Endodontic Biofilms and management..pptx

Contents
• Introduction
• Definitions
• Characterestics of biofilm
• Microbes in endodontic biofilm
• Stages of biofilm formation
3

Contents
• Types of endodontic biofilms
• Methods of identifying biofilms
• Methods to eradicate biofilms
• Conclusion
• References
4

Introduction
• Microoraganisms in the normal microbiota participate in many
beneficial physiological functions of the oral cavity.
5

Introduction
• In case, if the physical and biochemical conditions of these
tissues change, commensals can convert into opportunistic
pathogens.
• Microbes are the most essential agents for the development of
primary peri-redicular diseases and are most essential factors
responsible for the failure of endodontic therapy.
6

Introduction
• Hence the goal of primary endodontic therapy as well as
retreatment should be to eradicate the microbes from the root
canal system.
7

Introduction
It is not possible to eradicate the microbes completely from the
root canal system for following reasons:
• Bio-film formation inside the root canal system
• The relative antimicrobial resistance of microbes which are
continuously undergoing mutations to adapt to any situation.
• Bacterial penetration and persistence in dentinal tubules
8

Introduction
• Van Leeuwenhoek, using his simple microscopes, first
observed microorganisms on tooth surfaces and can be
credited with the discovery of microbial bio-films.
9

Types of Endodontic Infections
• There are two types of endodontic infections:
1. Intra radicular infections.
•Primary
•Secondary
•Persistent
2. Extra radicular infections.
10

Primary intra-radicular infections:
• Microorganisms that initially invade and colonize the necrotic
pulp tissue causes primary intraradicular infection.
• The involved microbiota is conspicuously dominated by Gram
negative anaerobic bacteria.
Porphyromonas
Prevotella
Fusobacterium
Campylobacter species
11

Secondary intra-radicular infections:
• Microorganisms that were not present in the primary infection
but that were introduced into the root canal system at some
time after professional intervention causes secondary
intraradicular infections.
• The entry can be during treatment, between appointments, or
even after root canal filling.
E.faecalis
Staphylococcus
E.coli
Pseudomonas aeruginosa
Candida species
12

Definitions
• Endodontic Biofilm is defined as a mode of microbial growth
where dynamic communities of interacting sessile cells are
irreversibly attached to a solid substratum, as well as each
other, and are embedded in a self-made matrix of extracellular
polymeric substances (EPS).
- Ingle’s Endodontics, 6th edition.
13

Definitions
• Biofilm can be defined as a sessile multicellular microbial
community characterized by cells that are firmly attached to a
surface and enmeshed in a self-produced matrix of
extracellular polymeric substance (EPS), usually
polysaccharide.
- Cohen, Pathways of pulp, 10th edition
14

Characteristics of biofilms
• Bacteria in a biofilm state show distinct capacity to survive
tough growth and environmental conditions.
– Biofilm structure protects the residing bacteria from
environmental threats.
– Structure of biofilm permits trapping of nutrients and metabolic
cooperativity between resident cells of same species and/or
different species.
15

• Biofilm structures display organized internal
compartmentalization, which allows bacterial species with
different growth requirements to survive in each compartment.
• Biofilms shows resistance to antimicrobial agents.
16

• Bacterial cells in a biofilm community may communicate and
exchange genetic materials to acquire new traits.
Communications between bacterial cells residing in a biofilm
is attained through the following processes:
• Quorum sensing
• Auto inducer system 2(AI -2)
17

Microbes in endodontic biofilms
• The most commonly involved bacteria are :
– E. faecalis
– Coagulase-negative staphylococcus
– Actinomyces species
– P. propionicum
• Others are P. aeuroginosa, Fungi, Fusobacterium nucleatum,
Porphromonas gingivalis, Tanerella forsythia.
18

Molecular studies have also showed the occurrence of
• Synergistes,
• Dialaster,
• Prevotella,
• Solobacterium,
• Olsenella,
• Eubacterium,
• Megaspheara, Vellionella, Selenomonas.
19

• Enterococcus faecalis is a microorganism commonly detected
in asymptomatic, persistent endodontic infections.
• Its prevalence in such infections ranges from 24% to 77%.
• Enterococci are gram positive cocci, facultative anaerobes that
can occur singly, in pairs, or as short chains.
20

• Enterococci survive very harsh environments including
extreme alkaline pH and salt concentrations.
• They can grow in the range of 10 to 45°C and survive a
temperature of 60°C for 30 min.
21

•
• E. faecalis in dentinal tubules has been shown to resist
intracanal dressings of calcium hydroxide for over 10 days.
• E. faecalis is able to form a biofilm that enable the bacteria to
become 1000 times more resistant to phagocytosis, antibodies,
and antimicrobials than nonbiofilm producing organisms
causing endodontic failure.
22

Stages of bio-filmformation
Biofilm is considered as community as it possesses following
criteria:
• Autopoiesis (must possess the abilities to self-organize)
• Haemostasis (resist environmental perturbations)
23

• Synergy (must be more effective in association than in
isolation).
• Communality (respond to environmental changes as a unit
rather than single individuals).
24

• Biofilm formation is a step-wise procedure and it requires 3
major elements:
• Microorganisms
• Solid surface
• Fluid medium
25

Step 1:
• It involves adsorption of inorganic and organic molecules, to
the solid surface which leads to the formation of conditioning
film.
26

Step 2:
• Adhesion of planktonic microbial cells to the conditioning
layer and colonization of microbes.
• Phase 1: Transport of the microbe to substrate
surface and its attachment.
• Phase 2: Initial non specific microbial and substrate
adherence phase
• Phase 3: Specific microbial–substrate adherence
phase
27

Step 3:
• Bacterial grow by co-adhesion of other organisms and biofilm
expansion.
• Monolayer of microorganisms attracts secondary colonizers to
form microcolonies by co-adhesion and co-aggregation.
28

• Step 4: Detachment of sessile microorganisms i.e. present
within biofilm into their surroundings by
• Seeding dispersal
• Clumping dispersal.
29

In vitro experiments have revealed distinct stages in the
development of E. faecalis biofilm on root canal dentine.
Stage 1:
• E. faecalis cells adheres and forms microcolonies on the root
canal dentine surface.
30

Stage 2:
• They induced bacterial-mediated dissolution of the mineral
fraction from the dentine substrate.
• This localized increase in the calcium and phosphate ions
promotes mineralization (or calcification) of the E.faecalis
biofilm.
31

• Stage 3: The mature biofilm structure formed after 6 weeks of
incubation showed signs of mineralization and subtle but distinct
compositional difference.
• The mineralized E.faecalis biofilm showed carbonated-apatite
structure as compared to natural dentine which had carbonated
fluorapatite structure.
32

Types of endodontic biofilms
Endodontic bacterial biofilms can be categorized as:
• Intra-canal biofilms
• Extra-radicular biofilms
• Peri-apical biofilms
• Biomaterial centered infections
33

Intracanal biofilms
• Intracanal microbial biofilms are microbial biofilms formed on
the root canal dentine of an endodontically infected tooth.
• A detailed description on the intracanal bacterial biofilm was
documented by Nair in 1987.
34

• Intracanal biofilms
• The intracanal microbiota existed as both loose collection and
biofilm structures, made up of cocci, rods, and filamentous
bacteria.
• Monolayer and/or multilayered bacterial biofilms were found
to adhere to the dentinal wall of the root canal.
35

• E. faecalis under nutrient-rich environment (aerobic and
anaerobic) produced typical biofilm structures with
characteristic surface aggregates of bacterial cells and water
channels.
36

• Under nutrient-deprived environment, irregular growth of
adherent cell clumps were observed.
37

Types of endodonticbiofilms
• Riccuci D et al (2010) evaluated the prevalence of bacterial
biofilms in untreated and treated root canals of teeth evincing
apical periodontitis.
• They stated that intraradicular biofilms were observed in the
apical segment of 77% of the root canals.
• Biofilms were also seen covering the walls of ramifications
and isthmuses.
• Extraradicular biofilms were observed in only 6% of the cases.
38
Ricucci D, Siqueira JF, Biofilms and apical periodontitis: study of prevalence and association with clinical and
histopathologic findings. J Endod. 2010 Aug;36(8):1277-88.

Extra-radicular biofilms
• Extraradicular microbial biofilms are formed on the root
(cementum) surface adjacent to the root apex of
endodontically infected teeth.
• They are also termed root surface biofilms.
39

• Extraradicular biofilms were reported in teeth with
– Asymptomatic periapical periodontitis
– Chronic apical abscesses associated with sinus tracts.
• Mature bacterial biofilms were found in many areas of the
apical root surfaces in all clinical specimens.
40

• The extraradicular biofilm structures were dominated by cocci
and short rods, with cocci attached to the tooth substrate.
• Filamentous and fibrillar forms were also observed in the
biofilm.
41

• Harn et al.., noticed calculus-like deposits on apical root
surface of tooth presented with lesion refractory to
conventional root canal treatment.
• These calcified biofilms were associated with periapical
inflammation and delayed periapical healing in spite of
adequate orthograde root canal treatment.
42

Periapical biofilms
• Periapical microbial biofilms are isolated biofilms found in the
periapical region of an endodontically infected teeth.
• Periapical biofilms may or may not be dependent on the root
canal.
43

• Members of the genus Actinomyces and the species P.
propionicum are seen in asymptomatic periapical lesions
refractory to endodontic treatment.
• These microorganisms have the ability to overcome host
defense mechanisms, thrive in the inflamed periapical tissue,
and subsequently induce a periapical infection.
44

• Actinomyces species in tissues grow in aggregates, up to a
diameter of 3 to 4mm.
• They are commonly referred to as ‘‘sulfur granules,’’ because
of the yellow granular appearance.
45

• Wang J et al (2012) investigated the bacterial flora and type
of film formed in persistent peri-radicular infections.
• Apical root samples from root-end surgery were collected
from 23 root-filled teeth with apical periodontitis.
• Samples were observed by SEM analysis.
Wang J, Jiang Y, Chen W, Zhu C, Liang J. Bacterial flora and extraradicular biofilm associated with
the apical segment of teeth with post-treatment apical periodontitis. J Endod. 2012 Jul;38(7):954-9.
46

• Extraradicular biofilm was present on the external root surface
of treated teeth with persistent periapical lesions.
• Actinomyces species and Propionibacterium are likely
important contributors to extraradicular biofilm formation and
persistent periapical infection.
47
Wang J, Jiang Y, Chen W, Zhu C, Liang J. Bacterial flora and extraradicular biofilm associated with
the apical segment of teeth with post-treatment apical periodontitis. J Endod. 2012 Jul;38(7):954-9.

Biomaterial-centered infection
• Biomaterial-centred infection (BCI) is caused when bacteria
adheres to an artificial biomaterial surface and forms biofilm
structures.
• BCI is one of the major complications associated with
prosthesis and/or implant-related infections.
48

• In endodontics, biomaterial-centered biofilms would form on
root canal obturating materials.
• These biofilms can be intraradicular or extraradicular
depending upon whether the obturating material is within the
root canal space or has it extruded beyond the root apex.
49

• BCI usually reveals opportunistic invasion by nosocomial
organisms.
• Coagulase-negative Staphylococcus, S. aureus, enterococci,
streptococci, P. aeruginosa, and fungi are commonly isolated
from infected biomaterial surfaces.
50

• Because biofilms are extremely resistant to host defense
mechanisms and antibiotic treatments.
• BCI are rarely resolved, and often the only solution to an
infected biomaterial such as obturating material is its surgical
removal.
51

• Takemura N et al (2004) reported the biofilm formation ability
in teeth with over filled guttapercha.
• They stated that Gram-positive facultative anaerobes (E.
faecalis) have the ability to colonize and form extracellular
matrices on gutta-percha points.
52

Ultra-structure of biofilm
• A glycocalyx matrix made up of extra-cellular polymeric
substances surrounds the microcolonies and anchors the
bacterial cell to the substrate.
• It is made up of 85% by volume is made up of polysacchride
matrix material, while 15% is made up of cells.
53

• A viable, fully hydrated biofilm appears as “tower” or
“mushroom” shaped structures adherent to a substrate.
54

• The water channels, which are regarded as a primitive
circulatory system in a biofilm, to establish connections
between the microcolonies.
55

• Presence of water channels facilitates efficient exchange of
materials between bacterial cells and bulk fluid, which in turn
helps to coordinate functions in a biofilm community.
56

Methods of identifying biofilms
• Traditional methods
• Recent methods
57

Traditional methods
1. Gram's stain
2. Culture methods
58

Disadvantages of Culturing Method
1. Unable to grow several microorganisms which can give
false negative results.
2. Strictly depend on mode of sample transport which must
allow growth of anaerobic bacteria.
3. Low sensitivity and specificity.
4. Time consuming.
59

Molecular Diagnostic Methods
• Molecular diagnostic methods identify the microorganisms
using gene as a target which are unique for each species.
– DNA-DNA hybridization method
– polymerase chain reaction method
60

DNA-DNA Hybridization Method
• This method uses DNA probes which target genomic DNA or
individual genes.
• This method helps in simultaneous determination of the
presence of a multitude of bacterial species in single or
multiple clinical samples.
61

• In this method, segments of labeled, single strand DNA locate
and bind to their complementary nucleic acid sequences.
• After washing, the presence of bound label indicates the
presence of the target DNA sequence.
62

• Polymerase Chain Reaction Method (PCR Method)
• PCR method involves in vitro replication of DNA, therefore it
is also called as "genetic xeroxing" method.
• Multiple copies of specific region of DNA are made by
repeated cycles or heating and cooling.
63

• PCR has remarkable sensitivity and specificity because each
distinct microbial species has unique DNA sequences.
• PCR can be used to detect virtually all bacterial species in a
sample.
• It is also used to investigate microbial diversity in a given
environment.
64

Terminal-RFLP method
• T-RFLP is a recent molecular approach that can assess genetic
differences between microbial strains as well as provide
insight into the structure and function of microbial
communities.
65

• rDNA from different species in a community is PCR amplified
using one of the PCR primers labeled with a fluorescent dyes
– 4, 7, 2′, 7′ tetrachloro-6-carboxyfluorescein (TET)
– Phosphoramidite fluorochrome 5-carboxyfluorescein (6-
FAM).
66

DNA Microarrays
• Microarray methods essentially consist of many probes that
are discretely located on a nonporous solid support, such as a
glass slide.
• Probes can be DNA fragments such as library clones or PCR
products.
• Printed arrays and high-density oligonucleotide arrays are the
most commonly used types of microarrays.
67

• Confocal Laser Scanning microscope and fluorescence in situ
hybridization are valuable tools for obtaining high resolution
images and 3-D reconstruction of a variety of biological
samples including their biofilm.
68

• The Calgary Biofilm Device (CBD) offers a new technology
for the rational selection of antibiotics, effective against
microbial biofilms and for the screening of new effective
antibiotic compounds.
69

Methods of eradicating endodontic
biofilms
70

Methods of eradicating endodontic biofilms
• Antimicrobial agents have been developed to remove the
bacteria in the biofilm.
• However, microbial communities in biofilms are remarkably
difficult to eradicate with antimicrobial agents and
microorganisms in mature biofilms can be notoriously resistant
for the following reasons
71

Mechanisms of anti-microbial resistance of biofilm
• The microorganisms grown in biofilms could be 1000-1500
times more resistant to antimicrobial agents that planktonic
bacteria.
– The nature of biofilm structure .
– Physiological characteristics of the resident
microorganisms.
72

73
•The polysaccharide matrix of biofilms can retard diffusion of antibiotics.
•Bacteria protect themselves by being located within the interior part of a
biofilm.
•Bacterial cells residing within a biofilm grow more slowly than planktonic
cells and as a result, antimicrobial agents act more slowly.
•Depletion of nutrients or accumulation of waste products can result in
bacteria entering a non-growing state

• Chemo-mechanical preparation
• It has been proved that bacteria might penetrate dentinal
tubules to depths of 200 mm or more.
• Complete uniform enlargement of a root canal by 200 mm is
not achieved with any mechanical instrumentation.
• So, a chemical irrigant is used as an adjunct to assist in
reducing the bacterial load and their toxic by-products.
74

Chemo-mechanical preparation
• All infected pulp tissue, bacteria and their by products should
be removed from the root canal.
• The root canal preparation should develop a continuously
tapering cone .
• Making the canal narrower apically and widest coronally.
• Avoid transportation of the foramen.
75

Chemo-mechanical preparation
• Keep the apical opening as small as possible.
• Procedure should be confined to the root canal space.
• Sufficient space for intracanal medicaments and irrigants
should be created.
76

Sodium hypochlorite
• Sodium hypochlorite (NaOCl) is an excellent antibacterial
agent, capable of dissolving necrotic tissue, vital pulp tissue,
and the organic components of dentin and biofilms.
• NaOCl ionizes in water into Na+2 and the hypochlorite ion,
OCl-
77

• NaOCl is commonly used in concentrations between 0.5% and
6%.
• 6% NaOCl was the only irrigant capable of both rendering
bacteria nonviable and physically removing the biofilm.
• The combined application of EDTA and NaOCl significantly
reduces the amount of intracanal biofilm.
78

• Its anti-bacterial effect is due to :
– Penetration into the bacterial cell wall.
– Chemical combination with the protoplasm of the bacteria
and disruption of DNA synthesis.
79

• Afzal A et al.., (2013) evaluated the antimicrobial efficacy of
5.25% sodium hypochlorite, 2% chlorhexidine and MTAD
against E. faecalis in the bio-film during the cleaning and
shaping of the canal system.
• 80 single-rooted mandibular premolars were taken.
• E. faecalis ATCC 29212 strain were inoculated into the teeth.
80
A Afzal, V Rajesh Gopal, Rajesh Pillai, Asha Sarah Jacob, S U-Nu, S Shan.. Antimicrobial
activity of various irrigants against E. faecalis biofilm: An in vitro study. Year : 2013 | Volume :
3 | Issue : 2 | Page : 103-108

• 5.25% Sodium hypochlorite showed the most effective
antimicrobial property among the three irrigants against E.
faecalis biofilm.
81
A Afzal, V Rajesh Gopal, Rajesh Pillai, Asha Sarah Jacob, S U-Nu, S Shan.. Antimicrobial
activity of various irrigants against E. faecalis biofilm: An in vitro study. Year : 2013 | Volume :
3 | Issue : 2 | Page : 103-108

Iodine Solutions
• Iodine in potassium iodide solution is a potential root canal
irrigant against E. faecalis.
• A 10-minute irrigant–dentine contact time on dentine infected
with E. faecalis was sufficient to prevent growth.
82

• A solution of 5% iodine in potassium iodide or Churchill’s
solution can be used.
• Churchill’s solution consists of
83
•Iodine (16.5 g)
•Potassium iodide (3.5 g)
•Distilled water (20 g)
•90% ethanol (60 g).

Chlorhexidine
• 0.2 - 2% CHX may be used as an irrigant that is effective
against E. faecalis.
• CHX permeates the microbial cell wall and attacks the
bacterial cytoplasmic or inner membrane.
• In high concentrations, CHX causes coagulation of
intracellular components.
84

• Chlorhexidine gluconate has good substantivity and has the
ability to adhere to hydroxyapatite crystals in dentine.
• Although bacteria may be killed by CHX, the biofilm and
other organic debris are not removed by it.
• So, 2% chlorhexidine is best used after irrigating with 5.25 %
NaOCL for 100% removal of endodontic biofilms
85

• Vianna et al.., (2004) investigated the antimicrobial activity of
0.2%, 1%, and 2% chlorhexidine gluconate (CHX gel and
CHX liquid), against endodontic pathogens.
• 2 % CHX gel effectively removed E.faecalis in 1 min.
• The timing required for 1.0% and 2.0% CHX liquid to
eliminate all microorganisms was the same required for 5.25%
NaOCl i.e., around 30 sec.
Vianna ME, Gomes BP, Berber VB, Zaia AA, Ferraz CC, de Souza-Filho FJ.. In vitro evaluation of
the antimicrobial activity of chlorhexidine and sodium hypochlorite.. Oral Surg Oral Med Oral Pathol
Oral Radiol Endod. 2004 Jan;97(1):79-84.
86

MTAD
• MTAD is a recently developed irrigating solution that consists
of tetracycline, citric acid and detergent (Tween 80).
• The MTAD should be used for 5 minutes as a final rinse after
hypochlorite and EDTA.
87

MTAD
• It is able to kill E. faecalis.
• It is effective for removing smear layer along the entire length
of the prepared root canal.
88

• Kamberi B et al.., (2012) assessed the antimicrobial efficacy
of Biopure MTAD against E. faecalis in contaminated root
canals.
• 42 single rooted extracted teeth were inoculated with E.
faecalis and incubated for four weeks.
Blerim Kamberi, Donika Bajrami, Miranda Stavileci, Shuhreta Omeragiq, Fatmir Dragidella, Ferit Koçan.
The Antibacterial Efficacy of Biopure MTAD in Root Canal Contaminated with Enterococcus faecalis
ISRN Dent. 2012
89

• The samples were divided experimental groups irrigated with
90
1.5% NaOCl
3% NaOCl;
BioPure MTAD;
1.5% NaOCl/17% EDTA
3% NaOCl/17% EDTA

• They stated that BioPure MTAD possesses superior
bactericidal activity compared with 1.5 % NaOCl and 17 %
EDTA against E. faecalis.
91
Blerim Kamberi, Donika Bajrami, Miranda Stavileci, Shuhreta Omeragiq, Fatmir Dragidella, Ferit Koçan.
The Antibacterial Efficacy of Biopure MTAD in Root Canal Contaminated with Enterococcus faecalis
ISRN Dent. 2012

• Mittal R et al.., (2012) evaluate and compare the antibacterial
efficiency of MTAD, Oxytetracycline, 5% NaOCl, and 2%
chlorhexidine when used as root canal irrigants against
Enterococcus faecalis.
• 50 extracted single rooted anterior teeth were selected.
Mittal R, Singla MG, Garg A, Gupta S, Dahiya V. Comparative evaluation of the antimicrobial efficacy of
MTAD, oxytetracycline, sodium hypochlorite and chlorhexidine against Enterococcus faecalis: An ex-vivo
study. Saudi Endod J 2012;2:70-4 92

• Oxytetracycline has a great potential as a root canal irrigating
agent because of its superior antimicrobial efficacy against E.
faecalis, easy availability and cost effectiveness.
93
Mittal R, Singla MG, Garg A, Gupta S, Dahiya V. Comparative evaluation of the antimicrobial efficacy of
MTAD, oxytetracycline, sodium hypochlorite and chlorhexidine against Enterococcus faecalis: An ex-vivo
study. Saudi Endod J 2012;2:70-4

Tetraclean
• Tetraclean is a newer irrigant similar to MTAD.
Composition
– Doxycycline (50 mg/5 ml)
– Citric acid
– Detergent (polypropylene glycol).
94

• Pappen FG et al..,(2010) investigated the antibacterial effect
of Tetraclean, MTAD and five experimental irrigants with
planktonic cultures and mixed-species in vitro biofilm model.
95
Pappen, F. G., et al. "In vitro antibacterial action of Tetraclean, MTAD and five experimental
irrigation solutions." International endodontic journal 43.6 (2010): 528-535.
Tetraclean
MTAD
MTAD + 0.01% cetrimide (CTR),
MTAD + 0.1% CTR,
MTAC-1 (Tween 80 replaced by 0.01% CTR in MTAD),
MTAC-2 (Tween 80 replaced by 0.1% CTR)
MTAD-D (MTAD without the Tween 80 and no CTR
added)

• Tetraclean was more effective than MTAD against E. faecalis
in both the cultures.
• CTR improved the antimicrobial properties of the solutions,
whereas Tween 80 seemed to have a neutral or negative impact
on their antimicrobial effectiveness.
Pappen, F. G., et al. "In vitro antibacterial action of Tetraclean, MTAD and five experimental
irrigation solutions." International endodontic journal 43.6 (2010): 528-535. 96

Qmix:
• It is effective in removal of smear layer containing bacteria
and necrotic tissue.
• It consists of
• EDTA
• chlorhexidine
• Detergent
97

• Stojicic S et al.., (2012) assesed the efficacy of a novel root
canal irrigant, QMiX, against E.faecalis and mixed plaque
bacteria in planktonic phase and biofilms.
Stojicic, S., Shen, Y., Qian, W., Johnson, B., & Haapasalo, M. (2012). Antibacterial and smear layer
removal ability of a novel irrigant, QMiX.International endodontic journal, 45(4), 363-37198
QMiX,
2% chlorhexidine (CHX),
MTAD
1% NaOCl for 5 s, 30 s and 3
min

• QMiX and NaOCl were superior to CHX and MTAD in killing
E.faecalis and plaque bacteria in planktonic
and biofilm culture.
99
Stojicic, S., Shen, Y., Qian, W., Johnson, B., & Haapasalo, M. (2012). Antibacterial and smear layer
removal ability of a novel irrigant, QMiX.International endodontic journal, 45(4), 363-371

• Ordinola R et al.., (2013) evaluated whether the use of
chelating agents with anti-microbial activity show similar
disinfection ability in comparison to conventional irrigants as
sodium hypochlorite or iodine potassium iodide against
biofilms developed on dentin.
Ordinola-Zapata, R., Bramante, C. M., Brandão Garcia, R., Bombarda de Andrade, F., Bernardineli, N.,
Gomes de Moraes, I., & Duarte, M. A. (2013). The antimicrobial effect of new and conventional endodontic
irrigants on intra-orally infected dentin. Acta Odontologica Scandinavica, 71(3-4), 424-431. 100
• MTAD,
• Qmix,
• Smear Clear,
• 7% maleic acid,
• 2% iodine potassium iodide,
• 4% peracetic acid,
• 2.5% and 5.25% sodium hypochlorite

101
• The irrigant solutions 4% peracetic acid and 2.5-5.25% sodium
hypochlorite decreases significantly the number of live
bacteria in biofilms, providing also cleaner dentin surfaces.
Ordinola-Zapata, R., Bramante, C. M., Brandão Garcia, R., Bombarda de Andrade, F., Bernardineli, N.,
Gomes de Moraes, I., & Duarte, M. A. (2013). The antimicrobial effect of new and conventional endodontic
irrigants on intra-orally infected dentin. Acta Odontologica Scandinavica, 71(3-4), 424-431.

• Recent advances in irrigation for effectively removing
biofilms:
102
•Ultrasonic irrigation
•Sonic irrigation
•Plasma dental probe
•Photo-activated disinfection
•Lasers
•Ozone

Ultrasonic irrigation
• Ultrasonics together with an irrigant, contribute to a better
cleaning of the root canal system than conventional irrigation.
• It works at a frequency of 25-30KHz.
• It is of 3 types:
• Active irrigation
• Passive irrigation
• Continous irrigation
103

• 2 main features:
• Cavitation
• Acoustic streaming
104

• de Almeida AP et al.., (2014) compared the effectiveness of
calcium hypochlorite (Ca[OCl]2) and sodium hypochlorite
(NaOCl) associated with passive ultrasonic irrigation in root
canals infected with Enterococcus faecalis.
• The root canals of 60 single-rooted bovine extracted teeth
inoculated with Enterococcus faecalis, and incubated for
30 days.
de Almeida AP1, Souza MA2, Miyagaki DC1, Bello YD1, Cecchin D3, Farina AP . Comparative Evaluation of Calcium
Hypochlorite and Sodium Hypochlorite Associated with Passive Ultrasonic Irrigation on Antimicrobial Activity of a
Root Canal System Infected with Enterococcus faecalis: An In Vitro Study. J Endod. 2014 Dec;40(12):1953-7
105
G1: no treatment;
G2: distilled water;
G3: 2.5% NaOCl;
G4: 2.5% Ca(OCl)2;
G5: 2.5% NaOCl with ultrasonic activation;
G6: 2.5% Ca(OCl)2 with ultrasonic activation.

• Ca(OCl)2 as well as passive ultrasonic irrigation can aid in
chemomechanical preparation, contributing in a significant
way to the reduction of microbial content during root canal
treatment.
106
de Almeida AP1, Souza MA2, Miyagaki DC1, Bello YD1, Cecchin D3, Farina AP . Comparative Evaluation of Calcium
Hypochlorite and Sodium Hypochlorite Associated with Passive Ultrasonic Irrigation on Antimicrobial Activity of a
Root Canal System Infected with Enterococcus faecalis: An In Vitro Study. J Endod. 2014 Dec;40(12):1953-7

Endo-activator
• The EndoActivator system, a sonic device, has recently been
developed for root canal irrigation.
• Special polymer tips can be driven sonically at three different
frequencies in order to activate the irrigant
107

• It is able to debride into deep lateral anatomy, remove the
smear layer and dislodge the simulated biofilms in curved
canals.
108

• Huffaker SK et al.., (2010) evaluated the ability of a new
passive sonic irrigation (sonic group) system (EndoActivator)
to eliminate cultivable bacteria from root canals in vivo and
compared it with that of standard syringe irrigation .
• They supported the multi-visit approach to the treatment of
apical periodontitis.
Huffaker, S. K., Safavi, K., Spangberg, L. S., & Kaufman, B. (2010). Influence of a passive sonic irrigation
system on the elimination of bacteria from root canal systems: a clinical study. Journal of endodontics, 36(8),
1315-1318. 109

Photo activated disinfection
• PAD is a unique combination of a photosensitizer solution and
low-power laser light.
• The low-power laser will destruct the target area and inactivate
the microbial invaders.
• The photosensitizer then binds to microbial cell walls or even
enters the cells.
• PAD needs a maximum of 150 seconds.
110

• Bago I (2013) evaluated the antimicrobial effect of a diode
laser irradiation, photo-activated disinfection (PAD),
conventional and sonic activated irrigation with 2.5% sodium
hypochlorite (NaOCl) on E. faecalis.
• Root canals of 120 human extracted teeth contaminated with
an E. faecalis suspension and incubated for 7 days.
Bago, I., Plečko, V., Gabrić Pandurić, D., Schauperl, Z., Baraba, A., & Anić, I. (2013). Antimicrobial efficacy
of a high‐power diode laser, photo‐activated disinfection, conventional and sonic activated irrigation during
root canal treatment. International endodontic journal, 46(4), 339-347.
111
G1, diode laser irradiation (2 W, 3 × 20 s);
G2, PAD (100 mW, 60 s);
G3, PAD with 3D Endoprobe (100 mW, 60 s);
G4, 30-gauge syringe irrigation with NaOCl (60 s);
G5, sonic agitation of NaOCl with the EndoActivator system (60 s);
G6, 30-gauge syringe irrigation with NaCl (60 s).

• PAD and EndoActivator system were more successful in
reducing the root canal infection than the diode laser and
NaOCl syringe irrigation alone.
112
Bago, I., Plečko, V., Gabrić Pandurić, D., Schauperl, Z., Baraba, A., & Anić, I. (2013). Antimicrobial efficacy
of a high‐power diode laser, photo‐activated disinfection, conventional and sonic activated irrigation during
root canal treatment. International endodontic journal, 46(4), 339-347.

• Plasma dental probe
• These plasma jets generate efficiently reactive plasma species
including ions, ozone, and oxygen radicals by energetic
collisions of electrons.
• The interaction of plasma species with the bacterial membrane
causes their disruption and consequently the death of bacterial
cells
113

• SEM shows complete destruction of endodontic biofilms for a
depth of 1 mm inside a root canal after plasma treatment for 5
min.
114

• Schaudinn C et al.., (2013) evaluated the efficacy of a
nonthermal plasma (NTP) probe on ex vivo biofilm in root
canals of extracted teeth.
• One group of teeth was treated with NTP, another with 6%
NaOCl and one set was left untreated.
• The nonthermal plasma displayed antimicrobial activity
against endodontic biofilms in root canals, but was not as
effective as the use of 6% NaOCl.
Schaudinn, C., Jaramillo, D., Freire, M. O., Sedghizadeh, P. P., Nguyen, A., Webster, P., ... & Jiang, C.
(2013). Evaluation of a nonthermal plasma needle to eliminate ex vivo biofilms in root canals of extracted
human teeth.International endodontic journal, 46(10), 930-937. 115

Lasers
• The commonly used laser for disinfection are Er:YAG laser.
• Lasers induces the thermal effect causing an alteration in the
bacterial cell wall leading to changes in the osmotic gradients
and cell death.
116

• Ordinola R (2014) compared the removal of biofilm utilizing
four irrigation techniques on a bovine root canal model.
• Needle irrigation,
• Endoactivator
• Passive ultrasonic irrigation
• Laser-activated irrigation
Ordinola‐Zapata, R., Bramante, C. M., Aprecio, R. M., Handysides, R., & Jaramillo, D. E. (2013). Biofilm
removal by 6% sodium hypochlorite activated by different irrigation techniques. International endodontic
journal. 117

• Laser activation of 6% sodium hypochlorite significantly
improved the cleaning of biofilm-infected dentine followed by
passive ultrasonic irrigation
118
Ordinola‐Zapata, R., Bramante, C. M., Aprecio, R. M., Handysides, R., & Jaramillo, D. E. (2013). Biofilm
removal by 6% sodium hypochlorite activated by different irrigation techniques. International endodontic
journal.

Intra-canal medicaments
The commonly used medicaments for removal of bio-film are:
• Calcium hydroxide
• Triple antibiotic paste
• Antibiotics
119

Calcium Hydroxide
• The use of calcium hydroxide in endodontics was introduced
by Hermann in 1920.
• In aqueous form, calcium hydroxide is poorly dissociated but
the hydroxyl ions liberated create the high pH (10-12) that is
required for bacterial killing.
120

The following reasons have been proposed to explain why E. faecalis is
able to survive intracanal treatment with calcium hydroxide:
• E. faecalis passively maintains pH homeostasis. This occurs as a
result of ions penetrating the cell membrane as well as the
cytoplasm’s buffering capacity.
• E. faecalis has a proton pump that provides an additional means of
maintaining pH homeostasis. This is accomplished by “pumping”
protons into the cell to lower the internal pH.
121

Iodine in Potassium Iodide
• Iodine compounds are powerful oxidizing agents that disrupt
bacterial cellular enzyme systems and inactivate them.
• Iodine in potassium iodide (IPI) has been as an intracanal
antiseptic agent in concentrations between 2 and 10% in
aqueous solution.
• The effectiveness of the medicament is short-lived and is not
suitable for periods of medication longer than 2 days.
122

• Commercially available preparations of iodoform and calcium
hydroxide, such as Metapex, are available.
• The material is highly radio-opaque and can be useful to
confirm that the entire root canal system has been obliterated
with medicament prior to sealing the access cavity
123

Chlorhexidine digluconate
• It can be used as an effective intracanal medicament,
• 2% CHX gel is used normally.
• Mixture of CHX and calcium hydroxide can also be used.
124

• de Lucena JM (2013) determine the viability of E.faecalis in
infected human root dentine in vitro after exposure to root
canal medicaments.
• Human root segments were infected with E. faecalis for
8 weeks.
• Calcium hydroxide
• 5% chlorhexidine gel
• 5% Octenidine gel
Lucena, J. M. V. M., Decker, E. M., Walter, C., Boeira, L. S., Löst, C., & Weiger, R. (2013). Antimicrobial
effectiveness of intracanal medicaments on Enterococcus faecalis: chlorhexidine versus
octenidine. International endodontic journal, 46(1), 53-61.
125

• Both CHX - and octenidine-based intracanal medicaments
were effective in decreasing the viability of E. faecalis.
• OCT showed the most favourable results and may have
potential as an endodontic medicament
126
Lucena, J. M. V. M., Decker, E. M., Walter, C., Boeira, L. S., Löst, C., & Weiger, R. (2013). Antimicrobial
effectiveness of intracanal medicaments on Enterococcus faecalis: chlorhexidine versus
octenidine. International endodontic journal, 46(1), 53-61.

Antibiotics
• Pastes such as Grossman’s polyantibiotic paste have been used
as a medicament in root canal treatment.
• The paste contained penicillin, but, as most of the bacterial
species found in the root canal are resistant due to their ability
to produce the enzyme beta-lactamase, it is relatively
ineffective.
127

Antibiotics
• Metronidazole is effective against Gram negative anaerobes.
• It has been advocated for use as a intracanal medicament.
128

Tetracyclines
• Tetracyclines have been used with some success in periodontal
treatment.
• Doxycycline combined with corticosteroids are ingredients of
Ledermix.
• Unfortunately, the range and duration of antimicrobial action
may be limited in the endodontic environment.
129

Clindamycin
• Clindamycin placed in teeth with necrotic pulps undergoing
root canal treatment offered no advantage over conventional
root canal dressings such as calcium hydroxide for elimination
of bacteria.
130

• Triple antibiotic paste (TAP)
• TAP should be used for 1-2 weeks for effective removal of
biofilm
– Metronidazole,
– Ciprofloxacin,
– Minocycline.
131

• TAP has a broad spectrum anti-microbial activity and highly
effective against anaerobic bacteria.
132

• Ordinola-Zapata R et al (2013) evaluate the antimicrobial activity
of calcium hydroxide, 2% chlorhexidine gel, and triantibiotic paste
(ie, metronidazole, minocycline, and ciprofloxacin) by using an
intraorally infected dentin biofilm model.
• Forty bovine dentin specimens were infected intraorally using a
removable orthodontic device in order to induce
the biofilm colonization of the dentin.
• Then, the samples were treated with the medications for 7 days.
Ordinola-Zapata, R., Bramante, C. M., Minotti, P. G., Cavenago, B. C., Garcia, R. B., Bernardineli, N., ...
& Hungaro Duarte, M. A. (2013). Antimicrobial activity of triantibiotic paste, 2% chlorhexidine gel, and
calcium hydroxide on an intraoral-infected dentin biofilm model. Journal of endodontics, 39(1), 115-118.
133

• Antibacterial nano-particles
• Nanoparticles are microscopic particles with one or more
particle dimensions in the range of 1–100 nm.
• The electrostatic interaction between positively charged
nanoparticles and negatively charged bacterial cells.
• The accumulation of a large number of nanoparticles on the
bacterial cell membrane, have been associated with the
increase in membrane permeability and rapid loss of
membrane function
134

– Zinc oxide
– Magnesium oxide
– Calcium oxide
• Recently, chitosan layered ZnO nanoparticles has shown 80-
95 % reduction in bacteria.
135

• Mozayeni MA et al (2014) evaluated the antimicrobial activity
of four intracanal medicaments on E. Faecalis.
– Calcium hydroxide (CH),
– 2% chlorhexidine gel (CHX),
– Triple antibiotic paste (TAP)
– Nanosilver (NS).
• Microbial samples were obtained from the roots after 7 days
136
.
Mozayeni MA1, Haeri A2, Dianat O1, Jafari AR Antimicrobial effects of four intracanal medicaments on
enterococcus faecalis: an in vitro study. Iran Endod J. 2014 Summer;9(3):195-8. Epub 2014 Jul 5

• NS gel was not efficient enough against E. Faecalis; however,
TAP and CHX gel showed better antibacterial efficacy than
CH.
.
Mozayeni MA1, Haeri A2, Dianat O1, Jafari AR Antimicrobial effects of four intracanal medicaments on
enterococcus faecalis: an in vitro study. Iran Endod J. 2014 Summer;9(3):195-8. Epub 2014 Jul 5
137

Bio-active glass
• Bioactive glass (BAG) consists of SiO2, Na2O, CaO2, and
P2O5 at different concentrations.
• The antibacterial mechanism of BAG to its high pH, osmotic
effects and Ca/P precipitation.
138

• Atila-Pektaş B et al.., (2013) compared the antimicrobial
activities of Activ Point, Calcium Hydroxide Plus Point,
calcium hydroxide, 1% chlorhexidine gel and bioactive glass
(S53P4) against Enterococcus faecalis and Streptococcus
mutans.
Atila‐Pektaş, B., Yurdakul, P., Gülmez, D., & Görduysus, Ö. (2013). Antimicrobial effects of root canal
medicaments against Enterococcus faecalis and Streptococcus mutans. International endodontic
journal, 46(5), 413-418. 139

• The medicaments containing chlorhexidine were effective
against both E. faecalis and S. mutans compared with the
medicaments having an antimicrobial effect because of their
alkaline pH.
140
Atila‐Pektaş, B., Yurdakul, P., Gülmez, D., & Görduysus, Ö. (2013). Antimicrobial effects of root canal
medicaments against Enterococcus faecalis and Streptococcus mutans. International endodontic
journal, 46(5), 413-418.

Conclusion
• The formation of biofilms carries particular clinical
significance because not only host defense mechanisms, but
also the therapeutic efforts including chemical and mechanical
anti-microbial treatment measures, have a most difficult task to
deal with organisms that are gathered in a biofilm.
141

References
• Text book of Endodontics, John I Ingle Leif K Bakland, 5th
edition
• Pathways of Pulp, Stephen Cohen, 8th edition
• Grossman’s Endodontic practise, 12 th edition
• Text book of endodontics, Nisha Garg, 2nd edition
142

References
• Mohammadi, Zahed, et al. "Microbial biofilms in endodontic infections: an
update review." Biomedical journal 36.2 (2013): 59.
• ZahedMohammadi,Mohammad Karim Soltani,and Sousan Shalavi. An Update
on the Management of Endodontic Biofilms Using Root Canal Irrigants and
Medicaments. Iran Endod J. 2014 Spring; 9(2): 89–97
• Usha, H. L. "Biofilm in endodontics: New understanding to an old
problem."International Journal of Contemporary Dentistry 1.3 (2010).
• Kanaparthy, Aruna, and Rosaiah Kanaparthy. "Biofilms-The Unforgiving
Film in Dentistry (Clinical Endodontic Biofilms)." Dentistry 2.145 (2012):
2161-1122.
143

References
• Ordinola‐Zapata, R., Bramante, C. M., Aprecio, R. M., Handysides, R., &
Jaramillo, D. E. (2013). Biofilm removal by 6% sodium hypochlorite
activated by different irrigation techniques. International endodontic
journal.
• de Almeida AP1, Souza MA2, Miyagaki DC1, Bello YD1, Cecchin
D3, Farina AP . Comparative Evaluation of Calcium Hypochlorite and
Sodium Hypochlorite Associated with Passive Ultrasonic Irrigation
on Antimicrobial Activity of a Root Canal System Infected with
Enterococcus faecalis: An In Vitro Study. J Endod. 2014 Dec;40(12):1953-
7
144

References
• Stojicic, S., Shen, Y., Qian, W., Johnson, B., & Haapasalo, M.
(2012). Antibacterial and smear layer removal ability of a
novel irrigant, QMiX.International endodontic journal, 45(4),
363-371
145

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