Biofilm-PDT (research attachment)

Targeting Endodontic Targeting Endodontic BiofilmBiofilm Bacteria Using Bacteria Using

Antimicrobial Photodynamic Antimicrobial Photodynamic TherapyTherapy

NUS Presentation Title 2001Outline of presentationOutline of presentation

Conventional endodontic therapyConventional endodontic therapy

Limitations of conventional endodontic therapyLimitations of conventional endodontic therapy

Microbial factors associated with failure of Microbial factors associated with failure of

endodontic therapy-Biofilm bacteriaendodontic therapy-Biofilm bacteria

Photodynamic therapy-Principle of actionPhotodynamic therapy-Principle of action

Application of Photodynamic therapy for root Application of Photodynamic therapy for root

canal disinfectioncanal disinfection

NUS Presentation Title 2001IntroductionIntroduction Endodontic infection

Endodontic therapy

Mechanical cleaning Use of Chemical Irrigants

Chemical irrigants

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 irrigantsCytotoxicity 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)

Removing the tissue to get rid of bacteria- effect on tissue structure and function

NUS Presentation Title 2001

Adapted from www.drcav.com/ images/tooth1.gif

Dentinal tubules

Root apex

Microbiological factors associated with failure of endodontic therapy

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

2. Biofilm mode of bacterial growth

Root canal lumen

Community of bacteriaHigh resistance to antimicrobial agentsOften associated with chronic infectionsMany clinical reports high light the presence of biofilm in persistent infectionsConventional 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.)

NUS Presentation Title 2001Biofilm and endodontic infectionsBiofilm and endodontic infections

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)


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 Human infections Involving biofilmbiofilm

NUS Presentation Title 2001What is a Biofilm?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.


Biofilm Biofilm Vs Vs Planktonic Planktonic

Community theory of Infection

Co operating community of various types of microorganism

Micro organisms arranged in micro colonies

Covering by matrix of ‘glycocalyx’or ‘slime’

Gradients of pH, Nutrients, and Oxygen tension

Quorum sensing-communication - ‘pheromones’

Increased resistance to antimicrobialsFluid channels in matrix

Germ theory of Infection

No community of Microorganisms

Free Living microbial cells

No glycocalyx production

No gradients of Nutrients, pH andOxygen

Less resistance to Antibiotics


How relevant is biofilm…..How relevant is biofilm…..

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

Scientists thought most of the Scientists thought most of the bacterial world was made up bacterial world was made up

of free-floating bacteriaof free-floating bacteria. They developed antibiotics and . They developed antibiotics and

vaccines using bacteria floating in a test tube. In many vaccines using bacteria floating in a test tube. In many

cases, the cases, the medicines just didn't workmedicines just didn't work —prostatitis, middle —prostatitis, middle

ear infections in children and periodontal disease to name a ear infections in children and periodontal disease to name a

few. As it turns out, scientists were few. As it turns out, scientists were targeting the wrong kind targeting the wrong kind

of bacteriaof bacteria”.”.

Kate Dalke… Genome News Net workKate Dalke… Genome News Net work


Biofilm-Structure & ComponentsBiofilm-Structure & Components

Solid Substratum

Fluid Phase

Micro Organisms

Micro Colonies

Fluid Channels

Protective Matrix

Together we stand, individual we fall…….


Stages of Biofilm FormationStages of Biofilm Formation

EPS production, Extra Protein expressions.

Modification by Mineral accumulation

Planktonic cells

Coaggregation and Coadhesion of planktonic

cells

Formation of elevated mushroom like structures of bacterial cells

Mature biofilm with new bacterial cells emerging

Phenotypic variation.

Disperse bacterial cells to the bulk fluid

NUS Presentation Title 2001Microbial InteractionsMicrobial Interactions

Bio

film

Physical

Genetic

Metabolic

Coaggregation and Coadhession

Facilitate gene transfer

Determine Spatial relationship

Specific gene activationGene transfer

across MOs

Increased rate of transformation

Tolerance to extremes of pH, salinity, Nutrients and Antibiotics

Nutrient exchange and sharing


Macromolecules

Ionic entities

Bacteria

Fluid phase

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.

Factors affecting biofilm formation on a solid surface

NUS Presentation Title 2001Resistance mechanisms in BFResistance mechanisms in BF

Bacteria in BF are resistant to Bacteria in BF are resistant to

AntibioticsAntibiotics

HeatHeat

Quaternary ammonium compoundsQuaternary ammonium compounds

Iodine, Chlorine etcIodine, Chlorine etc

Understanding various stages and method of Understanding various stages and method of

bacterial resistance mechanisms to antimicrobials bacterial resistance mechanisms to antimicrobials

forms the 1forms the 1stst step in developing a antibiofilm step in developing a antibiofilm

regimeregime


Matrix of the biofilm

Enzymes present in Matrix

Constituents of Matrix

Metabolic and genetic alteration of bacteria

Sites of Antimicrobial Resistance

Limited Diffusion through Matrix

GlycocalyxIonic interactionSieving effectIncreased viscosity

Neutralization of antimicrobials

a. Constituents of Glycocalyx eg neutralization of I2

b. Act as an ion exchange resin

Enzyme Mediated resistance

a. Reduction of cations to Metals

b. Action of detoxifying enzymes

Metabolic state of bacteria

a. Slow rate of growth

b. Active metabolism of antibiotics

c. Altered protein profile

d. Multi drug efflux pump

e. Gene transfer


New treatment New treatment conceptsconcepts


Anti biofilm coatingsAnti biofilm coatings

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

6622).

Possibility of using furanones as anti bacterial coating on Possibility of using furanones as anti bacterial coating on

biomaterials biomaterials

Furanones are the compounds isolated from sea weeds Furanones are the compounds isolated from sea weeds

((Delisea pulchra-Australian red algae)

Prevents Prevents Staphylococcus epidermisStaphylococcus epidermis adhesion and slime adhesion and slime

production on biomaterialproduction on biomaterial

Furanones target the quorum sensing agentsFuranones target the quorum sensing agents..


Surface modificationSurface modificationModify the solid surfaces to prevent bacterial adhesion. Eg using antibacterial nano particles

Replacement therapyReplacement therapyReplace potential pathogenic micro-organisms with genetically modified organisms that are less virulent

ImmunizationImmunizationThe aim is to inhibit adhesion or reduce the virulence of putative microbial etiologic agents.


Use of laser irradiation- Use of laser irradiation- Asta Richter et alAsta Richter et al

Pulsed nitrogen laser to the in vitro cultivated biofilmPulsed nitrogen laser to the in vitro cultivated biofilm

Damage to the surface substrate at higher power of laserDamage to the surface substrate at higher power of laser

Removing efficiency depends on the surface substrateRemoving efficiency depends on the surface substrate

matrix enhances the susceptibility to photodamage as matrix enhances the susceptibility to photodamage as

seen inseen in P. aeroginosa P. aeroginosa


Using photosensitizing agents- (Mark Wainwright)Using photosensitizing agents- (Mark Wainwright)

Photosensitizers based on Phenothiazinim chromophores have Photosensitizers based on Phenothiazinim chromophores have

broad spectrum antimicrobial action- broad spectrum antimicrobial action- suitable for eliminating suitable for eliminating

microbial communitymicrobial community

Least resistance to singlet oxygen by micro organisms unlike to Least resistance to singlet oxygen by micro organisms unlike to

antibioticsantibiotics- appropriate for treating biofilms since the indwellers are appropriate for treating biofilms since the indwellers are

resistant to antimicrobialsresistant to antimicrobials

Photosensitization can even cause Photosensitization can even cause EPS breakdownEPS breakdown


Involve the killing of microorganisms when a photosensitizer selectively accumulated in the target is activated by a visible light of appropriate wavelength.

Photodynamic therapy/ Light Activated TherapyPhotodynamic therapy/ Light Activated Therapy

cc c

c

c

c

Sensitized microbial cells Damaged cell Cell destruction

Light Irradiation

NUS Presentation Title 2001Mechanism of PhotosensitizationMechanism of PhotosensitizationPhotosensitizer +Light

The ability of a photosensitizer depends on the proportion

undergoing inter system crossing .

Highly fluorescent compound dissipating energy as fluorescence

will be less efficient.

Aromatic compoudns with π system makes long lived triplet state(Journal of Antimicrobial Chemotherapy (1998) 42, 13–28)

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 determined by environment and structure..

Emission of photons as fluorescence to restore the ground state/Inter system crossing with a spin flip…Triplet state ..higher half life


BiomoleculesO2

O**

PS

PS**

LIGHT

PS

BiomoleculesPS

PS**

LIGHT

PS

Type 1 mechanism

Type 2 mechanism (Photodynamic effect)

Type I - The pathway 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.

Mechanism….Mechanism….


Singlet OxygenSinglet OxygenThe 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.

NUS Presentation Title 2001The production of The production of 11OO22

(A) Absorption of light by the

photosensitiser

(B) Formation of the photosensitiser

triplet state; the quantum yield of

this process is the ISC efficiency or

triplet yield (FT)

(C) Trapping of the triplet state by

molecular oxygen within its

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

1O2

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+)


Singlet oxygen quantum yieldSinglet oxygen quantum yieldDefined as the number of molecules of Defined as the number of molecules of 1OO2 molecules generated for molecules generated foreach photon absorbed by a photosensitizer. each photon absorbed by a photosensitizer. Quantum efficiencyQuantum efficiency is an is anequivalent term.equivalent term.

Detection and Measurement of Singlet Oxygen Detection and Measurement of Singlet Oxygen Singlet oxygen luminescenceSinglet oxygen luminescence:- :- Based on the 1269 nm luminescence emitted in the Based on the 1269 nm luminescence emitted in the

radiative decay of O2(radiative decay of O2(1ΔΔgg++) ) Electron paramagnetic resonanceElectron paramagnetic resonance:- :- Energy transfer between the intrinsic magnetism of Energy transfer between the intrinsic magnetism of

unpaired electrons and an external magnetic field is measured with a sensitive unpaired electrons and an external magnetic field is measured with a sensitive microwave detection system microwave detection system

Photochemical reactions-Photochemical reactions- indirect measurement indirect measurement

NATA oxidation- The oxidation of tryptophanyl moiety is measured NATA oxidation- The oxidation of tryptophanyl moiety is measured fluorimetricallyfluorimetrically

DPBF oxidation- Measured spectrophotometricallyDPBF oxidation- Measured spectrophotometrically


Singlet oxygen and bacteriaSinglet oxygen and bacteria

Both gram positive and gram negative were killed on exposure to singlet oxygenKilling curves for gram negatives were indicative of multihit killing, whereas curves for

gram positive exhibited single-hit kineticsDirect action of singlet oxygen on gram positive Secondary radicals production from the LPS of gram negative

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


Gram negative cell surfaceGram negative cell surface

Outer membrane-reason for resistance

Check entry of the chemicals into the cells

Cations bind together the anionic LPS

Type of bacteria- Susceptibility to Photodynamic Therapy

Gram positive cell wallGram positive cell wall

Polysaccharides

LPS(Outer membrane

Glycan layer (GlcNAc & MurNAc)

Cell membrane

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


Antimicrobial Photodynamic Therapy Antimicrobial Photodynamic Therapy for Root Canal Disinfectionfor Root Canal Disinfection

Concept of APDT in Root Canal Disinfection

Infected tooth Access cavity Sensitization Light treatment Restored tooth


Anaerobic environment Anaerobic environment (Need of oxygen carrier)(Need of oxygen carrier)

Bacterial population Bacterial population

(Dye uptake)(Dye uptake)

Tissue penetration Tissue penetration

(Formulation)(Formulation)

Light Scattering Light Scattering

(refractive index (refractive index matching liquid)matching liquid)

Application of PDT in Endodontics- Concerns

NUS Presentation Title 2001Ideal formulation for LAT in root canal Ideal formulation for LAT in root canal infectioninfection

Ensure enough oxygen concentrationEnsure enough oxygen concentration

Maximum triplet of the dye and molecular oxygenMaximum triplet of the dye and molecular oxygen

Maximum abs wavelength which is minimally scattered by Maximum abs wavelength which is minimally scattered by

surrounding tissuessurrounding tissues

Maximum penetration through the dentine and biofilm Maximum penetration through the dentine and biofilm

Maximum penetration into bacterial cells and killingMaximum penetration into bacterial cells and killing


Use of EnhancersUse of Enhancers• Reduced Oxygen tensionReduced Oxygen tension

• Limitation in dye uptake by Limitation in dye uptake by

bacterial cellbacterial cell

• Limitation in dye diffusion Limitation in dye diffusion

across the dentine and across the dentine and

apical regionapical region

• Light propagation through Light propagation through

the dentinethe dentine

Use of oxygen carriersUse of oxygen carriers

Cationic dye and Cationic dye and

formulationformulation

Use of penetration Use of penetration

enhancersenhancers

Use of refractive index Use of refractive index

matching liquidmatching liquid

Type of ProblemType of Problem

Oxygen Requirement


Dependence on oxygenDependence on oxygen

Lessons from PDT of cancerCancer killing is dependent on the oxygen

concentration (Henderson 1990)Improvement of tumor response by

manipulation of tumor oxygenation during

photodynamic therapy Photochem Photobiol

2002;76:197–203Oxygen requirement is depend on the fluence

rate (Fig)Under high fluence rate the rate of oxygen

consumption increases and finally oxygen get

depletedAn oxygen carrier is required for a better PDT

effect in hypoxygenic sites.

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

PDT Efficiency decreased when O2 concentration fall below 3.4% and an advanced infection presents an hypoxygenic site


PerfluorcarbonsPerfluorcarbonsNon-polar highly fluorinated compounds Non-polar highly fluorinated compounds

Strong intramolecular bonding (C-F Strong intramolecular bonding (C-F

bonds are 485 kJ/mol, that is 84 kJ/mol bonds are 485 kJ/mol, that is 84 kJ/mol

more than a regular C-H bond),more than a regular C-H bond),

Are chemically and biochemically inert .Are chemically and biochemically inert .

Properties of PFCs includeProperties of PFCs include

The low surface tensions (<20 mN m-1), The low surface tensions (<20 mN m-1),

dielectric constants and refractive indicesdielectric constants and refractive indices

High densities, viscosities and gas High densities, viscosities and gas

solubilitysolubility

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 ).

Perfluorodecalin

Solubility of oxygen in PerfluorocarbonsSolubility of oxygen in Perfluorocarbons

Bacterial Population in Bacterial Population in Endodontic InfectionEndodontic Infection

NUS Presentation Title 2001Microorganisms present in RCMicroorganisms present in RC 10 and 50 bacterial

species.

Contain both Gram

positive and gram

negative organism

Almost equal distribution Almost equal distribution

of facultative and of facultative and

obligate anaerobesobligate anaerobes

Nature of microbial flora Nature of microbial flora

depend up on the quality depend up on the quality

of the treatment receivedof the treatment received

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

NUS Presentation Title 2001 F. nucleatum Streptococcus spp. P. micros P. F. nucleatum Streptococcus spp. P. micros P.

propionicum A. israelii P. alactolyticus.propionicum A. israelii P. alactolyticus.

P. intermedia,P. nigrescens, P. gingivalis, P. endodontalis P. intermedia,P. nigrescens, P. gingivalis, P. endodontalis

(Black Pigmented Bacteria)(Black Pigmented Bacteria) C. rectus, F. alocis, C. rectus, F. alocis,

EnterococcusEnterococcus

New species identified were..New species identified were..

Prevotella tannerae, Actinomyces radicidentis, Olsenella Prevotella tannerae, Actinomyces radicidentis, Olsenella

spp., Dialister pneumosintes, Tanerella forsynthensis, spp., Dialister pneumosintes, Tanerella forsynthensis,

Treponema maltophilum, T. amylovorum, T. medium, Treponema maltophilum, T. amylovorum, T. medium, andand

T. lecithinolyticum T. lecithinolyticum ( Spirochetes).( Spirochetes).


LAT against bacteriaLAT against bacteria

PPS 2004, Michael R Hamblin


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 radicalsSuggest the use of membrane permiabilizing agents(Journal of Photochemistry and Photobiology. B Biology. 1992.14,262-265)


Experimental DesignExperimental DesignMode of bacterial growth in the root canal for a better Mode of bacterial growth in the root canal for a better understanding of their persistenceunderstanding of their persistence

Defining the components for an effective Light Defining the components for an effective Light Activated killing of microbesActivated killing of microbes

Testing on biofilms formed in root canalTesting on biofilms formed in root canal


Incubation at 370C for different time interval

(1-4 weeks), under nutrient- rich and nutrient-

deprived condition

60 human teeth (Single rooted)

Tooth specimens prepared by removing crown and root tip

Cleaned and sterilized

Inoculated with Enterococcus faecalis

Split open longitudinally observed

with Scanning Electron Microscopy

Biofilm Dynamics-Morphology (SEM)Biofilm Dynamics-Morphology (SEM)


Incubation at 370C for 16 weeks

24 human teeth (Single rooted)

Tooth specimens prepared by removing crown and root tip

Cleaned and sterilized

Inoculated with Enterococcus faecalis

Cross-sectioned and subjected to different

microscopic techniques

•SEM coupled with EDX-Microanalysis (Micro structure and Calcium

content)

•Fluorescence microscopy after Acridine Orange staining

•Gram Staining- Light Microscopy and Polarization microscopy (Light

conductance)

•BacLight LIVE/DEAD staining and observation under Laser Confocal

Scanning Microscope for cell distribution

Characterization of Matured Biofilm Characterization of Matured Biofilm Formed at Root Canal WallFormed at Root Canal Wall

NUS Presentation Title 2001Further characterization of biofilm for Mineralization

Human dentine blocks were prepared and sterilized

Incubated under different condition with Enterococcus faecalis (ATCC

29212)

Incubated for different time intervals 2-6 weeks

The mineralization potential were evaluated using advanced material characterization techniques such as

FTIR and XRD

Clean and sterile Glass slides

Enterococcus faecalis (ATCC 29212)

incubated under medium supplemented with Calcium chloride

After 1 week of incubation slides were taken and washed with deionized

water

Von-Kossa staining conducted and observed under oil immersion light

microscope

FTIR and XRD of Biofilm Von-Kossa Staining of Biofilm


A B

C D

Biofilm development at the root canal wall under nutrient-deprived condition. 1-4 weeks

A B

C D

Biofilm development at the root canal wall under nutrient-Rich condition. 1-4 weeks

Biofilm Dynamics-Morphology Biofilm Dynamics-Morphology (Scanning Electron Microscopy)(Scanning Electron Microscopy)


Different stages of biofilm formationby Enterococcus

faecalis On root canal

dentine

Dentine Biophotonics Group

NUS Presentation Title 2001Characterization of bacteria-dentine interaction

Light Conductance

Cell Distribution

Internal Architecture

Polarisation Microscopy Digital microscopy

Scanning Electron Microscopy

LCSM Fluorescence microscope0

4

8

12

16

20

Dentine Group2 Group4

Different Groups

Ato

mic

per

cen

tag

e

Ca

P

Ca/P

EDX Microanalysis

Dentine Nutrient rich Nutrient deprived


0

20

40

60

80

100

120

600110016002100260031003600

wavenumber (cm-1)

T (

%)

Control

2 weeks3 weeks6 weeks

XRD spectra of biofilm grown on dentine surface for different periods. The hump at 22.5 is decreased over time and there is an increase in the peak corresponding to apatite peak indicating the precipitation and growth of fresh layer of crystals

FTIR reflectance spectra for E. faecalis biofilm on dentine under nutrient-rich incubated for different time intervals. A systematic increase in the transmittance intensity peak at 1448, 1394 and 985cm-1 with incubation period (2, 3 and 6 weeks) corresponds to the increase in carbonate and phosphate groups on the biofilm surface.

Mineralization potential of Mineralization potential of E. faecalisE. faecalis

(A. Kishen, S. George, R. Kumar. Bacterial mediated biomineralized biofilm formation on root canal dentine-JBMR)


Mineralization potential of Mineralization potential of E. faecalisE. faecalis

Figure -FTIR reflectance spectra for E. faecalis biofilm on dentine under nutrient-rich incubated for different time intervals. A systematic increase in the transmittance intensity peak at 1448, 1394 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).

0

20

40

60

80

100

120

600110016002100260031003600

wavenumber (cm-1)

T (%

)

Control

2 weeks3 weeks6 weeks

The Von-Kossa staining of biofilm formed on glass slides by E. faecalis, in media added with CaCl3. The figure shows dark patches corresponding to mineralization

Possible reason for mineralized bacterial structure at infected root Possible reason for mineralized bacterial structure at infected root tip?tip?


DentineBiofilm

Viable cells

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).

NUS Presentation Title 2001Photophysical, Photochemical and Photobiological Characterization of Methylene Blue Formulations for

Light Activated Root Canal Disinfection

Photophysical

Water, Glycerol PEG MIX

Photochemical

Photobiological

Absorption spectra

Dimmer formation

Fluorescence spectra

Absorption spectra

NATA oxidation

Singlet oxygen yield

Penetration into dentinal tubules

MB uptake by bacteria

Cytotoxicity to fibroblast cell line

Molecular mechanism of action

Disinfection potential on biofilm bacteria

MB dissolved in different formulations


-0.5

0

0.5

1

1.5

2

2.5

3

3.5

Abs

orba

nce Water

PEG

Glycerol

MIX

0

0.5

1

1.5

2

2.5

0 20 40 60 80 100

Concentration (uM)

Mo

no

mer

/Dim

er

WaterGlycerolPEGMIX

The photophysical characteristics

revealed that water is not a good

medium for light activated disinfection

using MB. Aggregation of MB molecules

was evident when dissolved in water.

0

200

400

600

800

1000

1 5 10 15 20 25

Concentration of MB (mM)

Fluo

resc

ence

Inte

nsity

W

G

PEG

MIX

Photo physical characteristics of MB in different mediaAbsorption Spectra Monomer:Dimer ratio

Fluorescent intensity @ 686nm

Dim

er p

eak

monomer peak


The photochemical characteristics revealed that MIX is the best medium in terms

of model substrate oxidation and singlet oxygen production.

Photochemical characteristics of MB in different media

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

0

2

4

6

8

10

12

0 5 10 15 20

Time in minutes

Con

cent

ratio

n of

NA

TA

(m

M)

Water

Glycerol

PEG

MIX

k- 0.19 (±0.09)

k- 0.004 (±0.003)

k- 0.021(±0.02)

k- 0.29(±0.04)

0

20

40

60

80

100

120

0 5 10 15 20

DP

BF

Con

cent

ratio

n (m

M)

Water

Glycerol

PEG

MIX

Time in minutes

k- 0.31(±0.05)

k- 0.37(±0.07) k- 0.29(±0.01)

k- 0.90(±0.03)

NUS Presentation Title 2001Photobiological characteristics of MB in different media

Extent of MB penetration across the dentinal tubules

0

10

20

30

40

50

60

70

80

90

Water Glycerol PEG MIX

% D

iffu

sion

Coronal region

Middle region

Apical region


Coronal Sections

Middle Sections

Apical Sections

MIX based MB formulation showed maximum

penetration into the dentinal tubules in all the

tested regions of root canal.

1 2 3

NUS Presentation Title 2001Photobiological characteristics of MB…..(dye uptake)

Dye uptake by bacteria

0

10

20

30

40

50

60

70

80

90


% D

ye U

ptak

e

E. faecalis

A.actinomycetumcomitans

The treatment of E. faecalis cells with divalent cations decreased the uptake of MB (50uM). 75% reduction in MB uptake if the cells are subjected to 50mM of CaCl2

Since the endodontic environment is rich in divalent cations higher MB concentrations should be used to achieve reasonable dye uptake by bacteria.

0

0.2

0.4

0.6

0.8

1

1.2

0 mM 6.25 mM 12.5 mM 25 mM 50 mM

Abs

orba

nce

at 6

64 n

m

CaCl2

MgCl2

EDTA

The effect of divalent cations and EDTA on MB uptake by E.faecalis cells

The graph shows the percentage of MB taken up from 100µM of original MB formulation by bacterial 108-109cells. There was significant variation in uptake of photosensitizer by bacterial cells when applied in different formulations. Except for water based formulation E. faecalis was found to have higher MB uptake (gram positive bacteria) compared to A. actinomycetemcomitans (gram negative) (p<0.05). Error bars show the standard deviation from average value.

NUS Presentation Title 2001Photobiological characteristics of MB…..(cytotoxicity)

0

20

40

60

80

100

120

Water Glycerol PEG MIX Hypo

% C

ell S

urvi

val

With Light

Without Light

Formulation effect on cytotoxicity of LAT

Cytotoxicity of LAT Vs Sodium hypochlorite

Meniscus of test solution

Tooth structure

Irradiation using optical fiber

Tissue culture plate (Lid)

Cell Line

DiodeLaser

The MTT staining pattern of cell line underlying the root canal of tooth subjected to (A) Sodium hypochlorite and (B) light activation of MB. The cells subjected to sodium-hypochlorite showed dye uptake and disrupted cell morphology which was relatively less in cells subjected to LAT

A B

C D

A B

The percentage survival of E. faecalis and fibroblast cells subjected to simultaneous treatment with increasing irradiation of MB in MIX. The dose required for complete elimination of E. faecalis showed only 36% fibroblast destruction.

0

20

40

60

80

100

1 min 5 min 10 min 20 min

% C

ell s

urvi

val

E. faecalis

Fibroblast

y= 95.939e-0.0764x

y= 137.48e-1.0088x

Time

Cytotoxicity of LAT Vs Antimicrobial Activity

NUS Presentation Title 2001Photobiological characteristics …..(mechanism of action)

Antimicrobial effect of LAT MB in water vs. MB in MIX

MB when dissolved in MIX produced significantly higher bacterial killing compared to MB dissolved in water(p<0.05).

Effect on membrane integrity

The ratio of fluorescence intensity at 530/630 measured as an index of membrane damage after staining with BacLight. The difference between the ratio was significant only in MIX based MB formulation p<0.001).

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MB in Water MB in MIXR

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MIX

The intensity of DNA band was reduced upon treatment with MB dissolved in MIX formulation even without irradiation (lane 4). The extensive DNA damage on irradiation is evident from lane 6 showing a faint band. The intensities of band is given in brackets.WL-- MB dissolved in water, ML--MB dissolved in MIX, WL+- MB in water irradiated, ML+- MB in MIX irradiated.

Marker Con WL- ML- WL+ ML+

DNA damage

The total membrane protein profile of E. faecalis subjected to LAT using MB dissolved in different solvent systems. The intensity of protein band was reduced upon treatment with MB dissolved in both water and MIX formulation.

Mark Con L+ WL- M L- WL+ M L+

Membrane protein damage


Preparation of tooth specimen

Incubation with bacterial culture to produce biofilm at root canal wall the root canal

Control specimen with undisrupted bacterial biofilm

Photosensitization (MB) and irradiation (diode laser, 30 mW)

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

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Photobiological characteristics …..(Disinfection potential)Photobiological characteristics …..(Disinfection potential)

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

Bactericidal action of LAT on biofilm grown in multiwell plate

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A. actinomycetemcomitans

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Control Laser alone Water Glycerol PEG MIX

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A. actinomycetemcomitans

Bactericidal action of LAT on biofilm grown in tooth blocks

MB in MIX showed maximum bacterial reduction

Light alone or media alone had no significant bacterial reduction (multi well plate)


ConclusionsThe photochemical assays showed that MIX based formulation had a better The photochemical assays showed that MIX based formulation had a better

photooxidation potential.photooxidation potential.

The MB diffusion into dentinal tubules and uptake by bacterial cells also revealed The MB diffusion into dentinal tubules and uptake by bacterial cells also revealed

the competence of MIX based formulation.the competence of MIX based formulation.

The improvement of photophysical and photochemical characteristics of MB in the The improvement of photophysical and photochemical characteristics of MB in the

MIX formulation, enhanced the bactericidal property of LAT on biofilm bacteria. MIX formulation, enhanced the bactericidal property of LAT on biofilm bacteria.

MIX based MB formulation could achieve better bacterial elimination from biofilms of MIX based MB formulation could achieve better bacterial elimination from biofilms of

gram negative (gram negative (A. actinomycetemcomitansA. actinomycetemcomitans) and gram positive () and gram positive (E. faecalisE. faecalis) bacteria. ) bacteria.

LAT causes destruction of the functionally intact membrane DNA and membrane LAT causes destruction of the functionally intact membrane DNA and membrane

proteins of proteins of E. faecalis E. faecalis cells. The extents of damage at these sites were highly cells. The extents of damage at these sites were highly

influenced by the photosensitizer formulation. MIX based MB formulation amplified influenced by the photosensitizer formulation. MIX based MB formulation amplified

the deleterious effect of LAT on the deleterious effect of LAT on E. faecalisE. faecalis cells. cells.

MIX based photosensitizer formulation was comparatively less cytotoxic to fibroblast MIX based photosensitizer formulation was comparatively less cytotoxic to fibroblast

cells. The cytotoxicity of NaOCl was significantly higher than that due to LAT.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 These experiment in this study, indicated the potential advantages of using ANILAD

to disinfect root canal system. to disinfect root canal system.

Biofilm-PDT (research attachment)

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