Professional Documents
Culture Documents
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)
Nair et al. Oral Surg Oral Med Oral Pathol Oral Radiol Endod
2005;99:231-52
Dentinal tubules
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
Modification by
Disperse Mineral
bacterial cells accumulation
to the bulk
fluid
Mature biofilm with new bacterial
cells emerging
Microbial Interactions
NUS Presentation Title 2001
Biofilm
Physical transfer
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
Antibiotics
Heat
Constituents of Matrix
New treatment
concepts
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
Light Irradiation
cc c c
c c
Photosensitizer +Light
Light on photosensitizer
PS Biomolecules
PS
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
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
Cell membrane
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
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.
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
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
Experimental Design
Mode of bacterial growth in the root canal for a better
understanding of their persistence
Mineralization
FTIR and XRD of Biofilm Von-Kossa Staining of Biofilm
Human dentine blocks were prepared Clean and sterile Glass slides
and sterilized
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
16
Atomic percentage
12 Ca
P
8 Ca/P
80
T (%)
60
40
20
0
3600 3100 2600 2100 1600 1100 600
wavenumber (cm-1)
(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)
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
Absorption spectra
Dimmer formation
Photophysical
Fluorescence spectra
Absorption spectra
Photochemical Singlet oxygen yield
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
PEG k- 0.29(±0.01)
DPBF Concentration (m M)
8 80 MIX k- 0.90(±0.03)
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
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)
Cell Line
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)
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
1 2 3
7 7
5 5
4 4
3 3
2 2
1 1
0 0
Control Laser alone Water Glycerol PEG MIX
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.