Novel Activated Microbubbles-based Strategy

to Coat Nanoparticles on Root Canal Dentin:

Fluid Dynamical Characterization

Li FC, Borkar S, Ramachandran A, Kishen A.

Journal of Endodontics.
2019 Jun 1;45(6):797-802.
1
 INTRODUCTION
• Predictable elimination of root canal biofilms and
reestablishment of the mechanical integrity of the remaining
tooth structure still remain as major challenges in
conventional root canal treatment.

2
• Therefore, developing treatment approaches that combine
the advantage of antibiofilm efficacy in root canals as well as
improved mechanical characteristics of the remaining root
dentin is imperative to enhance predictability in root canal
treatment.

3
 CHITOSAN NANOPARTICLES
• Chitosan is a natural polysaccharide that is obtained by
deacetylation of chitin, one of the most abundant
polysaccharides in nature that forms most of the external
skeleton of arthropods such as crabs and shrimps.

• Chemically, chitin is composed of (1-4)-linked 2-acetamido-2-

deoxy-β-D glucose.

4
• Chitosan with low molecular weight was considered to have
the ability to penetrate the bacterial cell membrane and then
to bind to the DNA, inhibiting its transcription and mRNA
synthesis

• Chitosan with high molecular weight was surmised to bind to

the negatively charged components of the bacterial cell wall,
forming an impermeable layer and blocking transportation
into the cell. 5
 Chitosan
• Chitosan is a hydrophilic biopolymer with a molecular structure similar to
extracellular matrix components.

• Previous studies have shown that

– Modified/ unmodified chitosan nanoparticles (CSnps) used as an intracanal
medicament would effectively eliminate/inactivate residual root canal
biofilm/endotoxins

– Cross-linked CSnps stabilize the dentin matrix by resisting host/ bacteria-

mediated enzymatic degradation and increasing the mechanical toughness of
the dentin

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• Effective delivery of nanoparticles in root canals is a
prerequisite to promote nanoparticle-dentin interaction.

• Root canal dentin surfaces coated with CSnps will aid in

effectively translating their antibacterial and mechanical
benefits in root canal treatment.

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 Activated microbubbles (MBs)
• Ability to potentiate bubble dynamics in microspaces.

• MBs are composed of microsized droplets with a

protein/surfactant shell, which are prepared by an oxygen
carrier and an oxidizer in an emulsion.

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 Activation of MBs

High particle velocity and stresses from bubble streaming, cavitation, and shock
waves

Can induce additional forces toward the wall in constrained microspaces, which
could presumably be optimized for the effective application of nanoparticles in
root canal treatment.

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 2 main objectives of this study
• To determine the fluid dynamical parameters such as velocity
and stress associated with ultrasonically, sonically, and
manually activated MBs in simulated root canals

• To assess the effectiveness of CSnp application within the

root canals using the previously described activation methods
in extracted teeth.

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MATERIALS AND METHODS

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 Characterization of Fluid Dynamics in
Simulated Root Canal Models
Simulated Root Canal Models
• Root canal models were prepared using polydimethylsiloxane
(PDMS) simulating a prepared F2 size main canal with a 50-mm-
width blocked-end side channel 4 mm below the orifice of the main
canal at 1 side of the main canal.

• Two sectional models were aligned under a stereomicroscope and

bonded with the plasma bonding technique to obtain a single canal
model with a canal space of 1-mm height

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• Eighteen models were used to assess the fluid dynamical
parameters in water and MB-based formulation with ultrasonic,
sonic, and manual activation methods.

• The velocity, wall stress, and the depth of side channel penetration
by the particles were determined.

• MBs were prepared using a modified combination of an oxygen

carrier and a nonionic detergent surfactant.

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Fluid Dynamical Analysis

• In the water manual (WM), water sonic (WS), and water

ultrasonic (WU) groups, the canal spaces were filled with
deionized water containing hollow glass beads at 106
particles/mL concentration by syringe delivery with a 30-G
needle.

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• In the MB manual (MM), MB sonic (MS), and MB ultrasonic
(MU) groups, the canal spaces were filled with MBs by syringe
delivery with a 30-G needle.

• In the WM and MM groups, a gutta-percha (GP) point

(ProTaper Universal) of size F2 was inserted to 1 mm from the
apical terminus, after which push-and-pull strokes were
applied (amplitude = 5 mm,frequency = 100 strokes/minute)

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• In the WS, MS, WU, and MU groups, the tip of the sonic and
activation devices was stabilized to 4 mm from the root canal
orifice and was agitated.

• The fluid flow was viewed on an inverted microscope with a

10x objective lens

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• The mean velocity and the wall stress generated by the fluid
movement in the main canal space as well as the depth of the
flow penetration were determined.

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18
 Assessing Nanoparticle Delivery and
Nanoparticle based Coating in Tooth
Model

• CSnps were synthesized using an ionic gelation method

• The CSnps obtained were approximately 120 nm and were

dispersed in water/or MBs at a concentration of 1.0 mg/mL.

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• Thirty-five freshly extracted human incisors were sectioned at
the cementoenamel junction

• Root canals were enlarged to F2 size

• Irrigated with 6 mL 2.5% sodium hypochlorite followed by

17% EDTA for 3 minutes.

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• The specimens were randomly divided into 7 groups (n = 5/group)

• In the control group, the root canal was enlarged without the application of CSnps.

• In the experimental groups, CSnps in deionized water were delivered into the root canals

using manual (WM), sonic (WS), and ultrasonic agitations (WU) for a total period of 5

minutes.

• In the remaining 3 groups, CSnps were dispersed in MBs and delivered into the root canals

using manual (MM), sonic (MS), and ultrasonic agitation (MU).

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• The root canal was then rinsed with water and dried gently with

paper points

• The specimens were split mesiodistally into 2 halves to expose

the root canal dentin surface

• Subjected to field emission scanning electron microscopy to

ascertain the coating/penetration of nanoparticles on dentin

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 STATISTICAL ANALYSIS
• Means and standard deviations were analyzed with 1-way
analysis of variance and the post hoc Dunnett T3 test.

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 RESULTS

Characterization of Fluid Dynamics in Simulated

Root Canal Models

The particle velocity and wall stress caused by the fluid/bubble

dynamics in proximity to the lateral aspect of the agitating tips
were significantly different for different activation methods

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• In the WM, MM, and MS groups, the fluid motion was
predominantly viscous in nature or was a combination of
viscous and inertial effects

• The inertial forces were dominant in the WS, WU, and MU

groups

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26
• The MU group consistently generated the highest velocity
(0.74 m/s) and wall stresses (658.45 Pa) and the deepest
penetration of particles into the side channel (149.14 mm)

• The WM group produced the lowest velocity (0.025 m/s) and

wall stress (0.51 Pa), and particles were not pushed into the
side channel during agitation.

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 Assessing Nanoparticle Delivery and
Nanoparticle based Coating in Tooth
Models
• A significant difference was found among groups of the percentages of
CSnp-covered dentin and dentinal tubule occlusion .

• Specimens from the MM, WU, and MU groups showed nanoparticle-based

coating for over 70% of the dentin surface covered.

• Although the specimens from the MM group showed the highest

percentage of dentinal tubule occlusion (74.82%), there was no
statistically significant difference between the MM, WU, and MU groups

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29
 DISCUSSION
• The fluid dynamic parameters associated with ultrasonic,
sonic, and manual agitations in water or MBs and the final
static evaluation of the effectiveness of CSnp delivery with
these agitation techniques/ media in root canals were
assessed in this study.

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• Soft lithography enabled accurate replication of instrumented
root canals with PDMS.

• This substrate presented substantial optical transparency,

allowing high-quality visualization and analysis.

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• The current goal for clinical translation of nanoparticle-guided
endodontics is to achieve a uniform coating on the root canal
dentin surface.

• A thin layer of nanoparticle-based coating over peritubular

and intertubular dentin surfaces with occluded dentinal
tubules would be ideal for their clinical benefits.

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• The examination of the physical parameters of fluid and the
surface coating of nanoparticles were assessed at the coronal
third of the root canal.

• Accordingly, the tip of the ultrasonic/sonic inserts, which

created a higher velocity and shear stress, was placed at the
coronal third for both PDMS and the tooth models

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• A higher degree of stress/strain distribution and stress
concentrations occurred at the cervical region of the root
dentin, particularly in the buccolingual direction

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• The velocity of the activated particles in water was highest in
the ultrasonic group followed by the sonic and manual groups
in the present study.

• This is because of the higher frequency in this group resulting

in a higher flow velocity

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• On the contrary, fluid dynamics generated by sonic agitation
was less effective compared with ultrasonic agitation, mainly
because of the lower frequency.

• The sonically activated insert displayed only 1 node and

antinode, and the oscillation mostly remained longitudinal
when movement was constrained within the microspace of
the root canal.
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• The particles/bubbles only moved back and forth following
the movement of the insert.

• The region in the simulated root canal below the sonic insert
was essentially motionless without any fluid
streaming/circulation.

• These factors resulted in the inadequate nanoparticle-based

coating in the MS group

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• The ultrasonically activated insert induced intense acoustic

microstreaming with multiple nodes and antinodes and with higher

vibration frequency.

• The mean velocity in MU was 3 times higher than that in WU.

• This could be caused by the intensified cavitation bubble dynamics and

the reduced threshold for bubble formation, resulting in the large/strong

bubble coalescence.

• The microjets generated from the collapsing cavitation bubbles may also

cause rapid fluid streaming and high stress.

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• In the current study, the highest wall stress and the
percentage of optimal nanoparticle-based surface coverage
were derived from manual agitation of MBs (the MM group).

• In MM, the motion of MB emulsion through the gap shears

the concentrated emulsion, inducing interdroplet interactions
that result in a phenomenon called shear-induced self-
diffusion
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• This improve the deposition rates of CSnps on the wall via
electrostatic interaction between cationic CSnps and anionic
dentin, leading to the observed uniform coating of CSnps on
dentin in the MM group

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• In addition, a consistent layer of coating was also observed on
the dentin surfaces after ultrasonic agitation in the WU and
MU groups

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 CONCLUSION
• Activated MBs enhanced fluid dynamics in simulated root
canals, whereas the application of manually activated MBs
aided in nanoparticle delivery and subsequent optimal
nanoparticle coating on root canal dentin.

42
 Chelating and antibacterial
properties of chitosan nanoparticles
on dentin.
del Carpio-Perochena A, Bramante CM, Duarte
MA, de Moura MR, Aouada FA, Kishen A.
Restorative dentistry & endodontics.
2015 Aug 1;40(3):195-201.

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 AIM
• To investigate the ability of bioactive CNPs to remove the
smear layer and inhibit bacterial recolonization on dentin.

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 Materials and Methods

• One hundred bovine dentin sections were divided into five groups (n = 20
per group) according to the treatment.

• The irrigating solutions used were 2.5% sodium hypochlorite (NaOCl) for
20 min, 17% ethylenediaminetetraacetic acid (EDTA) for 3 min and 1.29
mg/mL CNPs for 3 min.

• The samples were irrigated with either distilled water (control), NaOCl,
NaOCl-EDTA, NaOCl-EDTA-CNPs or NaOCl-CNPs.
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• After the treatment, half of the samples (n = 50) were used to
assess the chelating effect of the solutions using portable
scanning electronic microscopy, while the other half (n = 50)
were infected intra-orally to examine the post-treatment
bacterial biofilm forming capacity.

• The biovolume and cellular viability of the biofilms were

analysed under confocal laser scanning microscopy.

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 STATISTICAL ANALYSIS
• The Kappa test was performed for examiner calibration, and
the non-parametric Kruskal-Wallis and Dunn tests were used
for comparisons among the groups.

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 Results

• The smear layer was significantly reduced in all of the groups

except the control and NaOCl groups .

• The CNPs-treated samples were able to resist biofilm

formation significantly better than other treatment groups

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 DISCUSSION
• The antibacterial mechanism of chitosan has been attributed to its

polycationic nature that interacts with the negatively charged surface of

bacteria, altering cell permeability and resulting in the leakage of

intracellular components.

• In addition, CNPs are able to inhibit bacterial enzymatic degradation

reducing the possibility of bacterial penetration and dentinal micro-

fractures.

• This biopolymer is also capable of improving the mechanical properties of

the root dentin.

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 Conclusion

• CNPs could be used as a final irrigant during root canal

treatment with the dual benefit of removing the smear layer
and inhibiting bacterial recolonization on root dentin.

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Effectiveness of nanoparticles solutions
and conventional endodontic irrigants
against Enterococcus faecalis biofilm.

De Almeida J, Cechella BC, Bernardi AV, de

Lima Pimenta A, Felippe WT.
Indian Journal of Dental Research.
2018 May 1;29(3):347.

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 Aim
• This study aims to evaluate the effectiveness of experimental
solutions containing silver and zinc oxide nanoparticles (ZnO
Np) and conventional endodontic irrigants
against Enterococcus faecalis biofilm, in root canals.

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 Materials and methods
• Seventy-six extracted human teeth were biomechanically
prepared and sterilized.

• The root canal surface was exposed to E. faecalis suspension

to form a 7-day-old biofilm.

• Four teeth were analyzed by scanning electron microscopy

(SEM) to confirm the presence of biofilm.

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• The remaining teeth were randomly divided into 6 groups (n = 12) and
treated with passive ultrasonic irrigation and different solutions:

• G1 – 0.85% saline (control);

• G2 – 2% chlorhexidine gluconate (CHX);

• G3 – 5% sodium hypochlorite (NaOCl);

• G4 – 1% NaOCl;

• G5 – 1% silver nanoparticles (Ag Np) solution; and

• G6 – 26% ZnO Np solution.

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• The susceptibility of E. faecalis biofilms to disinfecting
solutions (n = 10) was determined by quantification of colony-
forming units.

• SEM analysis was also carried out to examine the biofilm

structure after treatments (n = 2).

• Data were analyzed by Kruskal–Wallis and Dunn post hoc tests

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 Results
All tested solutions showed superior effectiveness compared to
0.85% saline .

Overall, 2% CHX presented the most effective action against E.

faecalis biofilm, followed by 5% NaOCl, 1% Ag Np, 26% ZnO Np,
and 1% NaOCl.

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 DISCUSSION
• A current tendency in dentistry, which has presented positive
results with regard to antimicrobial and antibiofilm activities,
is the use of nanoparticles, such as Ag Np and ZnO Np

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• Ag Np with size range of 1–100 nm have a great bactericidal
potential against both Gram-positive and negative bacteria, which
could be linked to the high-contact, surface-dependent silver
antimicrobial action.

• Silver nanoparticles bind to the cell membrane leading to altered

cell permeability, thus affecting the transport system through the
plasmatic membrane.

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• Hence, silver ions, released due to the oxidation of
nanoparticles after binding to the cell membrane, are able to
penetrate inside the bacteria and react with specific proteins.

• This alters the bacterial metabolism and inhibits vital

enzymatic systems, such as respiratory process and cellular
division, resulting in cell death

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 Conclusion
• 1% Ag Np and 26% ZnO Np were effective against E.
faecalis biofilm similarly to conventional endodontic irrigants.

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