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J Dent Res 84(4):350-354, 2005
© 2005 International and American Associations for Dental Research

RESEARCH REPORTS
Biomaterials & Bioengineering

Continuing Etching of an All-in-One Adhesive in Wet Dentin Tubules

Y. Wang1,*, and P. Spencer1,2

1 Department of Oral Biology and
2 Department of Pediatric Dentistry, University of Missouri-Kansas City School of Dentistry, 650 E. 25th St., Kansas City, MO 64108, USA;

* corresponding author, Wangyo{at}umkc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Self-etch adhesives that etch and prime simultaneously are becoming more acidic. We hypothesized that the degree of acidic monomer conversion at the interface and within the tubules was high enough that the acidic reaction would be very self-limiting. Dentin surfaces prepared from extracted, unerupted human third molars were treated with Prompt L-Pop (3M ESPE). The prepared teeth were stored in normal saline, and specimens retrieved at intervals ≤ 4 wks were randomly selected for light, scanning electron microscopic and micro-Raman spectroscopic analysis. Morphologic and spectroscopic analyses indicated dentin demineralization and adhesive penetration throughout the demineralized layer and tubules. Increased dentin demineralization and loss of adhesive integrity were noted after aqueous storage. The degree of monomer conversion at the interface was consistently greater than conversion within the tubules. Fluid within the tubules may inhibit monomer conversion. The acidic characteristics of this adhesive may be retained and, thus, continue to affect/demineralize the surrounding dentin.

KEY WORDS: dentin • adhesive • spectroscopy • Raman


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent reports have indicated that conventional acid-etching and bonding techniques inhibited the formation of a seal at the adhesive/dentin interface, which might be associated with collapse of demineralized dentin (Pashley et al., 1993), over-etching (Walker et al., 2000), or discrepancy between the depths of demineralization and resin infiltration (Sano et al., 1995; Spencer et al., 2000; Wang and Spencer, 2002, 2003). One promising approach to preventing the collapse of demineralized dentin and simplifying the bonding procedure is to use acidic monomers or self-etching primers to etch through the smear layer into the underlying dentin, applying the adhesive without rinsing the etched surface (Watanabe et al., 1990).

There is a trend toward eliminating as many steps as possible in the bonding protocol, since this increases the efficiency of the procedure and reduces technique sensitivity. Similar to the above two-step self-etching systems, the all-in-one, single-step adhesives were recently introduced. The increased concentration of acidic resin monomers in these systems enabled them to etch the dentin and enamel simultaneously. Prompt L-Pop (3M ESPE), a representative of these new-generation dentin/enamel adhesives, has been reported to show very promising results when used on both dentin and enamel (Perdigão et al., 2000). Acidic monomers in Prompt L-Pop consist of methacrylated phosphoric acid mono- and diesters (Tay and Pashley, 2001), in which the phosphoric acid and methacrylate group are combined into one molecule that etches and primes simultaneously. There is a possibility that the more highly acidic and hydrophilic resin monomers may deeply penetrate not only intertubular dentin but also water-filled tubules. The water may interfere with acidic monomer polymerization. Ideally, the mineral components would neutralize the acidity of the self-etching monomers, and/or the degree of monomer conversion would be high enough that the acidic reaction will be self-limiting. The purpose of this study was to determine the degree of conversion of this commercial single-step adhesive at the dentin interface or within the tubules, and the effect of aqueous storage on the interfacial morphologic characteristics and structure. The hypothesis tested was that the unpolymerized acidic monomers in the water-filled tubules would retain their acidity and continue to etch the surrounding dentin.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Adhesive/Dentin Specimen Preparation
We used extracted unerupted human third molars stored in 0.9% w/v NaCl containing 0.002% sodium azide at 4°C. The teeth were collected after the patients’ informed consent had been obtained under a protocol approved by the University Adult Health Sciences IRB. The occlusal one-third of the crown was removed by means of a water-cooled low-speed diamond saw (Buehler Ltd, Lake Bluff, IL, USA). We created a smear layer by abrading the dentin with 600-grit SiC under water for 30 sec. Six prepared dentin specimens were selected for treatment with Prompt L-Pop adhesive (Lot 122981, 3M ESPE, Seefeld, Germany) according to manufacturer’s instructions. The adhesive solution was brushed onto the entire dentin surface and agitated evenly for 15 sec. After being gently air-dried, the adhesive-coated dentin surfaces were light-cured for 20 sec by means of a conventional halogen light unit (Spectrum, Dentsply, Milford, DE, USA). These specimens were stored for 24 hrs in normal saline at 25°C before being sectioned. Two rectangular slabs (10 x 2 x 1.5 mm) of the adhesive/dentin were prepared from these teeth. Another 2 slabs were prepared from the adjacent fraction of the treated cut teeth after storage in normal saline for 4 wks. Sections from each prepared tooth with/without aqueous storage were analyzed.

Differential Staining Technique
The rectangular (10 x 2 x 1.5 mm) slabs of the adhesive/dentin from the same tooth, with 24-hour and four-week aqueous storage, were mounted on a methacrylate support, and 3-µm-thick sections were cut from the face of the slab by means of a tungsten carbide knife mounted on a Polycut S ‘sledge’ microtome. Following recovery of the microtomed sections, the remaining fraction of each adhesive/dentin interface slab was used for SEM analysis. Thus, the same slab was used for both light microscopic and SEM analyses. Differential staining was accomplished with Goldner’s trichrome, and the sections were examined under a Nikon E 800 light microscope.

Scanning Electron Microscopy
Following light microscopic analysis, the specimens described above were prepared for SEM examination. The surfaces of both 24-hour and four-week specimens from the same tooth were treated, respectively, according to the following protocol: (1) 30 sec in 5 N HCl, followed by 30 min in 5% NaOCl; or (2) 30 min in 5% NaOCl. Both air and HMDS drying methods were used (Itou et al., 2003). After being dried, the prepared specimens were mounted on aluminum stubs and sputter-coated with ~ 20 nm of gold-palladium. Specimens were examined at a variety of magnifications and tilt angles in a Philips XL30 ESEM-FEG (Philips Inc., Eindhoven, Netherlands) at 10 kV.

Micro-Raman Spectroscopy
Separate, but adjacent, slabs from the same tooth were prepared for investigation by micro-Raman spectroscopy. The slabs were microtomed to different depths so that resin tags would be exposed. The micro-Raman spectrometer consisted of an argon ion laser beam (514.5 nm) focused through a 100X Olympus objective (NA 0.92) to a beam diameter of ~ 1 µm. Raman spectra were acquired at the adhesive/dentin interface and at different depths within the tubules. Two consecutive scans of spectra (with 60-second accumulation time each) were obtained from each site. The laser power was approximately 3 mW.

Prompt L-Pop Adhesive/Water Mixtures
We collected the Prompt L-Pop adhesive by cutting the red reservoir of the dose package containing methacrylated phosphates, initiators, and stabilizers. The adhesive was mixed with 0, 20, 35, 50, and 60 vol% distilled water in microcentrifuge tubes. The Prompt/water mixtures were cast on glass slides with wells, covered with mylar, and polymerized for 20 sec. Following polymerization, the mylar was removed, and the specimen was placed at the focus of a 50X objective. Raman spectra were collected from a minimum of 6 different sites on each mixture.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In the photomicrograph of a Goldner’s-trichrome-stained section of the adhesive/dentin interface after 24 hrs in normal saline, the bulk adhesive appeared as a pale yellow, while the mineralized dentin was green (Fig. 1AGo). A distinct, straight pale red region was clearly differentiated (Fig. 1AGo). The mean width of this red zone was 2.5 ± 0.4 µm. Both the adhesive and dentin portions were intact and connected by this zone. The resin tags were also visible (Fig. 1AGo). In the photomicrographs of a stained section of the adhesive/dentin interface from the same tooth as above, after 4 wks’ storage in normal saline (Fig. 1BGo), both the adhesive and dentin phases lost their integrity. After being sectioned, the adhesive and dentin portions just below the adhesive/dentin interface appeared to be very porous. The resin tags were broken into small pieces.



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  		Figure 1. Light micrographs of Prompt L-Pop/dentin interfaces stained with Goldner’s trichrome. (A,A') Sectioned after 24 hrs in normal saline: mineralized dentin (green), adhesive (pale beige), interface (pale red); arrow indicates adhesive penetration into tubule. (B,B') Sectioned after 4 wks in normal saline. The last step in the process, which is the light green component, was not used. It showed very poor structural integrity following 4 wks’ aqueous storage.

 
SEM micrographs recorded from the adhesive/dentin interface after 24 hrs’ and 4 wks’ aqueous storage are shown in Fig. 2Go. The 24-hour specimens were treated with 5 N HCl for 30 sec and 5% NaOCl for 30 min; this HCl-bleach preparatory procedure is commonly used to expose the hybrid layer and resin tags. The dentin was effectively etched, and adhesive readily flowed into the intertubular dentin and the open dentin tubules, creating a hybrid layer ~ 2–3 µm wide and adhesive tags > 200 µm long (Fig. 2AGo). These ‘spaghetti-like’ tags appeared to be very flexible. Both the 24-hour and 4-week specimens were treated with 5% NaOCl for 30 min. Following this treatment, any exposed or demineralized collagen would have been removed. The morphology of the 24-hour specimen did not change following this bleach treatment (Fig. 2BGo). The morphologic characteristics of the adhesive/dentin interface after 4 wks’ aqueous storage were dramatically different from those of the 24-hour specimen (Figs. 2CGo, 2DGo). After 4 wks’ aqueous storage, the adhesive tags were exposed following only NaOCl treatment, and these tags appeared to be separate or fractured at ~ 20–50 µm.



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  		Figure 2. Scanning electron micrographs of Prompt L-Pop/dentin interfaces of cross-sections from the same tooth with different aqueous storage times. (A) The interface was treated with 5 N HCl and 5% NaOCl after 24 hrs in normal saline. (B) The interface was treated only with 5% NaOCl after 24 hrs in normal saline. (C,D) The interface was treated only with 5% NaOCl after 4 wks in normal saline.

 
The micro-Raman spectra of Prompt L-Pop adhesive recorded at the spots close to the adhesive/dentin interface and at ~ 20 and 50 µm deep to the surface within the tubules are shown in Fig. 3Go. The measurement of degree of conversion (DC) was made on a relative basis by comparison of the vibrational band of the residual unpolymerized methacrylate C=C bond at 1640 cm–1 against the CH2 deformation band at 1453 cm–1 used as an internal standard. The DC of adhesive at the adhesive/dentin interface was ~ 93%. The DC decreased to ~ 88% at 20 µm deep in the tubules and decreased further to ~ 79% at 50 µm deep to the surface. When the spectra were compared (Fig. 3Go), the spectrum of the adhesive recorded close to the adhesive/dentin interface was found to have strong phosphate bands at 960 and ~ 1100 cm–1, compared with the spectra recorded in the dentin tubules.



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  		Figure 3. In situ micro-Raman spectra of Prompt L-Pop adhesive recorded at the spot close to the adhesive/dentin interface and ~ 20 and 50 µm deep to the surface within the tubules.

 
Raman spectra of the mixtures of Prompt L-Pop adhesive with different water content (from 0 to 60 vol%) following 20 sec of light-curing are shown in Fig. 4AGo. The spectrum of unpolymerized adhesive is also presented. The bands at 1640 and 1403 cm–1 are associated with unpolymerized methacrylate. The effect of water on the degree of conversion was studied (Fig. 4BGo). The DC was only ~ 77% in the absence of water; the DC increased to ~ 93% with 20 vol% water. The DC decreased with a further increase in water content, i.e., DC dropped from ~ 93% to ~ 36% as the water content increased from 20 to 60 vol% (Fig. 4BGo).



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  		Figure 4. Effect of water on degree of conversion of Prompt L-Pop adhesive following 20-second light curing. (A) The Raman spectra of the mixtures of Prompt L-Pop adhesive with different water contents (0–60 vol%). The spectrum of unpolymerized adhesive is also presented. (B) Degree of conversion as a function of water content in Prompt L-Pop. It was noted that the DC of Prompt L-Pop adhesive is not the highest in the absence of water. This may be associated with the effect of viscosity on transportation of monomers to propagating chains. Values are means ± standard deviation (n = 6 for each cell of data).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Both light and scanning electron microscopic studies have revealed that this all-in-one Prompt L-Pop self-etching adhesive has effectively etched the dentin surface and created a uniform hybrid layer and long resin tags (Figs. 1AGo, 2AGo). However, following aqueous storage, these self-etching adhesive/dentin interface specimens lost their structural integrity, especially in the region just below the adhesive/dentin interface (Figs. 1BGo, 2CGo, 2DGo). The specimens that had been stored for 4 wks were treated only with 5% NaOCl for 30 min. However, the resin tags were all exposed and appeared fractured; big gaps were readily seen at 20–50 µm underneath the interface, indicating that this dentin region had been demineralized, and exposed collagen was removed after treatment with 5% NaOCl. This morphological characteristic supported the hypothesis that the acidic monomers might retain their acidity and continue to etch the surrounding dentin. These results, corroborated by the micro-Raman data (Fig. 3Go), suggested that, as a result of incomplete polymerization, the acidic characteristics of this self-etching adhesive system were retained in the water-filled tubules, and that the walls of the dentin tubules were continuously etched during this aqueous storage period.

Tubules are normally filled with water unless teeth are thoroughly dried. Since fluid/water within the tubules may be one interfering factor, the DC was determined at different water concentrations. It has been reported that water can inhibit polymerization of dentin adhesive resins (Jacobsen and Söderholm, 1995). The DC dropped from ~ 93% to ~ 36% as water content increased from 20–60 vol% (Fig. 4Go). A prescribed amount of water could decrease the viscosity and help increase the DC of this water-compatible resin. Water is also an essential component to provide the medium for ionization of acidic monomers. In Prompt L-Pop, the phosphoric acid esters and water are distributed in a ratio of 80:20 by volume (Frey, 2000). However, the presence of water within the resin and the continuous supply of water within the dentin/tubules might cause incomplete polymerization of the adhesive, i.e., these water-soluble monomers could be diluted to an extent that there might not be adequate free radicals for polymer chain propagation. Tay et al.(2002) observed the water-treeing phenomenon in this type of adhesive and the patterns of nanoleakage within both adhesive and hybrid layers. The vertically oriented water trees were thought to be caused by outward movement of water from dentin tubules during etching. They speculated that the nanoleakage represented sites of incomplete water removal that led to suboptimal polymerization. In this study, the use of the high-resolution micro-Raman technique allowed us to determine the DC of resin in the area as small as 1 µm. Using the method shown in Fig. 3Go, we were able to detect the DC of resin at different depths in situ. The DC of resin tags (~ 2 µm in diameter) at 50 µm deep to the surface was only ~ 79%.

This all-in-one adhesive was developed based on the concept that dentin etching and priming could be accomplished simultaneously. The penetration and demineralization process neutralizes the acidic portions of the molecules. However, this may not necessarily be the case in the water-filled dentin tubules. The micro-Raman technique also provided significant information about neutralization of acidic methacrylated phosphoric acid esters in Prompt L-Pop (Fig. 3Go). In a comparison of spectra of Prompt L-Pop before and after reaction with dentin mineral, new bands appeared at ~ 1100 cm–1 and ~ 960 cm–1. These bands are associated with {nu}3 PO4 and {nu}1 PO4 of calcium phosphate complexes, respectively (Penel et al., 1999), which were the result of the reaction of acidic resins with the mineral. It was shown that the resin near the adhesive/dentin interface had reacted with the mineral, but that the resin tags in the tubules did not. SEM studies have revealed that Prompt L-Pop is as acidic and aggressive as 32–37% phosphoric acid (Croll and Berg, 2000). Because of the highly acidic nature of the resin tags, there is danger that these acidic resins could continue to etch the dentin. The acidity of resin also depends on the degree of conversion. Decreased DC within the tubules was detected by an in situ method (Fig. 3Go). In the presence of water within the dentin tubules, unpolymerized acidic monomers and poorly polymerized oligomers dissolved in water and continued to etch the surrounding dentin. Because of the very low viscosity, this adhesive readily penetrated the anastomosing tubules. The acidic monomers/oligomers continued to demineralize the surrounding dentin, including the walls of these small, interconnecting accessory tubules. The continued demineralization of the dentin tubules eliminated any structural support for the resin tags, causing the dentin portion to lose its structural integrity (Figs. 1Go, 2Go). In addition, it was suspected that the hydrolysis of the ester bond within the acidic monomer, over time, could also produce phosphoric acid strong enough to create demineralized dentin.

In summary, although this all-in-one product presents a very interesting approach to the problems associated with conventional etching and bonding to wet dentin, the results shown in this study indicated a serious limitation of this all-in-one adhesive—incomplete polymerization and continued demineralization of the adjacent dentin structure in the tubules. In an effort to make adhesives more user-friendly and to improve their adhesion to enamel, there is a trend toward increasing the aggressive, acidic nature of the contemporary self-etching primers, or the timely &;lsquo;all-in-one’ adhesives. For these adhesives to become more acidic, the formulations may have become more hydrophilic, thus allowing for deeper penetration. This study indicated that water is a major interfering factor in polymerization, and, as a result, unpolymerized acidic and aggressive monomers could continue to etch the dentin, leading to a detrimental impact on the bond. Since there is little control over the presence of water in the tooth, especially in dentin tubules, investigators must study more efficient water-compatible photoinitiators to address the problems associated with incomplete conversion in these sixth-generation self-etch adhesive systems.


   ACKNOWLEDGMENTS
 
This investigation was supported by USPHS Research Grants DE014392 (PS) and DE 015281 (YW) from the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892. The authors gratefully acknowledge 3M ESPE for donating the dentin adhesive products used in this study.

Received August 23, 2003; Last revision January 10, 2005; Accepted January 11, 2005


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Croll TP, Berg J (2000). Simplified adhesion of compomers in pediatric dentistry. Quintessence Int 31:563–569.[ISI][Medline]

Frey O (2000). Creating a reliable bond. An all-in-one system. Am J Dent 13:85D–87D.[ISI][Medline]

Itou K, Torii Y, Oyama F, Yoshiyama M, Pashley DH (2003). Effect of drying methods on hybrid layer thickness. Am J Dent 16:335–339.[ISI][Medline]

Jacobsen T, Söderholm K-J (1995). Some effects of water on dentin bonding. Dent Mater 11:132–136.[ISI][Medline]

Pashley DH, Ciucchi B, Sano H, Horner JA (1993). Permeability of dentin to adhesive agents. Quintessence Int 24:618–631.[Medline]

Penel G, Leroy N, Rey C, Lemaitre J, Van Landuyt P, Ghanty N, et al. (1999). Qualitative and quantitative investigation of calcium phosphate of biological interest by Raman microspectrometry. Recent Res Dev Appl Spectrosc 2:137–146.

Perdigão J, Frankenberger R, Rosa BT, Breschi L (2000). New trends in dentin/enamel adhesion. Am J Dent 13:25D–30D.[ISI][Medline]

Sano H, Takatsu T, Ciucchi B, Horner JA, Matthews WG, Pashley DH (1995). Nanoleakage: leakage within the hybrid layer. Oper Dent 20:18–25.[ISI][Medline]

Spencer P, Wang Y, Walker MP, Wieliczka DM, Swafford JR (2000). Interfacial chemistry of the dentin/adhesive bond. J Dent Res 79:1458–1463.[Abstract/Free Full Text]

Tay FR, Pashley DH (2001). Aggressiveness of contemporary self-etching systems. I: Depth of penetration beyond dentin smear layers. Dent Mater 17:296–308.[ISI][Medline]

Tay FR, Pashley DH, Yoshiyama M (2002). Two modes of nanoleakage expression in single-step adhesives. J Dent Res 81:472–476.[Abstract/Free Full Text]

Walker MP, Wang Y, Swafford J, Evans A, Spencer P (2000). Influence of additional acid etch treatment on resin cement dentin infiltration. J Prosthodont 9:77–81.[Medline]

Wang Y, Spencer P (2002). Quantifying adhesive penetration in adhesive/dentin interface using confocal Raman microspectroscopy. J Biomed Mater Res 59:46–55.[ISI][Medline]

Wang Y, Spencer P (2003). Hybridization efficiency of the adhesive dentin interface with wet bonding. J Dent Res 82:141–145.[Abstract/Free Full Text]

Watanabe I, Nikaido T, Nakabayashi N (1990). Effect of adhesion promoting monomers on adhesion to ground dentin. J Jpn Dent Mater 9:888–893.




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