Effects of final irrigation with CHX and EDTA based irrigant … · 2018. 11. 15. · The blocks were divided in CHX group: Irrigation with 6% NaOCl 17% EDTA 6 % NaOCl and 2% CHX,
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Effects of final irrigation with CHX and EDTA based irrigant combined with detergents οn the surface of
dentin using surface analytical methods
by
Myrto Piperidou
A thesis submitted in conformity with the requirements
Austria). The 12 dentin blocks were embedded in low viscosity epoxy resin (Epo-Thin, Buehler,
Lake Bluff, USA) for 24 hours. In order to expose the dentinal tubules in a perpendicular
orientation to the surface, the surface was further ground with either a diamond or glass
microtomes (Leica EM UC6/FC6 Ultra-cryomicrotome, Leica Microsystems GmbH, Vienna,
Austria).
Exposure to irrigation agents
The dentin block specimens were randomly divided into two groups. CHX Group: Six specimens
were immersed in 5 ml of 6% NaOCI (Sodium Hypochlorite, Lavo inc., Montreal, Quebec) for 1
min, immediately followed by 5 ml of 17% EDTA (EDTA, Vista Dental Products, Racine, WI,
USA) for 1 min, then in fresh 5 ml of 6% NaOCI for 1 min and a final immersion in 5 ml of 2%
CHX (chlorhexidine digluconate Vista Dental Products, Racine, WI, USA) for 1 min. Smear OFF
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Group: Six specimens were immersed in 5 ml of 6% NaOCI for 1 min, then in 5 ml of Smear OFF
for 1 min (as per manufacturer’s recommendation). After completion of the immersion protocol,
the samples were left on a bench top to dry overnight.
TOF-SIMS analysis
Three specimens from each group were analyzed using an IonTOF TOFSIMS V (IonTOF GmbH,
Münster, Germany) equipped with a bismuth (Bi) liquid metal ion gun. The Bi3 ++ cluster was used
as the primary ion source. The gun was operated in both high-mass and high-spatial resolution
modes. High mass resolution spectra were obtained on the initial surface (500 µm x 500 µm, 100
sec), followed by high mass resolution spectra on a 150 µm x 150 µm area, within a 450 µm x 450
µm cleaned area using an Ar cluster source. Images (256 x 256 pixels, 20 scans) were also obtained
from this region in high spatial resolution mode. Both positive and negative spectra were obtained.
XPS analysis
XPS spectra from the remaining three specimens were obtained on a ThermoFisher Scientific
ESCALAB 250Xi (Thermofisher Scientific, East Grinstead, UK). A monochromatic Al Kα source
with a spot size of 400 µm was used. Photoelectrons were collected at take-off angle of 90⁰ relative
to the specimen surface using a pass energy of 100 eV for the survey spectra and 20 eV for the
spectral regions. Charge compensation was applied using the combined Ar+/e-flood gun and the
peaks were shifted to place the main C 1s peak at 285.0 eV. Composition was obtained from the
latter spectra by integrating the peaks after subtracting a Shirley type background (27) and
applying the supplied sensitivity factors using the instrument’s software (Avantage 5.926—
Thermofisher Scientific, East Grinstead, UK). To ascertain depth of coverage, the surfaces were
Ar cluster sputter-cleaned followed by more intense Ar+ depth profiling using the dual-mode Ar
mono/cluster source provided with the instrument. The beam was rastered over a 2 mm x 2 mm
area. The XPS spectra were obtained from a 400 µm spot at the crater’s center.
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Results
Brown precipitate formation was detected in all specimens of the CHX group, whereas no
precipitate and no color change were observed in any specimens of the Smear OFF group.
TOF- SIMS analysis
The positive ion TOF-SIMS spectra for the CHX and the Smear OFF groups are shown in Fig. 1A
and 1B, respectively, for mass ranges 100 – 200 u and 500 – 510 u. The negative ion spectra for
CHX and Smear OFF groups, for mass range 0 – 100 u, are shown in Fig. 1C and 1D, respectively.
The position of the characteristic positive fragments for CHX (127 u, 153 u, 170 u and 195 u) and
that of the parent ion for CHX (505.24 u), are indicated in Figures 1A and B. It should be noted
that the peak at 127 u also corresponds to the main peak of PCA. Characteristic peaks in the
negative ion spectra, CN- and Cl for the additive (CHX or Smear OFF), and the phosphate peaks
PO2 - (63 u) and PO3 - (79 u) for the substrate, are indicated in Figures 1C and D.
For the CHX group, the characteristic peaks for CHX are clearly observed whereas the substrate
phosphate peaks are greatly reduced, indicative of substantial coverage. The reverse occurs for
Smear OFF in that the substrate peaks are prominent and peaks assignable to CHX are greatly
reduced.
This observation is confirmed in the high imaging resolution mode, for both the positive and the
negative ions. An irregular precipitate was observed occluding the dentinal tubules in specimens
from the CHX group (Figs. 2A and C), whereas no precipitate and patent dentinal tubules were
seen in specimens from the Smear OFF group (Figs. 2B and D).
XPS analysis
The XPS survey spectra for the Smear OFF group (Fig. 3B) show an increase in the relative
intensity of Ca + and PO4 - peaks arising from the dentin substrate, compared to the spectra for the
CHX group (Fig. 3A), suggesting that the dentin in the Smear OFF group is not covered by
precipitate or thick (> 10 nm) film.
From the molecular formula of CHX (C22H30Cl2N10 ) the ratio of N/Cl is 5 and in PCA it is 1.96
(14). The ratio of N/Cl in the CHX group in all samples was smaller than 5. While not conclusive,
26
this finding was consistent with the presence of PCA. For the Smear OFF group, this ratio was not
assessed since nitrogen from the substrate would also be present.
The Cl 2p spectra (Figs. 3C and D) show the presence of covalent Cl and the chloride (Cl -). For
the Smear OFF group, the amount of Cl -relative to the covalent Cl was greater than that for the
CHX group. This implied a much thinner layer, as did the presence Ca and P peaks in the spectrum
and indicated that the over layer was less than the escape depth of the photoelectrons (<10 nm)
(31).
The C 1s envelope for Smear OFF (Fig. 3F) implied the presence of CHX since a shake-up feature,
characteristic of aromatic structures, was observed and the overall peak shape was similar to that
of CHX (Fig. 3E).
Discussion
Interaction between CHX and NaOCl has gained attention with the controversial finding of the
formation of PCA (12-14, 17-20). Even if PCA does not form, mixing these two irrigation agents
produces a visible insoluble precipitate which has been shown to occlude dentinal tubules and
discolor teeth (23), in addition to the possibility of its being toxic (24, 25). Therefore, it is
imperative that any new CHX-containing irrigation agent applied as a final root canal flush after
use of NaOCl should be assessed for its interaction with NaOCl. This study examined the
interaction of NaOCl and Smear OFF by assessing by-product formation on the dentin surface.
Smear OFF is an irrigation agent containing CHX gluconate (<1% wt),
tetrasodiumethylenediaminetetraacetate dihydrate (18% wt) and a surface-active detergent as its
active components (33). According to the manufacturer, it is formulated to have antibacterial effect
and to remove the smear layer of the root canal walls. An earlier report on its interaction with
NaOCl suggested that NaOCl lost the free available chlorine after alternating with either Smear
OFF or with EDTA (33).
TOF-SIMS analysis provides the composition, distribution and molecular information of the
analyzed surface. Analysis of the fragmentation pattern can result in the identification of the
various components (26). In a previous study (34) from our group, the fingerprint of CHX was the
peaks of 127 u, 153 u, 170 u, 195 u and its parent 505 u. The characteristic peak of PCA was 127
27
u (34). Since the precipitate in the present study showed peaks at 127 u, 153 u, 170 u and 195 u,
the peak at 127 u could not solely be used as characteristic of PCA. Therefore, comparison of the
relative heights and ratios of these peaks was used. In the precipitate, the peaks of 153 u, 170 u
and 195 u were less intense than the 127 u, in contrast to CHX where the signal of 127 u was less
intense than the rest of the peaks. Normally, from the CHX spectra, the peak height ratio for 170
u/127 u is 3:1, and for 153 u/127 u it is 3:2. In the present study, the 153 u/127 u ratio was more
than 1:1 and the 170 u/127 u ratio was 3:2, indicating a stronger signal coming from the 127 u
component suggestive of PCA. This fragmentation pattern was absent from the Smear OFF and
presence of PO2 - (63 u) and PO3 - (79 u) was noted, suggesting that the dentin was not covered by
a thick layer or precipitate. While Kolosowski et al. (17) analyzed cross sections of the dentin with
TOF-SIMS to assess penetration of precipitate into dentinal tubules, a similar analysis was not
performed for Smear OFF in the present study because no precipitate was observed.
XPS analysis was utilized to further investigate the source of the chlorine in the Smear OFF group,
which could be either CHX or NaOCl. For both PCA and CHX, chlorine should appear as the
covalent species, whereas the chloride reflects the presence NaOCl. Even though small amounts
of CHX exist in Smear OFF, the observed combination of both forms of chlorine suggested that
CHX was concentrated on the surface. Specific profiling was performed using a combined
mono/cluster Ar source. The absence of detectable changes for the CHX group implied the
formation of a thick layer over the dentin surface, the quick reduction in the amount of Cl for the
Smear OFF group implied that the surface was covered with just a few monolayers. These results
were consistent with presence of Ca and P peaks in the survey spectra for the Smear OFF group
and not in the CHX group.
The lack of precipitate formation in the Smear OFF group could be attributed to various reasons.
The surfactant forms a tertiary structure with CHX which protects the CHX from hypochlorite
anion (OCl-) attack and eliminates the formation of a precipitate. Moreover, the layer that the
detergent forms may be the one analyzed by TOF-SIMS. However, lack of detection of chlorine
could also be attributed to the possible small amount of CHX in the irrigation agent.
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The results of the present in vitro study suggest that Smear OFF can be safely applied after NaOCl,
as a final irrigant, without the need of an extra step of saline rinse. However, assessment of its
antibacterial properties is a research question that is prudent to be assessed in future studies.
Conclusion:
Within the limitations of this in-vitro study, the exposure of dentin surfaces to NaOCl followed by
Smear OFF did not result in formation of precipitate or PCA.
Acknowledgements
We acknowledge the Ontario Centre for the Characterisation of Advanced Materials (OCCAM)
for the conduction of the experiments, as well as Dr. Anil Kishen for valuable contribution to the
study design and Dr. Shimon Friedman and Dr Calvin Torneck for assistance with the manuscript.
29
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3. Zehnder M. Root canal irrigants. J Endod 2006;32(5):389-398.
4. Hidalgo E, Bartolome R, Dominguez C. Cytotoxicity mechanisms of sodium hypochlorite in cultured human dermal fibroblasts and its bactericidal effectiveness. Chemico-Biological Interactions 2002;139(3):265-282.
5. Ng YL, Mann V, Gulabivala K. A prospective study of the factors affecting outcomes of nonsurgical root canal treatment: part 1: periapical health. Int Endod J 2011;44(7):583-609.
6. Souza M, Cecchin D, Farina AP, Leite CE, Cruz FF, da Cunha Pereira C, et al. Evaluation of Chlorhexidine Substantivity on Human Dentin: A Chemical Analysis. J Endod 2012;38(9):1249- 1252.
7. Komorowski R, Grad H, Yu Wu X, Friedman S. Antimicrobial Substantivity of Chlorhexidine-Treated Bovine Root Dentin. J Endod;26(6):315-317.
8. White RR, Hays GL, Janer LR. Residual antimicrobial activity after canal irrigation with chlorhexidine. J Endod;23(4):229-231.
9. Löe H, Rindom Schiøtt C. The effect of mouthrinses and topical application of chlorhexidine on the development of dental plaque and gingivitis in man. J Periodontal Res 1970;5(2):79-83.
10. Haapasalo M, Shen Y, Qian W, Gao Y. Irrigation in endodontics. Dent Clin North Am 2010;54(2):291-312.
11. Kuruvilla JR, Kamath MP. Antimicrobial activity of 2.5% sodium hypochlorite and 0.2% chlorhexidine gluconate separately and combined, as endodontic irrigants. J Endod 1998;24(7):472-476.
12. Basrani BR, Manek S, Fillery E. Using diazotization to characterize the effect of heat or sodium hypochlorite on 2.0% chlorhexidine. J Endod 2009;35(9):1296-1299.
13. Basrani BR, Manek S, Mathers D, Fillery E, Sodhi RN. Determination of 4-chloroaniline and its derivatives formed in the interaction of sodium hypochlorite and chlorhexidine by using gas chromatography. J Endod 2010;36(2):312-314.
14. Basrani BR, Manek S, Sodhi RN, Fillery E, Manzur A. Interaction between sodium hypochlorite and chlorhexidine gluconate. J Endod 2007;33(8):966-969.
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15. Krishnamurthy S, Sudhakaran S. Evaluation and Prevention of the Precipitate Formed on Interaction between Sodium Hypochlorite and Chlorhexidine. J Endod 2010;36(7):1154-1157.
16. Mortenson D, Sadilek M, Flake NM, Paranjpe A, Heling I, Johnson JD, et al. The effect of using an alternative irrigant between sodium hypochlorite and chlorhexidine to prevent the formation of para-chloroaniline within the root canal system. Int Endod J 2012;45(9):878-882.
17. Kolosowski KP, Sodhi RN, Kishen A, Basrani BR. Qualitative analysis of precipitate formation on the surface and in the tubules of dentin irrigated with sodium hypochlorite and a final rinse of chlorhexidine or QMiX. J Endod 2014;40(12):2036-2040.
18. Prado M, Santos Júnior HM, Rezende CM, Pinto AC, Faria RB, Simão RA, et al. Interactions between Irrigants Commonly Used in Endodontic Practice: A Chemical Analysis. J Endod 2013;39(4):505-510.
19. Nowicki JB, Sem DS. An In Vitro Spectroscopic Analysis to Determine the Chemical Composition of the Precipitate Formed by Mixing Sodium Hypochlorite and Chlorhexidine. J Endod 2011;37(7):983-988.
20. Thomas JE, Sem DS. An In Vitro Spectroscopic Analysis to Determine Whether Para- Chloroaniline Is Produced from Mixing Sodium Hypochlorite and Chlorhexidine. J Endod 2010;36(2):315-317.
21. Arslan H, Uygun AD, Keskin A, Karatas E, Seçkin F, Yıldırım A. Evaluation of orange-brown precipitate formed in root canals after irrigation with chlorhexidine and QMix and spectroscopic analysis of precipitates produced by a mixture of chlorhexidine/NaOCl and QMix/NaOCl. Int Endod J 2015;48(12):1199-1203.
22. Nocca G, Ahmed HMA, Martorana GE, Callà C, Gambarini G, Rengo S, et al. Chromographic Analysis and Cytotoxic Effects of Chlorhexidine and Sodium Hypochlorite Reaction Mixtures. J Endod 2017;43(9):1545-1552.
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24. Matsumoto M, Aiso S, Senoh H, Yamazaki K, Arito H, Nagano K, et al. Carcinogenicity and Chronic Toxicity of para-chloronitrobenzene in Rats and Mice by Two-Year Feeding. J Environ Pathol Toxicol Oncol. 2006;25(3):571-584.
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27. Ratner BD, Castner DG. Electron Spectroscopy for Chemical Analysis. In: Vickerman JC, Gilmore IS, editors. Surface Analysis: The Principal Techniques. 2nd. ed. Chichester, U.K.: John Wiley & Sons, Ltd; 2009.
28. Gotliv BA, Robach JS, Veis A. The composition and structure of bovine peritubular dentin: mapping by time of flight secondary ion mass spectroscopy. J Struct Biol 2006;156(2):320-333.
29. Gotliv BA, Veis A. The composition of bovine peritubular dentin: matching TOF-SIMS, scanning electron microscopy and biochemical component distributions. New light on peritubular dentin function. Cells Tissues Organs 2009;189(1-4):12-19.
30. Gotliv BA, Veis A. Peritubular dentin, a vertebrate apatitic mineralized tissue without collagen: role of a phospholipid-proteolipid complex. Calcif Tissue Int 2007;81(3):191-205.
31. Sodhi RN, Grad HA, Smith DC. Examination by X-ray photoelectron spectroscopy of the adsorption of chlorhexidine on hydroxyapatite. J Dent Res 1992;71(8):1493-1497.
32. Bukiet F, Couderc G, Camps J, Tassery H, Cuisinier F, About I, et al. Wetting Properties and Critical Micellar Concentration of Benzalkonium Chloride Mixed in Sodium Hypochlorite. J Endod;38(11):1525-1529.
33. Krishnan U, Saji S, Clarkson R, Lalloo R, Moule AJ. Free Active Chlorine in Sodium Hypochlorite Solutions Admixed with Octenidine, SmearOFF, Chlorhexidine, and EDTA. J Endod 2017;43(8):1354-1359.
34. Sodhi R, Manek S, Fillery E, Basrani B. Tof-SIMS studies on chlorhexidine and its reaction products with sodium hypochlorite to ascertain decomposition products. Surface and InterfaceAnalysis 2011;43(1-2):591-594.
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Legends to Figures
Figure 1: Selected TOF-SIMS high mass resolution spectra of the treated dentin: Positive ion
spectra for (A) CHX group; (B) Smear OFF group; and the corresponding negative ion spectra
for (C) CHX and (D) Smear OFF. Peaks assignable to PCA, CHX and the substrate are indicated
– see text for details.
Figure 2: Selected TOF-SIMS high spatial resolution images of dentin surfaces: Negative ion
images for (A) CHX group; (B) Smear OFF; and the corresponding positive ion images for (C)
CHX and (D) Smear OFF. Presence of precipitate is observed in the CHX group whereas open
and patent dentinal tubules are observed in the Smear OFF group.
Figure 3: Survey spectra with relative atomic percentages for (A) CHX; (B) Smear OFF. Also
shown are the fitted high resolution peaks for the Cl 2p region (C and D) and the C 1s region (E
and F) for CHX and Smear OFF respectively – see text for details.
33
Figure 1
34
Figure 2
35
Figure 3:
36
DISCUSSION
Aim and Methodology
This in vitro study was conducted in order to examine whether precipitation occurs after rinse with
NaOCl followed by final rinse of Smear OFF and if PCA can be detected in the precipitate. Up
until this manuscript was written, this was the first study assessing formation of precipitate or PCA
using Smear OFF as a final rinse protocol. Previous studies from our group have assessed the
interaction after rinse with NaOCl followed by CHX (131, 133-135) and after final rinse with
QMiX (135). The results revealed that precipitate with PCA occurred after final rinse with CHX
whereas in case of QMiX no precipitation occurred.
A pilot study was first performed in order to assess the end result of combining 6% NaOCl
followed by Smear OFF in a plastic cup according to the manufacturer’s protocol. A reaction was
noticeable, with bubbling action occurring, probably because of the surfactant that the irrigant
contains and a color change of the solution to a more yellowish hue (pic. 4). No precipitate was
noted. However, the end result of a mixture between two or more products is not necessarily only
related to the products themselves. Dentin, inorganic components and proteins contained within,
can act as a catalyst allowing a reaction to occur, where the reaction would not otherwise occur in
the absence of dentin. Hydrogenation reactions are examples where a surface can act as a catalyst.
Therefore, a decision was made to study the precipitate on dentin. Following Kolosowski’s study
(135), dentinal blocks were created in order to eliminate the different confounding factors such as
grooves, curvatures and irregularities in the topographical aspect of the surface which could
potentially be source of contaminants. In addition, TOF-SIMS analysis requires flat surface
otherwise topographical shape will influence the direction of the ions hitting the surface of the
sample and emitted from the surface and hence the number of the ions reaching the detector.
Therefore, upper maxillary teeth were chosen for the experiments, since the dentinal tubules
exhibit more consistency in terms of the direction (161, 162). More specifically, dentinal tubules
travel through the dentin exhibiting an S shape. The shape and the size of them depends on the
location. They are conical in shape with the base being close to the pulp and they present with a
right angle and size of 3 µm whereas in the junction of the enamel the size is less than 1µm (161,
163, 164). The cervical dentin was chosen in order to have the maximum size dimeter of the
dentinal tubules and better assess the results. In Kolosowski’s et al. study (135), regarding the
37
CHX group analysis of penetration of the precipitate with cross sections of the dentinal samples
was also performed and penetration of the precipitate was confirmed. In the present study, TOF –
SIMS analysis of the cross sections was not pursued since no precipitate was found. The age of
the teeth could be an influencing factor. In that study, a bank of teeth anonymously donated was
used. Aging, progresses from the apical to the coronal part of the teeth and it results to sclerosis.
Previous study from our group using midroot dentin has shown that the results among the samples
were similar, possibly meaning that the effect of age was not very substantial.
The irrigation protocol that was used in the control group was based on the outcome study of Ng
et al.(12), penultimate EDTA increases the success rate. Thus, the irrigation protocol used 6 %
NaOCl, 17 % EDTA, 6 % NaOCl and final rinse of 2% CHX, and for the experimental group 6%
NaOCl and Smear OFF as per manufacture instructions. The concentrations used for this study,
followed the protocol that is used for EDTA and CHX. Regarding the concentration of NaOCl,
there is a variability in the concentration being used among dentists and endodontists. The
maximum concentration of 6% was decided to be used in that study, in order to maximize the
precipitate formation, since the amount of precipitate has been shown to be proportional to the
concentration of NaOCl (131). All the reported irrigants were used for 1 minute each for
standardization purposes. However, clinical times vary among the different clinicians. Regarding
the surface that was analyzed, cross sections of the dentinal samples were not prepared, since in
the Smear OFF group precipitate was not found and the penetration in the CHX group of the
precipitate was tested from previous study (135).
During the preparation of the dentinal blocks, various technical steps took place, which could have
provoked contamination of the analyzed surface. TOF-SIMS can be performed in various modes.
In our study it was decided to use sputter erosion with an Ar cluster source in order to remove any
surface contamination thereby simplifying the mass spectrum and making the surface composition
clearer. PCA is a volatile in nature and in high temperatures, such as in a vacuum environment it
may be sublimated before being detected. Thus, liquid nitrogen was used in order to cool the
samples(165). However, it has been shown, that even in ambient temperature it could be detected.
In order to further investigate the source of the chlorine in the Smear OFF group, which could be
either CHX or NaOCl, XPS analysis was utilized as well. XPS is another surface sensitive
analytical tool, however, in contrast to TOF-SIMS, it is readily quantifiable. Another advantage in
38
comparison to the TOF-SIMS, is that the analyzed surfaces do not have to be so precisely polished,
allowing for minor handling inaccuracies not to result into significant artifacts. Theoretically both
in PCA and in CHX, chlorine should be covalent (Clo). The chloride (Cl-) reflects the presence
NaOCl. There is a chemical shift between these components with the covalent Cl appearing at a
higher binding energy. Thus, it was possible to confirm the presence of CHX in Smear OFF group.
Results
PCA detection in the precipitate was done by assessing the fragmentation pattern of the high mass
resolution analysis. Previous study from our group has shown that the fingerprint of CHX are the
peaks 127 u, 153 u, 170 u, 195 u as well as the parent ion at 505 u (165). The precipitate also
shows these features. It can be argued that since the peak of 127 is fragment of both CHX as well
as being a characteristic peak for PCA, it cannot solely be used to determine the presence of PCA
(165). Therefore, comparison of the relative heights and ratios of these peaks was used. More
specifically, in the precipitate, the peaks of 153 u, 170 u and 195 u were less intense than the 127
u on the contrary to the CHX alone where the signal of 127 u is less intense than the rest of the
peaks. Normally, from the CHX spectra, there is a ratio of peak height 3:1 170 u to 127 u and 3:2
looking at 153 u to 127 u. In my study, the ratio 153u to 127u was more 1:1 and ratio of 170 u to
127 u was 3:2 which indicates stronger signal coming from 127 u component (meaning PCA).
Interestingly, in the precipitate we could also identify the 505 u peak, which is parental peak of
CHX, showing that possibly there was residual CHX in the precipitate.
With reference to the XPS analysis, addition of NaOCl to the CHX group should result in the
presence of some amounts of chloride (Cl). This reacts with the CHX and forms PCA resulting in
reduction of Clo. Even though small amount of CHX exists in Smear OFF group the combination
of Cl- and Clo could be seen, suggesting that the small amount of CHX is concentrated on the
surface. Some profiling was performed using a combined mono/cluster source. No significant
changes were observed for CHX implying a thick layer whereas the amount of Cl- was quickly
reduced for Smear OFF group implying a few monolayers coverage. These results are consistent
with calcium and phosphate ions from the substrate being observed in the Smear OFF treated
samples and not in CHX treated samples. The fact that no precipitate was formed in the Smear
OFF group can be because of various reasons. Smear OFF is composed of CHX, EDTA, a
surfactant acidic polymer and water. The surfactant forms a tertiary structure with CHX which
39
protects the CHX from hypochlorite anion (OCl-) attack and eliminates the formation of a
precipitate. Moreover, the layer that the detergent formed could have been the one analyzed from
TOF – SIMS. However, lack of detection of chlorine in TOF-SIMS could also be attributed to the
possible small amount of CHX in the irrigant.
40
CONCLUSIONS & FUTURE DIRECTION:
- Within the limitations of this in vitro study utilizing dentin blocks, TOF-SIMS and XPS
analyses have shown that precipitate, resulting from NaOCl being mixed with CHX, may
form on the root canal dentin surface. These results, confirm previous study from our
group using TOF-SIMS solely to examine that.
- TOF-SIMS and XPS have indicated this precipitate to contain PCA, and to occlude the
tubules on the dentin of the root canal surface when dentin blocks are irrigated with 6%
NaOCl, followed by 17% EDTA, 6% NaOCl and 2% CHX.
- It has been noted that no precipitatation occured and no PCA was detected on root canal
dentin surface with TOF-SIMS and XPS analyses when dentin is irrigated with 6%
NaOCl, followed by final rinse with Smear OFF.
- The results of the present in vitro study suggest that Smear OFF can be safely applied after
NaOCl, as a final irrigant, without the need of an extra step of saline rinse. However,
assessment of its antibacterial properties is a research question that is prudent to be
examined in future studies.
41
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APPENDICES Appendix 1:
Letter of ethics approval from Health Sciences Research Ethics Board (REB), University
Toronto
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Appendix 2: Molecular structures
Figure A): Molecular structure of Chlorhexidine (CHX)
Figure B): Molecular structure of Ethylenediaminetetraacetic acid (EDTA)
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Appendix 3: Pictures
Picture 1: Sequence to create the dentin blocks
Picture 2: Dentin blocks in resin
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Picture 3: Dentin polishing with glass knife with Leica EM UC6/FC6 Ultracryomicrotome
Picture 4: Brown precipitate after irrigation with NaOCl followed by CHX in plastic cup & tooth
Picture 5: Minor change of color in a yellowish hue after combination of NaOCl and Smear OFF
in plastic cup & tooth
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Picture 6: Samples being analyzed in TOF - SIMS
Picture 7: Leica EM UC6/FC6 Ultracryomicrotome
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Picture 8: TOF- SIMS- ION – TOF GmbH
Picture 9: ThermoFisher Scientific Escalab 250Xi
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Appendix 4: Figures
Figures with the high mass resolutions and high spatial resolution from TOF-SIMS and survey
spectra, carbon and chlorine fits from XPS analyses