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Ultrastructure of Smear Layer-Covered Intraradicular Dentin After Irrigation with BioPure MTAD

Ultrastructure of Smear Layer-Covered Intraradicular Dentin After Irrigation with BioPure MTAD

Basic Research—Technology Ultrastructure of Smear Layer-Covered Intraradicular Dentin After Irrigation with BioPure MTAD Franklin R. Tay, BDSc (Hons)...

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Basic Research—Technology

Ultrastructure of Smear Layer-Covered Intraradicular Dentin After Irrigation with BioPure MTAD Franklin R. Tay, BDSc (Hons), PhD,* David H. Pashley, DMD, PhD,† Robert J. Loushine, DDS,‡ Michael D. Doyle, DDS,‡ W. Trent Gillespie, DMD, MPH,‡ R. Norman Weller, DMD, MS,‡ and Nigel M. King, BDS, MS, PhD* Abstract The structure of mechanically instrumented intraradicular dentin after irrigation with NaOCl as the initial rinse and BioPure MTAD as the final rinse were examined from the coronal, middle, and apical parts of root canal walls using transmission electron microscopy. Sterile distilled water and EDTA as final rinses were employed as the respective positive and negative controls under the same experimental conditions. There were 2 to 5 ␮m thick smear layers produced on mechanically instrumented root canal walls that were completely removed by EDTA and BioPure MTAD under agitation. Both irrigants created a zone of demineralized collagen matrices in eroded dentin and around the dentinal tubules, with the mildly acidic BioPure MTAD being more aggressive than EDTA. These demineralized dentin zones create the opportunity for dentin hybridization by infiltration of hydrophilic adhesives/sealers. However, the potential consequences of compaction of hydrophobic sealers against air-dried, collapsed collagen matrices, and hydrolytic degradation of incompletely infiltrated matrices remain unresolved. (J Endod 2006; 32:218 –221)

Key Words EDTA, endodontic smear layer, intraradicular dentin hybridization, MTAD, NaOCl

From the *Pediatric Dentistry and Orthodontics, The University of Hong Kong, Pokfulam, Hong Kong SAR, China; † Department of Oral Biology and Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, Georgia; ‡Department of Endodontics, School of Dentistry, Medical College of Georgia, Augusta, Georgia. Address requests for reprints to Dr. Franklin R. Tay, Prince Philip Dental Hospital, The University of Hong Kong, Pokfulam, 34 Hospital Road, Hong Kong SAR, China. E-mail address: [email protected] 0099-2399/$0 - see front matter Copyright © 2006 by the American Association of Endodontists. doi:10.1016/j.joen.2005.10.035

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echanical instrumentation of root canals invariably creates smear layers on the root canal walls that occlude tubules and prevent optimal penetration of medicaments, sealers, and root filling materials into the lateral canals and dentinal tubules (1–3). The alternating use of ethylenediaminetetraacetic acid (EDTA), a calcium-chelating agent, and sodium hypochlorite (NaOCl), a deproteinizing agent, has been recommended for efficient removal of these smear layers (4 – 6). However, there is concern that this combined irrigation regime causes inadvertent erosion of the intraradicular dentin (7–10). Although doxycycline, a tetracycline isomer, and citric acid have been employed separately for removing endodontic smear layers, the introduction of MTAD (11), an aqueous solution of 3% doxycycline, 4.25% citric acid, and 0.5% polysorbate 80 detergent (12), represents an advance in endodontic irrigation research (13). This biocompatible intracanal irrigant (14) is commercially available as a two-part set that is mixed upon demand (BioPure MTAD, DentsplyTulsa, Tulsa, OK). In this product, 4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,5,10,12,12a-pentahydroxy-6methyl-1,11-dioxy-2-naphthacenecarboxamide monohydrochloride hemiethanolate hemihydrate (doxycycline hyclate) is used instead of its free base, doxycycline monohydrate, to increase the water solubility of this broad spectrum antibiotic (15). MTAD has been reported to be effective in removing endodontic smear layers (10), eliminating microbes that are resistant to conventional endodontic irrigants and dressings (16), and providing sustained antimicrobial activity through the affinity of doxycycline to bind to dental hard tissues (17). Similar to EDTA, initial rinsing of instrumented root canals with dilute NaOCl is recommended for enhancing the efficacy of MTAD in dissolving endodontic smear layers (10). Unlike the use of EDTA as the final rinse, minimal erosion of intraradicular dentin has been reported when NaOCl and MTAD were employed in a similar sequence (10). To date, studies on the efficacy of smear layer removal and the aggressiveness of root dentin demineralization were conducted using scanning electron microscopy (SEM), often in the absence of critical point drying for preserving the integrity of soft, unsupported structural components that are susceptible to stresses induced by surface tension. When calcium chelators or mild acidic solutions are used as the final rinse, depletion of surface minerals (18, 19) result in the formation of demineralized collagen matrices on the surface of intraradicular dentin. Bonding research performed on crown dentin has shown that collapse of these unsupported collagen matrices occurs by intrafibrillar hydrogen bonding during air-drying (20), rendering them difficult to be identified by SEM examination. In light of the expanding trend to use bonding technologies in endodontics, there is also the need to identify the quality of endodontic smear layers and the extent of subsurface demineralization in intraradicular dentin. These issues may be more appropriately resolved with the use of transmission electron microscopy (TEM).

Materials and Methods Twenty-four single-rooted human premolars stored in 0.5% chloramine T at 4°C were used within 1 month after extraction. Access cavities were prepared using tapered diamond burs in a high-speed handpiece with water spray cooling. Patency was confirmed using a size #15 Flex-o-file (DentsplyTulsa). The working length was established 1-mm short of the apex. Instrumentation was performed with a crown-down technique,

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Basic Research—Technology using 0.06 taper nickel-titanium rotary instruments (Sequence, Brasseler, Savannah, GA) to ISO size 25. Although 1.3% sodium hypochlorite (NaOCl, pH 10.45) is recommended for use with MTAD, all canals were rinsed during instrumentation with 10 ml of 2.6% NaOCl (Clorox, Oakland, CA; pH 10.46) as an initial rinse (total time approximately 20 min), to ensure consistency of the control and experimental groups (n ⫽ 8). This was followed by rinsing of the canals with 10 ml of sterile distilled water, to minimize potential interactions of NaOCl with any acidic irrigant that was employed as the final rinse. The canals were irrigated on completion of instrumentation with 5 ml of one of the following solutions as the final rinse: 1. Sterile distilled water (positive control); 2. 17% EDTA (negative control); 3. BioPure MTAD (pH 1.83) that was used immediately upon mixing. There were 1 ml of each irrigant was delivered to within 2 mm of the working length using a 30-gauge ProRinse syringe tip (DentsplyTulsa). To enable direct contact of each irrigant with the root canal wall, a size 25, 0.04 taper resin-coated gutta-percha point (Ultradent, South Jordan, UT) was employed in an up and down motion to mechanically agitate the irrigant. Each irrigant was left in the root canal for 5 minutes and then removed with suction. The canal was then rinsed with the remaining 4 ml of each irrigant. After drying the root canals with multiple paper points, the teeth were fixed in Karnovsky’s fixative (2% paraformaldehyde and 2.5% glutaraldehyde; pH 7.2) for 1 hour.

TEM Processing To prevent inadvertent introduction of artifactual smear layers when sectioning through the root canals, shallow slits were prepared along the external root surfaces 90 degrees to the long axis without cutting into the root canals. These slits were made at 5, 10, and 15 mm from the root apices, to represent the apical, middle, and coronal parts of the root canals. The roots were fractured into segments using a sharp cutting blade and a mallet. The apical, middle, and coronal root segments were processed for TEM examination according to a previously reported protocol (21). There were 100 to 120 nm thick, undemineralized, epoxy resin-embedded sections prepared, collected on singleslot, carbon-and-formvar-coated copper grids, and examined unstained using a TEM (Philips EM208S, Eindhoven, The Netherlands) at 80 kV. In Situ Section Demineralization Conventional bulk demineralization TEM protocols can result in destruction of the integrity of endodontic smear layers, and masking irrigant-induced demineralization zones along the surfaces of unbonded dentin. Thus, an in situ section demineralization protocol was developed for the preparation of stained, demineralized sections. After examination, grids containing undemineralized sections of interest were floated upside down on drops of 5% formic acid for 5 minutes to completely demineralize the sections in situ. Each grid was then floated on five drops of distilled water in succession to rinse away the dissolved minerals. The grids were double-stained with 2% uranyl acetate and Reynold’s lead citrate using the in situ drop floatation technique. The stained, demineralized sections were re-examined with the same microscope.

Results The use of nickel-titanium rotary instruments resulted in the formation of smear layers on intraradicular dentin from the coronal (Fig. 1A), middle (Fig. 1B) and apical third (Fig. 1C) of the root canals. There were 2 to 5 ␮m of these layers retained after rinsing with distilled JOE — Volume 32, Number 3, March 2006

Figure 1. TEM micrographs of intraradicular dentin specimens that were shaped with nickel-titanium rotary instruments and irrigated with 2.6% NaOCl and distilled water (negative control). The latter was used as the final rinse. E, laboratory embedding epoxy resin; D, undemineralized intertubular root dentin; Dd, intertubular dentin demineralized in-situ. (A) An unstained, undemineralized section that is representative of the coronal portion of a root canal, where a high density of dentinal tubules were identified. A 2 to 5 mm thick endodontic smear layer (between open arrows) could be observed on the dentin surface. P, pertitubular dentin. (B) An unstained, undemineralized section taken from the middle portion of a root canal. A spherical structure (open arrowhead), probably representing the silhouette of a bacterium, was trapped within the endodontic smear layer (between open arrow) and the dentinal tubular orifice. (C) An unstained, undemineralized section taken from the apical portion of a root canal, where dentinal tubules (pointer) were sparse and occasionally sclerotic. Between open arrows: endodontic smear layer. (D). In-situ demineralized, stained section created from the undemineralized section examined in Fig. 1A. The smear layer (between open arrows) appeared to be more loosely arranged on the top and more compact at the bottom.

water under agitation (positive control). The porous nature of the endodontic smear layer could be seen in stained sections that were demineralized in situ (Fig. 1D). The use of EDTA as the final rinse (negative control) resulted in complete removal of the smear layer and the creation of 4 to 6 ␮m thick surface demineralized dentin zones in the coronal (Fig. 2A), middle (Fig. 2B), and apical third (Fig. 2C) of the root canal walls. Because of the profuse distribution of dentinal tubules in the coronal intraradicular dentin, coalescence of the demineralized zones around the periphery of the subsurface dentinal tubules resulted in a highly eroded appearance of the underlying mineralized dentin (Fig. 2A). This feature was less apparent in the middle part of the root canal as the dentinal tubules were more widely separated (Fig. 2B). Dentinal tubules in the apical third of the root canal were sparse and often sclerotic in appearance (Fig. 2C). Infiltration of epoxy resin into the EDTA-demineralized dentin zone created hybrid layers that became evident after in situ section demineralization and staining (Fig. 2D). The use of MTAD as the final rinse resulted in complete smear layer removal and the creation of 10 to 12 ␮m thick zones of partially demineralized dentin with fine, partially dissolved apatite crystallites (not shown) in the coronal (Fig. 3A), middle (Fig. 3B)

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Basic Research—Technology ness of hybrid layers produced in considerably more porous cariesaffected dentin (23). The acidity of BioPure MTA is similar to the mild self-etching primers used in dentin bonding (24). A 5-minute irrigation period is long compared with the 30 to 60 seconds etching time recommended for self-etching primers. Mechanical agitation for enhancing the efficacy of smear layer removal (25), and replenishing of fresh irrigants during subsequent rinsing may have also contributed to the formation of thick demineralized dentin zones. Conversely, mineralized dentin has excellent buffering capacities that can neutralize acidic solutions with calcium salts (26). Thus, the relation between the porous nature of crown versus root intertubular dentin and the aggressiveness of demineralization should be investigated in future studies by comparing the etching capacity of BioPure MTAD on these two substrates. It is also necessary to examine whether the experimental reduction in MTAD irrigation time to 2 min (10) is accompanied by a simultaneous reduction in demineralization of root canal walls. Because demineralized collagen matrices are susceptible to collapse after air-drying (27), their presence after endodontic irrigation

Figure 2. TEM micrographs of intraradicular dentin specimens that were shaped with nickel-titanium rotary instruments and irrigated with 2.6% NaOCl and 17% EDTA (positive control). The latter was used as the final rinse. E, laboratory embedding epoxy resin; D, undemineralized intertubular root dentin; Dd, intertubular dentin demineralized in-situ. (A) An unstained, undemineralized section that is representative of the coronal portion of a root canal, where profuse dentinal tubules were identified. The endodontic smear layer was completely removed and a 4 to 5 ␮m thick zone of completely demineralized dentin (between arrows) could be identified along the dentin surface. Circumferential demineralization of the subsurface dentinal tubules is also evident (open arrowhead). (B) An unstained, undemineralized section taken from the middle portion of a root canal, showing a 5-␮m thick demineralized zone along the dentin surface (between arrows). Pointer: lateral branches of the dentinal tubules. (C) An unstained, undemineralized section taken from the apical portion of a root canal, showing an 8-␮m thick demineralized zone along the dentin surface (between arrows). Dentinal tubules were sparse and were occasionally sclerotic (open arrowhead). (D) In situ demineralized, stained section created from the undemineralized section examined in Fig. 2A. Infiltration of the EDTAdemineralized dentin during specimen embedding by the epoxy resin resulted in the formation of a 5 to 6 ␮m thick, electron-dense hybrid layer (H). Circumferential hybrid layers (open arrowhead) could also be seen around the periphery of the subsurface dentinal tubules.

and apical third (Fig. 3C) of the root canals. Similar to EDTA, epoxy resin-infiltrated hybrid layers were evident in the stained demineralized MTAD sections (Fig. 3D).

Dicussion Under the same experimental conditions, 17% EDTA and BioPure MTAD are equal in their capacities to completely remove endodontic smear layers when these irrigants were utilized as the final rinses with mechanical agitation. BioPure MTAD is comparatively more aggressive in demineralizing intact intraradicular dentin, exposing collagen matrices that were 1.5 to 2 times as thick as those produced with EDTA. Subsurface demineralization fronts were not exposed and no surface erosions were observed for either irrigant, because NaOCl was not used as the final rinse to deproteinize the collagen matrices (22). The thickness of these demineralized dentin zones were comparable to those formed on crown dentin after phosphoric acid (pH ⫺0.1– 0.8) etching for 15 seconds, and may even approach the thick220

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Figure 3. TEM micrographs of intraradicular dentin specimens that were shaped with nickel-titanium rotary instruments and irrigated with 2.6% NaOCl and MTAD (experimental group). The latter was used as the final rinse. E, laboratory embedding epoxy resin; D, undemineralized intertubular root dentin; Dd, intertubular dentin demineralized in-situ. (A) An unstained, undemineralized section that is representative of the coronal portion of a root canal, where profuse dentinal tubules were identified. A 10 to 12 ␮m thick zone of MTAD-demineralized dentin could be identified along the dentinal surface (between arrows), extending circumferentially around the subsurface dentinal tubules. (B) An unstained, undemineralized section taken from the middle portion of a root canal where the distribution of the dentinal tubules was less profuse. A 10-␮m thick zone of demineralized dentin (between arrows) was created on the dentin surface after MTAD irrigation. (C) An unstained, undemineralized section taken from the apical portion of a root canal, showing a 10-␮m thick demineralized zone along the dentin surface (between arrows). Dentinal tubules were sparse and were occasionally sclerotic (pointer). (D) In situ demineralized, stained section created from the undemineralized section examined in (B). Expansion and subsequent infiltration of the epoxy resin into the demineralized collagen matrix during laboratory processing resulted in the formation of a 10-␮m thick, electron-dense hybrid layer (H). A 1-␮m thick hybrid layer was also seen circumferentially around the periphery of the subsurface dentinal tubules (open arrowhead).

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Basic Research—Technology challenge the concept of longevity of endodontic seals created using hydrophobic sealers (28), even when perfect three-dimensional seals are created initially. Concomitantly, these matrices offer an opportunity for dentin hybridization with hydrophilic methacrylate-based resins/ sealers without the adjunctive use of phosphoric acid-etching or selfetching primers. Although excellent coronal seals have been reported for MTAD-irrigated, AH Plus obturated root canals (29), the issue of incomplete resin infiltration of these thick, intraradicular collagen matrices (30), however, remains unresolved and also requires further investigation. Incomplete resin infiltration may result in leakage in nanometer dimensions within the collagen matrices (i.e. nanoleakage), as opposed to frank leakage within the filled root canals. Bonding studies performed on crown dentin have shown that denuded collagen matrices are susceptible to hydrolytic degradation. Even in the absence of leakage, slow degradation of these collagen matrices may occur through the release and activation of endogenous matrix metalloproteinases (MMPs) from partially demineralized dentin (31). Although “exogenous” MMP-8 identified from inflamed periapical tissues (32) is unlikely to diffuse into optimally root-filled canals, release of “endogenous” intracanal MMPs by irrigants that are not acidic enough to denature these enzymes may expedite potential collagenolytic and gelatinolytic activities within filled root canals, challenging the integrity of pre-established seals. The use of BioPure MTAD with doxycycline affinity to dentin (17) may provide a sustained MMP-inhibitory function, apart from its clearly established antimicrobial capacity. It is known that EDTA inhibits MMP activity by chelating zinc and calcium ions that are mandatory for functioning of these enzymes. However, EDTA does not exhibit sustained inhibition toward MMPs that are released subsequently from the underlying mineralized dentin. Sub-antimicrobial doses of chlorhexidine (33) and doxycycline (34, 35) are potent MMP inhibitors. Application of low concentrations of chlorhexidine to acid-etched dentin prohibited the degradation of demineralized collagen matrices (31). Thus, the hypothesis that BioPure MTAD exerts a sustained MMP-inhibiting effect on exposed intraradicular collagen matrices warrants testing and validation, possibly with the use of nonantimicrobial, chemically modified doxycycline analogs (e.g. CMT-8) (35) as controls to distinguish between its MMP-inhibitory function and antimicrobial activity.

Acknowledgments This study was supported by grant 10204604/07840/08004/ 324/01, Faculty of Dentistry, the University of Hong Kong, and by R01 grants DE 014911 and DE 015306 from the NIDCR, USA (PI. David Pashley). The authors are grateful to Michelle Barnes for secretarial support.

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6. Peters OA, Barbakow F. Effects of irrigation on debris and smear layer on canal walls prepared by two rotary techniques: a scanning electron microscopic study. J Endod 2000;26:6 –10. 7. Baumgartner JC, Mader CLA. Scanning electron microscopic evaluation of four root canal irrigation regimens. J Endod 1987;13:147–57. 8. C᝺ alt S, Serper A. Time-dependent effects of EDTA on dentin structures. J Endod 2002;28:17–9. 9. 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. 10. Torabinejad M, Cho Y, Khademi AA, Bakland LK, Shabahang S. The effect of various concentrations of sodium hypochlorite on the ability of MTAD to remove the smear layer. J Endod 2003;29:233–9. 11. Torabinejad M, Khademi AA, Babagoli J, et al. A new solution for the removal of the smear layer. J Endod 2003;29:170 –5. 12. Torabinejad M, Johnson WB Irrigation solution and methods for use. US Patent & Trademark Office. United States Patent Application 20030235804; December 25, 2003. 13. Torabinejad M, Shabahang S, Bahjri K. Effect of MTAD on postoperative discomfort: a randomized clinical trial. J Endod 2005;31:171– 6. 14. Zhang W, Torabinejad M, Li Y. Evaluation of cytotoxicity of MTAD using the MTTtetrazolium method. J Endod 2003;29:654 –7. 15. Bogardus JB, Blackwood RK Jr. Solubility of doxycycline in aqueous solution. J Parm Sci 1979;68:188 –94. 16. Shabahang S, Torabinejad M. Effect of MTAD on Enterococcus faecalis-contaminated root canals of extracted human teeth. J Endod 2003;29:576 –9. 17. Baker PJ, Evans RT, Coburn RA, Genco RJ. Tetracycline and its derivatives strongly bind to and are released from the tooth surface in active form. J Periodontol 1983;54:580 –5. 18. Verdelis K, Eliades G, Oviir T, Margelos J. Effect of chelating agents on the molecular composition and extent of decalcification at cervical, middle and apical root dentin locations. Endod Dent Traumatol 1999;15:164 –70. 19. Ari H, Erdemir A. Effects of endodontic irrigation solutions on mineral content of root canal dentin using ICP-AES technique. J Endod 2005;31:187–9. 20. Pashley DH, Carvalho RM, Tay FR, Agee KA, Lee KW. Solvation of dried dentin matrix by water and other polar solvents. Am J Dent 2002;15:97–102. 21. Tay FR, Moulding KM, Pashley DH. Distribution of nanofillers from a simplified-step adhesive in acid conditioned dentin. J Adhes Dent 1999;1:103–17. 22. Prati C, Chersoni S, Pashley DH. Effect of removal of surface collagen fibrils on resin-dentin bonding. Dent Mater 1999;15:323–31. 23. Yoshiyama M, Tay FR, Doi J, et al. Bonding of self-etch and total-etch adhesives to carious dentin. J Dent Res 2002;81:556 – 60. 24. Tay FR, Pashley DH. Aggressiveness of contemporary self-etching systems. I: Depth of penetration beyond dentin smear layers. Dent Mater 2001;17:296 –308. 25. Chan KM, Tay FR, King NM, Imazato S, Pashley DH. Bonding of mild self-etching primers/adhesives to dentin with thick smear layers. Am J Dent 2003;16:340 – 6. 26. Camps J, Pashley DH. Buffering action of human dentin in vitro. J Adhes Dent 2000;2:39 –50. 27. Gwinnett AJ. Chemically conditioned dentin: a comparison of conventional and environmental scanning electron microscopy findings. Dent Mater 1994;10:150 –5. 28. Economides N, Liolios E, Kolokuris I, Beltes P Long-term evaluation of the influence of smear layer removal on the sealing ability of different sealers. J Endod 1999;25:123–5. 29. Park DS, Torabinejad M, Shabahang S. The effect of MTAD on the coronal leakage of obturated root canals. J Endod 2004;30:890 –2. 30. Tay FR, Gwinnett JA, Wei SH. Micromorphological spectrum from overdrying to overwetting acid-conditioned dentin in water-free acetone-based, single-bottle primer/adhesives. Dent Mater 1996;12:236 – 44. 31. Pashley DH, Tay FR, Yiu C, et al. Collagen degradation by host-derived enzymes during aging. J Dent Res 2004;83:216 –21. 32. Wahlgren J, Salo T, Teronen O, Luoto H, Sorsa T, Tjäderhane L. Matrix metalloproteinase-8 (MMP-8) in pulpal and periapical inflammation and periapical root-canal exudates. Int Endod J 2002;11:897–904. 33. Gendron R, Grenier D, Sorsa T, Mayrand D. Inhibition of the activities of matrix metalloproteinases 2, 8, and 9 by chlorhexidine. Clin Diagn Lab Immunol 1999;6:437–9. 34. Golub LM, Lee HM, Ryan ME, Giannobile WV, Payne J, Sorsa T. Tetracyclines inhibit connective tissue breakdown by multiple non-antimicrobial mechanisms. Adv Dent Res 1998;12:12–26. 35. Liu Y, Ramamurthy N, Marecek J, et al. The lipophilicity, pharmacokinetics, and cellular uptake of different chemically-modified tetracyclines (CMTs). Curr Med Chem 2001;8:243–52.

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