ConfidentiaConfidentiall

DiscussionDiscussion

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

ConfidentiaConfidentiall

DiscussionDiscussion

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

ConfidentiaConfidentiall

ConclusionConclusion

‘‘Matured’ bacterial biofilm, that is Matured’ bacterial biofilm, that is generally resistant to antimicrobial generally resistant to antimicrobial agents, can be eradicated by PDT agents, can be eradicated by PDT using PF4using PF4

Thank YouThank You