Keywords
antimicrobial - chlorhexidine - calcium hydroxide -
Enterococcus faecalis
- nanographene - root canal therapy
Introduction
Pulpal and periapical pathology are primarily caused by microbial insults and the
presence of microorganisms in dentinal tubules leading to the recurrence of apical
periodontitis. Hence, the objective of nonsurgical root canal treatment is to eliminate
microorganisms from the root canal system to prevent failure. The combined use of
instrumentation and irrigants helps to reduce the microorganisms. After instrumentation,
35% or more of the canal's surface area remain unchanged, which could be the limitation
of the instrumentation technique.[1] The mechanical cleaning with irrigation using antimicrobials could significantly
decrease the bacterial load in the root canal system,[2] but residual bacteria could still flourish and multiply in between appointments
if no antimicrobial agent was applied in the root canal system. Hence, intracanal
medicament plays a key role in root canal treatment.[3]
Failures of nonsurgical root canal therapy are frequently associated with the presence
of Enterococcus faecalis in root canal systems.[4]
[5] Close to the pulp and outer borders of the tooth, the diameter of dentinal tubules
are 2.5 µm and 0.95 µm, respectively. However, the mean diameter of E. faecalis is between 0.8 and 1 µm, which allows the organism to enter and survive within the
dentinal tubules and form biofilm on walls of root canals.[6]
[7]
[8]
[9] Maximum penetration of E. faecalis was found to be 1,483.33 µm. Hence, the penetrating ability of the traditional antimicrobial
agents needs to be higher to reduce the remaining bacteria inside the dentinal tubules.[10]
Calcium hydroxide (Ca(OH)2) and chlorhexidine (CHX) gluconate are commonly used as intracanal medicaments in
endodontic therapy. The mode of action of Ca(OH)2 allows it to be a physical barrier, disturbing remaining nutrient resources for bacteria,[8] and most importantly a high pH of 12.5 causes lysis of bacterial cell membrane and
proteins.[11] CHX's mode of action is through penetration of negatively charged bacteria cell
wall by the positively charged CHX molecules that are toxic to bacteria.[11]
[12]
Graphene is an atomically thin two-dimensional layer. It has a large specific surface
area and is highly suited to be a promising drug carrier.[13]
[14]
[15] It also has unique chemical and physical properties that make it possible to react
with molecules and modify the surfaces leading to important molecules like graphene
oxide (GO) and carboxylated graphene.[16]
[17] Graphene, an allotropic type of carbon, was first successfully prepared by Novoselov
et al in 2004.[18] Graphene is constituted of single-atom-thick carbon nanosheets with a honeycomb
structure.
Singular graphene units are called nanographenes (NGs); NGs are graphene fragments
with a diameter of less than 100 nm, while graphene should exceed 100 nm in both directions.
The production of NG is quite complicated: it is done by a process called dehydrogenation.
Dehydrogenation is achieved by selectively removing hydrogen atoms from organic molecules
consisting of carbon and hydrogen.
Potential applications of graphene and its derivatives in biomedical fields such drug
delivery, tissue engineering, and biosensors have been investigated. Applications
of nanotechnology in endodontics include the incorporation of bio-ceramic nanoparticles
such as bioglass, zirconia, and glass ceramics in endodontic sealers. It has been
found that the use of nanoparticles enhances the adaptation of the adhesive to nano-irregularities,
in addition to its fast setting time in comparison to conventional sealers, its dimensional
stability, insolubility in tissue fluid, chemical bond to tooth tissue, and osseo
conductivity.[19] The use of nanoparticles in dentistry is gaining popularity for various applications.[20]
[21] Numerous studies have investigated the effect of NG in dentistry including endodontics.[22]
[23]
The effectiveness of NG oxide as intracanal medications has been evaluated in earlier
research.[21] Nevertheless, no research has been done on the use of NG as an intracanal irrigant.
The objective of this study was to compare the antimicrobial efficacy of NG particles
compared to CHX and Ca(OH)2 against E. faecalis in contaminated extracted teeth using colony-forming unit (CFU).
Materials and Methods
A randomized controlled in vitro study was designed. The study protocol was approved by the institutional research
ethical committee (IMU-JC NO 361/2016). The minimum inhibitory concentration (MIC)
of NG against E. faecalis was determined using serial dilutions of 0.1%, 2% CHX, and 3% sodium hypochlorite
(NaOCl). Cultures of E. faecalis was prepared to obtain the 0.5 McFarland turbidity. The MIC was defined as the lowest
concentration of each irrigant in which no visible turbidity was noted.
NG particles powder (Sigma Aldrich Gold nano urchins) was mixed in saline to form
a suspension.
Each treatment (untreated × 3; NG 1,000 µg/mL; NG 750 µg/mL; NG 500 µg/mL; NG 250
µg/mL; NG 200 µg/mL; NG 150 µg/mL; NG 100 µg/mL; NG 50 µg/mL) was then added accordingly
to the corresponding wells.
Sample Preparation for Tooth Model
Forty extracted human mandibular premolar teeth were used for this project. A pilot
study was conducted in our experimental research to calculate the sample size accurately
using G*power (Version 3.1)
Accordingly, a total of 40 extracted human mandibular premolars were used in this
study that were further divided into 4 main groups consisting of 10 samples for each
group, in accordance with the previously calculated sample size.
The external surfaces of the teeth were cleaned using an ultrasonic scaler. A rotary
diamond disk was used to obtain the middle-third of the root (6 mm) by decoronating
the teeth below the cementoenamel junction and the apical part of the root. The accuracy
of the slices was checked with calipers. The inner diameter of the teeth was made
consistent using #3 Gates Glidden (GG) (Mani Inc, Tochigi-ken, Japan). To remove debris,
the teeth samples were placed in an ultrasonic bath containing 17% ethylene diamine
tetra-acetic acid (PD Dental) for 5 minutes followed by 3% NaOCl (Coltene). Samples
were placed in an ultrasonic distilled water bath for three cycles of 20 minutes.
Lastly, the teeth samples were sterilized by autoclave (Infitek Class B benchtop sterilizer).
Sample Contamination
E. faecalis (ATCC 29212) cells were grown in Tryptic soy broth (TSB) overnight. After incubation,
the culture was diluted in 5 mL of TSB and its turbidity adjusted to 0.5 McFarland
standards (1 × 106 CFU/mL). Each dentine block was immersed in presterilized microcentrifuge tubes containing
TSB. To contaminate the dentine blocks, 50 µL of the overnight E. faecalis culture was transferred into each of the microcentrifuge tubes. The purity of the
culture was checked by subculturing 5 µL of the broth from the incubated dentine blocks
on Tryptic soy agar (TSA) plates. Dentine blocks were contaminated with E. faecalis for 21 days, where media was replaced daily.
Antibacterial Assessment
After incubation (21 days), the tooth samples were washed with 5 mL of sterile saline
to remove excess culture on the surface of the specimens. The teeth samples were randomly
divided into 4 groups with 10 specimens each: Group I, untreated saline (control);
Group II, Ca(OH)2; Group III, CHX; and Group IV, NG.
Preparation of NG gel was done by mixing 1 mg of NG powder with 1 mL of hydroxypropyl
methylcellulose in a magnetic stirrer. Each treatment group was introduced into the
teeth samples and paraffin wax was used to seal both ends of the dentine blocks. Incubation
was carried out at 37°C for 24 hours. At the end of days 1, 3, and 7, an assessment
of bacterial growth was carried out by harvesting dentine chips using GG #4 (200µm)
into Eppendorf tubes containing 0.5 mL saline. Homogenization of the suspension was
done through vortexing for 10 seconds. After this process, 0.05 mL from each Eppendorf
tube was introduced into separate Eppendorf tubes of 0.45 mL saline. This process
continued up to three micro dilutions for all treatment groups. The Eppendorf tubes
holding dilutions 1, 2, and 3 for all variables had 0.02 mL of their contents drawn
out and seeded onto the TSA plate in a triplicate pattern. The plates were then left
to dry and incubated at 37°C for 24 hours. The colonies were physically counted and
tabulated after 24 hours from seeding.
Statistical Analysis
The collected data were analyzed with SPSS Version 20.0 by analysis of variance and
Tukey's post hoc test. A p-value of less than 0.05 was considered to be statistically significant.
Results
The average droplet and particle size distribution were analyzed using Zetasizer (Nano-ZS90,
Malvern Instruments, Worcestershire, UK) ([Table 1]). The surface charge of the formulations was also measured with the same instrument.
As indicated previously, thermodynamic stability investigations on the NG were conducted.
The graphene particle size analysis was done using Malvern Zetasizer ZS90, and the
polydispersity index indicates the homogenous size distribution of the particles ([Fig. 1A]). If the value is around 0.5, it shows relative homogeneity. Zeta potential is the
surface charge of a particle ([Fig. 1B]). If the observed surface charge is higher than –20, it indicates high stability
of the particle.
Table 1
Physical characterization of nanographene
|
Nano formulation
|
Average particle size
|
Polydispersity index
|
Zeta potential
|
|
Nanographene
|
403.1 nm
|
0.541
|
–27.2 ± 0.7 mV
|
Fig. 1 (A) Size distribution intensity. (B) Zeta potential curve.
Antibacterial Assessment
The contaminated dentine blocks irrigated with NG (0.5 µg) and 2% CHX (0 ± 0; p < 0.001) did not have any growth of E. faecalis colonies compared to blocks of Ca(OH)2 (10 ± 21) and saline (927 ± 455). All concentrations of NG (0.5 and 1.0 µg) showed
effectiveness higher (p < 0.001) than 2% CHX when measured by the zone of inhibition against E. faecalis ([Fig. 2]).
Fig. 2 Mean plot of Enterococcus faecalis at 200 µm at varying time intervals. Ca(OH)2, calcium hydroxide; CHX, chlorhexidine; NG, nanographene.
Real-time polymerase chain reaction (PCR) was performed using a thermal cycler (7900
HT RT-PCR, Applied Biosystem, UK)[22] with SYBR™ Green fluorophore. The reaction mix was prepared to a final volume of
20 μL and loaded in an optical 96 well plate, which was then covered with an optical
adhesive sheet. E. faecalis was identified using PCR amplification of 16S rRNA gene sequences. The oligonucleotide
species-specific primers for E. faecalis were 5¢GTT TAT GCC GCA TGG CAT AAG AG3¢ (forward primer, located at base position
165 to 187 of the E. faecalis 16S rDNA) and 5¢CCG TCA GGG GAC GTT CAG¢3 (reverse primer, located at base position
457 to 474 of the E. faecalis 16S rDNA).
Biocompatibility Assay
[Figure 3] shows that the biocompatibility assay using epithelial and fibroblast cell lines
was done using Trypan blue exclusion. Dilution of a cell sample in Trypan blue dye
of an acid azo exclusion medium prepared by a 1:1 dilution of the cell suspension
using a 0.4% Trypan blue solution was done. Nonviable cells will be blue while viable
cells will be unstained. The total number of cells overlying 1 mm2 was between 20 and 50 cells/square. The Trypan blue dye stains the dead cells blue
that can be counted versus the live cells and the ratio can be used to see the percentage
of dead cells (a small percentage of dead cells are likely; however, untreated sample
and the treated sample should be compared to optimize the data).
Fig. 3 Cells in the presence and absence of nanographene in an NIH-3T3 (mouse fibroblast).
Discussion
Successful root canal therapy may be characterized as absence of clinical symptoms,
regression of periapical lesion, tight seal canal and coronal spaces, and recovery
of tooth function.[4] This can be achieved by root canal preparation, stringent chemo-mechanical debridement,
and intracanal dressing placement, which aim to disrupt bacteria biofilm and their
by-product along with neutralizing bacterial invasion within the complex root canal
system. It is later followed by adequate sealing of canal spaces and restoration of
root treated teeth to complete the treatment.[8]
[10]
[21] Different types of antimicrobials either single or in combination have been used
in root canal disinfection. These antimicrobials act by promoting slow destruction
of the biofilm structure by destroying persistent cell or quorum sensing signals,
which then allows it to diffuse into the biofilm structure, signaling destruction
of both the matrix and resident bacteria in the structure.[8] On the contrary, the increase in resistance against antimicrobials leads to persistence
of certain species of bacteria namely E. faecalis, which have been commonly found to cause chronic inflammation in the root canal spaces
and ultimately leading to endodontic failures.[7]
[11]
This study investigated the antibacterial efficacy of NG against E. faecalis in comparison to Ca(OH)2 and CHX. Just like the cumulative nature of science, dentistry is ever evolving,
into a new era of nanotechnology. New silver nanotechnology chemistry has also been
proven to be effective against biofilms such as Escherichia coli, Streptococcus pneumoniae, Staphylococcus aureus, and Aspergillus niger. It is reported that silver has a high affinity against negatively charged side groups
found distributed all over microbial cells and it acts by attacking multiple sites
within the cell to inactivate its critical physiological functions. On top of that,
it was also discovered that all it takes is as little as one part per billion of silver
to be effective in preventing certain types of bacteria growth.[23]
Our results show that contaminated dentin blocks irrigated with nanographene or CHX
did not have any growth as compared to Ca(OH)2 and saline ([Fig. 2]). The results reported are similar to studies that have evaluated the antibacterial
efficacy of Ca(OH)2 using molecular method. Majority of the studies showed that although bacterial counts
were significantly reduced, it was not completely eradicated.[24] It has been suggested that this could be due to the ability of microorganisms to
stay hidden within the complexity of radicular canal spaces that have made the evasion
of close contact with lethal hydroxyl ions possible.[25]
[26] On the contrary, in terms of clinical outcome, controversial results have been reported
as there were no significant differences in healing modalities between single visits
and multiple visits with or without interappointment Ca(OH)2 dressing.[27]
[28] In the present study, all concentrations of NG have shown effectiveness against
E. faecalis and were found to be significantly higher than CHX. Similarly, in a study conducted
by Wu et al, the antibacterial efficacy of GO was evaluated by fabricating a calcium
phosphate cement-chitosan-GO scaffold and was tested against E. faecalis.[29] The results have shown that in the presence of graphene particles, it possesses
high antibacterial activity against E. faecalis, hence rendering it a promising root canal sealer in endodontic treatment. Other
than that, its versatility to undergo different surface modification and functionalization
to reduce cytotoxicity along with its ability to be produced easily, and on large
scale with low cost, makes GO a suitable antibacterial agent against multiresistant
bacteria.[29]
In our study, the antibacterial effect of CHX was found to be inferior to that of
NG. This might be due to the resistance and poor penetration of CHX through the dense
exopolysaccharides-matrix encased E. faecalis.[30]
As opposed to NG, it has been found to exhibit strong antibacterial properties and
this could be attributed to the ability of NG to impale the bacteria via physical
and chemical mechanisms.
These mechanisms include the following:
-
Physically damage the bacterial membrane via direct contact.
-
Reactive oxygen species production causing disruption and deactivation of cell metabolism.
-
Electron transfer from bacterial membrane leading to compromised membrane integrity.[31]
The application of nanoparticles into dentistry has been gaining popularity due to
its excellent antibacterial properties. Its large surface to volume ratio, ultrasmall
sizes, and excellent chemical and physical properties give graphene good bonding capabilities
and surface chemistry as compared to conventional materials.[32] As a result, the interaction between the positively charged, increased surface area
of nanoparticles and negatively charged bacterial cells leads to increased antibacterial
activity.
To date, NaOCl remains the gold standard for root canal irrigation due to its potent
antimicrobial and tissue dissolving properties; however, accidental apical extrusion
may lead to inflammation and tissue cytotoxicity.[32]
Furthermore, it has been discovered that the antibacterial effect of graphene remains
the same by incorporating it into silver nanoparticles while concurrently preserving
its cytotoxic effect to surrounding tissues and bones. This can be supported by the
findings from our study in which the epithelial and fibroblast cells treated with
NG were found to be viable hence biocompatible to surrounding tissue structure ([Figs. 3] and [4]). Multiple studies have also shown that monolayer graphene or GO films were found
biocompatible to mouse fibroblasts (NIH-3T3), human osteoblasts-like cell line, and
A549 cell line.[33]
[34] However, Zhang et al reported in a study that biocompatibility of NG platelets may
be dose-dependent with 10 µg/mL−1 as its critical concentration.[35] In addition to this, Chang et al[33] has reported that the cytotoxicity of graphene-based material may be affected by
its shape and size in which the smaller the size of GO particles, the higher the cytotoxicity
it expresses.
Fig. 4 Dead cells appearing in the image shown treated with chlorhexidine.
From this we can deduce that even though smaller-sized graphene may enhance its ability
to penetrate and impale bacteria, it may also be less biocompatible. Hence, more research
should be conducted in formulating a size and dosage appropriate graphene-based material
to be safely used in the human body. In our study, 0.5 µg/mL of NG has been shown
to express potent antimicrobial activity against E. faecalis and was found to be biocompatible against mouse fibroblast cells (NIH-3T3) ([Fig. 5]). From this, we can conclude that NG may be used as a promising antimicrobial agent
in root canal treatment. Therefore, more research should be carried out to investigate
the effect of NG against other dental pathogens such as Streptococci or Porphyromonas in in vivo or clinical settings.
Fig. 5 Cell viability is measured by live/dead staining of NIH-3T3 cells after incubation
on each substrate for 24 and 48 hours. Live and dead cells were counted and the percentage
of live cells was plotted. NG, nanographene.
Conclusion
It may be concluded that NG is effective against growth of E. faecalis and may be used as a promising antimicrobial agent during root canal treatment. However,
further studies should be done to investigate the effect of NG against other dental
pathogens.
Limitations and Future Recommendations
The limitations of the current study are that it assesses the antibacterial activity
of NG in single-rooted premolars, and complex canal anatomies of multirooted teeth
has not been evaluated. Future studies can assess the antibacterial effect of NG in
multirooted teeth to assess the penetration into complex canal anatomy. Future studies
can also investigate the mechanism of action and interaction with other intracanal
medicaments and irrigants.
