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Biomaterials & Bioengineering |
Incompatibility of Oxalate Desensitizers with Acidic, Fluoride-containing Total-etch Adhesives
C.K.Y. Yiu1,
N.M. King1,
B.I. Suh2,
L.J. Sharp2,
R.M. Carvalho3,
D.H. Pashley4 and
F.R. Tay1,*
1 Paediatric Dentistry and Orthodontics, Faculty of Dentistry, The University of Hong Kong, 34 Hospital Road, Hong Kong SAR, China;
2 Bisco Inc., Schaumburg, IL, USA;
3 Department of Operative Dentistry, Endodontics and Dental Materials, Bauru School of Dentistry, University of São Paulo, Bauru, SP, Brazil; and
4 Department of Oral Biology and Maxillofacial Pathology, School of Dentistry, Medical College of Georgia, Augusta, GA, USA;
Correspondence: * corresponding author, kfctay{at}netvigator.com
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ABSTRACT
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The use of oxalate desensitizers on acid-etched dentin prior to adhesive application can result in subsurface tubular occlusion by calcium oxalate crystals. However, the solubility of calcium oxalate increases in acidic solution. We hypothesized that total-etch adhesives can, depending upon their pH, interact with oxalate-desensitizer-treated dentin in an adverse manner. Acid-etched human dentin treated with 2 oxalate desensitizers (BisBlock and Super Seal) was bonded with 4 simplified total-etch adhesives: One-Step (OS), Single Bond (SB), OptiBond Solo Plus (OB), and Prime&Bond NT (PB). Composite-dentin beams were examined by SEM and TEM, both of which revealed numerous spherical globules on OB- and PB-bonded, desensitizer-treated dentin, but not in OS or SB samples. Bond strengths produced by OB and PB were significantly lower in oxalate-treated specimens than those produced by OS or SB. These surface globules may have interfered with hybridization of demineralized dentin with OB and PB resins and caused compromised bond strengths.
Key Words: oxalate desensitizer • acid-etch • single-bottle adhesives • pH • fluoride.
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INTRODUCTION
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Post-operative sensitivity is frequently encountered with the use of adhesives that require the acid-etching of vital dentin (Akpata and Sadiq, 2001; Unemori et al., 2001). The application of these total-etch adhesives can be technique-sensitive, since wet bonding is required for full expansion and optimal hybridization of the demineralized collagen matrix (Gwinnett, 1992; Kanca, 1992). The difficulty in bonding is further complicated by the intrinsic wetness of vital deep dentin after removal of the smear layer (Itthagarun and Tay, 2000), and the increased permeability associated with the simplified version of these adhesives (Tay et al., 2003a). Incomplete sealing and continuous transudation of dentinal fluid through open dentinal tubules before polymerization of the adhesive may result in entrapment of water-filled blisters along the adhesive interface (Tay et al., 1996). Compression of these blisters during mastication may cause, within the dentinal tubules (Brännström and Johnson, 1970), rapid fluid movement that activates the intradental A nerve fibers (Närhi et al., 1994), which results in post-operative sensitivity.
One way of relieving post-operative sensitivity clinically is the adjunctive use of oxalate desensitizers on acid-etched dentin prior to adhesive application (Pashley et al., 2001; Tay et al., 2003b). Depletion of calcium ions from the surface dentin forces the oxalate ions to diffuse further down into the dentinal tubule, until calcium ions are encountered for reaction. The calcium oxalate crystals that are formed result in subsurface tubular occlusion and reduction in the hydraulic conductance of dentin. However, the solubility of calcium oxalate is affected by pH, since the anion is the conjugate base of a weak acid (Kotz and Treichel, 1999). Preliminary screening of the compatibility of oxalate desensitizers with total-etch adhesive systems revealed complete compatibility with One-Step (Bisco) and Single Bond (3M ESPE) and poor compatibility with Prime&Bond NT (Dentsply) and OptiBond Solo Plus (Kerr). This led to a more detailed investigation seeking the reasons for compatibility vs. incompatibility. The purpose of this study was to determine if the acidity of total-etch adhesives may influence their bonding to oxalate-desensitizer-treated acid-etched dentin.
Thus, the objectives of this study were: (1) to compare the microtensile bond strengths of 4 single-bottle total-etch adhesives of different acidities to oxalate-desensitizer-treated acid-etched dentin; and (2) to examine, with the use of scanning and transmission electron microscopy, the ultrastructure of the bonded interface. The null hypothesis tested was that there is no difference in the bonding of single-bottle total-etch adhesives of different acidities to oxalate-desensitizer-treated acid-etched dentin.
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MATERIALS & METHODS
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Eighty-four non-carious human third molars, that were stored in a 0.5% chloramine T solution at 4°C, were used within 1 mo following extraction. The teeth were collected after the patients’ informed consent had been obtained under a protocol approved by the Institutional Review Board of the Medical College of Georgia, Augusta, USA. The occlusal enamel was removed with the use of a slow-speed saw with a diamond-impregnated disk (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water cooling. A 180-grit silicon carbide paper was used under running water to create a clinically relevant smear layer on the dentin surface.
Experimental Design
Four single-bottle total-etch adhesives and 2 oxalate desensitizers were investigated. The adhesives were: One-Step (OS; Bisco Inc., Schaumburg, IL, USA), Single Bond (SB; 3M ESPE, St. Paul, MN, USA), OptiBond Solo Plus (OB; Kerr Co., Orange, CA, USA), and Prime&Bond NT (PB; Dentsply De Trey, Konstanz, Germany). The oxalate desensitizers were: BisBlock (BB; Bisco Inc.) and Super Seal (SS, Phoenix Dental Inc., Fenton, MI, USA). Each adhesive was divided into 3 experimental groups with 7 teeth each. Five restored teeth were used for microtensile bond strength evaluation and 2 teeth for ultrastructural examination.
Microtensile Bond Strength (µTBS)
Bonding was performed on the occlusal surfaces of deep, coronal dentin. The 3 experimental groups were:
Group 1: The surface to be bonded was etched with a 32% phosphoric acid gel (Uni-Etch, Bisco Inc.) for 15 sec and rinsed with water for 20 sec before bonding occurred.
Group 2: After the surface was acid-etched, BisBlock was applied with a rubbing motion for 30 sec and rinsed with water for 60 sec before bonding occurred.
Group 3: After the surface was acid-etched, Super Seal was applied with a rubbing motion for 30 sec and rinsed with water for 60 sec before bonding occurred.
The treated teeth were bonded according to the manufacturers’ instructions. Bonded surfaces were air-dried and light-cured for 10 sec. Composite build-ups were performed with the use of a light-cured composite (Z250, 3M ESPE) in 5 1-mm increments and individually light-cured for 40 sec. The teeth were stored in distilled water at 37°C for 24 hrs. Bonded teeth were then sectioned occluso-gingivally into 0.9 x 0.9-mm composite-dentin beams (Shono et al., 1999). Eight beams were retrieved from the 2 widest slabs of each tooth. Five teeth from each group yielded 40 beams for bond strength evaluation. Specimens were stressed to failure under tension in a Bencor Multi-T device (Danville Engineering, San Ramon, CA, USA) with the use of a universal testing machine, Model 4440 (Instron, Inc., Canton, MA, USA), at a crosshead speed of 1 mm/min. Beams with premature bond failure were assigned a null bond strength value and were included in the compilation of the mean bond strength. The data collected were analyzed with SigmaStat Version 2.03 (SPSS, Chicago, IL, USA). Since the µTBS data were not normally distributed (Kolmogorov-Smirnof test), the data were analyzed by Kruskal-Wallis one-way ANOVA on ranks and Dunn’s multiple-comparison tests, with statistical significance set at  = 0.05.
Scanning Electron Microscopy
Four representative fractured beams from each group with µTBS close to the mean bond strength of that group were selected for fractographic analysis by scanning electron microscopy (SEM). The dentin sides of the fractured specimens were air-dried, coated with gold/palladium, and examined with a SEM (Cambridge Stereoscan 440, Cambridge, UK), operating at 10–20 kV.
Transmission Electron Microscopy
Two teeth from each group were acid-etched and similarly treated with the oxalate desensitizers in the manner as previously described. The teeth were sectioned into 0.9-mm slabs. The 2 widest slabs were coated with 2 layers of nail varnish applied 1 mm from the bonded interfaces. They were immersed in a 50 wt% ammoniacal silver nitrate solution for 24 hrs, according to the silver impregnation protocol reported by Tay et al.(2002). After reduction of the diamine silver ions, the silver-impregnated slabs were processed for transmission electron microscopy (TEM) without further demineralization, and were examined, unstained, by TEM (Philips EM208S, Philips, Eindhoven, The Netherlands) at 80 kV.
Measurement of pH and F Concentration of Adhesives
Water-free adhesives that are dissolved in polar solvents do not usually dissociate into ionic species. To circumvent this problem, we dispensed 2-mL specimens of each adhesive into clean glass vials containing 3 mL of 70% ethanol and 30% distilled water. The pH values of the 4 adhesive solutions were measured at ambient temperature (22–25°C) by means of a digital pH meter (Model 501, Orion Research, Inc., Beverly, MA, USA). Three readings were taken for each adhesive, and the mean pH value was calculated for each adhesive.
Similarly to the pH measurement, a 2-mL specimen of each simplified-step adhesive was dispensed into a clean glass vial containing 3 mL of 70% ethanol and 30% distilled water. The solution was buffered with TISAB III (Orion Research Inc.). The fluoride concentrations were determined at ambient temperature by an ion-selective electrode (Cat. No. 96, Orion Research Inc.).
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RESULTS
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Microtensile Bond Strength
The µTBS of the 4 adhesives, and their measured pH values and F concentrations are summarized in the Table . Both OB and PB showed significant reduction in µTBS when applied to oxalate-treated acid-etched dentin (p < 0.05). In contrast, SB and OS showed no significant differences in bond strength when applied to oxalate-treated and untreated acid-etched dentin (p > 0.05). No statistically significant difference was noted between BisBlock and Super Seal when used with the 4 adhesives.
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Table. pH Values and F Concentrations of the 4 Total-etch, Two-step Adhesives and Their Microtensile Bond Strengths after Bonding to Oxalate-desensitizer-treated Acid-etched Dentin
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SEM Results
Adhesive failure occurred predominantly along the adhesive-dentin interface in PB-SS (Fig. 1A). The dentin surfaces were characterized by spherical globules (diameter, 2–4 µm) located preferentially at the orifices of the dentinal tubules. Spherical globules could also be seen in the voids of the adhesive layers of PB-BB (Fig. 1B). Similarly to PB, numerous spherical globules were evident on the fractured dentin surfaces of OB-BB (Fig. 1C). In contrast, the failure mode of the SB and OS-oxalate subgroups was predominantly "mixed", and no spherical globules were seen (Fig. 1D).
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Figure 1. SEM micrographs of the dentin side of a fractured beam following application of single-bottle total-etch adhesives to oxalate-desensitizer-treated acid-etched dentin. (A) Spherical globules (arrowheads) with sizes ranging from 2 to 4 µm were preferentially found at the orifices of the dentinal tubules in the PB-SS subgroup. Clusters of smaller globules (arrow) were found between the larger globules. Patent dentinal tubules (pointer) were frequently observed. (B) Spherical globules (pointers) up to 12 µm were also observed in the adhesive layer of the PB-BB subgroup. (C) The dentin side of the OB-BB subgroup was covered with a continuous layer of spherical globules with sizes ranging from 0.5 to 3 µm. Patent dentinal tubules (arrowheads) were evident from the dentin (D) below. (D) No spherical globules could be found on the dentin surface of the SB-SS subgroup. Numerous fractured resin tags (arrowheads) could be seen at the base of the hybrid layer (H).
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Fluoride Analysis
Ion-specific fluoride analysis of the 4 adhesive systems revealed relatively low levels in OS and SB (70 and 130 ppm, Table ), compared with very high levels in OB and PB (4527 and 3641 ppm, Table), respectively.
TEM Results
Bonded interfaces from PB-SS specimens revealed extensive nanoleakage of silver within the hybrid layer, and electronlucent spherical globules with silver deposits were observed in the adhesive layer. Discontinuous electronlucent spaces could be identified along the adhesive-dentin interface (Fig. 2A). Nanoleakage was also observed within the entire hybrid layer from OB-BB (Fig. 2B). The dentinal tubules were filled with silver deposits, with no subsurface oxalate crystals identified. Discontinuous spaces with scalloped margins were found between the hybrid and adhesive layers in OB-BB. No major differences could be recognized between the 2 oxalate desensitizers after the application of OB and PB. In contrast to OB and PB, isolated silver deposits were found in only some areas of the hybrid layer of SB-SS (Fig. 2C) and OS-BB (Fig. 2D).
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Figure 2. Unstained, undemineralized TEM micrographs of oxalate-desensitizer-treated acid-etched dentin specimens that were bonded with single-bottle total-etch adhesives. The bonded specimens were immersed in an ammoniacal silver nitrate tracer solution before laboratory dehydration and epoxy resin embedding. U: undemineralized dentin. (A) A specimen from the PB-SS subgroup, showing extensive nanoleakage (open arrowheads) in the hybrid layer (H) and the dentinal tubule (T). Electronlucent spherical globules (pointer) were depicted in the adhesive layer (A). Silver deposits were occasionally seen within the spherical globules. Discontinuous electronlucent structures with a scalloped margin could be identified along the adhesive-hybrid layer interface (open arrow). Electronlucent crystals, probably representing the calcium oxalate crystals (arrow), were also seen in the dentinal tubules at a position that was 8–10 µm from the dentin surface. (B) A specimen from the OB-BB subgroup, showing fairly extensive nanoleakage (arrowhead) within the hybrid layer (H). Discontinuous electronlucent deposits, with scalloped margins resembling conglomerates of spherical globules (pointer), could be identified along the adhesive-dentin interface. Subsurface electronlucent deposits (asterisk) were also observed in the dentinal tubules (T). (C) A high-magnification view of the hybrid layer in the SB-BB subgroup. Only isolated spots of silver grains (arrowhead) were observed in the hybrid layer (H). No globular structures were identified on the surface of the hybrid layer. The dentinal tubules (beneath the position of the hybrid layer) were blocked by subsurface electronlucent deposits (asterisk). P: polyalkenoic acid copolymer component of the adhesive. (D) A high-magnification view of subsurface angular crystalline deposits (asterisk) within the dentinal tubule (T) in the OS-BB subgroup. No surface globular structures could be identified. Silver deposits (arrowhead) were occasionally observed within the hybrid layer (H). A: adhesive.
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DISCUSSION
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The application of oxalate desensitizers to acid-etched dentin prior to adhesive application represents one way of avoiding post-operative sensitivity, since this clinical complication is highly product-related. Better sealing ability of conventional three-step total-etch and two-step self-etch adhesives (De Munck et al., 2005; Frankenberger and Tay, 2005) may similarly block the effects of hydraulic pressure and reduce post-operative sensitivity. The results of this study showed that effective bonding to the desensitizer-treated acid-etched dentin is adhesive-specific. Since both the microtensile bond strengths and ultrastructural features of the simplified total-etch adhesives were different when bonded to oxalate-desensitizer-treated acid-etched dentin, we have to reject the null hypothesis tested.
The bond strengths of OS and SB were not compromised with the adjunctive use of the oxalate desensitizers. Optimal resin infiltration and hybrid layer formation occurred in the presence of bi-pyramidal calcium oxalate dihydrate crystals in the dentinal tubules. These crystals can be seen more clearly in cryofractured specimens (Fig. 3). In contrast, significantly lower bond strengths were observed in OB and PB when applied to oxalate-desensitizer-treated dentin, that could be attributed to the presence of spherical globules along the adhesive-dentin interface. These spherical globules closely resemble the loosely bound spherical calcium fluoride (CaF2)-like material formed on enamel (Dijkman et al., 1983; Nelson et al., 1983, 1984) and demineralized dentin surfaces (Itota et al., 2002) following topical fluoride treatment. These spherical globules were removed by the KOH extraction method (Caslavska et al., 1975), confirming that the globules were KOH-soluble fluoride (Appendix). Regardless of the composition of the spherical globules found on the surfaces of oxalate-treated specimens bonded with the 2 incompatible adhesive systems (OB and PB), their presence at the bonded interface and in the adhesive layer could serve as stress-raisers that would create debonding at lower stresses than would occur in their absence. TEM examination of the bonded interface also revealed discontinuous electronlucent spaces with distinct "scalloped" margins. These were not true gaps along the adhesive-dentin interfaces, since they were not infiltrated by silver deposits. The "scalloped" margins correlated nicely with the shapes of spherical globules.
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Figure 3. SEM micrograph of cryofractured phosphoric-acid-etched dentin following the application of an oxalate desensitizer. Bi-pyramidal calcium oxalate crystals (open arrowhead) were identified in the dentinal tubules, at 8–10 µm beneath the dentin surface, where calcium ions were available from the adjacent mineralized dentin (D). In contrast, the demineralized collagen matrix (asterisk) was almost completely devoid of these angular crystals. Since no acidic, fluoride-containing dentin adhesive was further applied to the desensitizer-treated dentin, the etched dentin surface was devoid of the spherical calcium-fluoride-like structures. Only demineralized collagen fibrils (pointer) were identified from the surface of the acid-etched dentin.
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Variability was seen in size and distribution densities of these globular structures following the application of PB and OB to oxalate-desensitizer-treated dentin. This could be due to the differences in concentration of fluoride, pH values of PB and OB, and the availability of calcium ions on the dentin surface (Arends et al., 1988; Saxegaard and Rölla, 1988). Both PB and OB are fluoride-containing adhesives; it is reasonable to assume that the free fluoride ions from the adhesives may interact with calcium and phosphate ions on dentin surfaces to precipitate CaF2-like material. The greater intensity of fluoride deposition in OB may be explained by the higher F concentration in OB (4527 ppm) vs. PB (3641 ppm, Table). Conversely, no globular structures were formed following the application of OS and SB to oxalate-treated dentin, since these adhesives contain minimum amounts of fluoride (Table). The presence of spherical globules on the surface of the desensitizer-treated dentin may hinder the adhesive infiltration and hybridization of demineralized dentin. This is supported by the TEM results, where extensive accumulation of silver deposits was observed in the porous hybrid layers following the application of PB and OB to oxalate-treated dentin. Similar effects have recently been reported following the application of hydrofluoric acid gel to acid-etched dentin (Szep et al., 2000; Pioch et al., 2003).
Although the presence of fluoride ion is necessary for formation of the spherical globules in OB- and PB-bonded specimens, the availability of calcium ions on the dentin surface is also critical. Since the dentin surface was completely deprived of calcium phosphate following phosphoric-acid-etching (Van Meerbeek et al., 2003), the calcium ions could have been derived from the dissolution of calcium oxalate crystals in the dentinal tubules. The solubility of calcium oxalate (CaC2O4) is affected by pH, since the anion is the conjugate base of a weak acid. The low pH values of OB and PB may increase the solubility of calcium oxalates in the dentinal tubules. According to Le Châtelier’s principle (Silberberg, 2003), as calcium oxalate crystals are exposed to more H3O+, more calcium oxalate dissolves into calcium and oxalate ions, to compensate for the depletion of oxalate ions and maintain the equilibrium constant. This is supported by the SEM observation of considerably fewer, and smaller, calcium oxalate crystals in the dentinal tubules following the application of OB and PB to oxalate-desensitizer-treated dentin.
In conclusion, the results of the present in vitro study indicated that the bond strength of simplified total-etch adhesives to oxalate-desensitizer-treated acid-etched dentin may be compromised by the acidity and the availability of fluorides from some total-etch adhesives. In vivo studies are needed to confirm these in vitro observations. Clinicians should be aware of the potential drop in bond strength with the use of PB and OB on oxalate-desensitizer-treated acid-etched dentin.
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ACKNOWLEDGMENTS
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This study was based on the work partially performed by Cynthia Yiu for the fulfillment of the degree of Doctor of Philosophy, the University of Hong Kong. The authors express their gratitude to Bill Lee of the Electron Microscopy Unit, the University of Hong Kong, for technical assistance, and to Jenny Wang of Bisco Inc. for advice. The products examined in this study were generously sponsored by the manufacturers. This study was supported by RCG CERG grant 10204604/07840/08004/324/01, Faculty of Dentistry, The University of Hong Kong, by grants DE 014911 and DE 015306 from the National Institute of Dental and Craniofacial Research, USA (P.I., David Pashley), and by grants 300481/95-0 and 474226/03-4 from CNPq, Brazil. The authors thank Frances Chow and Michelle Barnes for secretarial support. Byoung Suh is President of Bisco, Inc., the company that manufactures BisBlock, the oxalate desensitizer used in this study. Louis Sharp is an employee of Bisco, Inc. Drs. Carvalho and Pashley are the inventors of the patented oxalate desensitizer used in this study. The other authors have no financial interest in any of the products.
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FOOTNOTES
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A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.
Received for publication August 27, 2004.
Revision received May 12, 2005.
Accepted for publication May 13, 2005.
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Journal of Dental Research, Vol. 84, No. 8,
730-735 (2005)
DOI: 10.1177/154405910508400809
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ABSTRACT
INTRODUCTION