Biofilm-pdt (research Attachment)

Eradicating Biofilm Bacteria Using Antimicrobial Photodynamic Therapy

Dentine Biophotonics Group

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)

NUS Presentation Title 2001 Microbiological 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.)

NUS Presentation Title 2001 Biofilm 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)

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

Biofilm-Structure & NUS Presentation Title 2001 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, Extra Protein expressions.

Phenotypic variation. Coaggregation and Coadhesion of planktonic cells

Planktonic cells Formation of elevated mushroom like structures of bacterial cells

Disperse bacterial to the fluid

Modification by Mineral accumulation

cells bulk Mature biofilm with new bacterial cells emerging

Microbial Interactions NUS Presentation Title 2001

Coaggregation and Coadhession

Nutrient exchange and sharing

Tolerance to extremes of pH, salinity, Nutrients and Antibiotics

Biofilm

Metabolic

Physical

Genetic

Increased rate of Specific gene transformation Gene transfer activation across MOs

Facilitate gene transfer

Determine Spatia relationship

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.

NUS Presentation Title 2001

Microscopic examinations of Biofilms

Atomic Force Microscopy

Fluorescent microscope

Polarization microscope

Biofilm

Light microscope

Scanning Electron microscope

Laser Confocal Scanning microscope

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 1st step in developing a antibiofilm regime

NUS Presentation Title 2001 Sites of Antimicrobial Resistance

Matrix of the biofilm Constituents of Matrix Enzymes present in Matrix Metabolic and genetic alteration of bacteria

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

NUS Presentation Title 2001

Limited Diffusion through Matrix Glycocalyx Ionic interaction Sieving effect Increased viscosity

Enzyme Mediated resistance

Neutralization of antimicrobials a. Constituents of Glycocalyx eg neutralization of I2 b. Act as an ion exchange resin

Metabolic state of bacteria

a. Reduction of cations to Metals

a. Slow rate of growth

b. Action of detoxifying enzymes

b. Active metabolism of antibiotics c. Altered protein profile d. Multi drug efflux pump e. Gene transfer

NUS Presentation Title 2001

Limited Diffusion through Matrix a. Glycocalyx b. Ionic interaction ls a bi o c. Sieving effect cr i im t n Increased viscosity Ad.

NUS Presentation Title 2001

a bi

ls

o Enzyme Mediatedicrresistance

im t a. Reduction of An cations to Metals

b. Action of detoxifying enzymes

NUS Presentation Title 2001

Neutralization of antimicrobials

ls a bi a. Constituents of Glycocalyx eg o r ic I neutralizationimof 2 t An b. Act as an ion exchange resin

NUS Presentation Title 2001

Metabolic state of bacteria a. Slow rate of growth b. Active metabolism of antibiotics ls a c. Altered proteinbiprofile o r c i d. Multi drugtimefflux pump An 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 pulchraAustralian 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

In summary current RCT  Cannot attain disinfection of anatomically complicated root canals  Cannot reach microorganisms penetrating deep into the dentinal tubules  Is not effective in eliminating Bacterial biofilms from peri apex  Use of caustic irrigants degrades the dentine and is also cytotoxic  The expense of treatment and economic loss due to failure of treatment counts to billions

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

c

cc c c Sensitized microbial cells

Damaged cell

c

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

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)

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

Mechanism…. Presentation Title 2001 TypeNUS 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. 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.

Presentation Title 2001 TheNUSproduction of 1O2

(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

O2

1

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+)

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

NUS Presentation Title 2001 Type of bacteria- Susceptibility to Photodynamic Therapy

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 infection

NUS Presentation Title 2001

 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

Potential Problems of Applying LAT in Endodontic Environment

NUS Presentation Title 2001

Reduced oxygen tension

Light Scattering

Limited Photosensitizer (PS) diffusion-Dentine& Bacterial cells

Use of Oxygen Carrier

Use of Surfactant based media and Poly amino acid or antibody conjugated Photosensitizer

Refractive index matching liquid

Successful LAT in Root Canal Disinfection

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 • Limitation in dye diffusion across the dentine and

formulation  Use of penetration enhancers

apical region • Light propagation through the dentine

 Use of refractive index 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)

NUS Presentation Title 2001

Perfluorcarbons 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

Solubility of oxygen in Perfluorocarbons

High densities, viscosities and gas solubility

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. propionicum A. israelii P. alactolyticus.

micros

P.

 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

Tooth specimens prepared by removing crown and root tip

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

Cleaned and sterilized Inoculated with Enterococcus faecalis

Split open longitudinally observed with Scanning Electron Microscopy

Characterization of Matured Biofilm Formed at Root Canal Wall

NUS Presentation Title 2001

24 human teeth (Single rooted) Tooth specimens prepared by removing crown and root tip Cleaned and sterilized Inoculated with Enterococcus faecalis

Incubation at 370C for 16 weeks

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

Further characterization of biofilm for Mineralization

NUS Presentation Title 2001

FTIR and XRD of Biofilm 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

Von-Kossa Staining of Biofilm 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

Biofilm Dynamics-Morphology (Scanning Electron Microscopy)

NUS Presentation Title 2001

A

B

C

D

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

A

B

C

D

Biofilm development at the root canal wall under 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

Polarisation Microscopy

Internal Architecture

Digital microscopy

Scanning Electron Microscopy

Cell Distribution

EDX Microanalysis Atomic percentage

20 16 12

Ca P

8

Ca/P

4 0 Dentine

Group2

Group4

Different Groups

LCSM

Fluorescence microscope Dentine

Nutrient rich

Nutrient deprived

NUS Presentation Title 2001

Mineralization potential of E. faecalis 6 weeks

3 weeks 2 weeks Control

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.

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

(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 Control

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-

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

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 Characterization of Methylene Blue Formulations for Light Activated Root Canal Disinfection

NUS Presentation Title 2001

Absorption spectra

Photophysical

Dimmer formation Fluorescence spectra

MB dissolved in different formulations

NATA oxidation

Photochemical

Water,

Glycerol

PEG

Absorption spectra

Singlet oxygen yield

Penetration into dentinal tubules

MIX

Photobiological

MB uptake by bacteria Cytotoxicity to fibroblast cell line Molecular mechanism of action Disinfection potential on biofilm bacteria

Optimization of LAT components for root canal disinfection NUS Presentation Title 2001 Water Glycerol PEG MIX

2.5

120

90 Waterk-

100

0.31(±0.05)

80

Glycerol

k- 0.37(±0.07)

2 80

MIX

k- 0.29(±0.01) k- 0.90(±0.03)

60

40

0.5

% Dye Uptake

DPBF Concentration (µM)

1

60 50 40 30 20

20

10

0 0

20

Water Glycerol

40

60

80

0

100

0

PEG MIX Concentration (uM)

5

10

Time in minutes

15

20

9 Coronal Sections

E. faecalis

8

A. actinomycetemcomitans

7

Middle Sections

Log number of bacteria surviving

Monomer/Dimer

1.5

E. faecalis A.actinomycetumcomitans

70

PEG

6 5 4 3 2 1

Apical Sections

0 Control Laser alone

Water

Glycerol

PEG

MIX

0 Water

Glycerol

PEG

MIX

NUS Presentation Title 2001characteristics of MB in different media Photo physical Monomer:Dimer ratio

Absorption Spectra 3.5

monomer peak

Dimer peak

2.5

PEG MIX Glycerol

Absorbance

2

2 Monomer/Dimer

3

Water Glycerol PEG MIX

2.5

Water

1.5 1

1.5

1

0.5 0.5

W av el

-0.5

34 9. 5 39 9. 5 44 9. 5 49 9. 5 54 9. 5 59 9. 5 64 9. 5 69 9. 5 74 9. 5 79 9. 5 84 9. 5 89 9. 5

en gt h

nm

.

0

0

Fluorescent intensity @ 686nm W G PEG MIX

800 600 400 200 0 1

5

10

15

Concentration of MB (µM)

20

20

40

60

80

100

Concentration (uM)

1000

Fluorescence Intensity

0

25

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.

NUS Presentation Title 2001characteristics of MB in different media Photochemical

Model substrate (NATA ) oxidation

DPBF oxidation (singlet oxygen measurement) 120

12

100

10

k- 0.37(±0.07) Glycerol

PEG k- 0.29(±0.01) DPBF Concentration (µM)

Concentration of NATA ( µ M)

k- 0.31(±0.05) Water

8 6

Water

4

k- 0.004 (±0.003) Glycerol k- 0.021(±0.02)

PEG

2

k- 0.19 (±0.09)

80

MIXk- 0.90(±0.03)

60

40

k- 0.29(±0.04)

MIX

20

0

0

0

5

10

15

20

0

5 Time in minutes10

15

20

Time in minutes

The photochemical characteristics revealed that MIX is the best medium in terms of model substrate oxidation and singlet oxygen production.

NUS Presentation Title 2001 characteristics of MB in different media Photobiological

Extent of MB penetration across the dentinal tubules

1

2

3

Water

Glycerol

PEG

MIX

Coronal Sections

90 Coronal region Middle region Apical region

80

% Diffusion

70

Middle Sections

60 50 40 30 20 10 0 Water

Glycerol

PEG

MIX

MIX based MB formulation showed maximum penetration into the dentinal tubules in all the tested regions of root canal.

Apical Sections

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

Dye uptake by bacteria

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

90 80

% Dye Uptake

70 60

E. faecalis A.actinomycetumcomitans

50

1.2

40 30 20 10 0 Water Glycerol PEG MIX The graph shows the percentage of MB taken up from 100µM of original MB formulation by bacterial 108109cells. 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.

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

1

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

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

Cytotoxicity of LAT Vs Sodium hypochlorite

Formulation effect on cytotoxicity of LAT

120 100

Tooth structure Tissue culture plate (Lid) Meniscus of test solution Cell Line

Diode Laser

80 60

AA

BB

C

D

40 20 0 Water

Glycerol

PEG

MIX

Hypo

Cytotoxicity of LAT Vs Antimicrobial Activity 100 80 % Cell survival

% Cell Survival

Irradiation using optical fiber

With Light Without Light

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

y= 95.939e-0.0764x

60

E. faec alis

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

Fibroblast subjected

40 y= 137.48e-1.0088x

20 0 1 min

Time

5 min

10 min

20 min

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

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

Effect on membrane integrity

Water

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

Ratio of intact to damaged cells

10 9

Without Light

8

With Light

7 6 5

MIX

4 3 2 1 0

DNA damage

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

Membrane protein damage

Marker Con WL- ML- WL+ ML+

Mark Con L+

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.

WL- M L- WL+ M L+

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.

Photobiological characteristics …..(Disinfection potential) NUS Presentation Title 2001 Preparation of tooth specimen 1

Control specimen with undisrupted bacterial biofilm

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

2

3

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

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

Bactericidal action of LAT on biofilm grown in tooth blocks 9 E. faecalis

8

A. actinomycetemcomitans

Log number of bacteria surviving

7 6 5 4 3 2

7 6 5 4 3 2 1

0

0

La l se ra lo ne W ate rL W ate rL G + ly ce ro lL G ly ce ro lL + PE G LPE G L+ M IX LM IX L+

1

Co nt ro

Log number of bacteria surviving

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

Control

Laser alone

Water

Glycerol

PEG

MB in MIX showed maximum bacterial reduction Light alone or media alone had no significant bacterial reduction (multi well plate)

MIX

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.