- Email: [email protected]
Investigation into the nature of dentin resin tags: a scanning electron microscopic morphological analysis of demineralized bonded dentin
Investigation into the nature of dentin resin tags: A scanning electron microscopic morphological analysis of demineralized bonded dentin Luca Giachetti, MD, DMD,a Fabio Bertini, MD, DMD,b and Daniele Scaminaci Russo, DDSc Department of Dentistry, Faculty of Medicine and Surgery, University of Florence, Florence, Italy Statement of problem. While the formation of the dentin/adhesive hybrid layer has been generally established, the infiltration and flow of the adhesive resin inside the acid treated dentinal tubules remains controversial. Purpose. The aim of the present study was to investigate and review the current interpretation of resin tags by means of scanning electron microscopic (SEM) observation. Material and methods. Eight noncarious, human third molars were cut transversally and then longitudinally to obtain 8 middle-to-deep dentinal surfaces. The dentin was etched with 37% phosphoric acid (H3PO4) gel for 10 seconds and then rinsed with water for 20 seconds. The dentin was kept moist by removing the excess water with a damp cotton pellet. The conditioned dentin was treated with a dentin bonding agent (Single Bond) and was light-polymerized for 20 seconds. A 0.2- to 0.5-mm layer of flowable composite (Tetric Flow) was then applied to the bonded dentin followed by 2 layers (2 mm each) of composite (Z 250). Each composite was lightpolymerized for 40 seconds. Subsequently, the specimens were cut lengthwise into 2 halves and randomly divided into 4 groups (n=4), according to the surface preparation modality of the sectioned surface: Group EA: ethylenediamine tetraacetic acid, Group PA3: H3PO4, Group PA120: H3PO4 1 NaOCl, and Group CA: HCl 1 NaOCl. Two additional teeth (Group N) were cut lengthwise into 2 halves and served as the control. The sectioned surfaces were treated with HCl and NaOCl. All specimens were processed for SEM observation. Results. Specimens from Groups EA, PA3, PA120, CA, and N showed filamentous structures that were tens of microns long. Some filaments presented split-ends with hollow structures and very thin walls. Others made sharp hairpin turns indicating they were soft and compliant. Conclusions. Conventional SEM techniques, which are currently used to detect resin tags, actually identified filamentous organic structures, supposedly glycosaminoglycans, which were resistant to conventional specimen preparation techniques. The organic component showed a strong resemblance to the lamina limitans contained within the dentinal tubules. Over-reliance on SEM morphology has led to much confusion about the depth of penetration of resin tags. (J Prosthet Dent 2004;92:233-8.)
CLINICAL IMPLICATIONS The presence of glycosaminoglycans in the etched dentin could jeopardize the efficacy of the adhesive systems.
T
he literature reports that so called ‘‘resin tags,’’ ‘‘resin tails,’’ or ‘‘resin strings’’ are the result of adhesive resin penetration inside the dentinal tubules.1,2 However, current literature provides several and occasionally contradictory interpretations regarding the formation of these filament structures.3-9 To achieve an optimal dentin bond, the adhesive must penetrate the demineralized dentin tubules and branches before polymerization. The importance given to the infiltration and flow of the adhesive resin inside the acid-treated dentinal tubules remains controversial. The relative contribution a
Assistant and Chair of Dental Materials. Adjunct faculty. c Adjunct faculty. b
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of resin tags to bond strength must include where the bond is made (superficial, middle, or deep dentin) and whether the dentinal tubules are perpendicular or parallel to the prepared dentin surface.10 Resin tags may contribute about 30% to the total strength of the adhesivedentin bond.11 Tao and Pashley12 indicate that no link exists between the bond strength and the formation of the resin tags. Furthermore, resin penetration within the dentinal tubules should hermetically seal the dentin-pulp complex, thus minimizing permeability and pulpal irritation.13 The shrinkage that results from polymerization may, however, cause the separation of the adhesive resin from the walls of the tubules, thus allowing the passage of fluids.10 One of the procedures most commonly used in vitro for evaluation of the thickness of the resin-infiltrated THE JOURNAL OF PROSTHETIC DENTISTRY 233
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dentin layer is the partial or total removal of dentin that occurs by dissolving the mineral component with strong acids and the organic matrix with sodium hypochlorite (NaOCl).3,14 Specimens sequentially treated with acids and NaOCI show numerous sinuous filaments that are tens or hundreds of microns long.3,5 The morphology, length, and density of so-called resin tags are used by some for the qualitative and/or semiquantitative evaluation of the efficacy of adhesive systems.15,16 Nevertheless, careful examination of the morphological characteristics of the resin tags may result in confusion regarding the nature of these structures. In fact, the length of the resin tags often exceeds the area where dentin has been demineralized by the acids used in either the total-etch or self-etch technique.17,18 Occasionally resin tags may assume a hollow, tubular form, with thin walls. In the specimens where dentin has been completely dissolved, the tags often show a contorted form, sometimes with sharp bends, which do not correspond to that of the dentinal tubules. The aim of the present study was to investigate and review the current interpretation of resin tags by means of scanning electron microscopic (SEM) observation.
MATERIAL AND METHODS Ten noncarious, human third molars, stored in 0.5% chloramine T (Sigma-Aldrich Co, St. Louis, Mo) at 4°C, were used within 1 month after extraction. Eight of these teeth were subjected to occlusal enamel removal using a slow-speed, water-cooled diamond saw (Isomet 1000; Buhler, Lake Bluff, NY). The dentin was yellow with no evidence of translucency (North Carolina Dentin Sclerosis Scale of 1).19 The prepared flat dentin specimens were etched with 37% phosphoric acid (H3PO4) gel (Uni-Etch; Bisco, Itasca, Ill) for 10 seconds and then rinsed with water for 20 seconds. The dentin was kept moist by removing the excess water with a damp cotton pellet. The conditioned dentin was treated with a dentin bonding agent (Single Bond; 3M ESPE, St. Paul, Minn) as recommended by the manufacturers and was light-polymerized (US Ø 11 mm wand, 600 mW/cm2, Optilux 500; Demetron Research Corp/Kerr, Danbury, Conn) for 20 seconds with the tip as near as possible, without touching the bonded surface. A 0.2- to 0.5-mm layer of flowable composite (Tetric Flow; Ivoclar Vivadent, Schaan, Liechtenstein) was then applied to the bonded dentin followed by 2 layers (2 mm each) of composite (Z 250; 3M ESPE). Each composite was light-polymerized for 40 seconds following the previously described procedure. The roots were cut off each bonded specimen just below the cemento-enamel junction. This was achieved by transversally cutting the roots with a water-cooled diamond saw. Each specimen was then sectioned into 2 halves with a slow-speed, water-cooled diamond saw 234
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(Isomet 1000; Buhler). The cut surface was polished with sandpaper (600- and 1200-grit, Wetordry sheet; 3M ESPE). The 16 specimens obtained were randomly assigned to 4 groups. For group EA specimens, the sectioned, polished surface was treated with 15 wt % ethylenediamine tetraacetic acid (EDTA) (Largal Ultra; Septodont, Cedex, France) for 60 seconds, followed by an air-water spray rinse for 15 seconds. For group PA3 specimens, 37% H3PO4 gel (Uni-Etch; Bisco) was applied to the section surface for 3 seconds and followed by an air-water spray rinse for 15 seconds. The sectioned surface of group PA120 specimens was treated with 37% H3PO4 gel (Uni-Etch; Bisco) for 120 seconds and then rinsed with air-water spray for 30 seconds. Subsequently, the specimens were immersed in NaOCl (7% 6 2%) for 120 seconds and rinsed again with tap water for 30 seconds. Group CA specimens were stored in l N hydrochloric acid (HCl) for 48 hours (changing the solution every 24 hours) to completely demineralize the dentin. Specimens were then rinsed with tap water for 6 hours. To dissolve all of the collagen dentinal tissue, the specimens were immersed in NaOCl (7% 6 2%) for 24 hours, and then rinsed again with tap water for 6 hours. The 2 remaining molars were used as the control group (Group N). Their roots were removed, and the crowns were sectioned into 2 halves with a slow-speed, water-cooled diamond saw (Isomet 1000; Buhler). The cut surface was then polished with sandpaper (600- and 1200-grit; Wetordry sheet; 3M ESPE). Each specimen was stored in l mol/L hydrochloric acid (HCl) for 300 seconds to demineralize the dentin; the specimens were then rinsed with tap water for 5 minutes. Subsequently, the specimens were immersed in NaOCl (7% 6 2%) for 5 minutes and were rinsed again with tap water for 5 minutes. All specimens were dehydrated in a graded ethanol series and critical-point dried in acetone-CO2 in a Critical Point Drier Unit20 (CPD 030; BAL-TEC AG, Balzers, Liechtenstein). Subsequently, all specimens were mounted on aluminum stubs with colloidal silver paint and sputter-coated ˚ gold-palladium (SCD 005; BAL-TEC AG) with 200 A alloy (Foil Target AU; BAL-TEC AG). Each specimen was examined by SEM (Philips 515; Philips Co, Amsterdam, The Netherlands) at a 15-KV accelerating voltage. The images were achieved with a computerized program (Analysis 2.1; Soft Imaging System GmbH, Munster, Germany).
RESULTS Specimens from Group EA showed a 2- to 3-mm– thick hybrid layer, as well as so-called ‘‘resin tags’’ which were characterized by 2 distinct sections (Fig. 1, A). Thick resin tags filled the acid-etched dentinal tubules. VOLUME 92 NUMBER 3
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Fig. 1. A, SEM microphotograph (33700) of dentin-restoration interface in specimen surface treated with 15% EDTA for 60 seconds. So-called ‘‘resin tags’’ are visible. Distinct chromatic discontinuity near adhesive layer is evident. B, SEM microphotograph (Original magnification 31150) of dentin-restoration interface in specimen surface treated with 37% H3PO4 for 3 seconds. Several filaments are visible. Appearance is column-like and perpendicularly oriented to bonded surface. Portions within black and white frames are magnified in Fig. 1, C and D, respectively. C, SEM microphotograph (Original magnification 34580), higher magnification of portion within white frame in Fig. 1, B. Due to specimen preparation some tags presented splitends with hollow structures (arrow). D, SEM microphotograph (Original magnification 34580), higher magnification of portion within black frame in Fig.1, B. This portion is approximately 40 mm from hybrid layer. Partial dissolution of peritubular dentin shows thin fibers, presumably of collagen origin (black arrow). Fibers are subtended between intertubular dentin and filament structure. Some structures present split-ends with very thin walls.
Fig. 2. SEM microphotographs of dentin-restoration interface in specimen surface treated with 37% H3PO4 for 120 seconds and NaOCl for 120 seconds. Dissolution of peritubular and intertubular dentin shows several fibrous structures tens of microns long. A, These structures, initially funnel shaped, subsequently become thinner until taking on cylindrical and irregular sinuous form (Original magnification 31100). Results of stronger magnification include several lateral branches (highlighted). B, Original magnification 32200; C, Original magnification 31150; D, Original magnification 32300: Various fibrous structures are visible. Structures are tens of microns long and show irregular sinuous form (arrows). Just below hybrid layer, some plugs may show brittle fractures (arrowhead ).
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Fig. 3. SEM microphotograph of bonded surface in specimen treated with 1 N HCl for 48 hours and NaOCl for 24 hours. A, Several filamentous structures can be observed on surface of restoration (Original magnification 3263). Filaments extend sinuously for tens of microns to form type of ‘‘carpet.’’ B, Filaments are characterized by initial vertical conical-trunk portion followed by thinner portion, which abruptly bends and continues for tens of microns on surface of restoration (Original magnification 34780).
Fig. 4. SEM microphotographs of specimen section surface treated with 1M HCl for 300 seconds and NaOCl for 300 seconds. A, Dissolution of peritubular and intertubular dentin shows several fibrous structures that are tens of microns long and have cylindrical form (Original magnification 32200). B, Some filaments present split-ends with hollow structures and very thin walls (Original magnification 38800).
The PA3 group specimens had numerous column-like filaments which were perpendicularly set to the bonded surface and were more clearly visible (Fig. 1, B). These filament structures showed a linear form where sustained by mineralized tissue (Fig. 1, B). As a result of the specimen preparation, some tags presented split-ends with hollow structures and very thin walls (Fig. 1, C and D). Furthermore, the partial dissolution of the peritubular dentin showed thin fibers, presumably of collagen origin. These fibers were subtended between the intertubular dentin and the filament structure (Fig. 1, D). The specimens of the PA120 group showed numerous fibrous structures that were tens of microns long (Fig. 2, A). These structures, initially funnel shaped, subsequently became thinner until they took on a cylindrical form. With stronger magnification, numerous lateral branches were highlighted (Fig. 2, B). Where the intertubular and peritubular dentin had been removed, resin tags occasionally showed an irregular sinuous form (Fig. 2, C and D). Just below the hybrid layer some of these tags showed brittle fractures (Fig. 2, C and D). 236
In Group CA, several filament structures could be observed on the surface of the restoration. These filaments extended sinuously for tens of microns to form a type of ‘‘carpet’’ (Fig. 3, A). These filaments were characterized by an initial vertical conical-trunk portion followed by a thinner portion, which abruptly bent and continued for tens of microns on the surface of the restoration (Fig. 3, B). Group N specimens showed numerous fibrous structures which were similar to those found in Group PA3 and PA120 specimens (Fig. 4, A). These structures, which were tens of microns long, were characterized by a cylindrical form. Some filaments presented split-ends with hollow structures and very thin walls (Fig. 4, B).
DISCUSSION The flow of adhesive resins within the dentinal tubules is unlikely to occur, due to both the surface tension of the adhesive resin and to the low surface free energy of the tubular walls in those areas which have not been conditioned by the etchant.6 There is a general consensus VOLUME 92 NUMBER 3
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that resin tags have the capacity to adhere to the tubular surface only where they can infiltrate the interfibrillar spaces of surrounding demineralized intertubular dentin.9 Deeper resin tags may occupy the tubular liner but do not adhere to the fibular walls.3 Upon careful observation, resin tags show 2 different segments: 1 in or near the hybrid layer and another that is more distant (Figs. 2 and 3, B). The former is a few microns long and has a conical trunk shape as a result of the penetration of the adhesive resin in the tubular lumen. The latter is tens of microns long and appears thinner and cylindrical. It has been suggested that this shape may be due to the shrinkage of the adhesive system, which does not sufficiently bond to the nonconditioned dentin and, thus, tends to detach from the walls of the tubule.6 Various interpretations have been proposed regarding the hollow appearance and very thin walls which characterize the final portion of the resin tags.7,9 When the bonding agent adheres tenaciously to the walls of the tubules, the shrinkage produced by the polymerization process will produce hollow resin tags.7,8 On the other hand, if the bonding agent does not adhere sufficiently to the walls of the tubule, polymerization will create solid tags.7,8 This theory however, does not account for the presence of hollow resin tags in deeper areas where the peritubular dentin is not etched and, therefore, does not allow the adhesion of the bonding agent to the walls of the tubule. Another theory claims that the adhesive resin flows along the tubular walls lined with lamina limitans, creating a thin sheath around the extremity of the odontoblast process.9 According to this hypothesis, the hollow aspect should only occur at the distal extremity of the resin tag. However, hollow structures were observed even in the area of the adhesive layer (Fig. 1). Therefore, the hollow shape with thin walls was not only visible at the distal extremity of the resin tags, but also along the entire length of these filaments. If the resin tags were made of polymerized adhesive, they would have the same form as the dentinal tubules, without sharp bends. However, demineralized specimens often showed filaments with a contorted form (Fig. 2, C and D) that, in some situations, rest on the hybrid layer to form a ‘‘carpet’’ of filaments (Fig. 3), whereas others suddenly change direction (Fig. 2, C and D). These filamentous structures showed a linear form where they were supported by mineralized tissue (Fig. 1, B). Moreover, the processes of decalcification, deproteinization, and in particular, the tap water rinse, which were performed to prepare the specimens for SEM visualization, could cause brittle fractures in these thin resinous polymerized structures. These can only be observed in those parts of the resin tags that are close to the hybrid layer (Fig. 3). The section surface examination of Group EA specimens treated with 15% EDTA for 60 seconds SEPTEMBER 2004
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showed a discontinuity in the resin tags (Fig. 1, A). When the tags were in the proximity of the adhesive, these filaments took on the same characteristics as the hybrid layer, whereas the appearance changed as the distance from the adhesive increased, thus demonstrating a different nature. A previous study,13 which utilized SEM examination and vital staining with Alcian Blue, indicated that the resin tags were a combination of resin and lamina limitans. Dentinal tubules are lined throughout their length with an organic structure, the lamina limitans, that has a high content of glycosaminoglycans (GAGs).21 Vital staining demonstrated the presence of short and long string-like tag projections containing GAGs.13 Given that the glycosaminoglycanic component of the organic matrix is resistant to strong acids and NaOCl,21 this residual structure could lead to the formation of resin tags. This hypothesis provides a more convincing explanation of the morphological aspects of the resin tags. The remarkable length of such filaments may be the result of residual lamina limitans. The thin cylindrical shape of the resin tags is due to the collapse (in the centripetal direction) of the lamina limitans, which is no longer supported by the peritubular dentin.21 SEM analysis performed in this study confirms that since the lamina limitans is an organic and flexible structure, it collapses on itself when dentinal support is lacking, thus justifying the sinuous form and the absence of fractures in the resin tags. Furthermore, the persistence of the lamina limitans in the dentinal tubules may be responsible for the hollow structure of these filaments. The hypothesis that the glycosaminoglycanic component of the organic matrix could lead to the formation of resin tags is further supported by another micromorphological characteristic. The partial dissolution of the peritubular dentin showed several thin fibrils (Fig. 1, D) which link the intertubular dentin and the structure stored within the dentinal tubules. These collagen fibers have the function of anchoring the lamina limitans to the walls of the tubules.20 Control specimens (Group N) confirmed this study’s hypothesis that the filament structures observed by SEM are not entirely made of resin. Although control specimens were neither etched nor bonded, they still presented filament structures that were morphologically similar to the tags found in the bonded specimens. Control specimen preparation was based on the procedure used by Thomas21 in a study of the lamina limitans morphology. The current confusion caused by relying only on SEM morphology to identify ‘‘resin tags’’ is reminiscent of controversy in the 1980s over the extent of the odontoblastic process in dentinal tubules. Several SEM reports indicated that the cytoplastic process extended from the pulp to the DEJ. However, careful SEM and transmission electronic microscopy (TEM) correlative 237
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studies indicated that the tubular structures seen by SEM and erroneously identified as odontoblastic processes were shown by TEM to be hollow tubules created by the lamina limitans.22,23 Therefore, further investigation, using different specimen preparation techniques for SEM analysis along with TEM, is necessary to confirm the results of this study and to clarify the nature of the so-called ‘‘resin tags.’’ Since the morphological information was gathered from specimens prepared in vitro without pulpal fluid and under physiological pulpal pressure, further research under clinical conditions is needed.
CONCLUSIONS Within the limitations of this study, the results confirmed the difficulty in defining the nature of resin tags using current specimen preparation techniques for SEM analysis. The morphological and structural characteristics of ‘‘resin tags’’ that were observed in demineralized bond dentin showed a strong resemblance to lamina limitans. This organic structure which lines the dentinal tubules appears to be responsible for the formation of resin tags. This study does not prove that the resin tags are entirely made of glycosaminoglycans, rather, there is no evidence that resin tags are entirely made of resin. REFERENCES 1. Gwinnett AJ, Jendresen MD. Micromorphologic features of cervical erosion after acid conditioning and its relation with composite resin. J Dent Res 1978;57:543-9. 2. Perdigao J, Swift EJ Jr, Denehy GE, Wefel JS, Donly KJ. In vitro bond strengths and SEM evaluation of dentin bonding systems to different dentin substrates. J Dent Res 1994;73:44-55. 3. Prati C, Chersoni S, Mongiorgi R, Pashley DH. Resin-infiltrated dentin layer formation of new bonding systems. Oper Dent 1998;23:185-94. 4. Brady JM, Clarke-Martin JA. Penetration of etched enamel and dentin cavity surfaces by bonding agent/composite resin. Clin Prev Dent 1990;12: 30-3. 5. Gwinnett AJ, Kanca J 3rd. Interfacial morphology of resin composite and shining erosion lesions. Am J Dent 1992;5:315-7. 6. Chappell RP, Cobb CM, Spencer P, Eick JD. Dentinal tubule anastomosis: a potential factor in adhesive bonding? J Prosthet Dent 1994;72:183-8. 7. Fusayama T. New adhesive resin restoration (Material science and clinical use). In: Fusayama T. New concepts in operative dentistry. Chicago: Quintessence; 1980. p. 61-156.
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8. Suh BI. All-Bond–fourth generation dentin bonding system. J Esthet Dent 1991;3:139-47. 9. Tay FR, Gwinnett AJ, Pang KM, Wei SH. Structural evidence of a sealed tissue interface with a total-etch wet-bonding technique in vivo. J Dent Res 1994;73:629-36. 10. Pashley DH. Clinical correlations of dentin structure and function. J Prosthet Dent 1991;66:777-81. 11. Gwinnett AJ. Quantitative contribution of resin infiltration/hybridization to dentin bonding. Am J Dent 1993;6:7-9. 12. Tao L, Pashley DH. Shear bond strengths to dentin: effects of surface treatments, depth and position. Dent Mater 1988;4:371-8. 13. Titley K, Chernecky R, Chan A, Smith D. The composition and ultrastructure of resin tags in etched dentin. Am J Dent 1995;8:224-30. 14. Prati C, Chersoni S, Mongiorgi R, Montanari G, Pashley DH. Thickness and morphology of resin-infiltrated dentin layer in young, old, and sclerotic dentin. Oper Dent 1999;24:66-72. 15. Ferrari M, Vichi A, Grandini S. Efficacy of different adhesive techniques on bonding to root canal walls: an SEM investigation. Dent Mater 2001;17: 422-9. 16. Dagostin A, Ferrari M. In vivo bonding mechanism of an experimental dual-cure enamel-dentin bonding system. Am J Dent 2001;14:105-8. 17. Van Meerbeek B, Inokoshi S, Braem M, Lambrechts P, Vanherle G. Morphological aspects of the resin-dentin interdiffusion zone with different dentin adhesive systems. J Dent Res 1992;71:1530-40. 18. Van Meerbeek B, De Munck J, Yoshida Y, Inoue S, Vargas M, Vijay P, et al. Buonocore memorial lecture. Adhesion to enamel and dentin: current status and future challenges. Oper Dent 2003;28:215-35. 19. Heymann HO, Bayne SC. Current concepts in dentin bonding: focusing on dentinal adhesion factors. J Am Dent Assoc 1993;124:26-36. 20. Thomas HF, Carella P. A scanning electron microscope study of dentinal tubules from un-erupetd human teeth. Arch Oral Biol 1983;28:1125-30. 21. Thomas HF. The lamina limitans of human dentinal tubules. J Dent Res 1984;63:1064-6. 22. Thomas HF. The dentin-predentin complex and its permeability: anatomical overview. J Dent Res 1985;64:607-12. 23. Weber DF, Zaki AE. Scanning and transmission electron microscopy of tubular structures presumed to be human odontoblast processes. J Dent Res 1986;65:982-6. Reprint request to: Dr LUCA GIACHETTI UNIVERSTIY OF FLORENCE DEPARTMENT OF DENTISTRY (DIPARTIMENTO VIALE MORGAGNI 85-50134 FIRENZE ITALY FAX: 139055411798 E-MAIL: [email protected]
DI
ODONTOSTOMATOLOGIA)
0022-3913/$30.00 Copyright Ó 2004 by The Editorial Council of The Journal of Prosthetic Dentistry doi:10.1016/j.prosdent.2004.06.021
VOLUME 92 NUMBER 3
CLINICAL IMPLICATIONS The presence of glycosaminoglycans in the etched dentin could jeopardize the efficacy of the adhesive systems.
T
he literature reports that so called ‘‘resin tags,’’ ‘‘resin tails,’’ or ‘‘resin strings’’ are the result of adhesive resin penetration inside the dentinal tubules.1,2 However, current literature provides several and occasionally contradictory interpretations regarding the formation of these filament structures.3-9 To achieve an optimal dentin bond, the adhesive must penetrate the demineralized dentin tubules and branches before polymerization. The importance given to the infiltration and flow of the adhesive resin inside the acid-treated dentinal tubules remains controversial. The relative contribution a
Assistant and Chair of Dental Materials. Adjunct faculty. c Adjunct faculty. b
SEPTEMBER 2004
of resin tags to bond strength must include where the bond is made (superficial, middle, or deep dentin) and whether the dentinal tubules are perpendicular or parallel to the prepared dentin surface.10 Resin tags may contribute about 30% to the total strength of the adhesivedentin bond.11 Tao and Pashley12 indicate that no link exists between the bond strength and the formation of the resin tags. Furthermore, resin penetration within the dentinal tubules should hermetically seal the dentin-pulp complex, thus minimizing permeability and pulpal irritation.13 The shrinkage that results from polymerization may, however, cause the separation of the adhesive resin from the walls of the tubules, thus allowing the passage of fluids.10 One of the procedures most commonly used in vitro for evaluation of the thickness of the resin-infiltrated THE JOURNAL OF PROSTHETIC DENTISTRY 233
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dentin layer is the partial or total removal of dentin that occurs by dissolving the mineral component with strong acids and the organic matrix with sodium hypochlorite (NaOCl).3,14 Specimens sequentially treated with acids and NaOCI show numerous sinuous filaments that are tens or hundreds of microns long.3,5 The morphology, length, and density of so-called resin tags are used by some for the qualitative and/or semiquantitative evaluation of the efficacy of adhesive systems.15,16 Nevertheless, careful examination of the morphological characteristics of the resin tags may result in confusion regarding the nature of these structures. In fact, the length of the resin tags often exceeds the area where dentin has been demineralized by the acids used in either the total-etch or self-etch technique.17,18 Occasionally resin tags may assume a hollow, tubular form, with thin walls. In the specimens where dentin has been completely dissolved, the tags often show a contorted form, sometimes with sharp bends, which do not correspond to that of the dentinal tubules. The aim of the present study was to investigate and review the current interpretation of resin tags by means of scanning electron microscopic (SEM) observation.
MATERIAL AND METHODS Ten noncarious, human third molars, stored in 0.5% chloramine T (Sigma-Aldrich Co, St. Louis, Mo) at 4°C, were used within 1 month after extraction. Eight of these teeth were subjected to occlusal enamel removal using a slow-speed, water-cooled diamond saw (Isomet 1000; Buhler, Lake Bluff, NY). The dentin was yellow with no evidence of translucency (North Carolina Dentin Sclerosis Scale of 1).19 The prepared flat dentin specimens were etched with 37% phosphoric acid (H3PO4) gel (Uni-Etch; Bisco, Itasca, Ill) for 10 seconds and then rinsed with water for 20 seconds. The dentin was kept moist by removing the excess water with a damp cotton pellet. The conditioned dentin was treated with a dentin bonding agent (Single Bond; 3M ESPE, St. Paul, Minn) as recommended by the manufacturers and was light-polymerized (US Ø 11 mm wand, 600 mW/cm2, Optilux 500; Demetron Research Corp/Kerr, Danbury, Conn) for 20 seconds with the tip as near as possible, without touching the bonded surface. A 0.2- to 0.5-mm layer of flowable composite (Tetric Flow; Ivoclar Vivadent, Schaan, Liechtenstein) was then applied to the bonded dentin followed by 2 layers (2 mm each) of composite (Z 250; 3M ESPE). Each composite was light-polymerized for 40 seconds following the previously described procedure. The roots were cut off each bonded specimen just below the cemento-enamel junction. This was achieved by transversally cutting the roots with a water-cooled diamond saw. Each specimen was then sectioned into 2 halves with a slow-speed, water-cooled diamond saw 234
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(Isomet 1000; Buhler). The cut surface was polished with sandpaper (600- and 1200-grit, Wetordry sheet; 3M ESPE). The 16 specimens obtained were randomly assigned to 4 groups. For group EA specimens, the sectioned, polished surface was treated with 15 wt % ethylenediamine tetraacetic acid (EDTA) (Largal Ultra; Septodont, Cedex, France) for 60 seconds, followed by an air-water spray rinse for 15 seconds. For group PA3 specimens, 37% H3PO4 gel (Uni-Etch; Bisco) was applied to the section surface for 3 seconds and followed by an air-water spray rinse for 15 seconds. The sectioned surface of group PA120 specimens was treated with 37% H3PO4 gel (Uni-Etch; Bisco) for 120 seconds and then rinsed with air-water spray for 30 seconds. Subsequently, the specimens were immersed in NaOCl (7% 6 2%) for 120 seconds and rinsed again with tap water for 30 seconds. Group CA specimens were stored in l N hydrochloric acid (HCl) for 48 hours (changing the solution every 24 hours) to completely demineralize the dentin. Specimens were then rinsed with tap water for 6 hours. To dissolve all of the collagen dentinal tissue, the specimens were immersed in NaOCl (7% 6 2%) for 24 hours, and then rinsed again with tap water for 6 hours. The 2 remaining molars were used as the control group (Group N). Their roots were removed, and the crowns were sectioned into 2 halves with a slow-speed, water-cooled diamond saw (Isomet 1000; Buhler). The cut surface was then polished with sandpaper (600- and 1200-grit; Wetordry sheet; 3M ESPE). Each specimen was stored in l mol/L hydrochloric acid (HCl) for 300 seconds to demineralize the dentin; the specimens were then rinsed with tap water for 5 minutes. Subsequently, the specimens were immersed in NaOCl (7% 6 2%) for 5 minutes and were rinsed again with tap water for 5 minutes. All specimens were dehydrated in a graded ethanol series and critical-point dried in acetone-CO2 in a Critical Point Drier Unit20 (CPD 030; BAL-TEC AG, Balzers, Liechtenstein). Subsequently, all specimens were mounted on aluminum stubs with colloidal silver paint and sputter-coated ˚ gold-palladium (SCD 005; BAL-TEC AG) with 200 A alloy (Foil Target AU; BAL-TEC AG). Each specimen was examined by SEM (Philips 515; Philips Co, Amsterdam, The Netherlands) at a 15-KV accelerating voltage. The images were achieved with a computerized program (Analysis 2.1; Soft Imaging System GmbH, Munster, Germany).
RESULTS Specimens from Group EA showed a 2- to 3-mm– thick hybrid layer, as well as so-called ‘‘resin tags’’ which were characterized by 2 distinct sections (Fig. 1, A). Thick resin tags filled the acid-etched dentinal tubules. VOLUME 92 NUMBER 3
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Fig. 1. A, SEM microphotograph (33700) of dentin-restoration interface in specimen surface treated with 15% EDTA for 60 seconds. So-called ‘‘resin tags’’ are visible. Distinct chromatic discontinuity near adhesive layer is evident. B, SEM microphotograph (Original magnification 31150) of dentin-restoration interface in specimen surface treated with 37% H3PO4 for 3 seconds. Several filaments are visible. Appearance is column-like and perpendicularly oriented to bonded surface. Portions within black and white frames are magnified in Fig. 1, C and D, respectively. C, SEM microphotograph (Original magnification 34580), higher magnification of portion within white frame in Fig. 1, B. Due to specimen preparation some tags presented splitends with hollow structures (arrow). D, SEM microphotograph (Original magnification 34580), higher magnification of portion within black frame in Fig.1, B. This portion is approximately 40 mm from hybrid layer. Partial dissolution of peritubular dentin shows thin fibers, presumably of collagen origin (black arrow). Fibers are subtended between intertubular dentin and filament structure. Some structures present split-ends with very thin walls.
Fig. 2. SEM microphotographs of dentin-restoration interface in specimen surface treated with 37% H3PO4 for 120 seconds and NaOCl for 120 seconds. Dissolution of peritubular and intertubular dentin shows several fibrous structures tens of microns long. A, These structures, initially funnel shaped, subsequently become thinner until taking on cylindrical and irregular sinuous form (Original magnification 31100). Results of stronger magnification include several lateral branches (highlighted). B, Original magnification 32200; C, Original magnification 31150; D, Original magnification 32300: Various fibrous structures are visible. Structures are tens of microns long and show irregular sinuous form (arrows). Just below hybrid layer, some plugs may show brittle fractures (arrowhead ).
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Fig. 3. SEM microphotograph of bonded surface in specimen treated with 1 N HCl for 48 hours and NaOCl for 24 hours. A, Several filamentous structures can be observed on surface of restoration (Original magnification 3263). Filaments extend sinuously for tens of microns to form type of ‘‘carpet.’’ B, Filaments are characterized by initial vertical conical-trunk portion followed by thinner portion, which abruptly bends and continues for tens of microns on surface of restoration (Original magnification 34780).
Fig. 4. SEM microphotographs of specimen section surface treated with 1M HCl for 300 seconds and NaOCl for 300 seconds. A, Dissolution of peritubular and intertubular dentin shows several fibrous structures that are tens of microns long and have cylindrical form (Original magnification 32200). B, Some filaments present split-ends with hollow structures and very thin walls (Original magnification 38800).
The PA3 group specimens had numerous column-like filaments which were perpendicularly set to the bonded surface and were more clearly visible (Fig. 1, B). These filament structures showed a linear form where sustained by mineralized tissue (Fig. 1, B). As a result of the specimen preparation, some tags presented split-ends with hollow structures and very thin walls (Fig. 1, C and D). Furthermore, the partial dissolution of the peritubular dentin showed thin fibers, presumably of collagen origin. These fibers were subtended between the intertubular dentin and the filament structure (Fig. 1, D). The specimens of the PA120 group showed numerous fibrous structures that were tens of microns long (Fig. 2, A). These structures, initially funnel shaped, subsequently became thinner until they took on a cylindrical form. With stronger magnification, numerous lateral branches were highlighted (Fig. 2, B). Where the intertubular and peritubular dentin had been removed, resin tags occasionally showed an irregular sinuous form (Fig. 2, C and D). Just below the hybrid layer some of these tags showed brittle fractures (Fig. 2, C and D). 236
In Group CA, several filament structures could be observed on the surface of the restoration. These filaments extended sinuously for tens of microns to form a type of ‘‘carpet’’ (Fig. 3, A). These filaments were characterized by an initial vertical conical-trunk portion followed by a thinner portion, which abruptly bent and continued for tens of microns on the surface of the restoration (Fig. 3, B). Group N specimens showed numerous fibrous structures which were similar to those found in Group PA3 and PA120 specimens (Fig. 4, A). These structures, which were tens of microns long, were characterized by a cylindrical form. Some filaments presented split-ends with hollow structures and very thin walls (Fig. 4, B).
DISCUSSION The flow of adhesive resins within the dentinal tubules is unlikely to occur, due to both the surface tension of the adhesive resin and to the low surface free energy of the tubular walls in those areas which have not been conditioned by the etchant.6 There is a general consensus VOLUME 92 NUMBER 3
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that resin tags have the capacity to adhere to the tubular surface only where they can infiltrate the interfibrillar spaces of surrounding demineralized intertubular dentin.9 Deeper resin tags may occupy the tubular liner but do not adhere to the fibular walls.3 Upon careful observation, resin tags show 2 different segments: 1 in or near the hybrid layer and another that is more distant (Figs. 2 and 3, B). The former is a few microns long and has a conical trunk shape as a result of the penetration of the adhesive resin in the tubular lumen. The latter is tens of microns long and appears thinner and cylindrical. It has been suggested that this shape may be due to the shrinkage of the adhesive system, which does not sufficiently bond to the nonconditioned dentin and, thus, tends to detach from the walls of the tubule.6 Various interpretations have been proposed regarding the hollow appearance and very thin walls which characterize the final portion of the resin tags.7,9 When the bonding agent adheres tenaciously to the walls of the tubules, the shrinkage produced by the polymerization process will produce hollow resin tags.7,8 On the other hand, if the bonding agent does not adhere sufficiently to the walls of the tubule, polymerization will create solid tags.7,8 This theory however, does not account for the presence of hollow resin tags in deeper areas where the peritubular dentin is not etched and, therefore, does not allow the adhesion of the bonding agent to the walls of the tubule. Another theory claims that the adhesive resin flows along the tubular walls lined with lamina limitans, creating a thin sheath around the extremity of the odontoblast process.9 According to this hypothesis, the hollow aspect should only occur at the distal extremity of the resin tag. However, hollow structures were observed even in the area of the adhesive layer (Fig. 1). Therefore, the hollow shape with thin walls was not only visible at the distal extremity of the resin tags, but also along the entire length of these filaments. If the resin tags were made of polymerized adhesive, they would have the same form as the dentinal tubules, without sharp bends. However, demineralized specimens often showed filaments with a contorted form (Fig. 2, C and D) that, in some situations, rest on the hybrid layer to form a ‘‘carpet’’ of filaments (Fig. 3), whereas others suddenly change direction (Fig. 2, C and D). These filamentous structures showed a linear form where they were supported by mineralized tissue (Fig. 1, B). Moreover, the processes of decalcification, deproteinization, and in particular, the tap water rinse, which were performed to prepare the specimens for SEM visualization, could cause brittle fractures in these thin resinous polymerized structures. These can only be observed in those parts of the resin tags that are close to the hybrid layer (Fig. 3). The section surface examination of Group EA specimens treated with 15% EDTA for 60 seconds SEPTEMBER 2004
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showed a discontinuity in the resin tags (Fig. 1, A). When the tags were in the proximity of the adhesive, these filaments took on the same characteristics as the hybrid layer, whereas the appearance changed as the distance from the adhesive increased, thus demonstrating a different nature. A previous study,13 which utilized SEM examination and vital staining with Alcian Blue, indicated that the resin tags were a combination of resin and lamina limitans. Dentinal tubules are lined throughout their length with an organic structure, the lamina limitans, that has a high content of glycosaminoglycans (GAGs).21 Vital staining demonstrated the presence of short and long string-like tag projections containing GAGs.13 Given that the glycosaminoglycanic component of the organic matrix is resistant to strong acids and NaOCl,21 this residual structure could lead to the formation of resin tags. This hypothesis provides a more convincing explanation of the morphological aspects of the resin tags. The remarkable length of such filaments may be the result of residual lamina limitans. The thin cylindrical shape of the resin tags is due to the collapse (in the centripetal direction) of the lamina limitans, which is no longer supported by the peritubular dentin.21 SEM analysis performed in this study confirms that since the lamina limitans is an organic and flexible structure, it collapses on itself when dentinal support is lacking, thus justifying the sinuous form and the absence of fractures in the resin tags. Furthermore, the persistence of the lamina limitans in the dentinal tubules may be responsible for the hollow structure of these filaments. The hypothesis that the glycosaminoglycanic component of the organic matrix could lead to the formation of resin tags is further supported by another micromorphological characteristic. The partial dissolution of the peritubular dentin showed several thin fibrils (Fig. 1, D) which link the intertubular dentin and the structure stored within the dentinal tubules. These collagen fibers have the function of anchoring the lamina limitans to the walls of the tubules.20 Control specimens (Group N) confirmed this study’s hypothesis that the filament structures observed by SEM are not entirely made of resin. Although control specimens were neither etched nor bonded, they still presented filament structures that were morphologically similar to the tags found in the bonded specimens. Control specimen preparation was based on the procedure used by Thomas21 in a study of the lamina limitans morphology. The current confusion caused by relying only on SEM morphology to identify ‘‘resin tags’’ is reminiscent of controversy in the 1980s over the extent of the odontoblastic process in dentinal tubules. Several SEM reports indicated that the cytoplastic process extended from the pulp to the DEJ. However, careful SEM and transmission electronic microscopy (TEM) correlative 237
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studies indicated that the tubular structures seen by SEM and erroneously identified as odontoblastic processes were shown by TEM to be hollow tubules created by the lamina limitans.22,23 Therefore, further investigation, using different specimen preparation techniques for SEM analysis along with TEM, is necessary to confirm the results of this study and to clarify the nature of the so-called ‘‘resin tags.’’ Since the morphological information was gathered from specimens prepared in vitro without pulpal fluid and under physiological pulpal pressure, further research under clinical conditions is needed.
CONCLUSIONS Within the limitations of this study, the results confirmed the difficulty in defining the nature of resin tags using current specimen preparation techniques for SEM analysis. The morphological and structural characteristics of ‘‘resin tags’’ that were observed in demineralized bond dentin showed a strong resemblance to lamina limitans. This organic structure which lines the dentinal tubules appears to be responsible for the formation of resin tags. This study does not prove that the resin tags are entirely made of glycosaminoglycans, rather, there is no evidence that resin tags are entirely made of resin. REFERENCES 1. Gwinnett AJ, Jendresen MD. Micromorphologic features of cervical erosion after acid conditioning and its relation with composite resin. J Dent Res 1978;57:543-9. 2. Perdigao J, Swift EJ Jr, Denehy GE, Wefel JS, Donly KJ. In vitro bond strengths and SEM evaluation of dentin bonding systems to different dentin substrates. J Dent Res 1994;73:44-55. 3. Prati C, Chersoni S, Mongiorgi R, Pashley DH. Resin-infiltrated dentin layer formation of new bonding systems. Oper Dent 1998;23:185-94. 4. Brady JM, Clarke-Martin JA. Penetration of etched enamel and dentin cavity surfaces by bonding agent/composite resin. Clin Prev Dent 1990;12: 30-3. 5. Gwinnett AJ, Kanca J 3rd. Interfacial morphology of resin composite and shining erosion lesions. Am J Dent 1992;5:315-7. 6. Chappell RP, Cobb CM, Spencer P, Eick JD. Dentinal tubule anastomosis: a potential factor in adhesive bonding? J Prosthet Dent 1994;72:183-8. 7. Fusayama T. New adhesive resin restoration (Material science and clinical use). In: Fusayama T. New concepts in operative dentistry. Chicago: Quintessence; 1980. p. 61-156.
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8. Suh BI. All-Bond–fourth generation dentin bonding system. J Esthet Dent 1991;3:139-47. 9. Tay FR, Gwinnett AJ, Pang KM, Wei SH. Structural evidence of a sealed tissue interface with a total-etch wet-bonding technique in vivo. J Dent Res 1994;73:629-36. 10. Pashley DH. Clinical correlations of dentin structure and function. J Prosthet Dent 1991;66:777-81. 11. Gwinnett AJ. Quantitative contribution of resin infiltration/hybridization to dentin bonding. Am J Dent 1993;6:7-9. 12. Tao L, Pashley DH. Shear bond strengths to dentin: effects of surface treatments, depth and position. Dent Mater 1988;4:371-8. 13. Titley K, Chernecky R, Chan A, Smith D. The composition and ultrastructure of resin tags in etched dentin. Am J Dent 1995;8:224-30. 14. Prati C, Chersoni S, Mongiorgi R, Montanari G, Pashley DH. Thickness and morphology of resin-infiltrated dentin layer in young, old, and sclerotic dentin. Oper Dent 1999;24:66-72. 15. Ferrari M, Vichi A, Grandini S. Efficacy of different adhesive techniques on bonding to root canal walls: an SEM investigation. Dent Mater 2001;17: 422-9. 16. Dagostin A, Ferrari M. In vivo bonding mechanism of an experimental dual-cure enamel-dentin bonding system. Am J Dent 2001;14:105-8. 17. Van Meerbeek B, Inokoshi S, Braem M, Lambrechts P, Vanherle G. Morphological aspects of the resin-dentin interdiffusion zone with different dentin adhesive systems. J Dent Res 1992;71:1530-40. 18. Van Meerbeek B, De Munck J, Yoshida Y, Inoue S, Vargas M, Vijay P, et al. Buonocore memorial lecture. Adhesion to enamel and dentin: current status and future challenges. Oper Dent 2003;28:215-35. 19. Heymann HO, Bayne SC. Current concepts in dentin bonding: focusing on dentinal adhesion factors. J Am Dent Assoc 1993;124:26-36. 20. Thomas HF, Carella P. A scanning electron microscope study of dentinal tubules from un-erupetd human teeth. Arch Oral Biol 1983;28:1125-30. 21. Thomas HF. The lamina limitans of human dentinal tubules. J Dent Res 1984;63:1064-6. 22. Thomas HF. The dentin-predentin complex and its permeability: anatomical overview. J Dent Res 1985;64:607-12. 23. Weber DF, Zaki AE. Scanning and transmission electron microscopy of tubular structures presumed to be human odontoblast processes. J Dent Res 1986;65:982-6. Reprint request to: Dr LUCA GIACHETTI UNIVERSTIY OF FLORENCE DEPARTMENT OF DENTISTRY (DIPARTIMENTO VIALE MORGAGNI 85-50134 FIRENZE ITALY FAX: 139055411798 E-MAIL: [email protected]
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0022-3913/$30.00 Copyright Ó 2004 by The Editorial Council of The Journal of Prosthetic Dentistry doi:10.1016/j.prosdent.2004.06.021
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