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Qualitative Time-of-Flight Secondary Ion Mass Spectrometry Analysis of Root Dentin Irrigated with Sodium Hypochlorite, EDTA, or Chlorhexidine
Basic Research—Technology
Qualitative Time-of-flight Secondary Ion Mass Spectrometry Analysis of Root Dentin Irrigated with Sodium Hypochlorite, EDTA, or Chlorhexidine Kamil P. Kolosowski, HBSc, DDS, MSc,* Rana N.S. Sodhi, BSc, MSc, PhD,† Anil Kishen, BDS, MDS, PhD,* and Bettina R. Basrani, DDS, MSc, PhD* Abstract Introduction: Sodium hypochlorite (NaOCl), chelating agents, and chlorhexidine (CHX), which are commonly used irrigants during endodontic treatment, have the potential to alter the physical and chemical properties of the dentin structure. The aim of this study was to use time-of-flight secondary ion mass spectrometry to qualitatively evaluate the chemical characteristics of dentin surface and compare it with dentin exposed to NaOCl, EDTA, or CHX. Methods: Four blocks of dentin from a root of a human maxillary molar were embedded in resin and trimmed with a microtome to expose the dentin. Samples were randomly assigned to 4 treatment groups: (1) no irrigation treatment (sample A), (2) 2.5% NaOCl (sample B), (3) 17% EDTA (sample C), and (4) 2% CHX (sample D). Dentin surfaces were analyzed by timeof-flight secondary ion mass spectrometry, which allowed characterization of dentin surface chemistry by both imaging and mass spectroscopic analysis obtained in high mass and spatial resolution modes. Results: Sample A revealed intense peaks characteristics of hydroxyapatite in addition to Na+, K+, CH4N+, CN , CNO , Mg+, F , and HCO2 peaks. Sample B showed severely decreased CH4N+ and increased intensity of Cl . Sample C lacked Ca+ and Mg+ and showed decreased PO2 and PO3 . Sample D exhibited a distinct presence of CHX. The spectral image of sample A displayed even distribution of Na+ and Ca+ on a smeared surface. The surfaces of samples B and D had patent dentinal tubules, whereas sample D showed an intense CHX signal. Sample C had some patent dentinal tubules and lacked Ca+. Conclusions: NaOCl removed protein components from the dentin matrix, EDTA removed calcium and magnesium ions from the dentin, and CHX formed an adsorbed layer on the dentin surface. (J Endod 2015;-:1–6)
Key Words Chlorhexidine, dentin, EDTA, sodium hypochlorite, time-of-flight secondary ion mass spectrometry
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odium hypochlorite (NaOCl) is the root canal irrigant most commonly used in nonsurgical root canal therapy performed in North America. The other common irrigants used in endodontics are EDTA and chlorhexidine digluconate (CHX) (1). NaOCl is effective in killing microorganisms and removing necrotic organic tissue from the root canal (2, 3). In aqueous solution, NaOCl is in equilibrium with sodium hydroxide (NaOH) and hypochlorous acid (HOCl), which, in turn, are dissociated into their respective ions as follows: sodium (Na+), hydroxyl (OH ), hydrogen (H+), and hypochlorite (OCl ) (4). When dentin is subjected to NaOCl, it experiences a reduction in microhardness, flexural strength, and modulus of elasticity (5, 6). This alteration in the mechanical properties is mostly caused by degradation of collagen and glycosaminoglycans present in the dentin matrix (7). Irreversible erosion of the dentin microstructure is also detected, which may detrimentally affect the physical characteristics of the post-treated root dentin (8, 9). EDTA (C10H16N2O8) is a chelating and demineralizing agent and is used to remove the smear layer left behind on the root canal wall after instrumentation (10). When exposed to heavy metals or calcium ions, EDTA forms a ring-shaped structure that is stably bonded with a centrally positioned metal ion (11). When dentin is exposed to EDTA, it sequesters calcium and removes it from the solution, promoting dissolution of calcium hydroxyapatite and demineralization (11). Concentrations of 0.03% EDTA show some degree of decalcifying effect on the dentin, whereas with 10% EDTA the effect is sizable (12). CHX (C22H30Cl2N10) is used as an antimicrobial irrigating solution (13). Because of the cationic nature of the CHX molecule, it can be absorbed onto hydroxyapatite present in teeth. At low concentrations (<0.02%), a stable monolayer of CHX is formed on the surface. Above that concentration, a multilayer of CHX is formed from which it can be slowly released with time (14, 15). The uptake and release of CHX from a substrate is a reversible action and may explain its longterm substantivity (16). Nevertheless, unlike NaOCl, organic tissue dissolution does not occur when CHX irrigation is used (17, 18). Dentin is composed of approximately 70% (by weight) inorganic material, most of which is calcium hydroxyapatite (Ca10[PO4]6[OH]2) (19). Other minerals and elements found in dentin include nitrogen (2.42%–3.64%), carbonate (2.99%– 3.25%), zinc (300 ppm), and fluoride (F ) (100–500 ppm) (20, 21). About 20%
From the *Discipline of Endodontics, Faculty of Dentistry and †Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada. Address requests for reprints to Dr Bettina R. Basrani, Department of Endodontics, University of Toronto, Toronto, ON M5G 1G6, Canada. E-mail address: bettina. [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.06.010
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Basic Research—Technology of dentin consists of organic materials, which includes various collagenous and noncollagenous proteins, and proteoglycans. The remaining 10% is water in free or bound form (19). Scanning electron microscopic analysis (8–10) and dentin hardness testing (5, 6) are often used to study the ultrastructure and mechanical effects of irrigation agents on dentin. However, these tests fail to provide information on the chemical compositional changes in dentin. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) combines the advantages of scanning electron microscopy in providing high-resolution images of the test surface and mass spectroscopy in providing highly sensitive profiles of the chemical constituents. Analysis can be used to distinguish and identify inorganics such as calcium hydroxyapatite, organic proteins, and other chemical compounds (22–24). In dentistry, TOF-SIMS has been used to study the elemental constituents of dentin (25–27) and determine the composition of a precipitate formed when NaOCl is mixed with CHX (28). A previous study mapped the chemical characteristics of bovine dentin using TOF-SIMS technology (25–27). Another recent study analyzed human dentin with TOF-SIMS after irrigation with NaOCl and CHX for precipitate formation on the dentin surface and in the dentinal tubules (DTs) (29). It was highlighted that TOFSIMS explores the chemical elements and compounds present on the surface of dental hard tissues (27). TOF-SIMS was also used to study bone, dentin, and the ion charge distribution of certain elements and functionalities such as calcium (Ca), sodium (Na), and phosphates (PO2 and PO3) in those tissues (25–27). In TOF-SIMS, a focused, pulsed primary ion beam is directed at the surface of the target under study and rastered over the area. The beam penetrates the target surface to produce a collision cascade. The energy
transfer results in atomic and molecular fragments being emitted from the top 1 to 2 monolayers of the surface, a small fraction of which are ionized. These are mass analyzed by measuring the time of flight of the ion to an ion detector. The heavier the fragment, the longer it takes to reach the detector. Typical primary ion sources are bismuth (Bin+) liquid metal ion guns, which allow for high spatial resolution as well as high mass resolution. Identifying characteristic fragments associated with the various substances present on the surface allows a mapping of the distribution of the various components (24). The aim of the current study was to use TOF-SIMS to qualitatively evaluate the surface of untreated root dentin and compare it with root dentin treated with NaOCl, EDTA, or CHX. The information obtained from this study will be crucial to understand the effect of chemicals on dentin surface (14, 15, 29) and their role in bonding, sealing (30), cell adhesion, and healing potential of endodontic therapy (31, 32).
Materials and Methods Preparation of Samples The study was approved by the University Health Sciences Research Ethics Board. A noncarious human upper molar was sectioned at the midroot level to obtain a 2-mm-thick horizontal slice using a diamond coated saw (Leica EM TXP Target Sectioning System; Leica Microsystems GmbH, Vienna, Austria). The slice was further sectioned to create 4 dentin blocks, which were then embedded in low-viscosity epoxy resin (Epo-Thin; Buehler, Lake Bluff, IL) prepared in accordance with the manufacturer’s instructions and allowed to set for 24 hours. Blocks were then placed in a microtome (Leica EM UC6/FC6
Figure 1. TOF-SIMS positive ion mass spectra of dentin surfaces with high mass resolution. (A) Sample A (NT, no irrigation), (B) sample B (NaOCl), (C) sample C (EDTA), and (D) sample D (CHX). Marked are the positions of peaks assignable to Na+ at 23u, Mg+ at 24u, C4HN+ at 30u, K+ at 39u, Ca+ at 40u, CaOH+ at 57u, and CHX at 505u. Note the lack of C4HN+ in B (NaOCl), the lack of Ca+ and Mg+ in C (EDTA), and the abundance of CHX in D (CHX).
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Basic Research—Technology Ultra-cryomicrotome, Leica Microsystems GmbH) and sectioned to expose the root canal aspect of the dentin for further analysis.
Irrigation of Samples Dentin blocks were chosen at random for different treatment as follows: Sample A: No irrigation treatment was used (NT). Sample B: Dentin was immersed in 5 mL 2.5% NaOCl (Lavo Inc, Montreal, Quebec, Canada) for 3 minutes (NaOCl). Sample C: Dentin was immersed in 5 mL 17% EDTA (Vista Dental Products, Racine, WI) for 1 minute (EDTA). Sample D: Dentin was immersed in 5 mL 2% CHX (Chlorhexidine Digluconate BP; Medisca, Montreal, Quebec, Canada) for 1 minute (CHX). All the samples were left on the bench top overnight before TOFSIMS analysis.
TOF-SIMS Analysis In this study, dentin surface analysis was performed by TOF-SIMS (TOF-SIMS IV; ION-TOF GmbH, M€unster, Germany). Bi3++ cluster primary ion source was used with a bismuth (Bi) liquid metal ion gun operated in a high mass resolution bunched mode over an area of 500 mm 500 mm for 100 seconds. Positive and negative polarity spectra were obtained. Additionally, a high spatial resolution imaging mode (‘‘burst alignment’’) was used to obtain positive polarity spectral images (256 pixels 256 pixels) from 20 scans over an area of 150 mm 150 mm (24). A pulsed electron flood gun was used for charge neutral-
ization. The calibration of the mass scale was performed using standard, identifiable, and well-spaced peaks found in all the spectra.
Results TOF-SIMS: High Mass Resolution Analysis Sample A (NT). The positive ion analysis of dentin revealed intense peaks of Na+ (23u), K+ (39u), Ca+ (40u), and CH4N+ (30u); a peak that could be attributed to CaOH+ (57u), and a minor peak of Mg+ (24u) (Fig. 1A). The negative ion analysis of NT dentin revealed intense peaks of CN (26u), CNO (42u), PO2 (63u), and PO3 (79u) in addition to minor F (19u) and HCO2 (45u) peaks (Fig. 2A). Sample B (NaOCl). The positive ion analysis of NaOCl-treated dentin showed an intense peak of Na+ (23u) with decreased detection of K+ (39u), Ca+ (40u), CaOH+ (57u), and Mg+ (24u) and severely decreased CH4N+ (30u) (Fig. 1B) compared with NT. Negative ion analysis of sample B dentin (NaOCl) revealed relatively unchanged peak distribution when compared with NT, except for the increased intensity, indicating the presence of Cl (35 and 37u) (Fig. 2B). Sample C (EDTA). The positive ion analysis of EDTA-treated dentin showed an intense Na+ (23u) peak and less intense K+ (39u), CaOH+ (57u), and CH4N+ (30u) than NT. Ca+ (40u) and Mg+ (24u) were not detected in significant amounts (Fig. 1C). Additionally, in negative ion analysis of sample C (EDTA), PO2 (63u) and PO3 (79u) detection was decreased, with relatively unchanged intensity of CN (26u), CNO (42u), F (19u), and HCO2 (45u) (Fig. 2C). Sample D (CHX). Compared with NT, CHX-treated dentin showed a slight decrease in K+ (39u), Ca+ (40u), and CaOH+ (57u) with unchanged intensity of Na+ (23u), Mg+ (24u), and CH4N+ (30u) in positive ion
Figure 2. TOF-SIMS negative ion mass spectra of dentin surfaces with high mass resolution. (A) Sample A (NT, no irrigation), (B) sample B (NaOCl), (C) sample C (EDTA), and (D) sample D (CHX). Marked are the positions of peaks assignable to F at 19u, CN at 26u, Cl at 35u and 37u, CNO at 42u, HCO2 at 45u, PO2 at 63u, and PO3 at 79u. Note the increase in Cl in B (NaOCl) and D (CHX) and the decrease of PO2 and PO3 in C (EDTA).
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Basic Research—Technology analysis (Fig. 1D). There was also an appearance of peaks characteristic of CHX (505u) in this case (Fig. 1D inset). In the negative ion analysis, sample D (CHX) displayed an increased intensity of Cl (35 and 37u) and relatively unchanged intensities of peaks attributable to CN (26u), CNO (42u), PO2 (63u), PO3 (79u), F (19u), and HCO2 (45u) (Fig. 2D).
TOF-SIMS: High Spatial Resolution Analysis In positive polarity spectra images of sample A (NT), the DTs that were rather obstructed were observed on the dentin surface.
Na+ and Ca+ were evenly distributed on the surface in significant amounts (Fig. 3A). Patent DTs were clearly visualized on the surface of sample B (NaOCl), and Na+ intensity appeared to be increased on the lower portion of multiple DTs, whereas Ca+ was evenly distributed (Fig. 3B). Sample C (EDTA) displayed a surface with some patent DTs. Multiple tubules were not patent. Ca+ was scarce, and Na+ was evenly distributed on the dentin surface (Fig. 3C). Sample D (CHX) surface exhibited multiple patent DTs, and Ca+ was evenly distributed around the DTs; Na+ is concentrated in a few isolated DTs and in several isolated linear
Figure 3. High spatial resolution TOF-SIMS images of positive ion distribution on dentin surface. (A) Sample A (NT, no irrigation), (B) sample B (NaOCl), (C) sample C (EDTA), and (D) sample D (CHX). ‘‘Total’’ shows raw image; ClC6H4NH2+ shows distribution of CHX. Note the lack of visible tubules in A (NT) and the decrease in visible tubules in C (EDTA).
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Basic Research—Technology areas. An intense signal originating from CHX was evenly distributed on the dentin surface (Fig. 3D).
Discussion The advantages of using TOF-SIMS on dentin included its high sensitivity, its ability to analyze the chemical structure of 1 to 2 monolayers, its high spatial and mass resolution, and its ability to provide a chemical map of the surface (22–24). However, good mass resolution can present a problem if the surface of the sample is contaminated. In this study, the intent was to analyze the surface of untreated and treated dentin and avoid contamination by careful handling and storage of the specimens. Before this study, there were no published studies that examined human dentin and human dentin treated with NaOCl, EDTA, or CHX by TOF-SIMS analysis. A criticism of the current study may be the sample size, which consisted of only 1 specimen in each treatment group. However, this is consistent with other studies that have used TOF-SIMS analysis (22, 25–27). In addition, we used dentin from only 1 tooth. Although it afforded the study a measure of standardization, it did not account for variations in chemical composition that may be present among different teeth. To avoid measurement of artifacts, our high mass resolution size was 500 mm 500 mm; we obtained relatively even distribution of ions on the entire surface. This can be visualized in 150 mm 150 mm high spatial resolution views. The analysis of dentin not treated with irrigation (NT) was consistent with the findings from other studies (20, 21) showing that dentin contains Na+ (23u), K+ (39u), Ca+ (40u), Mg+ (24u), and F (19u) and hydroxyapatite phosphorous oxides groups PO2 (63u) and PO3 (79u). Another study using TOF-SIMS also characterized dentin to possess intensive levels of Na+, K+, Ca+, Mg+, CN , CNO , PO2 , PO3 , and HCO2 (25–27). Gotliv et al (25–27) found a differential distribution of ions with regard to their position in peritubular and intertubular dentin, which appeared in part in NaOCl-treated dentin in our study. However, Gotliv et al (25–27) used bovine dentin with a 5-time higher resolution imaging than the present study. The NaOCl-treated dentin showed an expected increased intensity of Na+ (23u) and Cl (35 and 37u). Signal from the common dentinal protein fragment CH4N+ (30u) almost disappeared, indicating that degradation of proteins present on the dentin surface occurred. This was consistent with previous studies (7) although the presence of CN (26u) and CNO (42u) showed that some protein fragments were still present on the surface. As shown by a previous study (11), treatment with EDTA removed most of the Ca+ (40u) and Mg+ (24u) and to a moderate extent PO2 (63u) and PO3 (79u) on the dentin surface, changes that were typical of demineralization. On the other hand, CHX treatment of the dentin resulted in a CHX (505u) signal detected on the surface, which was consistent with the current theory that CHX forms an adsorbed layer on the dentin surface when it is used in endodontic treatment (14, 15). The increase in Cl signal (35 and 37u) subsequent to CHX and NaOCl treatments was also expected and was attributed to the chlorine component of both irrigants. A uniformly distributed smearlike layer was present on the surface of untreated dentin (NT) and was most likely produced during specimen preparation. Surprisingly, DTs were visible after the dentin was treated with CHX, which is classified as neither a hard or organic tissue removing agent. The use of an organic tissue removing agent (NaOCl) allowed patent tubules to be clearly seen, whereas the hard tissue removing agent (EDTA) left a reduced number of patent tubules. It is likely that the protein components present inside the DTs hindered the EDTA effect within the DTs. JOE — Volume -, Number -, - 2015
The findings from this study showed a distinct difference in the chemical characteristics of surface dentin after treatment with different root canal irrigants. Changes in the chemical composition of the dentin after root canal irrigation can affect the viability of cells seeded onto the surface of the dentin (31, 32) and therefore should be of concern in pulp regenerative treatment. Increased intensities of Na+ (23u) and Cl (35 and 37u), which were NaOCl breakdown products (4) observed after irrigation with NaOCl, could be accountable for the reduced viability of cells (31, 32). It was also possible that the residual layer of CHX noted in the current study could create an unfavorable environment for cell viability and cell adhesion (31). This may perhaps explain why CHX irrigation results in a poorer treatment outcome in one study (33). It would be of interest to have future studies analyzing higher spatial resolution images of the dentin surface after irrigation with NaOCl and EDTA, in addition to the combined irrigation with both NaOCl and EDTA. This could allow a closer inspection of chemical changes occurring around the tubule during dissolution of soft and hard tissue during root canal therapy. In summary, TOF-SIMS of untreated dentin (NT) shows the presence of Na+, K+, Ca+, Mg+, CH4N+, PO2 , PO3 , CN , CNO , HCO2 , and F . After treatment with NaOCl, there is a degradation of matrix protein with the removal of CH4N+. EDTA removes Ca+ and Mg+ from dentin, and CHX treatment leaves behind a layer of CHX on the dentin surface.
Acknowledgments The authors thank the Surface Interface Ontario for the assistance in the conduct of these experiments and Dr Torneck for his feedback on the manuscript. Supported by a grant from Canadian Academy of Endodontics Endowment Fund and Endo Tech. The authors deny any conflicts of interest related to this study.
References 1. Dutner J, Mines P, Anderson A. Irrigation trends among American Association of Endodontists members: a web-based survey. J Endod 2012;38:37–40. 2. Bystr€om A, Sundqvist G. Bacteriologic evaluation of the effect of 0.5 percent sodium hypochlorite in endodontic therapy. Oral Surg Oral Med Oral Pathol 1983;55: 307–12. 3. Gordon TM, Damato D, Christner P. Solvent effect of various dilutions of sodium hypochlorite on vital and necrotic tissue. J Endod 1981;7:466–9. 4. Estrela C, Estrela CR, Barbin EL, et al. Mechanism of action of sodium hypochlorite. Braz Dent J 2002;13:113–7. 5. Zaparolli D, Saquy PC, Cruz-Filho AM. Effect of sodium hypochlorite and EDTA irrigation, individually and in alternation, on dentin microhardness at the furcation area of mandibular molars. Braz Dent J 2012;23:654–8. 6. Slutzky-Goldberg I, Maree M, Liberman R, Heling I. Effect of sodium hypochlorite on dentin microhardness. J Endod 2004;30:880–2. 7. Oyarzun A, Cordero AM, Whittle M. Immunohistochemical evaluation of the effects of sodium hypochlorite on dentin collagen and glycosaminoglycans. J Endod 2002; 28:152–6. 8. Niu W, Yoshioka T, Kobayashi C, Suda H. A scanning electron microscopic study of dentinal erosion by final irrigation with EDTA and NaOCl solutions. Int Endod J 2002;35:934–9. 9. Qian W, Shen Y, Haapasalo M. Quantitative analysis of the effect of irrigant solution sequences on dentin erosion. J Endod 2011;37:1437–41. 10. Baumgartner JC, Mader CL. A scanning electron microscopic evaluation of four root canal irrigation regimens. J Endod 1987;13:147–57. 11. H€ulsmann M, Heckendorff M, Lennon A. Chelating agents in root canal treatment: mode of action and indications for their use. Int Endod J 2003;36:810–30. 12. Patterson SS. In vivo and in vitro studies of the effect of the disodium slat of ethylenediamine tetra-acetate on human dentine and its endodontic implications. Oral Surg Oral Med Oral Pathol 1963;16:83–103. 13. Jordan RA, Holzner AL, Markovic L, et al. Clinical effectiveness of basic root canal treatment after 24 months: a randomized controlled trial. J Endod 2014;40: 465–70.
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Basic Research—Technology 14. Emilson CG, Ericson T, Heyden G, Magnusson BC. Uptake of chlorhexidine to hydroxyapatite. J Periodontal Res Suppl 1973;12:17–21. 15. Lin S, Levin L, Weiss EI, et al. In vitro antibacterial efficacy of a new chlorhexidine slow-release device. Quintessence Int 2006;37:391–4. 16. White RR, Hays GL, Janer LR. Residual antimicrobial activity after canal irrigation with chlorhexidine. J Endod 1997;23:229–31. 17. Naenni N, Thoma K, Zehnder M. Soft tissue dissolution capacity of currently used and potential endodontic irrigants. J Endod 2004;30:785–7. 18. Okino LA, Siqueira EL, Santos M, et al. Dissolution of pulp tissue by aqueous solution of chlorhexidine digluconate and chlorhexidine digluconate gel. Int Endod J 2004; 37:38–41. 19. Linde A, Goldberg M. Dentinogenesis. Crit Rev Oral Biol Med 1993;4:679–728. 20. Coklica V, Brudevold F, Amdur BH. The distribution and composition of density fractions from human crown dentine. Arch Oral Biol 1969;14:451–60. 21. Foreman PC, Soames JV. Comparative study of the composition of primary and secondary dentine. Caries Res 1989;23:1–4. 22. Eriksson C, Malmberg P, Nygren H. Time-of-flight secondary ion mass spectrometric analysis of the interface between bone and titanium implants. Rapid Commun Mass Spectrom 2008;22:943–9. 23. Malmberg P, Nygren H. Methods for the analysis of the composition of bone tissue, with a focus on imaging mass spectrometry (TOF-SIMS). Proteomics 2008;8: 3755–62. 24. Sodhi RN. Time-of-flight secondary ion mass spectrometry (TOF-SIMS):–versatility in chemical and imaging surface analysis. Analyst 2004;129:483–7.
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25. 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;156:320–33. 26. Gotliv BA, Veis A. Peritubular dentin, a vertebrate apatitic mineralized tissue without collagen: role of a phospholipid-proteolipid complex. Calcif Tissue Int 2007;81: 191–205. 27. Gotliv BA, Veis A. The composition of bovine peritubular dentin: matching TOFSIMS, scanning electron microscopy and biochemical component distributions. New light on peritubular dentin function. Cells Tissues Organs 2009;189:12–9. 28. Basrani BR, Manek S, Sodhi RN, et al. Interaction between sodium hypochlorite and chlorhexidine gluconate. J Endod 2007;33:966–9. 29. 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;40:2036–40. 30. Eldeniz AU, Erdemir A, Belli S. Shear bond strength of three resin based sealers to dentin with and without the smear layer. J Endod 2005;31:293–6. 31. Trevino EG, Patwardhan AN, Henry MA, et al. Effect of irrigants on the survival of human stem cells of the apical papilla in a platelet-rich plasma scaffold in human root tips. J Endod 2011;37:1109–15. 32. Huang X, Zhang J, Huang C, et al. Effect of intracanal dentine wettability on human dental pulp cell attachment. Int Endod J 2012;45:346–53. 33. 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: 583–609.
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Qualitative Time-of-flight Secondary Ion Mass Spectrometry Analysis of Root Dentin Irrigated with Sodium Hypochlorite, EDTA, or Chlorhexidine Kamil P. Kolosowski, HBSc, DDS, MSc,* Rana N.S. Sodhi, BSc, MSc, PhD,† Anil Kishen, BDS, MDS, PhD,* and Bettina R. Basrani, DDS, MSc, PhD* Abstract Introduction: Sodium hypochlorite (NaOCl), chelating agents, and chlorhexidine (CHX), which are commonly used irrigants during endodontic treatment, have the potential to alter the physical and chemical properties of the dentin structure. The aim of this study was to use time-of-flight secondary ion mass spectrometry to qualitatively evaluate the chemical characteristics of dentin surface and compare it with dentin exposed to NaOCl, EDTA, or CHX. Methods: Four blocks of dentin from a root of a human maxillary molar were embedded in resin and trimmed with a microtome to expose the dentin. Samples were randomly assigned to 4 treatment groups: (1) no irrigation treatment (sample A), (2) 2.5% NaOCl (sample B), (3) 17% EDTA (sample C), and (4) 2% CHX (sample D). Dentin surfaces were analyzed by timeof-flight secondary ion mass spectrometry, which allowed characterization of dentin surface chemistry by both imaging and mass spectroscopic analysis obtained in high mass and spatial resolution modes. Results: Sample A revealed intense peaks characteristics of hydroxyapatite in addition to Na+, K+, CH4N+, CN , CNO , Mg+, F , and HCO2 peaks. Sample B showed severely decreased CH4N+ and increased intensity of Cl . Sample C lacked Ca+ and Mg+ and showed decreased PO2 and PO3 . Sample D exhibited a distinct presence of CHX. The spectral image of sample A displayed even distribution of Na+ and Ca+ on a smeared surface. The surfaces of samples B and D had patent dentinal tubules, whereas sample D showed an intense CHX signal. Sample C had some patent dentinal tubules and lacked Ca+. Conclusions: NaOCl removed protein components from the dentin matrix, EDTA removed calcium and magnesium ions from the dentin, and CHX formed an adsorbed layer on the dentin surface. (J Endod 2015;-:1–6)
Key Words Chlorhexidine, dentin, EDTA, sodium hypochlorite, time-of-flight secondary ion mass spectrometry
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odium hypochlorite (NaOCl) is the root canal irrigant most commonly used in nonsurgical root canal therapy performed in North America. The other common irrigants used in endodontics are EDTA and chlorhexidine digluconate (CHX) (1). NaOCl is effective in killing microorganisms and removing necrotic organic tissue from the root canal (2, 3). In aqueous solution, NaOCl is in equilibrium with sodium hydroxide (NaOH) and hypochlorous acid (HOCl), which, in turn, are dissociated into their respective ions as follows: sodium (Na+), hydroxyl (OH ), hydrogen (H+), and hypochlorite (OCl ) (4). When dentin is subjected to NaOCl, it experiences a reduction in microhardness, flexural strength, and modulus of elasticity (5, 6). This alteration in the mechanical properties is mostly caused by degradation of collagen and glycosaminoglycans present in the dentin matrix (7). Irreversible erosion of the dentin microstructure is also detected, which may detrimentally affect the physical characteristics of the post-treated root dentin (8, 9). EDTA (C10H16N2O8) is a chelating and demineralizing agent and is used to remove the smear layer left behind on the root canal wall after instrumentation (10). When exposed to heavy metals or calcium ions, EDTA forms a ring-shaped structure that is stably bonded with a centrally positioned metal ion (11). When dentin is exposed to EDTA, it sequesters calcium and removes it from the solution, promoting dissolution of calcium hydroxyapatite and demineralization (11). Concentrations of 0.03% EDTA show some degree of decalcifying effect on the dentin, whereas with 10% EDTA the effect is sizable (12). CHX (C22H30Cl2N10) is used as an antimicrobial irrigating solution (13). Because of the cationic nature of the CHX molecule, it can be absorbed onto hydroxyapatite present in teeth. At low concentrations (<0.02%), a stable monolayer of CHX is formed on the surface. Above that concentration, a multilayer of CHX is formed from which it can be slowly released with time (14, 15). The uptake and release of CHX from a substrate is a reversible action and may explain its longterm substantivity (16). Nevertheless, unlike NaOCl, organic tissue dissolution does not occur when CHX irrigation is used (17, 18). Dentin is composed of approximately 70% (by weight) inorganic material, most of which is calcium hydroxyapatite (Ca10[PO4]6[OH]2) (19). Other minerals and elements found in dentin include nitrogen (2.42%–3.64%), carbonate (2.99%– 3.25%), zinc (300 ppm), and fluoride (F ) (100–500 ppm) (20, 21). About 20%
From the *Discipline of Endodontics, Faculty of Dentistry and †Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada. Address requests for reprints to Dr Bettina R. Basrani, Department of Endodontics, University of Toronto, Toronto, ON M5G 1G6, Canada. E-mail address: bettina. [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.06.010
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Basic Research—Technology of dentin consists of organic materials, which includes various collagenous and noncollagenous proteins, and proteoglycans. The remaining 10% is water in free or bound form (19). Scanning electron microscopic analysis (8–10) and dentin hardness testing (5, 6) are often used to study the ultrastructure and mechanical effects of irrigation agents on dentin. However, these tests fail to provide information on the chemical compositional changes in dentin. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) combines the advantages of scanning electron microscopy in providing high-resolution images of the test surface and mass spectroscopy in providing highly sensitive profiles of the chemical constituents. Analysis can be used to distinguish and identify inorganics such as calcium hydroxyapatite, organic proteins, and other chemical compounds (22–24). In dentistry, TOF-SIMS has been used to study the elemental constituents of dentin (25–27) and determine the composition of a precipitate formed when NaOCl is mixed with CHX (28). A previous study mapped the chemical characteristics of bovine dentin using TOF-SIMS technology (25–27). Another recent study analyzed human dentin with TOF-SIMS after irrigation with NaOCl and CHX for precipitate formation on the dentin surface and in the dentinal tubules (DTs) (29). It was highlighted that TOFSIMS explores the chemical elements and compounds present on the surface of dental hard tissues (27). TOF-SIMS was also used to study bone, dentin, and the ion charge distribution of certain elements and functionalities such as calcium (Ca), sodium (Na), and phosphates (PO2 and PO3) in those tissues (25–27). In TOF-SIMS, a focused, pulsed primary ion beam is directed at the surface of the target under study and rastered over the area. The beam penetrates the target surface to produce a collision cascade. The energy
transfer results in atomic and molecular fragments being emitted from the top 1 to 2 monolayers of the surface, a small fraction of which are ionized. These are mass analyzed by measuring the time of flight of the ion to an ion detector. The heavier the fragment, the longer it takes to reach the detector. Typical primary ion sources are bismuth (Bin+) liquid metal ion guns, which allow for high spatial resolution as well as high mass resolution. Identifying characteristic fragments associated with the various substances present on the surface allows a mapping of the distribution of the various components (24). The aim of the current study was to use TOF-SIMS to qualitatively evaluate the surface of untreated root dentin and compare it with root dentin treated with NaOCl, EDTA, or CHX. The information obtained from this study will be crucial to understand the effect of chemicals on dentin surface (14, 15, 29) and their role in bonding, sealing (30), cell adhesion, and healing potential of endodontic therapy (31, 32).
Materials and Methods Preparation of Samples The study was approved by the University Health Sciences Research Ethics Board. A noncarious human upper molar was sectioned at the midroot level to obtain a 2-mm-thick horizontal slice using a diamond coated saw (Leica EM TXP Target Sectioning System; Leica Microsystems GmbH, Vienna, Austria). The slice was further sectioned to create 4 dentin blocks, which were then embedded in low-viscosity epoxy resin (Epo-Thin; Buehler, Lake Bluff, IL) prepared in accordance with the manufacturer’s instructions and allowed to set for 24 hours. Blocks were then placed in a microtome (Leica EM UC6/FC6
Figure 1. TOF-SIMS positive ion mass spectra of dentin surfaces with high mass resolution. (A) Sample A (NT, no irrigation), (B) sample B (NaOCl), (C) sample C (EDTA), and (D) sample D (CHX). Marked are the positions of peaks assignable to Na+ at 23u, Mg+ at 24u, C4HN+ at 30u, K+ at 39u, Ca+ at 40u, CaOH+ at 57u, and CHX at 505u. Note the lack of C4HN+ in B (NaOCl), the lack of Ca+ and Mg+ in C (EDTA), and the abundance of CHX in D (CHX).
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Basic Research—Technology Ultra-cryomicrotome, Leica Microsystems GmbH) and sectioned to expose the root canal aspect of the dentin for further analysis.
Irrigation of Samples Dentin blocks were chosen at random for different treatment as follows: Sample A: No irrigation treatment was used (NT). Sample B: Dentin was immersed in 5 mL 2.5% NaOCl (Lavo Inc, Montreal, Quebec, Canada) for 3 minutes (NaOCl). Sample C: Dentin was immersed in 5 mL 17% EDTA (Vista Dental Products, Racine, WI) for 1 minute (EDTA). Sample D: Dentin was immersed in 5 mL 2% CHX (Chlorhexidine Digluconate BP; Medisca, Montreal, Quebec, Canada) for 1 minute (CHX). All the samples were left on the bench top overnight before TOFSIMS analysis.
TOF-SIMS Analysis In this study, dentin surface analysis was performed by TOF-SIMS (TOF-SIMS IV; ION-TOF GmbH, M€unster, Germany). Bi3++ cluster primary ion source was used with a bismuth (Bi) liquid metal ion gun operated in a high mass resolution bunched mode over an area of 500 mm 500 mm for 100 seconds. Positive and negative polarity spectra were obtained. Additionally, a high spatial resolution imaging mode (‘‘burst alignment’’) was used to obtain positive polarity spectral images (256 pixels 256 pixels) from 20 scans over an area of 150 mm 150 mm (24). A pulsed electron flood gun was used for charge neutral-
ization. The calibration of the mass scale was performed using standard, identifiable, and well-spaced peaks found in all the spectra.
Results TOF-SIMS: High Mass Resolution Analysis Sample A (NT). The positive ion analysis of dentin revealed intense peaks of Na+ (23u), K+ (39u), Ca+ (40u), and CH4N+ (30u); a peak that could be attributed to CaOH+ (57u), and a minor peak of Mg+ (24u) (Fig. 1A). The negative ion analysis of NT dentin revealed intense peaks of CN (26u), CNO (42u), PO2 (63u), and PO3 (79u) in addition to minor F (19u) and HCO2 (45u) peaks (Fig. 2A). Sample B (NaOCl). The positive ion analysis of NaOCl-treated dentin showed an intense peak of Na+ (23u) with decreased detection of K+ (39u), Ca+ (40u), CaOH+ (57u), and Mg+ (24u) and severely decreased CH4N+ (30u) (Fig. 1B) compared with NT. Negative ion analysis of sample B dentin (NaOCl) revealed relatively unchanged peak distribution when compared with NT, except for the increased intensity, indicating the presence of Cl (35 and 37u) (Fig. 2B). Sample C (EDTA). The positive ion analysis of EDTA-treated dentin showed an intense Na+ (23u) peak and less intense K+ (39u), CaOH+ (57u), and CH4N+ (30u) than NT. Ca+ (40u) and Mg+ (24u) were not detected in significant amounts (Fig. 1C). Additionally, in negative ion analysis of sample C (EDTA), PO2 (63u) and PO3 (79u) detection was decreased, with relatively unchanged intensity of CN (26u), CNO (42u), F (19u), and HCO2 (45u) (Fig. 2C). Sample D (CHX). Compared with NT, CHX-treated dentin showed a slight decrease in K+ (39u), Ca+ (40u), and CaOH+ (57u) with unchanged intensity of Na+ (23u), Mg+ (24u), and CH4N+ (30u) in positive ion
Figure 2. TOF-SIMS negative ion mass spectra of dentin surfaces with high mass resolution. (A) Sample A (NT, no irrigation), (B) sample B (NaOCl), (C) sample C (EDTA), and (D) sample D (CHX). Marked are the positions of peaks assignable to F at 19u, CN at 26u, Cl at 35u and 37u, CNO at 42u, HCO2 at 45u, PO2 at 63u, and PO3 at 79u. Note the increase in Cl in B (NaOCl) and D (CHX) and the decrease of PO2 and PO3 in C (EDTA).
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Basic Research—Technology analysis (Fig. 1D). There was also an appearance of peaks characteristic of CHX (505u) in this case (Fig. 1D inset). In the negative ion analysis, sample D (CHX) displayed an increased intensity of Cl (35 and 37u) and relatively unchanged intensities of peaks attributable to CN (26u), CNO (42u), PO2 (63u), PO3 (79u), F (19u), and HCO2 (45u) (Fig. 2D).
TOF-SIMS: High Spatial Resolution Analysis In positive polarity spectra images of sample A (NT), the DTs that were rather obstructed were observed on the dentin surface.
Na+ and Ca+ were evenly distributed on the surface in significant amounts (Fig. 3A). Patent DTs were clearly visualized on the surface of sample B (NaOCl), and Na+ intensity appeared to be increased on the lower portion of multiple DTs, whereas Ca+ was evenly distributed (Fig. 3B). Sample C (EDTA) displayed a surface with some patent DTs. Multiple tubules were not patent. Ca+ was scarce, and Na+ was evenly distributed on the dentin surface (Fig. 3C). Sample D (CHX) surface exhibited multiple patent DTs, and Ca+ was evenly distributed around the DTs; Na+ is concentrated in a few isolated DTs and in several isolated linear
Figure 3. High spatial resolution TOF-SIMS images of positive ion distribution on dentin surface. (A) Sample A (NT, no irrigation), (B) sample B (NaOCl), (C) sample C (EDTA), and (D) sample D (CHX). ‘‘Total’’ shows raw image; ClC6H4NH2+ shows distribution of CHX. Note the lack of visible tubules in A (NT) and the decrease in visible tubules in C (EDTA).
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Basic Research—Technology areas. An intense signal originating from CHX was evenly distributed on the dentin surface (Fig. 3D).
Discussion The advantages of using TOF-SIMS on dentin included its high sensitivity, its ability to analyze the chemical structure of 1 to 2 monolayers, its high spatial and mass resolution, and its ability to provide a chemical map of the surface (22–24). However, good mass resolution can present a problem if the surface of the sample is contaminated. In this study, the intent was to analyze the surface of untreated and treated dentin and avoid contamination by careful handling and storage of the specimens. Before this study, there were no published studies that examined human dentin and human dentin treated with NaOCl, EDTA, or CHX by TOF-SIMS analysis. A criticism of the current study may be the sample size, which consisted of only 1 specimen in each treatment group. However, this is consistent with other studies that have used TOF-SIMS analysis (22, 25–27). In addition, we used dentin from only 1 tooth. Although it afforded the study a measure of standardization, it did not account for variations in chemical composition that may be present among different teeth. To avoid measurement of artifacts, our high mass resolution size was 500 mm 500 mm; we obtained relatively even distribution of ions on the entire surface. This can be visualized in 150 mm 150 mm high spatial resolution views. The analysis of dentin not treated with irrigation (NT) was consistent with the findings from other studies (20, 21) showing that dentin contains Na+ (23u), K+ (39u), Ca+ (40u), Mg+ (24u), and F (19u) and hydroxyapatite phosphorous oxides groups PO2 (63u) and PO3 (79u). Another study using TOF-SIMS also characterized dentin to possess intensive levels of Na+, K+, Ca+, Mg+, CN , CNO , PO2 , PO3 , and HCO2 (25–27). Gotliv et al (25–27) found a differential distribution of ions with regard to their position in peritubular and intertubular dentin, which appeared in part in NaOCl-treated dentin in our study. However, Gotliv et al (25–27) used bovine dentin with a 5-time higher resolution imaging than the present study. The NaOCl-treated dentin showed an expected increased intensity of Na+ (23u) and Cl (35 and 37u). Signal from the common dentinal protein fragment CH4N+ (30u) almost disappeared, indicating that degradation of proteins present on the dentin surface occurred. This was consistent with previous studies (7) although the presence of CN (26u) and CNO (42u) showed that some protein fragments were still present on the surface. As shown by a previous study (11), treatment with EDTA removed most of the Ca+ (40u) and Mg+ (24u) and to a moderate extent PO2 (63u) and PO3 (79u) on the dentin surface, changes that were typical of demineralization. On the other hand, CHX treatment of the dentin resulted in a CHX (505u) signal detected on the surface, which was consistent with the current theory that CHX forms an adsorbed layer on the dentin surface when it is used in endodontic treatment (14, 15). The increase in Cl signal (35 and 37u) subsequent to CHX and NaOCl treatments was also expected and was attributed to the chlorine component of both irrigants. A uniformly distributed smearlike layer was present on the surface of untreated dentin (NT) and was most likely produced during specimen preparation. Surprisingly, DTs were visible after the dentin was treated with CHX, which is classified as neither a hard or organic tissue removing agent. The use of an organic tissue removing agent (NaOCl) allowed patent tubules to be clearly seen, whereas the hard tissue removing agent (EDTA) left a reduced number of patent tubules. It is likely that the protein components present inside the DTs hindered the EDTA effect within the DTs. JOE — Volume -, Number -, - 2015
The findings from this study showed a distinct difference in the chemical characteristics of surface dentin after treatment with different root canal irrigants. Changes in the chemical composition of the dentin after root canal irrigation can affect the viability of cells seeded onto the surface of the dentin (31, 32) and therefore should be of concern in pulp regenerative treatment. Increased intensities of Na+ (23u) and Cl (35 and 37u), which were NaOCl breakdown products (4) observed after irrigation with NaOCl, could be accountable for the reduced viability of cells (31, 32). It was also possible that the residual layer of CHX noted in the current study could create an unfavorable environment for cell viability and cell adhesion (31). This may perhaps explain why CHX irrigation results in a poorer treatment outcome in one study (33). It would be of interest to have future studies analyzing higher spatial resolution images of the dentin surface after irrigation with NaOCl and EDTA, in addition to the combined irrigation with both NaOCl and EDTA. This could allow a closer inspection of chemical changes occurring around the tubule during dissolution of soft and hard tissue during root canal therapy. In summary, TOF-SIMS of untreated dentin (NT) shows the presence of Na+, K+, Ca+, Mg+, CH4N+, PO2 , PO3 , CN , CNO , HCO2 , and F . After treatment with NaOCl, there is a degradation of matrix protein with the removal of CH4N+. EDTA removes Ca+ and Mg+ from dentin, and CHX treatment leaves behind a layer of CHX on the dentin surface.
Acknowledgments The authors thank the Surface Interface Ontario for the assistance in the conduct of these experiments and Dr Torneck for his feedback on the manuscript. Supported by a grant from Canadian Academy of Endodontics Endowment Fund and Endo Tech. The authors deny any conflicts of interest related to this study.
References 1. Dutner J, Mines P, Anderson A. Irrigation trends among American Association of Endodontists members: a web-based survey. J Endod 2012;38:37–40. 2. Bystr€om A, Sundqvist G. Bacteriologic evaluation of the effect of 0.5 percent sodium hypochlorite in endodontic therapy. Oral Surg Oral Med Oral Pathol 1983;55: 307–12. 3. Gordon TM, Damato D, Christner P. Solvent effect of various dilutions of sodium hypochlorite on vital and necrotic tissue. J Endod 1981;7:466–9. 4. Estrela C, Estrela CR, Barbin EL, et al. Mechanism of action of sodium hypochlorite. Braz Dent J 2002;13:113–7. 5. Zaparolli D, Saquy PC, Cruz-Filho AM. Effect of sodium hypochlorite and EDTA irrigation, individually and in alternation, on dentin microhardness at the furcation area of mandibular molars. Braz Dent J 2012;23:654–8. 6. Slutzky-Goldberg I, Maree M, Liberman R, Heling I. Effect of sodium hypochlorite on dentin microhardness. J Endod 2004;30:880–2. 7. Oyarzun A, Cordero AM, Whittle M. Immunohistochemical evaluation of the effects of sodium hypochlorite on dentin collagen and glycosaminoglycans. J Endod 2002; 28:152–6. 8. Niu W, Yoshioka T, Kobayashi C, Suda H. A scanning electron microscopic study of dentinal erosion by final irrigation with EDTA and NaOCl solutions. Int Endod J 2002;35:934–9. 9. Qian W, Shen Y, Haapasalo M. Quantitative analysis of the effect of irrigant solution sequences on dentin erosion. J Endod 2011;37:1437–41. 10. Baumgartner JC, Mader CL. A scanning electron microscopic evaluation of four root canal irrigation regimens. J Endod 1987;13:147–57. 11. H€ulsmann M, Heckendorff M, Lennon A. Chelating agents in root canal treatment: mode of action and indications for their use. Int Endod J 2003;36:810–30. 12. Patterson SS. In vivo and in vitro studies of the effect of the disodium slat of ethylenediamine tetra-acetate on human dentine and its endodontic implications. Oral Surg Oral Med Oral Pathol 1963;16:83–103. 13. Jordan RA, Holzner AL, Markovic L, et al. Clinical effectiveness of basic root canal treatment after 24 months: a randomized controlled trial. J Endod 2014;40: 465–70.
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Basic Research—Technology 14. Emilson CG, Ericson T, Heyden G, Magnusson BC. Uptake of chlorhexidine to hydroxyapatite. J Periodontal Res Suppl 1973;12:17–21. 15. Lin S, Levin L, Weiss EI, et al. In vitro antibacterial efficacy of a new chlorhexidine slow-release device. Quintessence Int 2006;37:391–4. 16. White RR, Hays GL, Janer LR. Residual antimicrobial activity after canal irrigation with chlorhexidine. J Endod 1997;23:229–31. 17. Naenni N, Thoma K, Zehnder M. Soft tissue dissolution capacity of currently used and potential endodontic irrigants. J Endod 2004;30:785–7. 18. Okino LA, Siqueira EL, Santos M, et al. Dissolution of pulp tissue by aqueous solution of chlorhexidine digluconate and chlorhexidine digluconate gel. Int Endod J 2004; 37:38–41. 19. Linde A, Goldberg M. Dentinogenesis. Crit Rev Oral Biol Med 1993;4:679–728. 20. Coklica V, Brudevold F, Amdur BH. The distribution and composition of density fractions from human crown dentine. Arch Oral Biol 1969;14:451–60. 21. Foreman PC, Soames JV. Comparative study of the composition of primary and secondary dentine. Caries Res 1989;23:1–4. 22. Eriksson C, Malmberg P, Nygren H. Time-of-flight secondary ion mass spectrometric analysis of the interface between bone and titanium implants. Rapid Commun Mass Spectrom 2008;22:943–9. 23. Malmberg P, Nygren H. Methods for the analysis of the composition of bone tissue, with a focus on imaging mass spectrometry (TOF-SIMS). Proteomics 2008;8: 3755–62. 24. Sodhi RN. Time-of-flight secondary ion mass spectrometry (TOF-SIMS):–versatility in chemical and imaging surface analysis. Analyst 2004;129:483–7.
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25. 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;156:320–33. 26. Gotliv BA, Veis A. Peritubular dentin, a vertebrate apatitic mineralized tissue without collagen: role of a phospholipid-proteolipid complex. Calcif Tissue Int 2007;81: 191–205. 27. Gotliv BA, Veis A. The composition of bovine peritubular dentin: matching TOFSIMS, scanning electron microscopy and biochemical component distributions. New light on peritubular dentin function. Cells Tissues Organs 2009;189:12–9. 28. Basrani BR, Manek S, Sodhi RN, et al. Interaction between sodium hypochlorite and chlorhexidine gluconate. J Endod 2007;33:966–9. 29. 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;40:2036–40. 30. Eldeniz AU, Erdemir A, Belli S. Shear bond strength of three resin based sealers to dentin with and without the smear layer. J Endod 2005;31:293–6. 31. Trevino EG, Patwardhan AN, Henry MA, et al. Effect of irrigants on the survival of human stem cells of the apical papilla in a platelet-rich plasma scaffold in human root tips. J Endod 2011;37:1109–15. 32. Huang X, Zhang J, Huang C, et al. Effect of intracanal dentine wettability on human dental pulp cell attachment. Int Endod J 2012;45:346–53. 33. 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: 583–609.
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