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Journal of Dental Research
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Biomaterials & Bioengineering

Dissociation States of Collagen Functional Groups and their Effects on the Priming Efficacy of HEMA Bonded to Collagen

N. Nishiyama1,*, K. Suzuki2, A. Nagatsuka1, I. Yokota1 and K. Nemoto1

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;

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Applying 2-hydroxyethylmethacrylate (HEMA) solution to etched dentin enhances the bonding of resin to dentin. However, the principal adhesion mechanisms have not yet been identified. In this study, we examined the dissociation states of the collagen functional groups of the side-chain amino acid residues and their effects on the bond strength of resin to etched dentin primed by the HEMA solution. The bond strength was strongly dependent upon the dissociation state of the collagen functional groups. Inhibiting the dissociation of the carboxylic acid or the amine of a collagen functional group resulted in increased bond strength of resin to collagen. By understanding the significance of inhibiting the dissociation state, we can better design and develop more effective and efficient primer and bonding agents.

Key Words: dentin adhesion mechanism • dentin primer • HEMA • dentinal collagen • collagen functional group


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
For dentin adhesion, applying HEMA solution as a dentin primer to collagen exposed by acid-etching results in increased bond strength of resin (Nakabayashi and Takarada, 1992; Suzuki and Nakai, 1994; Pashley, 1996). Previously, we examined the pH effects of N-methacryloyl glycine (NMGly) solution as a dentin primer on the bond strength of resin to etched dentin (Nishiyama et al., 1996). The application of an NMGly solution whose pH was below the 3.5 pKa of the NMGly intramolecular carboxylic acid provided noticeably higher bond strengths of 15 MPa. However, despite the formation of a hybrid layer at the resin-dentin interface, when NMGly solutions with pH values of above 3.5 were applied, the bond strength dramatically dropped to 3 MPa. This result suggests that the principal adhesion mechanism of resin to etched dentin is not solely micromechanical interlocking. For dentin adhesion, it is important to determine how the dentin primer promotes the bonding of resin to the collagen fiber.

To understand how NMGly enhances bonding at the resin-dentin interface, we examined the interactions between NMGly and collagen (Nishiyama et al., 1998, 1999). The amide or carboxylic acid group in the NMGly formed a hydrogen bond with the undissociated carboxylic acid of the collagen functional group. Analysis of these data clearly demonstrated that the hydrogen-bonded NMGly species enhanced the bonding of resin to collagen fiber.

The purpose of this study was to examine the dissociation states of the carboxylic acid or amine of collagen functional groups and their effects on the bond strength of resin to etched dentin by varying the pH values for the HEMA aqueous solution.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Collagen Powder
Collagen powder was prepared as per our previous paper (Nishiyama et al., 1998). Crown dentin from a fresh bovine tooth, after being frozen by liquid nitrogen, was reduced to powder by a ball agate mill. The obtained dentin particles were then demineralized by 40 mass% phosphoric acid for 15 min at 0°C. Next, the insoluble collagen was repeatedly decanted with distilled and de-ionized water until the pH value of the supernatant solution equaled 6. The insoluble collagen was then air-dried in a thermostabilized room at 20°C for one day.

Analysis of the Collagen
After 70 mg of the collagen was suspended in 20 mass% deuterium oxide aqueous solution (pH 5.57), the collagen suspension was then heated at 80°C for 30 min. The 13C NMR observations for the collagen, before and after being heated, were then performed at 25°C by means of an EX 270 spectrometer (JEOL, Tokyo, Japan).

Determination of the Average pKa for the Collagen Functional Group
To determine the average pKa for the carboxylic acids of the collagen functional group, we dispersed 70 mg of collagen into 600 mg of aqueous solutions whose pH values, which had been adjusted by the addition of hydrochloric acid or sodium hydroxide, ranged from 0.89 to 12.39. After the collagen suspension was vibrated for 30 sec, the pH of the collagen suspension was immediately measured at 20°C. The average pKa for the carboxylic acids of the collagen functional group was determined from a titration curve.

To determine the average pKa for the amines of the collagen functional group, we added 30 mg of collagen to 600 mg of aqueous solutions whose pH values ranged from 11.73 to 12.59. To prevent any pH changes due to the hydrolysis of the -CO-NH- bond of the main chain in the collagen macromolecule, we measured the pH of the collagen suspension immediately after vibration. The average pKa for the amines was then determined.

Preparation of HEMA Solution
HEMA of 30 mass% was dissolved into ionized aqueous solutions whose pH values had been adjusted to 1.5, 2.0, 3.0, 9.0, 10.0, 12.0, and 12.5, and also into a non-ionized aqueous solution with a pH of 6.6. These HEMA solutions were immediately used for the adhesion tests, since the ester portion in the HEMA could become hydrolyzed in an acidic or basic aqueous solution.

Adhesion Test
The specimen for the adhesion test was prepared as per our previous paper (Nishiyama et al., 1996). Grounded crown dentin from a fresh bovine tooth was etched for 30 sec with phosphoric acid of 40 mass%. The surface was then rinsed with water. After the surface was air-dried, a polyethylene ring, with an internal diameter of 3.8 mm, was mounted onto the etched-dentin surface. Next, the surface inside the ring was primed with a HEMA solution for 30 sec and then air-dried. Immediately thereafter, Clearfil New Bond (Kuraray, Osaka, Japan) was applied to the primed dentin surface. The surface was then air-dried. The ring was then filled with Clearfil SC-II. The specimen was left to harden at room temperature and then stored in water at 37°C. After one day, the tensile bond strength of resin to dentin was measured by means of a tensile testing machine at a crosshead speed of 2 mm/min. There were 10 specimens for each experiment.

The bond strength of resin to ground dentin conditioned with Clearfil SE Bond primer was measured.

Statistical Analysis of Bond Strength
Bond strengths were analyzed by Fisher’s Protected LSD test.

Scanning Electron Microscope (SEM) Examination of the Resin-Dentin Interface
After being prepared as per the previously discussed adhesion test procedures, the specimens were cross-sectioned perpendicular to the resin-dentin interface by a diamond disk under a stream of water. The cross-sectioned surface was then polished with #1000 silicon carbide paper under a stream of water. Next, the surface was etched with 40 mass% of phosphoric acid for 5 sec and rinsed with water. The specimens were immersed in Neo Cleaner (Neo Chemical Industry, Tokyo, Japan) for 1 hr. They were then rinsed with water and air-dried at 25°C for one day. The samples were mounted onto aluminum stubs and sputter-coated with gold. The specimens were examined at numerous magnifications and tilt angles in a scanning electron microscope (Hitachi S-430, Tokyo, Japan) at 15 kV.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NMR Spectra of the Collagen
Fig. 1AGo shows the 13C NMR spectra for the collagen, before and after heat treatment. When heat treatment was applied to the collagen, the carbonyl carbon peaks and the methine, methylene, or methyl peaks attributed to the amino acid residues in the collagen macromolecule were detected at 170-180 ppm and 15-70 ppm, respectively (lower spectrum). These peaks were observable due to an increase in the molecular motion of the amino acid residues in the collagen macromolecule. These results were attributed to the deformation of the second-order structure of the collagen macromolecule, from a triple helix to a random coil structure (Nishiyama et al., 1995). However, when the collagen was not heated, the 13C NMR peaks specifically attributed to the collagen were not detected, since the molecular motion of the amino acid residues in the collagen macromolecule was limited because the triple helix structure in the collagen was maintained.


Figure 1A
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  		Figure 1A. Liquid-state 13C NMR spectra of the collagen, before and after heat treatment. The accumulation and repetition times were 84,000 and 3.8 sec, respectively. The hexamethyl-di-siloxane was used as an external reference. The upper spectrum was obtained before heat treatment. The lower spectrum was obtained after heat treatment.

 
The pKa of the Collagen Functional Group
Fig. 1BGo shows titration curves for the carboxylic acid (I) and amine (II) of the collagen functional group. The average pKa values for the carboxylic acid and amine are 4.1 and 9.3, respectively. Dissociation states for the two collagen functions are summarized in Fig. 1CGo. When the collagen was suspended in an aqueous solution with a pH of 6.6 (control), the pH of the collagen suspension equaled 4.1. At this pH value, half of the carboxylic acids of the collagen function are dissociated, and all of the amines are protonated.


Figure 1B
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  		Figure 1B. Titration curves for the functional group of the side-chain amino acid residues in the collagen macromolecule. (I) Titration curve for the carboxylic acid of the collagen functional group. (II) Titration curve for the amine of the collagen functional group.

 

Figure 1C
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  		Figure 1C. Dissociation states for the carboxylic acid and amine of the side-chain amino acid residues in the collagen macromolecule.

 
Bond Strength of Resin to Etched Dentin Primed with HEMA Solutions of Different pH Values
Fig. 2Go shows the bond strengths of resin to dentin. Statistical analyses for the bond strengths are summarized in the TableGo. When Clearfil SE Bond primer was applied to ground dentin, the bond strength equaled 21 MPa.


Figure 2
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  		Figure 2. Bond strength of resin to etched dentin primed by HEMA solutions with various pH values of the aqueous solution for the HEMA primer. Error bar shows SD. Dashed line represents the bond strength when Clearfil New Bond was applied to etched dentin without any primer pre-treatment. Solid line represents the bond strength when the dentin was conditioned with Clearfil SE Bond primer instead of 40% phosphoric acid.

 

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  		Table. Statistical Analysis of Bond Strengthsa of Resin to Acid-etched Dentin Primed with HEMA Solutions of Different pH Values
 
When Clearfil New Bond was applied directly to etched dentin without any primer pre-treatment, the bond strength was 5.0 MPa. However, the application of a HEMA solution, with a pH of 6.6 (control) for the aqueous solution, resulted in increased bond strength to 11.3 MPa. The bond strength increased when the pH of the aqueous solution for the HEMA primer was decreased. The maximum bond strength of 15.5 MPa was observed when the pH of the aqueous solution equaled 2.0.

Conversely, when the pH of the aqueous solution was increased from 6.6 to 9.0, the bond strength decreased to a minimum value of 9.3 MPa. However, when the pH was increased from 9.0 to 12.5, the bond strength increased to 14.2 MPa.

Effects from HEMA Solutions of Different pHs on the Thickness of the Hybrid Layer
Fig. 3Go shows SEM views of the resin-dentin interface. When Clearfil SE Bond primer was applied to ground dentin, the thickness of the hybrid layer was approximately 1 µm.


Figure 3
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  		Figure 3. SEM views of the resin-dentin interface with various pH values of the aqueous solution for HEMA primer. Magnification: 5000 times. CR: Composite resin. H: Hybrid layer. Non-treated: Clearfil New Bond was applied to etched dentin without any primer pre-treatment. Clearfil SE Bond primer: After the ground dentin was conditioned with Clearfil New Bond primer, Clearfil New Bond and Clearfil SE-II were applied to the primed dentin. Treated: Etched dentin primed with HEMA solutions of different pH values. pH = 1.5: Distilled water ionized by HCl. pH = 6.6: Distilled and de-ionized water was used. pH = 12.0: Distilled water ionized by NaOH.

 
When Clearfil New Bond was applied directly to etched dentin, it was difficult to identify the formation of any hybrid layer. However, when the HEMA solution was applied to etched dentin, the creation of a hybrid layer was observed for all specimens. The thickness of the hybrid layer was approximately 3 µm, even though the pH of the aqueous solution for the HEMA primer was varied.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is well-understood that a HEMA solution applied to etched dentin facilitates bonding at the resin-dentin interface. In this study, to understand how HEMA enhances the bonding of resin to collagen fiber, we examined the dissociation effects of collagen functional groups, such as carboxylic acid and amine, on the bond strength to resin.

To investigate the effects of the collagen functional group on the bond strength of resin to collagen primed with HEMA solution, we determined the pKa values for the functional group of the side-chain amino acid residues in the collagen macromolecule. The dissociation states of the collagen functional group were strongly dependent upon the pH of the aqueous solution (Fig. 1CGo).

The application of the HEMA solutions resulted in an increase in the bond strength from 5 MPa to over 9 MPa. However, even though the thickness of the hybrid layer was the same, the bond strength was strongly dependent on the pH of the aqueous solution for the HEMA primer. Previously, we examined the adsorption characteristics of the HEMA to collagen (Nishiyama et al., 2002). The strength of the interaction that exists between the ester carbonyl portion in the HEMA and the undissociated carboxylic acid of the collagen functional group became stronger when the pH of the collagen suspension was decreased from 4.1 to 1.7. This increased the probability that the ester carbonyl portion in the HEMA hydrogen-bonded with the carboxylic acid of the collagen functional group. This was possible since a decrease in the pH of the collagen suspension to 1.7 inhibited the dissociation of the carboxylic acid of the collagen functional group. Hence, the observed increase in the bond strength, while the pH of the aqueous solution for the HEMA primer was decreased, was perhaps due to the HEMA species hydrogen-bonding with the undissociated carboxylic acids of the collagen functional group. This probably allowed for the observed tight bonding of resin to collagen fiber, similar to the results observed when N-methacryloyl-{omega}-amino acid primer was applied to the collagen (Nishiyama et al., 1999).

When Clearfil SE Bond primer, comprised of the MDP and HEMA, was applied to ground dentin, the highest bond strength was obtained. This was possible since the phosphoric acid in the MDP, which had not formed any acid-base interaction with the calcium cation in the dentin, dissociated, and the resultant proton inhibited the dissociation of the carboxylic acids of the collagen functional group. This allowed for hydrogen bonding between the ester carbonyl portion in the HEMA, contained in the Clearfil SE Bond primer, and the undissociated carboxylic acids of the collagen functional group. As a result, the bond strength of the resin increased as observed with the application of an acidic HEMA solution.

Conversely, when the pH of the aqueous solution for the HEMA primer was increased from 6.6 to 9.0, the bond strength decreased to 9.3 MPa. This decrease was due to the deformation in hydrogen bonding, as discussed, since most of the carboxylic acids of the collagen functional group became dissociated at this pH value. Here, we are most likely observing the efficacy of the hydroxyl group, as a collagen functional group, on the bond strength to resin. However, by increasing the pH value of the aqueous solution for the HEMA primer above the pKa for the amine of the collagen functional group, the bond strength increased to 14.2 MPa.

From these results, it can be concluded that inhibiting the dissociation of the carboxylic acid or the amine of the collagen functional group resulted in enhancing the bonding of resin to collagen fiber.


   ACKNOWLEDGMENTS
 
This work was supported by a grant-in-aid for Developmental Scientific Research from the Ministry of Education, Science and Culture in Japan (#11671955). David Mukai, Tokyo, is acknowledged for proofreading the manuscript.

Received for publication September 27, 2001. Revision received December 9, 2002. Accepted for publication January 9, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  • Nakabayashi N, Takarada K (1992). Effect of HEMA on bonding to resin. Dent Mater 8:125–130.[Medline] [Order article via Infotrieve]
  • Nishiyama N, Asakura T, Suzuki K, Horie K, Nemoto K (1995). Effects of a structural change in collagen upon binding to conditioned dentin studied by 13C NMR. J Biomed Mater Res 29:107–111.[Medline] [Order article via Infotrieve]
  • Nishiyama N, Suzuki K, Asakura T, Nakai H, Yasuda S, Nemoto K (1996). The effects of pH of N-methacryloyl glycine primer on bond strength to acid-etched dentin. J Biomed Mater Res 31:379–384.[Medline] [Order article via Infotrieve]
  • 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] [Order article via Infotrieve]
  • Nishiyama N, Asakura T, Suzuki K, Komatsu K, Nemoto K (1999). Bond strength of resin to acid-etched dentin studied by 13C NMR. J Dent Res 79:1–6.
  • Nishiyama N, Suzuki K, Komatsu K, Yasuda S, Nemoto K (2002). A 13C NMR study on the adsorption characteristics of HEMA to dentinal collagen. J Dent Res 81:469–471.
  • Pashley DH (1996). The effects of dentin bonding procedures on the dentin/pulp complex. Quintessence 31:193–201.
  • Suzuki K, Nakai H (1994). Adhesion of restorative resin to tooth substance. Jpn J Dent Mater 12:34–44.

Journal of Dental Research, Vol. 82, No. 4, 257-261 (2003)
DOI: 10.1177/154405910308200403
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