Dental Research

Scanning electron microscopic evaluation of resin-dentin and calcium hydroxide-dentin interface with resin composite restorations
Guido Goracci*/Giovanni Mori*
Abstract Calcium hyclro.xide has been used as a liner in resin composite restorations to protect the pulp. Recent research has demonstrated thai pulpal inflammation is caused by microleakage of restorations and by the .subsequent passage of bacteria. The present study involved scanning electron microscopic observation of crosssections of resin composite-dentin interfaces after the interposition of a layer of calcium hydroxide. A tiew-generation adhesive system that involves etching of the dentin was used. Ultrastructural analysis indicated that polymerization shrinkage ofihe resin composite caused he separation of the calcium hydroxide from the dentinal surface, forming 8- to 15-iim-wide interfacial gaps in 100% of the areas studied. Where the adhesive was applied directly to dentin, it adhered closely, forming a gap-free attachment with evidence of an acid-resistant hybrid layer (4 to 6 ^m in thickness) and resin tags of various lengths thai hermetically sealed the dentinal tubules. (Ouintessence lnt 996:27:29-35.)

Clinical relevance Calcium hydroxide-based linings do not adhere to the dentinal surface whereas those placed under resin composite with dentin bonding agents tend to pull away from the cavity surfaces, leaving a gap between the lining and dentin. In turn, a gap-free attachment, considered to be the best protection for the pulp, is produced where the adhesive is put directly on the dentinal surface.

Introduction

For a long time, the chemical action of restorative materials was considered to be the main cause of pulpal irritation. Lefkowitz et al' concluded that particular chemical substances contained in numerous resins have a toxic action on the odontoblasts and the underlying layers of the pulp. They even considered the residual monomers after polymerization of the resins as possible causes of puipal irritation.' In 1967, Stanley et al' used resin composites and
' Departmem of Operative Deiilistr>. Universit> La Sapienza, Rums. Italy. Reprinl requesis: Ptof Guido Goracci. Via Tagliamenlo. 50. 00198 Rome, Italy.

observed a pulpal reaction that is similar to that obtained when silicate cements are used, although the chemical properties of both materials are different. It was therefore considered necessary to protect the pulpodentinal organ with liner materials based on calcium hydroxide, thus isolating the dentin from the irritant action of resins. Brnnstrm and Nyborg,^ in a study on the pulpal response following the placement of resin composite restorations, observed that the inflammatory process is always associated with the presence of bacteria under the restorations.' In subsequent studies, zinc oxide-eugenol cement was applied over those restorative materials considered to be toxic, such as silicate cement and resin composite, to eliminate bacterial leakage. After this precaution, no puipal irritation was reported; thus it has been shown that microinfiltration between the cavity walls and the restoration represents the main cause of puipal irritation.^ Further in vivo studies-^"'^ have demonstrated that dental materials such as silicate cement, zinc phosphate cement, resin composite, and amalgam are biologically compatible, even if applied directly to the exposed dental pulp, provided that the latter is hermetically sealed and protected from any subsequent bacterial leakage. Moreover, the dental pulp has been shown to have its own reparative capacity, capable not only of healing but also of producing a dentinal bridge

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in the absence of calcium hydroxide.^ However, the problem of marginai infiltration is still unresolved because resin composites contract during polymerization, thus detaching from the walls of the cavity. It has therefore been necessary to isolate the dentin with intermediate materials, so thai, after the passage of fiuids in the contraction gap. there is no possibiiity of bacterial penetration inside tbe tubules, causing postoperatory sensitivity, puipai complications, and secondary caries. New adhesive systems have been recently introduced to improve the adhesion of resin composites to the dentinal surface and thereby to counteract the formation of marginai defects. These adhesive systems provide etching of the dentin and the combined use of monomers with bifunctional groups (primers) as well as hydrophilic resins able to penetrate the dentinal tubules and to chemically and mechanically bind to the perituhular and intertubular dentin." Further, acid etching of the dentin eliminates the smear iayer'*' and demineralizes the dentinal surface, thus allowing the penetration of resin tags in the tubules and the formation ofa demineralized resin-dentin interdifhision zonethe hybrid layer.-'--In spite of these considerations, many dentists continue to have doubts about the toxicity of the acid etching of vitai dentin and about the sealing provided and prefer to protect the deepest parts of the cavity with intermediate materials based on calcium hydroxide, beiieving that caicium ions confer protection against dentinai etching and stimulate the deposition of reparative and sclerotic dentins. The aim of the present study was therefore to observe, by scanning electron microscopy, the interface between the adhesive resin and the etched dentinal surface and the interface between calcium hydroxide and untreated dentin following apphcation and polymerization of the resin composite.

silicon carbide paper, in decreasing grits (240, 400, and 600), in an Ecomet grinder (Buehler). A light layer of self hardening calcium hydroxide (Dycal, LD Caulk) was applied over the treated dentinal surfaces, adjacent to the pulp. Tbe dentinal surfaces were then treated with the Scotchbond MP (3M Dental) dentinal adhesive system in accordance with the manufacturer's instructions. Thereafter, a layer of hybrid resin composite (ZIOO, 3 M Dental) of not more than 2 mm was applied to the dentinal surfaces and then light cured for 40 seconds (XL3000, 3M Dental}. The teeth were embedded in a self-curing epoxy resin and sectioned with a microtome (Leitz 1600 Microtome) along the longitudinal axis, thus passing through the center of the restorations (Fig 1 ). Section surfaces were cleaned with 10% orthophosphoric acid for 3 to 5 seconds and quickly rinsed with air-water spray for 15 seconds to remove the smear layer. AH the specimens were dehydrated through increasirtg concentrations of ethyl alcohol (30%, 50%, 70%, 90%, and 100%) and critical point dried (substitution of alcohol by carbon dioxide). Specimens were mounted with silver paste on metallic stubs, and coated with about 20 A of platinum (Edwards Sputtering 150 S). They were observed with a Cambridge 150 A scanning electron microscope (Cambridge Instruments) used at an acceleration voltage of 7 to 10 kV Scanning electron micrographs were made along the resin compositedentin margin and subsequently colored with a previously described method.^-'
Results

Method and materials

Ten noncarious extracted human third molars belonging to subjects ranging in age between 18 and 22 years were used in the present study. Teeth were fixed by immersion in 10% neutralized formalin immediately after the avulsion. After 48 hours, 2-mm-deep heels were made on the buccal and lingual surfaces at the level of the ccmcntocnamel junction of each tooth. The specimens were then ground fiat with a water-cooled orthodontic cast trimmer (Whip-Mix) until the heels disappeared. The dentin was then ground with wet

The scanning electron microscopic analysis was initialiy carried out to evaluate the relationships among the resin composite, calcium hydroxide, and dentinal surface (Fig 2). The layer of calcium hydroxide had a thickness of about 200 laminall the specimens (Fig 3). At a higher magnification, it was possible to observe the good adhesion of the resin composite to the calcium hydroxide ( interfacial gaps of 2 to 3 |j.m were observed in approximately 10% of the areas studied) and to note the presence ofa fissure between calcium hydroxide and dentin in 100% of the areas studied (Fig 4). In all the specimens, polymerization shrinkage of the resin composite caused the detachment of the calcium hydroxide from the dentinal surface. Thus, it was possible to observe the opened dentinal tubules inside the microfissure (Fig 5). The detachment of the calcium hydroxide occurred all along the dentinal

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Fig 1 Schematic representation of a specimen after iongitudinai sectioning, Caicium hydroxide is placed in the proximity of the pulp.

Fig 2 Composite resin (R, calcium hydroxide (C], and dentin (D) of about 1 mm in thickness, and the pulpal chamber |P].

Fig 3 Caicium hydroxide is about 200 \im thick in all the specimens.

Fig 4 Cise adhesion of resin composite (R) to calcium hydroxide (C) (interfaciai gaps of 2 to 3 |im were observed in oniy approximateiy lO^c of the areas studied). Formation of an interfacial gap between caicium hydroxide and dentin (D).

Fig 5 Partiaiiy cohesive detachment of caicium hydroxide from the dentinai surlace.

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Fig 6 in some zones, the gap between dentin and calcium hydroxide is 20 xm

Fig 7 Calcium hydroxide (C) interposed between resin composite (R) and dentin (D) on the lell; resin composite applied directly to tfie denlin on the right.

FJg 8 Wherever there is no caicium hydroxide (C). the resin composite (R) adheres perfectly to the dentinai surface (Dl by means of the formation of a hybrid iayer (H].

Fig 9 Characteristic bond following the use of Scotchbond MP in direct contacf with the dentin Structurai continuity between resin composite (R) and dentin can be observed. Note the hybrid iayer (H) and ihe resin tags penetrating the tubules (T).

surface, and the gap thus formed reached 20 [xm (Fig 6), The point of passage between calcium hydroxidetreated zones and those in which the adhesive was applied directly to the dentinal surface (Fig 7) revealed that, when the calcium hydroxide layer finished, the resin composite was able to adhere perfectly to the dentinal surface, forming a hybrid layer (Fig 8}. The relationship between the resin composite and the dentinal surface in the outermost areas where calcium hydroxide was not applied was also examined. Excellent adhesion of the composite resin-adhesive complex to the dentin, mediated by a hybrid layer, was

evident; the dentin appeared to be crossed by resin tags that penetrated the tubules for about 20 \im (Figs 9 and 10). In fact, the treatment of the sectioned surfaces with phosphoric acid involved the superficial demineralization of the untreated dentin for a depth of 2 \im, to expose the resin tags that had penetrated the tubules and to uncover a transitional acid-resistant zone between resin and dentinthe hybrid layer. At a higher magnification, the structural continuity between resin composite and dentin, as well as the hybrid layer, which was about 5 |im thick, could be clearly observed (Fig 11).

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Fig 10 lieft to nghH The reconstructive material, an acid-resjstant hybrid layer, the dentjn, and the tesin tags penetrating the tubules lor about 20 ^m

Fii 11 Higher iTiagnificatton showing Ihe junction between resin composite (R) and dentin treated with Scotchbond MP adhesive system. The hybrid layer (H) presents a thickness ol about 5 |im and continues wilh the resin tags penetrating
the tubules (T).

Discussion

Good marginal adaptation of restorative rnaterials reduces inicroleakage, staining, pulpal irritation, and rectirrent caries,-''Recently, newadhcsivesysterns with higher bond strengths have been introduced to resist contraction stresses developed by the resin composite during polymerization, thus preventing the detachment of the resin from the dentinal surface. The new enamel-dentinal adhesive systems include the acid conditioning of not only the enamel but also Ihe dentin, with the complete removal of smear layer and smear plug considered obstacles to adhesion.-""'^ The application of etchant to the enarnel removes the interprismatic substance: at the level of the dentin, the etchant opens the dentinal tubules and detnineralizes the dentinal surface, thus exposing the collagen fibers for a depth of 3 to 10 |xm, depending on Pka, concentration, and time of application of the selected acid,-*" The subsequent use of an intermediate agent (primer), to be applied to dentin before the piacement of an adhesive resin, promotes the wettabMity of the resin, which must undergo intimate adhesive relationships with the dentina! tissue and establish the conditions for a real resin-dentin chemical bond. In fact, the commonly used primers contain biflinctional molecules {hydroxyethyl methacrylate, hydrophilic monomers), able to bind to both the dentinal surface and the methacr>'late ( hydrophobic) groups of the resins, thus modifying the dentinal surface frorn a hydropiiiiic to a

hydrophobic one,-^' Successively, the adhesive fluid resin (bonding agent) is applied and light cured. Scanning electron microscopic examination of cross sections ofthe resin-dentin interface revealed, in 100% of the areas studied, a gap-free attachment between resin composite adhesive and detitin and a 4 to 6-[im hybrid layer. Thus, a chemical and micromechanical bond was obtained, based on both the penetration of resin tags inside the tubules and the impregnation of the collagen fibers by the adhesive monomer. This demineralized dentin-resin interdifHtsion zone is acid resistant and has the double function of binding the restoration to the dentin and rendering it caries resistant,-' This resin-dentin interdiusion zone not only allows adequate mechanical retention of the restoration but also is likely to be provided with a coefficient of elasticity that contributes to the maintenance of the bond. Such an elastic zone should be able to compensate for and uniformly distribute the stress formed by the contraction of the resin composite in relation to the rigid dentinal substrate, thus enhancing the marginal adaptation of the restoration,^* The present study showed that intetiacial gaps of 8 to 15 |irn wide were coincident with the presence of calcium hydroxide. In tact, the research dernonstrated that, due to the poor adhesion of calcium hydroxide to the dentinal surface, the polymerization shrinkage of the resin composite caused detachment of the calcium hydroxide, leading to the formation of an interfacial gap between calcium hydroxide and dentin. Therefore, it

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could be concluded that the application of calcium hydroxide underneath resin composite restorations not only is useless but also could be harmful if it leads to the formation of a microfissure in the zones where, due to the number and orientation of the tubules favoring the passage of bacteria, it would be more appropriate to obtain hermetic seal capable of protecting the pulp. In fact, under the calcium hydroxide in all the specimens, unsealed dentinal tubules were present, as well as microfissures of about 10 \im. In contrast, in the zones in which the adhesive had been applied in direct contact with the dentin, an excellent adhesion of the resin composite to the surface was evident. This result is in agreement with that of McConnel et ai-' who reported that a material such as V.L.C. Dycal placed under resin composite with a bonding agent tends to be pulled away from the cavity surface, leaving a gap between the lining and the dentin. Reinhardt and Chalkey-*" reported that calcium hydroxide does not adhere to the smear iayer or to the dentinal tubule complex. The present data showed that the bond strength of adhesive resin to calcium hydroxide was higher than the bond strength of calcium hydroxide to dentin. Interfacial gaps only 2 to 3 jim wide were observed between calcium hydroxide and adhesive in approximately 10% of the areas studied, whereas 8 to 15-nm interfacial gaps between calcium hydroxide and dentin were observed in 100% of the cases. According to Brnnstrm et al.-" such a microfissure will be quickly colonized by pulpal fluids, and, because of inward and outward fluid movement, most of the calcium hydroxide paste will disappear with time. In addition, the dissolution will happen more quickly, the greater the width of the marginal fissure.' This observation agrees with clinical reports on disappearing Dycal.-'' In fact, the fluid present in the dentinal tubules of vital teeth is in a continuous outward now because of a gradient of pressure. The presence of Gram-positive bacteria inside these spaces has been reported; therefore, calcium hydroxide does not constitute an adequate long-term protection in the presence of a marginal defect because it does not present an obstacle to the passage of bacteria or their metabolic products to the pulp.^' Therefore, according to the present results, the negative consequences of the interposition of a layer of calcium hydroxide between dentin and adhesive are evident, because the layer will detach from the dentinal

surface and will notably reduce the dentina! surface available for adhesion of the restoration. In the present in vitro study excellent and continuous attachment was evident wherever adhesive resin was directly applied to flat surfaces of conditioned dentin. Further in vivo studies using wide, disk-shaped cavities are necessary. In more concave cavities, contraction becomes hindered in the three dimensions, shrinkage stress will be less compensated for by flow, and not only calcium hydroxide-dentin but also the resin composite-dentin bond may be disrupted.

References
1. Letlowilz W. Seelig A. Zachinshy L. PJIP response lo self-turing acrylie niling material. NY Dent J 1949; 15:373-386. 2. Stanley HR. Swerdlow H. Stanwich L. Snare; C A comparison of the biologieal effeets of filbng malerials with recommendations for pulp protection. Am Acad Goid Foil Oper 19A7; 12:55-63. 3. Brannstrm M. Kyborg H. Pulpa] reaction to composite resin restorations. J Prostliet Dem 1973:27:181-189. 4. Briinsitm M. Dentin and Pulp in Restorative Dentistr>'. London: Wolfe Medical. 1982. 5. Asmussen li. Uno S. Adhesion of restorative resins to denlin: Chemical and physiochemical aspects. Oper Dent 1992:(SLLppl 5):6S-74. 6. Beri;enholtz G. Cox CF. LoescheWJ, Syed SA. Bacterial leakage around dental restorations: Its effect on the dental pulp. J Oriil Palhol 19S2:11.439-450. 7. Cox CF, Bergenhoiti G, Fitzgerald M. Heys DR, ileys RJ. Baktr JA. Pulp capping of dental pulp mechanically exposed to oral microflora: A 5 week observation of wound healing in the monkey. J Oral Pathol l982;ll:327-339. 8. Cox CF, Bergenholtz G, Heys DR, Syed SA, Fitzgerald M, Heys RJ. Pulp capping of dental pulp mechanically exposed to oral microflora: A 1-2 year observation of wound healing in the monkey. J Oral Palhol I985;I4:156-I68. 9. Cox CF. Biocompatibility of dental materials in absence of bacteria! infection. Oper Dent 1987; 12:146-153. lU. Cot CF. Feltoii D. Bergcnholti G. Histopathological response of infected cavities treated with Gluma and Scotchbond dentin bonding agents. AmJ Dent 1988;1;189-194. 11. Felton DA, Cox CF. Odom M, Kanoy BE. Pulpal response to chemically cured and experimental light-cured glass ionomer cavity liners. J Prosthet Dent 199h65:704-7l2. 12. Fusayama T. New Concepts in Operative Dentistry. Chicago; Quintessence, 1980. 13. Fusayama T. Factors and prevention of pulp irritation by adhesive composite resin reslorations. Quintesseiiee Inl 1987:18:633-641. 14. Inokoshi S, Iwaku M. Fusayania T. Pulpal response to a new adhesive restorative rebin ] Dent Res 19a2;61:1014-1019. 15. Kaiika J. Pulpal studies: Bioeompatibility or effectiveness of marginal seal? Quintessence lnt l99O;2l:775-779. 16. Pashley DH. The effects of acid-etching on the pulpodentin eomplex. Oper Dent l992;!7;229-242. 17. Stanley HR. Pulpal consideration of adhesive materials Oper Dent I992;(suppl 5); 151-164.

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18. Torslenson B. Nordenvall KJ. Brnnstrm M. Puipai reaelion and microorganisms under t-learfil eomposile resin in deep e avilie s with acid elched dentin. Swed DeniJ 1982;6:167-176. 19. GoracciG. Mori G, Casa de'ManinisL.BazziiechiM.Olturazioni in eomposilo ed adesione alle strullure dentarie. Milan: Ed Masson. 1994. 20. Pashley DH. Tlie smear layer: Physiological considerations. Oper Dent 19S4;(sijppl 31:13-19, 21. Nakabajiashi N. The hybrid layer: A resin-dintin eoinposile. Proe FtnDertSoc l992;SS(suppl l|;323-329. 22. Van Meetbeek B, Diiem A. Goret-Nieaise M, BraemM. Lambrechts P. Vanherie G. Comparative SEM and TEM examination of the ultrastrtieture of the resin-denlin interdi Rus ion zone. J Dem Res 1993:72:495-501. 23. Goracci G. Ba^zucchi M. Mori G. Casa de' Martinis L. In vivo and in viiro analysis ofa bonding agent. Qtiintessenee Inl 1994i25:627635. 24. Nakabayashi N. Adhesive bonding .th 4-META. Oper Dent I992;{siippl5):125-13.

25. KankaJ, A method for bonding to tuoth struetiire using phosphoric acid as a dentin enamel conditioner. Quintessence Int 199l;22:285-290. 26. FusayatTia T, Optimal cavity treatmenl for adhesive restorations. J Esthel Dem 1990:2:95-99, 27. Nakabayashi N. Nakamiira M. Yasuda N. Hybrid layer as a dentin bonding meciianism. J Esthct Dent I99l;3:l33-i38, 28. Watanabe I. Nakabayashi N. Bonding durability of photocured phcnyl-P in TEGDMA lo smear I ayer-retained bovine dentin. Otiintessenee Int l993;24:335-342. 29. McConnellRJ.BoksmanL. Hunter JK.Gratton DR. The effect of restorative materials on the adaptation of two bases and a dentin bonding agent to internal cavity walls. Quintessence Int 1986; 17:703-710 30. Reinhardt JW, Chalkey Y, Softening efl'ects of bases on composite resins. Chn Prev Dent 1983:5:5-12. 3 I. Brannstrm M. Mattson B. Torstenson B. Material techniques for lining composite resin restorations: A critical approach. J Dem 1991:19:71-79. 32. Akester J. Disappearing Dycal, Br Dent J 1979:147:369-375. D

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