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A ¹³C NMR Study on the Adsorption Characteristics of HEMA to Dentinal Collagen

¹ Department of Dental Materials, Nihon University School of Dentistry at Matsudo, 870-1 Sakaecho, Nishi 2, Matsudo, Chiba 271-8587, Japan; and
² Department of Biomaterials, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikadacho, Okayama, Okayama 700-8525, Japan;

* corresponding author, norihiro{at}mascat.nihon-u.ac.jp

	ABSTRACT

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To develop a more effective primer, we must understand how 2-hydroxyethylmethacrylate, the HEMA primer, enhances bonding at the resin-dentin interface. In this study, to obtain an insight into the adhesion mechanisms of adhesive resin to etched dentin through HEMA, we examined the adsorption characteristics of HEMA to dentinal collagen by using the ¹³C NMR technique. The addition of dentinal collagen to the HEMA solution resulted in a decrease in T₁ values of carbons attributed to the HEMA, thus reflecting an interaction between HEMA and collagen. Specifically, a reduction in the T₁ value in the ester carbonyl carbon attributed to HEMA greater than that in the other carbons suggested the formation of a hydrogen bond between the ester carbonyl group in HEMA and the dentinal collagen.

KEY WORDS: dentin adhesion mechanism • dentin primer • HEMA • dentinal collagen • collagen function • ¹³C NMR

	INTRODUCTION

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For dentin adhesion, it is well-understood that an application of a HEMA solution to etched dentin which has been air-dried allows for bonding at the resin-dentin interface (Pashley, 1990; Sugizaki, 1991; Nakabayashi and Takarada, 1992; Van Meerbeek et al., 1992; Nakabayashi and Pashley, 1998). This is due to the fact that the methacrylate monomer in a bonding agent could diffuse into the collagenous layer which had been restored by the HEMA, resulting in the formation of a hybrid layer on the subsurface of the intertubular dentin (Nakabayashi et al., 1982).

Suzuki and Nakai (1994) determined the amount of HEMA adsorbed onto the Type I collagen by utilizing FT-IR. They reported as follows: (1) The amount of HEMA adsorbed onto the collagen was dependent on the HEMA concentration in the aqueous solution, and (2) the adsorbed amount of HEMA was strongly correlated to the bond strength of the resin to the etched dentin primed with the HEMA solution.

Xu et al. (1997) examined the adsorption characteristics of HEMA onto collagen by utilizing FT-Raman spectroscopy. They suggested that the ester portion in HEMA reacts with the functional groups in collagen as either (1) the formation of hydrogen bonds or (2) the formation of a new functional group, such as an amide function, -CO-NH-.

Nishiyama et al. (1998, 2000, 2001) conducted studies on interactions between an N-methacryloyl--amino acid (NMA) primer and dentinal collagen by utilizing the ¹³C NMR technique. As a result, the amide or carboxylic acid group in the NMA formed hydrogen bonds with the carboxylic acid group of the side-chain of the amino acid residues in the collagen. The strength of the interactions between NmA and collagen exhibited strong correlations to the bond strength of the resin to the etched dentin primed with the NMA solution. The ¹³C NMR method is a very powerful technique for investigating the details of interactions that exist between primer and collagen.

In this study, to determine which types of interactions exist between HEMA and dentinal collagen that has been exposed by acid-etching, we studied the adsorption characteristics of HEMA to collagen using the ¹³C NMR technique, including the observation of spin-lattice relaxation times, T₁.

	MATERIALS & METHODS

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Preparation of Dentinal Collagen
After extraction, the crown dentin of bovine teeth was cross-sectioned by a diamond cutter under a stream of water. The sliced dentin disks, after being frozen in liquid nitrogen, were reduced to powder by means of a ball agate mill. These particles were demineralized by 40 mass% phosphoric acid for 15 min at 0°C. Insoluble collagen was decanted with de-ionized water to remove any soluble dentinal component. This process was repeated until the pH of the supernatant solution increased to 6. Insoluble collagen fragments, which had aggregated among themselves, were reduced to powder by means of an agate mortar. Dentinal collagen powder was obtained after being dried at 20°C (Nishiyama et al., 2000)

NMR Observation of Spin-lattice Relaxation Time
The 20 mass% deuterium oxide (99.8 atom%, CEA, Paris, France) solution was prepared as a non-acidic solution. The pH of this solution was 6.3. Acidic solution with a pH of 1.0 was prepared after the addition of hydrochloric acid to the non-acidic solution. Dentinal collagen (70 mg) was then dispersed into 0.600 g of the non-acidic or the acidic solution. The addition of collagen to the non-acidic solution (pH = 6.3) decreased the pH to 4.1, while addition of collagen to the acidic solution increased the pH from 1.0 to 1.7. This pH change was due to the protonation of the amino group and the dissociation of the carboxylic acid group of the side-chain of the amino acid residues in the collagen. When the pH of the collagen suspension was 1.7, the dissociation of the carboxylic acid groups in the collagen was limited, due to the pKa of these carboxylic acids being 3.9-4.3. Conversely, when the pH of the collagen suspension was 4.1, half of these carboxylic acids dissociated. At this point, the amino groups in the collagen became protonated under both of these conditions, since the pKa of the amines is 9-10 (Stryer, 1975).

After HEMA (6.73 x 10^-5 mol) was dissolved in both collagen suspensions, the T₁ of the carbons attributed to the HEMA were then observed by means of an EX-270 spectrometer (JEOL, Tokyo, Japan) operating at 67.80 MHz at 25°C. The (180°--90°-5T₁)_n pulse sequence was used, where the and the accumulation were 0.05-45.0 sec and 120-400 scans, respectively. The T₁ values of the collagen/HEMA solutions were then compared with those of the collagen-free HEMA solutions. The non-acidic (pH = 6.3) and the acidic aqueous solutions (pH = 1.7) were used for collagen-free HEMA solution. The amount of HEMA added to these solutions, 0.600 g, was 6.73 x 10^-5 mol. Hexamethyldisiloxane (HMDSO) was used as an external reference. The T₁ observation was conducted twice for each experiment.

	RESULTS

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The Fig. shows the ¹³C NMR spectra of HEMA in the absence (A) or the presence (B, pH = 4.1; C, pH = 1.7) of dentinal collagen. In both NMR spectra, B and C in the presence of collagen, the ¹³C NMR peaks attributed only to HEMA were detected. If the denaturation of the collagen from a triple-helix structure to a random-coil structure had occurred, the NMR peaks assigned to the amino acid residues in the collagen which takes on a random-coil structure should have been detected. This is because the molecular motion of the collagen having a random-coil structure becomes higher than that of a triple-helical collagen.

View larger version (20K): [in this window] [in a new window]   Figure. 13C NMR spectra of HEMA in the absence or in the presence of dentinal collagen. (A) NMR spectrum of HEMA in the absence of dentinal collagen at pH = 6.3. (B) NMR spectrum of HEMA in the presence of dentinal collagen at pH = 4.1. (C) NMR spectrum of HEMA in the presence of dentinal collagen at pH = 1.7.

However, when the aqueous HEMA solution, in the presence of collagen, had a pH of 1.7, the T₁ values of the carbons assigned to HEMA became dramatically smaller than the observed T₁ values of the corresponding carbons in the collagen-free HEMA solution. Specifically, a significant reduction in the T₁ value of the ester carbonyl carbon was observed. When the pH of the collagen suspension was increased to 4.1, the T₁ values for the majority of the carbons attributed to HEMA became greater than the observed T₁ values of the corresponding carbons in the presence of collagen with a pH of 1.7. The T₁ ratios of the ester carbonyl carbon also increased from 0.47 to 0.68. However, the T₁ ratio for the -methylene carbon bonded to the hydroxy group was almost the same, even though the pH of the collagen suspension had changed from 1.7 to 4.1.

	DISCUSSION

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It is understood that the application of a HEMA solution to etched dentin increases the bond strength of resin to dentin. However, the principal adhesion mechanism of resin to etched dentin primed with the HEMA solution is still not well-understood.

In this study, to obtain an insight into the type of interaction that occurs between HEMA and collagen, we conducted an NMR analysis of HEMA in the absence or in the presence of dentinal collagen.

The addition of dentinal collagen to a HEMA solution (pH = 1.7) resulted in a decrease in T₁ values of the carbons attributed to the HEMA. Specifically, the T₁ value of the ester carbonyl carbon decreased, indicating that the ester carbonyl group in HEMA directly formed a hydrogen bond with the carboxylic acid group in the collagen. This finding is the same as the results obtained with NMA forming hydrogen bonds with the carboxylic acid group in collagen (Nishiyama et al., 1998, 2000, 2001). However, when the pH of the collagen suspension was increased from 1.7 to 4.1, the T₁ ratio of the ester carbonyl carbon attributed to HEMA increased from 0.47 to 0.68. This increase was most likely due to the decrease in probability that the ester carbonyl group in HEMA would form a hydrogen bond with the carboxylic acid group in the collagen. This result was achieved, since half of the carboxylic acids in the collagen dissociated at a pH of 4.1.

Muramatsu and Nishiyama (1999) studied the adsorption characteristics of 3-methacryloyloxy propionic acid, MPA, on dentinal collagen at a pH of 1.7. The major difference between MPA and HEMA is the type of hydrophilic group. The MPA contains a carboxylic acid group, whereas HEMA contains a hydroxy group. The carboxylic acid or the ester carbonyl group in the MPA formed hydrogen bonds with the collagen. A significant reduction in the T₁ value of the -methylene carbon bonded to the carboxylic acid group was observed, reflecting the formation of hydrogen bonds between MPA and collagen. This was because the segmental motion of the -methylene carbon was restricted, and the T₁ ratio for a -methylene carbon was 0.57. This result suggested that we could determine further details of the interaction between the hydroxy group in the HEMA and the collagen function by observing the changes in the segmental motion of the -methylene carbon.

In contrast to the -methylene carbon in the MPA, the observed T₁ value of the -methylene carbon in HEMA in the presence of collagen at a pH of 1.7 was not significantly lower than that in HEMA without collagen. Furthermore, when the pH of the collagen suspension was increased from 1.7 to 4.1, the pH dependency in the T₁ ratio of the -methylene carbon was not observed. These results suggested that a fairly low probability exists, thus suggesting that the hydroxy group in HEMA would form a hydrogen bond with the functional groups in collagen.

Further, if HEMA reacted with the collagen function and if the ester portion formed an amide function as reported by Xu et al. (1997), HEMA should have been hydrolyzed, and an ethylene glycol should have been produced as a subproduct. However, the ¹³C NMR peak of the methylene carbon attributed to ethylene glycol was not detected in both ¹³C NMR spectra, B and C.

Based on the results obtained from the ¹³C NMR analysis and from the papers previously referenced, the hypotheses of the adhesion mechanisms of resin to etched dentin through HEMA are postulated as follows: (1) HEMA facilitates the restoration of the collagenous layer in which the collagen fiber arrangement has collapsed during an air-drying process, and the ester carbonyl group in HEMA forms hydrogen bonds with the undissociated carboxylic acid in the collagen; and (2) the hydrogen-bonded HEMA species promotes the hybridization of the adhesive resin to dentinal collagen fibers, and thus enhances bonding at the resin-dentin interface.

	ACKNOWLEDGMENTS

 
Part of this study was supported by a grant-in-aid for Developmental Scientific Research from the Ministry of Education, Science and Culture in Japan (#11671955). This paper was edited by David L. Mukai, Tokyo, Japan.

Received January 30, 2001; Last revision January 28, 2002; Accepted May 9, 2002

	REFERENCES

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Muramatsu Y, Nishiyama N (1999). Analysis of interaction mechanisms of dentin adhesive primer. Jpn J Dent Mater 19:1–10.

Nakabayashi N, Pashley DH (1998). Hybridization of dental hard tissues. 1st rev. ed. Osaka, Japan: Quintessence Publishing Co, Ltd.

Nakabayashi N, Takarada K (1992). Effect of HEMA on bonding to dentin. Dent Mater 8:125–130.[Medline]

Nakabayashi N, Kojima K, Masuhara E (1982). The promotion of adhesion by the infiltration of monomers into tooth substrates. J Biomed Mater Res 16:265–273.[Medline]

Nishiyama N, Asakura T, Suzuki K, Sato T, Nemoto K (1998). Adhesion mechanisms of resin to etched dentin primed with N-methacryloyl glycine studied by ¹³C NMR. J Biomed Mater Res 40:458–463.[Medline]

Nishiyama N, Asakura T, Suzuki K, Komatsu K, Nemoto K (2000). Bond strength of resin to acid-etched dentin studied by ¹³C NMR. J Dent Res 79:806–811.[Abstract/Free Full Text]

Nishiyama N, Suzuki K, Asakura T, Komatsu K, Nemoto K (2001). Adhesion studies of N-methacryloyl--amino acid primers to collagen analyzed by ¹³C NMR. J Dent Res 80:855–859.[Abstract/Free Full Text]

Pashley DH (1990). Interactions of dentinal materials with dentin. Trans Acad Dent Mater 3:55–73.

Stryer L (1975). Biochemistry. 1st rev. ed. San Francisco: W.H. Freeman and Company.

Sugizaki J (1991). The effect of the various primers on the dentin adhesion of resin composite. Jpn J Conserv Dent 34:228–265.

Suzuki K, Nakai H (1994). Adhesion of restorative resin to tooth substance. Treatment of acid-etched dentin by aqueous solution of HEMA. Jpn J Dent Mater 12:34–44.

Van Meerbeek B, Inokoshi S, Braem M, Lambrechts P, Vanherle G (1992). Morphological aspects of the resin-dentin interdiffusion zone with different dentin adhesive systems. J Dent Res 71:1530–1540.[Abstract/Free Full Text]

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