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Targeting Endodontic

Biofilm Bacteria Using


Antimicrobial Photodynamic
Therapy
NUS Presentation Title 2001
Outline of presentation

Conventional endodontic therapy

Limitations of conventional endodontic therapy

Microbial factors associated with failure of


endodontic therapy-Biofilm bacteria

Photodynamic therapy-Principle of action

Application of Photodynamic therapy for root


canal disinfection
NUS Presentation Title 2001
Introduction Endodontic infection
Endodontic therapy

Chemical
irrigants

Mechanical cleaning Use of Chemical Irrigants

Removing the tissue to get rid of bacteria- effect on tissue structure and function
5.25% NaOCl reduced the elastic modulus and flexural strength of dentine. (Sim et al Int Endod J, 34 120 , 120–132,
2001)
Saturated Ca(OH)2 reduced the flexural strength of dentine but not the modulus of elasticity (Grigoratos et al Int Endod J
34,113–119, 2001)

Cytotoxicity of Endodontic irrigants


Detrimental effect of NaOCl and Chlorhexidine on cultured Periodontal Ligament cells (Chang et al (Oral Surg Oral Med
Oral Pathol Oral Radiol Endod 2001;92:446-50)
Microbiological
NUS Presentation Title 2001
factors associated with failure of
endodontic therapy
Root canal lumen

1. Site of bacterial growth


Studies by Nair et al showed the inefficiency of contemporary
instruments and irrigation alone in removing microbes from the
anatomical complexity of the root canal system .

Nair et al. Oral Surg Oral Med Oral Pathol Oral Radiol Endod
2005;99:231-52

Dentinal tubules

2. Biofilm mode of bacterial growth Adapted from www.drcav.com/ images/tooth1.gif

Community of bacteria Root apex


High resistance to antimicrobial agents
Often associated with chronic infections
Many clinical reports high light the presence of biofilm in persistent infections
Conventional PDT may not be effective in removing biofilm formed at apical
foramen (Leonardo et al 2002, J Enfof 28(12):815-818
Can lead to persistence of endodontic infection (Nair et al. 1990Journal of Endodontics 16, 580
8 and Oral Surgery, Oral Medicine, Oral Pathology,Oral Radiology and Endodontics 87, 617 27.)
Biofilm and endodontic infections
NUS Presentation Title 2001

Apical periodontitis is a sequel to microbial


infection of the root-canal space of teeth
(Nair 2004).
The principal cause of failure of root canal
treatment is the persistence of bacteria within
the endodontic system (Nair et al. 1990, 1999)

How does biofilm contribute to persistence?


Cells growing in biofilm are defending themselves against the action of the
complement system, avoiding destruction by phagocytes, causing
immunosuppression, changing antigenic coats, and inducing proteolysis of
antibody molecules (Siqueira 2001)
NUS Presentation Title 2001

Extra radicular biofim – one of


the main reason for endodontic
failure.
(International Endodontic
Journal,34, 1–10, 2001)

Journal Of Endodontics,28, 815-818, 2002

Biofilm present at the root apex of untreated teeth with


chronic peri radicular lesion.
International Endodontic Journal,34, 216-220, 2001
Human infections Involving biofilm
NUS Presentation Title 2001
NUS Presentation Title 2001
What is a Biofilm?

Biofilm is a mode of microbial growth


where a community of microorganisms
adhere to a solid non-shedding
surfaces and embedded in a self
made matrix . Biofilm can form on
diverse materials such as metals,
plastics, medical implant materials,
and hard tissues.
NUS Presentation Title 2001
Biofilm Vs Planktonic

Community theory of Infection

Co operating community of various


types of microorganism Germ theory of Infection

Micro organisms arranged in micro colonies No community of Microorganisms

Covering by matrix of ‘glycocalyx’or ‘slime’ Free Living microbial cells


Gradients of pH, Nutrients, and
Oxygen tension
No glycocalyx production

Quorum sensing-communication - ‘pheromones’ No gradients of Nutrients, pH and


Oxygen
Increased resistance to antimicrobials
Fluid channels in matrix Less resistance to Antibiotics
NUS Presentation Title 2001

How relevant is biofilm…..

“Until the late seventies, no one even knew biofilms existed.


Scientists thought most of the bacterial world was made up of
free-floating bacteria. They developed antibiotics and vaccines
using bacteria floating in a test tube. In many cases, the
medicines just didn't work —prostatitis, middle ear infections in
children and periodontal disease to name a few. As it turns out,
scientists were targeting the wrong kind of bacteria”.

Kate Dalke… Genome News Net work


NUS Presentation Title 2001
Biofilm-Structure & Components

Together we stand, individual we fall…….

Fluid Phase

Protective Matrix
Micro Organisms
Micro Colonies
Fluid Channels

Solid Substratum
NUS Presentation Title 2001
Stages of Biofilm Formation

EPS production,
Phenotypic Extra Protein
variation. expressions.
Coaggregation and
Coadhesion of planktonic
cells

Planktonic cells

Formation of elevated mushroom like


structures of bacterial cells

Modification by
Disperse Mineral
bacterial cells accumulation
to the bulk
fluid
Mature biofilm with new bacterial
cells emerging
Microbial Interactions
NUS Presentation Title 2001

Nutrient exchange Coaggregation and


and sharing Coadhession

Metabolic Facilitate gene

Biofilm
Physical transfer

Tolerance to extremes Determine Spatia


of pH, salinity, Nutrients relationship
and Antibiotics

Genetic

Increased rate of
transformation Specific gene
Gene transfer activation
across MOs
NUS Presentation Title 2001
Factors affecting biofilm formation on a
solid surface

Bacteria

Ionic entities

Fluid phase
Macromolecules

Solid surface

The picture shows different factors influencing biofilm formation by


bacteria on a surface. The final outcome of bacteria adsorbing to any
surface is determined by the inter play of different factors such as the
ionic composition of the medium, charge on the bacterial surface,
charge on the solid surface, roughness of the surface, presence of
conditioning layer, etc.
Resistance mechanisms in BF
NUS Presentation Title 2001

Bacteria in BF are resistant to

Antibiotics

Heat

Quaternary ammonium compounds

Iodine, Chlorine etc

Understanding various stages and method of bacterial


resistance mechanisms to antimicrobials forms the 1 st
step in developing a antibiofilm regime
Sites of Antimicrobial Resistance
NUS Presentation Title 2001
Matrix of the biofilm

Constituents of Matrix

Enzymes present in Matrix

Metabolic and genetic


alteration of bacteria

Metabolic state of bacteria


a. Slow rate
Enzyme of growth
Mediated resistance
Limited Diffusion through Matrix
Neutralization
b.
a. Active
Reduction of of antimicrobials
metabolism of to
cations antibiotics
Metals
Glycocalyx
a.
c. Constituents
b. Altered
IonicAction of Glycocalyx
protein
of eg
profile enzymes
detoxifying
interaction
neutralization of I2
Sieving
d. Multieffect
drug efflux pump
b. Act as an
Increased ion exchange resin
viscosity
e. Gene transfer
NUS Presentation Title 2001

New treatment
concepts
NUS Presentation Title 2001

Anti biofilm coatings


Use of Furanones- ( Bavega et al, Givskov et al., J Bacteriol. 1996; 178:6618-
6622).

Possibility of using furanones as anti bacterial coating on


biomaterials

Furanones are the compounds isolated from sea weeds


(Delisea pulchra-Australian red algae)

Prevents Staphylococcus epidermis adhesion and slime


production on biomaterial

Furanones target the quorum sensing agents.


NUS Presentation Title 2001

Surface modification
Modify the solid surfaces to prevent bacterial
adhesion. Eg using antibacterial nano particles

Replacement therapy
Replace potential pathogenic micro-organisms with
genetically modified organisms that are less virulent

Immunization
The aim is to inhibit adhesion or reduce the virulence
of putative microbial etiologic agents.
NUS Presentation Title 2001

Use of laser irradiation- Asta Richter et al

Pulsed nitrogen laser to the in vitro cultivated biofilm

Damage to the surface substrate at higher power of laser

Removing efficiency depends on the surface substrate

matrix enhances the susceptibility to photodamage as


seen in P. aeroginosa
NUS Presentation Title 2001

Using photosensitizing agents- (Mark Wainwright)


Photosensitizers based on Phenothiazinim chromophores have
broad spectrum antimicrobial action- suitable for eliminating
microbial community

Least resistance to singlet oxygen by micro organisms unlike to


antibiotics- appropriate for treating biofilms since the indwellers are
resistant to antimicrobials

Photosensitization can even cause EPS breakdown


NUS Presentation Title 2001
Photodynamic therapy/ Light Activated Therapy
Involve the killing of microorganisms when a
photosensitizer selectively accumulated in the target is
activated by a visible light of appropriate wavelength.

Light Irradiation

cc c c

c c

Sensitized microbial cells Damaged cell Cell destruction


Mechanism of Photosensitization
NUS Presentation Title 2001

Photosensitizer +Light
Light on photosensitizer

Electron jumps to higher


vibronic level without
change in spin

Comes back to lower state


by Internal conversion…
energy dissipated as heat
to give fluorescent state

Fate of the molecule is


The ability of a photosensitizer depends on the proportion determined by
environment and
undergoing inter system crossing . structure..

Highly fluorescent compound dissipating energy as fluorescence


Emission of photons as
will be less efficient. fluorescence to restore the
ground state/Inter system
Aromatic compoudns with π system makes long lived triplet state crossing with a spin
(Journal of Antimicrobial Chemotherapy (1998) 42, 13–28) flip…Triplet state ..higher
half life
Mechanism….
TypeNUS Presentation
I - The pathway Title 2001
in which a photosensitiser triplet state reacts first with a substrate
other than molecular oxygen.
Type II pathway- The photosensitiser triplet state reacts first with molecular oxygen
and Type II photosensitisation of a biological system is referred to as photodynamic
action.
LIGHT
Type 1 mechanism
PS**

PS Biomolecules
PS

Type 2 mechanism (Photodynamic effect)


LIGHT

O**
PS**

PS PS O2 Biomolecules
NUS Presentation Title 2001

Singlet Oxygen
The presence and property of singlet oxygen was originally
demonstrated in 1931 by Hans Kautsky

The higher energy excited state is 1εg+.


In this state two paired electrons
occupy two different pg MOs . The
excitation energy is 1.63 eV (37.5
kcal/mole) and the decay lifetime is 7
seconds.
TheNUSproduction
Presentation Title 2001
of 1O2

(A) Absorption of light by the


photosensitiser
(B) Formation of the photosensitiser
triplet state; the quantum yield of
1
O2
this process is the ISC efficiency or
triplet yield (FT)
(C) Trapping of the triplet state by
molecular oxygen within its The triplet energy of the sensitiser relative to the 1S0 ground state
must exceed the 0.98 eV excitation energy of O2(1 δ g+)
lifetime; the fraction of trapped
triplet states in a given system is
designated by fT

(D) Energy transfer from the triplet


state to molecular oxygen
NUS Presentation Title 2001
Singlet oxygen quantum yield
Defined as the number of molecules of 1O2 molecules generated for
each photon absorbed by a photosensitizer. Quantum efficiency is an
equivalent term.

Detection and Measurement of Singlet Oxygen


Singlet oxygen luminescence:- Based on the 1269 nm luminescence emitted in the
radiative decay of O2(1Δg+)
Electron paramagnetic resonance:- Energy transfer between the intrinsic magnetism of
unpaired electrons and an external magnetic field is measured with a sensitive
microwave detection system

Photochemical reactions- indirect measurement


NATA oxidation- The oxidation of tryptophanyl moiety is measured
fluorimetrically
DPBF oxidation- Measured spectrophotometrically
NUS Presentation Title 2001
NUS Presentation Title 2001
Singlet oxygen and bacteria

Dahl et al. Journal Of Bacteriology, Apr. 1989, p. 2188-2194

Both gram positive and gram negative were killed on exposure to singlet oxygen
Killing curves for gram negatives were indicative of multihit killing, whereas curves for
gram positive exhibited single-hit kinetics
Direct action of singlet oxygen on gram positive
Secondary radicals production from the LPS of gram negative
Type of bacteria- Susceptibility to Photodynamic Therapy
NUS Presentation Title 2001

Polysaccharides

LPS(Outer membrane

Glycan layer (GlcNAc &


MurNAc)

Cell membrane

Gram positive cell wall Gram negative cell surface

Outer membrane-reason for resistance


Check entry of the chemicals into the
cells
Cations bind together the anionic LPS

Net negative charge on the bacterial cell-Due to LPS and Polysaccharides


NUS Presentation Title 2001

Antimicrobial Photodynamic Therapy


for Root Canal Disinfection
Concept of APDT in Root Canal
Disinfection

Infected tooth Access cavity Sensitization Light treatment Restored tooth


Application of PDT in Endodontics- Concerns
NUS Presentation Title 2001

Anaerobic environment
(Need of oxygen carrier)

Bacterial population
(Dye uptake)

Tissue penetration
(Formulation)

Light Scattering
(refractive index matching
liquid)
Ideal formulation for LAT in root canal
NUS Presentation Title 2001

infection
 Ensure enough oxygen concentration

 Maximum triplet of the dye and molecular oxygen

 Maximum abs wavelength which is minimally scattered by


surrounding tissues
 Maximum penetration through the dentine and biofilm

 Maximum penetration into bacterial cells and killing


NUS Presentation Title 2001

Type of Problem
Use of Enhancers
• Reduced Oxygen tension  Use of oxygen carriers
• Limitation in dye uptake by  Cationic dye and
bacterial cell formulation
• Limitation in dye diffusion  Use of penetration
across the dentine and enhancers
apical region
• Light propagation through  Use of refractive index
the dentine matching liquid
Oxygen Requirement
NUS Presentation Title 2001

Dependence on oxygen
PDT Efficiency decreased when O concentration fall below 3.4% and an advanced
2
infection presents an hypoxygenic site
Lessons from PDT of cancer
Cancer killing is dependent on the oxygen
concentration (Henderson 1990)
Improvement of tumor response by
manipulation of tumor oxygenation during
photodynamic therapy Photochem Photobiol
2002;76:197–203
Oxygen requirement is depend on the fluence
rate (Fig)
Under high fluence rate the rate of oxygen
consumption increases and finally oxygen get
depleted
An oxygen carrier is required for a better PDT
effect in hypoxygenic sites.

Hassan et al in Radiation oncology. Chapter40- PDT of Cancer 605-622)


Perfluorcarbons
NUS Presentation Title 2001

Non-polar highly fluorinated compounds


Strong intramolecular bonding (C-F
bonds are 485 kJ/mol, that is 84 kJ/mol
more than a regular C-H bond),

Perfluorodecalin
Are chemically and biochemically inert .
Properties of PFCs include
The low surface tensions (<20 mN m-1),
dielectric constants and refractive indices
High densities, viscosities and gas
solubility
Solubility of oxygen in Perfluorocarbons

Used as….
Blood substitutes, oxygen therapeutics, anti-tumural agents, perfusates for isolated
organs, surgical tools for ophthalmology, lubrication and cushioning for articular
disorders, cell culture media supplements and drug formulations and delivery (Dias et al ).
Bacterial Population in
Endodontic Infection
Microorganisms present in RC
NUS Presentation Title 2001

 10 and 50 bacterial
species.
 Contain both Gram
positive and gram
negative organism
 Almost equal distribution
of facultative and
obligate anaerobes
 Nature of microbial flora
depend up on the quality
of the treatment received

Tronstad & Sunde, Endodontic Topics 2003, 6, 57–77


NUS Presentation Title 2001
 F. nucleatum Streptococcus spp. P. micros P.
propionicum A. israelii P. alactolyticus.
 P. intermedia,P. nigrescens, P. gingivalis, P. endodontalis
(Black Pigmented Bacteria) C. rectus, F. alocis,
Enterococcus
New species identified were..
 Prevotella tannerae, Actinomyces radicidentis, Olsenella
spp., Dialister pneumosintes, Tanerella forsynthensis,
Treponema maltophilum, T. amylovorum, T. medium, and
T. lecithinolyticum ( Spirochetes).
NUS Presentation Title 2001

LAT against bacteria

PPS 2004, Michael R Hamblin


NUS Presentation Title 2001

Gram positives were easily killed compared to gram negative ( 3±30-fold higher concentrations of TB and MB).
Attributed to the difference in the outer membrane

In contrast with Gram-positive bacteria, the Gram-negative bacteria (Escherichia


coli or Pseudomonas aeruginosa) are not affected by porphyrins and light alone.
Outer membrane prevents or buffers the singlet oxygen and hydroxyl radicals
Suggest the use of membrane permiabilizing agents
(Journal of Photochemistry and Photobiology. B Biology. 1992.14,262-265)
NUS Presentation Title 2001

Experimental Design
Mode of bacterial growth in the root canal for a better
understanding of their persistence

Defining the components for an effective Light


Activated killing of microbes

Testing on biofilms formed in root canal


NUS Presentation Title 2001

Biofilm Dynamics-Morphology (SEM)

60 human teeth (Single rooted) Incubation at 370C for


different time interval Split open
longitudinally observed
Tooth specimens prepared by removing (1-4 weeks), under with Scanning
crown and root tip nutrient- rich and nutrient- Electron Microscopy
deprived condition
Cleaned and sterilized

Inoculated with Enterococcus faecalis


Characterization of Matured Biofilm
NUS Presentation Title 2001

Formed at Root Canal Wall

24 human teeth (Single rooted) Incubation at 370C for 16 Cross-sectioned and


weeks subjected to different
microscopic techniques
Tooth specimens prepared by removing
crown and root tip
•SEM coupled with EDX-Microanalysis (Micro structure and Calcium
content)
Cleaned and sterilized
•Fluorescence microscopy after Acridine Orange staining
•Gram Staining- Light Microscopy and Polarization microscopy (Light
Inoculated with Enterococcus faecalis conductance)
•BacLight LIVE/DEAD staining and observation under Laser Confocal
Scanning Microscope for cell distribution
Further characterization of biofilm for
NUS Presentation Title 2001

Mineralization
FTIR and XRD of Biofilm Von-Kossa Staining of Biofilm
Human dentine blocks were prepared Clean and sterile Glass slides
and sterilized

Enterococcus faecalis (ATCC 29212)


Incubated under different condition
incubated under medium supplemented
with Enterococcus faecalis (ATCC
with Calcium chloride
29212)

After 1 week of incubation slides were


Incubated for different time intervals
taken and washed with deionized
2-6 weeks
water

Von-Kossa staining conducted and


The mineralization potential were observed under oil immersion light
evaluated using advanced material microscope
characterization techniques such as
FTIR and XRD
Biofilm Dynamics-Morphology
NUS Presentation Title 2001

(Scanning Electron Microscopy)


A B
A B

C D
C D

Biofilm development at the root canal wall under nutrient- Biofilm development at the root canal wall under
deprived condition. 1-4 weeks nutrient-Rich condition. 1-4 weeks
NUS Presentation Title 2001

Different stages of
biofilm formation
by Enterococcus
faecalis
On root canal
dentine
Characterization of bacteria-dentine interaction
NUS Presentation Title 2001

Light Conductance
Internal Architecture

Polarisation Microscopy Digital microscopy


Scanning Electron Microscopy

Cell Distribution EDX Microanalysis


20

16

Atomic percentage
12 Ca
P
8 Ca/P

LCSM Fluorescence microscope


0
Dentine Group2 Group4
Dentine Nutrient rich Nutrient deprived
Different Groups
NUS Presentation Title 2001

Mineralization potential of E. faecalis


6 weeks 3 weeks
2 weeks 120
Control
100

80

T (%)
60

40

20

0
3600 3100 2600 2100 1600 1100 600

wavenumber (cm-1)

FTIR reflectance spectra for E. faecalis biofilm on


dentine under nutrient-rich incubated for different XRD spectra of biofilm grown on dentine surface for
time intervals. A systematic increase in the different periods. The hump at 22.5 is decreased over
transmittance intensity peak at 1448, 1394 and time and there is an increase in the peak
985cm-1 with incubation period (2, 3 and 6 weeks) corresponding to apatite peak indicating the
corresponds to the increase in carbonate and precipitation and growth of fresh layer of crystals
phosphate groups on the biofilm surface.

(A. Kishen, S. George, R. Kumar. Bacterial mediated biomineralized biofilm formation on root canal dentine-JBMR)
NUS Presentation Title 2001
Mineralization potential of E. faecalis
6 weeks 3 weeks
2 weeks 120

Control

100

80

T (%)
60

40

20

0
3600 3100 2600 2100 1600 1100 600

wavenumber (cm-1)

Figure -FTIR reflectance spectra for E. faecalis biofilm on dentine


The Von-Kossa staining of biofilm formed on glass slides by E.
under nutrient-rich incubated for different time intervals. A
faecalis, in media added with CaCl3. The figure shows dark
systematic increase in the transmittance intensity peak at 1448, 1394
patches corresponding to mineralization
and 985cm-1 with incubation period (2, 3 and 6 weeks) corresponds to
the increase in carbonate and phosphate groups on the biofilm
surface. A prominent hump was also noticed in the spectrum below
877cm-1, which extended beyond 600cm-1. This increase can be
attributed to carbonate in apatite structure (873cm-1), apatite (865cm-
1), P-O and PO4 (620cm-1, 600cm-1).

Possible reason for mineralized bacterial structure at infected root


NUS Presentation Title 2001

Viable cells

Dentine
Biofilm

The Laser Confocal Scanning Microscopy of the honey-comb like structure after staining with LIVE/DEAD BacLight
Staining. The superimposed images show the presence of viable cells inside the biofilm structure. The honey-comb like
structure is also found to stain with Syto 9 and propidium iodide giving a green and a red fluorescence background.
(Observation under 100X oil immersion lens).
Photophysical, Photochemical and Photobiological
NUS Presentation Title 2001

Characterization of Methylene Blue Formulations for


Light Activated Root Canal Disinfection

Absorption spectra

Dimmer formation
Photophysical
Fluorescence spectra

MB dissolved in different formulations NATA oxidation

Absorption spectra
Photochemical Singlet oxygen yield

Penetration into dentinal


tubules
Water, Glycerol PEG MIX
Photobiological MB uptake by bacteria

Cytotoxicity to fibroblast
cell line

Molecular mechanism of
action

Disinfection potential on
biofilm bacteria
Photo physical
NUS Presentation Title 2001characteristics of MB in different media

Monomer:Dimer ratio
Absorption Spectra
3.5
Water
monomer peak 2.5
3 Glycerol

Dimer peak
PEG PEG
MIX MIX
2.5
Glycerol 2

Monomer/Dimer
2
Water
Absorbance

1.5
1.5

1 1

0.5
0.5

0
-0.5
0 20 40 60 80 100
Concentration (uM)
Fluorescent intensity @ 686nm
W
1000
G The photophysical characteristics
PEG
800 MIX revealed that water is not a good
Fluorescence Intensity

600 medium for light activated disinfection


400 using MB. Aggregation of MB molecules
200 was evident when dissolved in water.
0
1 5 10 15 20 25
Concentration of MB (m M)
Photochemical
NUS Presentation Title 2001characteristics of MB in different media

Model substrate (NATA ) oxidation DPBF oxidation (singlet oxygen measurement)


120
12
Water k- 0.31(±0.05)

10 100 Glycerol k- 0.37(±0.07)


Concentration of NATA ( m M)

PEG k- 0.29(±0.01)

DPBF Concentration (m M)
8 80 MIX k- 0.90(±0.03)

6 Water k- 0.19 (±0.09)


60

Glycerol k- 0.004 (±0.003)


4 40
PEG k- 0.021(±0.02)

k- 0.29(±0.04)
2 MIX 20

0 0
0 5 10 15 20 0 5 10 15 20

Time in minutes
Time in minutes

The photochemical characteristics revealed that MIX is the best medium in terms
of model substrate oxidation and singlet oxygen production.
Photobiological
NUS Presentation Title 2001 characteristics of MB in different media

Extent of MB penetration across the dentinal tubules Water Glycerol PEG MIX

1 2 3
Coronal
Sections

90
80 Coronal region
Middle region
70 Apical region Middle
60 Sections
% Diffusion

50
40
30
20
10
0
Water Glycerol PEG MIX

MIX based MB formulation showed maximum Apical


Sections
penetration into the dentinal tubules in all the
tested regions of root canal.
Photobiological
NUS Presentation Title 2001 characteristics of MB…..(dye uptake)

Dye uptake by bacteria The effect of divalent cations and EDTA on MB


uptake by E.faecalis cells
90 1.2

80
1
70 E. faecalis
A.actinomycetumcomitans
0.8
60

Absorbance at 664 nm
% Dye Uptake

50 CaCl2
0.6
MgCl2
40 EDTA
0.4
30

20 0.2

10
0
0 0 mM 6.25 mM 12.5 mM 25 mM 50 mM
Water Glycerol PEG MIX
The treatment of E. faecalis cells with divalent cations
The graph shows the percentage of MB taken up from
decreased the uptake of MB (50uM). 75% reduction in MB
100µM of original MB formulation by bacterial 108-
uptake if the cells are subjected to 50mM of CaCl2
109cells. There was significant variation in uptake of
photosensitizer by bacterial cells when applied in
different formulations. Except for water based Since the endodontic environment is rich in divalent cations
formulation E. faecalis was found to have higher MB higher MB concentrations should be used to achieve
uptake (gram positive bacteria) compared to A. reasonable dye uptake by bacteria.
actinomycetemcomitans (gram negative) (p<0.05).
Error bars show the standard deviation from average
value.
Photobiological
NUS Presentation Title 2001 characteristics of MB…..(cytotoxicity)

Cytotoxicity of LAT Vs Sodium hypochlorite


Formulation effect on cytotoxicity of
120
LAT With Light
Irradiation using optical fiber

100 Without Light Diode


Tooth structure
Laser
Tissue culture plate (Lid)
80 Meniscus of test solution
% Cell Survival

Cell Line

60 AA BB The MTT staining pattern of


cell line underlying the root
40
canal of tooth subjected to
20
(A) Sodium hypochlorite and
(B) light activation of MB.
0 The cells subjected to
Water Glycerol PEG MIX Hypo sodium-hypochlorite showed
dye uptake and disrupted
C D cell morphology which was
Cytotoxicity of LAT Vs Antimicrobial relatively less in cells
subjected to LAT
Activity
100

80
y= 95.939e-0.0764x
% Cell survival

60
E. faecalis
Fibroblast The percentage survival of E. faecalis and fibroblast cells
40 subjected to simultaneous treatment with increasing
irradiation of MB in MIX. The dose required for complete
y= 137.48e-1.0088x elimination of E. faecalis showed only 36% fibroblast
20
destruction.

0
1 min 5 min 10 min 20 min
Time
Photobiological
NUS Presentation Title 2001characteristics …..(mechanism of action)

Antimicrobial effect of LAT MB in water vs. MB in MIX Effect on membrane integrity


Water
10
9 Without Light
With Light

Ratio of intact to damaged cells


8
7
6
5
MIX
4
3
2
MB when dissolved in MIX produced significantly higher 1
bacterial killing compared to MB dissolved in water
(p<0.05). 0
MB in Water MB in MIX
The ratio of fluorescence intensity at 530/630 measured as an index of
membrane damage after staining with BacLight. The difference
DNA damage between the ratio was significant only in MIX based MB formulation
p<0.001).
Marker Con WL- ML- WL+ ML+ Membrane protein damage
Mark Con L+ WL- M L- WL+ M L+

The intensity of DNA band was reduced The total membrane protein profile of E.
upon treatment with MB dissolved in faecalis subjected to LAT using MB
MIX formulation even without irradiation dissolved in different solvent systems.
(lane 4). The extensive DNA damage on The intensity of protein band was
irradiation is evident from lane 6 reduced upon treatment with MB
showing a faint band. The intensities of dissolved in both water and MIX
band is given in brackets. formulation.
WL-- MB dissolved in water, ML--MB
dissolved in MIX, WL+- MB in water
irradiated, ML+- MB in MIX irradiated.
Photobiological characteristics …..(Disinfection potential)
NUS Presentation Title 2001

Preparation of tooth specimen

1 2 3

Control specimen with Photosensitization (MB) and


Incubation with bacterial culture
undisrupted bacterial biofilm irradiation (diode laser, 30 mW)
to produce biofilm at root canal
wall the root canal

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
Photobiological
NUS Presentation Title 2001 characteristics …..(Disinfection potential)

Bactericidal action of LAT on biofilm grown in tooth blocks


Bactericidal action of LAT on biofilm grown in multiwell plate
9 9
E. faecalis
E. faecalis
8 A. actinomycetemcomitans 8
A. actinomycetemcomitans
Log number of bacteria surviving

7 7

Log number of bacteria surviving


6 6

5 5

4 4

3 3

2 2

1 1

0 0
Control Laser alone Water Glycerol PEG MIX

MB in MIX showed maximum bacterial reduction


Light alone or media alone had no significant bacterial reduction (multi well
plate)
NUS Presentation Title 2001

Conclusions
The photochemical assays showed that MIX based formulation had a better
photooxidation potential.
The MB diffusion into dentinal tubules and uptake by bacterial cells also revealed the
competence of MIX based formulation.
The improvement of photophysical and photochemical characteristics of MB in the
MIX formulation, enhanced the bactericidal property of LAT on biofilm bacteria.
MIX based MB formulation could achieve better bacterial elimination from biofilms of
gram negative (A. actinomycetemcomitans) and gram positive (E. faecalis) bacteria.
LAT causes destruction of the functionally intact membrane DNA and membrane
proteins of E. faecalis cells. The extents of damage at these sites were highly
influenced by the photosensitizer formulation. MIX based MB formulation amplified
the deleterious effect of LAT on E. faecalis cells.
MIX based photosensitizer formulation was comparatively less cytotoxic to fibroblast
cells. The cytotoxicity of NaOCl was significantly higher than that due to LAT.
These experiment in this study, indicated the potential advantages of using ANILAD
to disinfect root canal system.

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