pdt-biofilm and intracellular bacteria
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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
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