Layering mechanism of MDP-Ca salt produced in demineralization of enamel and dentin apatite

Highlights

  • One-step self-etch adhesives containing different amounts of MDP are designed.

  • Amounts of MDP-Ca salts produced by enamel and dentin depend on the MDP concentration.

  • Dentin develops a greater amount of MDP-Ca salts than enamel by 1.5 times.

  • Type of the molecular species of MDP-Ca salts differs between enamel and dentin.

  • Species of MDP-Ca salts forming a layered structure differs between enamel and dentin.

Abstract

Objective

The 10-methacryloyloxydecyl dihydrogen phosphate (MDP) (EX adhesives)-based one-step self-etch adhesives have become widely utilized due to their simplified application procedures. The aim of this study was to determine the type of the molecular species of calcium salts of MDP (MDP-Ca salts) that form a layered structure and to understand the layering mechanism of MDP-Ca salts.

Methods

The EX adhesives were prepared by varying the amounts of MDP (25.6, 49.9, 80.5 and 116.1 mg) added in 1 g of the EX adhesive. Enamel and dentin reactant residues were obtained after the reaction of each EX adhesive to enamel or dentin particles for 30 s. The chemical analyses of both reactant residues were then performed.

Results

The molecular species of MDP-Ca salts that form a layered structure were determined as mono-calcium salt (MCS-MD) and di-calcium salts of the MDP dimer (DCS-MD). The dentin sample showed two types of characteristic XRD peaks assigned to the layer structure, since the dentin produced DCS-MD along with MCS-MD in contrast to the enamel sample. A mono-calcium salt of the MDP monomer (MCS-MM), a predominant molecular species, was not contributed to a layered-structure formation, since the intensities of characteristic XRD peaks are limited by the production of DCS-MD and MCS-MD.

Significance

The self-assembled layering of MCS-MD and DCS-MD is associated by a hydrophobic bond between two 10-methylene groups in MCS-MD and DCS-MD. The MCS-MD may form a more tightly-packed layered structure than DCS-MD by the hydrogen bonded interaction between hydroxy groups bonded to each phosphorous atom.

Introduction

Two- and one-step self-etch adhesives have been developed to simplify application procedures and reduce technique sensitivity. These self-etch adhesives have been widely accepted by dentists, since they show good bonding performance .

Studies have been performed to understand the adhesion mechanism of self-etch adhesives to the tooth through acidic monomers, such as 10-methacryloyloxydecyl dihydrogen phosphate (MDP), 2-methacryloyloxyethyl phenyl hydrogen phosphate (Phenyl-P) or 4-methacryloyloxyethyl trimellitic acid (4-MET) . Yoshida et al. reported that MDP yields a more chemically stable calcium salt than 4-MET and phenyl-P and bonds electrostatistically to hydroxyapatite. Fukegawa et al. established that the MDP bonded to the hydroxyapatite surface is accompanied by the formation of an intermediary layer of MDP. It consists of two MDP molecules with their methacrylate groups directed toward each other and their phosphate groups directed away from each other and calcium salts were deposited between the layers of their phosphate groups. Furthermore, Yoshihara et al. reported that the higher bonding performance of MDP-based self-etch adhesive is contributed to the formation of an intermediary layer of MDP on the hydroxyapatite and its thickness. However, these studies have lack on which type of the molecular species of MDP-Ca salts produced forms a layered structure and why the molecules of MDP-Ca salts form a layered structure.

To gain an insight on the type of the molecular species of MDP-Ca salts that had been produced during the application of the EX adhesive to enamel and dentin, the enamel and dentin reactant residues of the experimental one-step self-etch adhesives containing different amounts of MDP (EX adhesive) were analyzed using phosphorous-31 nuclear magnetic resonance ( 31 P NMR) and X-ray diffraction (XRD) techniques. The molecular species of MDP-Ca salt that form a layered structure was determined and the layering mechanism of MDP-Ca salts was then discussed. The null hypotheses tested were that: the type of molecular species of MDP-Ca salts (1) that has been produced, and (2) that form a layered structure, differs between enamel and dentin.

Materials and methods

All chemical reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan), unless otherwise indicated.

Preparation of EX adhesives

A series of 4 types of EX adhesives was prepared by varying the amount of MDP added. In brief, the EX adhesives consisted of MDP (purity = 97.0%), a base monomer, catalysts, inhibitor, filler, acetone and water . The base monomer was prepared by mixing 10.0 g urethane dimethacrylate (Negamikogyo, Ishikawa, Japan), 10.0 g triethylene glycol dimethacrylate (Shin-Nakamura Chemical Co., Wakayama, Japan) and 9.4 g 4-methacryloyloxyethyl trimellitic anhydride (Sun Medical, Shiga, Japan). The mixed monomer was then prepared by adding different amounts of MDP (3.0, 6.0, 10.0 or 15.0 g) as an acidic monomer to 29.4 g of the base monomer. Thereafter, 4.26 g of colloidal silica (R-972, Nihon Aerosil, Tokyo, Japan) were filled in each mixed monomer.

A series of 4 types of EX adhesives was then prepared after each resin past was diluted with an acetone aqueous solution, consisting of 69.3 g acetone and 11.2 g distilled water. The quantities of MDP in 1.0000 g of the EX adhesive were 25.6, 49.9, 80.5 and 116.1 mg. The pH values of these EX adhesives were 1.75, 1.59, 1.44 and 1.38, respectively.

Preparation of enamel and dentin particles

The pulp was removed from 2 to 2.5 years old bovine teeth. The bovine crown enamel and dentin was then cut by using an air turbine with a diamond point (#105R, Shofu, Kyoto, Japan) under a stream of cooling water, respectively. After being cut from the 100 bovine teeth, the enamel and dentin particles were obtained by decanting the cooling water collected and then rinsed 3 times with distilled and deionized water. After being dried at 20° C for 1 day, the crown enamel and dentin particles were stored in freezer at −80 °C.

Preparation of enamel and dentin reactant residues with the EX adhesive

Preparation of enamel and dentin reactants was described previously . In brief, 0.200 g of the enamel and dentin particles were, after being thawed, suspended in each EX adhesive (1.000 g), and the suspensions were then vibrated for 30 s at 20 °C. After the reaction, 30 ml of ethanol (approximately 24 g) were added to each suspension to stop further reaction of MDP with the enamel and dentin. Each suspension was centrifuged at 3500 rpm for 20 min, and the supernatant was then decanted. Thereafter, each reactant residue was rinsed 3 times with 30 ml ethanol. The enamel and dentin reactant residues were then dried for 3 h at 20 °C. Enamel and dentin reactant residues were prepared 3 times.

Observation of solid-state 31 P NMR spectra

The 31 P NMR spectra of enamel and dentin reactant residues were observed using an NMR spectrometer (EX-270, JEOL, Tokyo, Japan). The contact, repetition and accumulation times were 2000 μs, 20.05 s and 120 times. The 31 P NMR chemical shifts are expressed in ppm, with 85% H 3 PO 4 as an external reference.

The curve-fitting analyses of the corresponding 31 P NMR spectra were performed using OriginPro ® 9.1 Data Analysis and Graphing Software (OriginLab Co., Northampton, MA, USA) in order to assume the DCPD is also produced along with several types of MDP-Ca salts as a byproduct as described previously . The intensity of each simulated peak used for the curve-fitting analyses of the enamel and dentin reactant residues was then determined.

Determination of production amount of MDP-Ca salts

The peak intensity for MDP-Ca salts was determined by totaling the relative intensity ratio of the simulated peak assigned to each MDP-Ca salt in each experimental group. The amount of MDP that had been consumed yielding several types of MDP-Ca salts was determined by assuming that the peak intensity for MDP-Ca salts was 2.641 when the 116.1 mg MDP in 1.0000 g of the EX adhesive had completely yielded MDP-Ca salts as described previously .

Determination of production rate of MDP-Ca salts

The regression line between the amount of MDP added in the EX adhesive and the consumption amount of MDP for yielding MDP-Ca salts was determined using a least-square method. The slope of the regression line was determined as the production rate of MDP-Ca salts. The regression slopes were compared between the enamel and dentin by one-way ANOVA and Scheffé’s multiple comparison tests. The level of statistical significance was set at 0.05.

Observation of XRD patterns

The XRD patterns of enamel and dentin reactant residues were recorded using an X-ray diffractometer (RINT2000, Rigaku Co., Tokyo, Japan). The experimental conditions were as follows: 50 kV accelerating voltage, 300 mA current, CuKα wavelength of 0.1542 nm, 1.0°/min scan speed, 1/2° divergence slit, 0.73 mm scattering slit, 0.3 mm receiving slit, 0.02° step width, 20.0 rpm rotation rate, 1.6–70° scanning range (2 θ degree) and a graphite crystal monochromator.

Statistical analysis

The amount of MDP-Ca salts produced was analyzed by one-way analysis of variance and Tukey-Kramer post-hoc test and a standard t-test with Bonferroni correction. In addition, the amount of DCPD produced was also analyzed using the same procedure. The level of statistical significance was set at 0.05.

Materials and methods

All chemical reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan), unless otherwise indicated.

Preparation of EX adhesives

A series of 4 types of EX adhesives was prepared by varying the amount of MDP added. In brief, the EX adhesives consisted of MDP (purity = 97.0%), a base monomer, catalysts, inhibitor, filler, acetone and water . The base monomer was prepared by mixing 10.0 g urethane dimethacrylate (Negamikogyo, Ishikawa, Japan), 10.0 g triethylene glycol dimethacrylate (Shin-Nakamura Chemical Co., Wakayama, Japan) and 9.4 g 4-methacryloyloxyethyl trimellitic anhydride (Sun Medical, Shiga, Japan). The mixed monomer was then prepared by adding different amounts of MDP (3.0, 6.0, 10.0 or 15.0 g) as an acidic monomer to 29.4 g of the base monomer. Thereafter, 4.26 g of colloidal silica (R-972, Nihon Aerosil, Tokyo, Japan) were filled in each mixed monomer.

A series of 4 types of EX adhesives was then prepared after each resin past was diluted with an acetone aqueous solution, consisting of 69.3 g acetone and 11.2 g distilled water. The quantities of MDP in 1.0000 g of the EX adhesive were 25.6, 49.9, 80.5 and 116.1 mg. The pH values of these EX adhesives were 1.75, 1.59, 1.44 and 1.38, respectively.

Preparation of enamel and dentin particles

The pulp was removed from 2 to 2.5 years old bovine teeth. The bovine crown enamel and dentin was then cut by using an air turbine with a diamond point (#105R, Shofu, Kyoto, Japan) under a stream of cooling water, respectively. After being cut from the 100 bovine teeth, the enamel and dentin particles were obtained by decanting the cooling water collected and then rinsed 3 times with distilled and deionized water. After being dried at 20° C for 1 day, the crown enamel and dentin particles were stored in freezer at −80 °C.

Preparation of enamel and dentin reactant residues with the EX adhesive

Preparation of enamel and dentin reactants was described previously . In brief, 0.200 g of the enamel and dentin particles were, after being thawed, suspended in each EX adhesive (1.000 g), and the suspensions were then vibrated for 30 s at 20 °C. After the reaction, 30 ml of ethanol (approximately 24 g) were added to each suspension to stop further reaction of MDP with the enamel and dentin. Each suspension was centrifuged at 3500 rpm for 20 min, and the supernatant was then decanted. Thereafter, each reactant residue was rinsed 3 times with 30 ml ethanol. The enamel and dentin reactant residues were then dried for 3 h at 20 °C. Enamel and dentin reactant residues were prepared 3 times.

Observation of solid-state 31 P NMR spectra

The 31 P NMR spectra of enamel and dentin reactant residues were observed using an NMR spectrometer (EX-270, JEOL, Tokyo, Japan). The contact, repetition and accumulation times were 2000 μs, 20.05 s and 120 times. The 31 P NMR chemical shifts are expressed in ppm, with 85% H 3 PO 4 as an external reference.

The curve-fitting analyses of the corresponding 31 P NMR spectra were performed using OriginPro ® 9.1 Data Analysis and Graphing Software (OriginLab Co., Northampton, MA, USA) in order to assume the DCPD is also produced along with several types of MDP-Ca salts as a byproduct as described previously . The intensity of each simulated peak used for the curve-fitting analyses of the enamel and dentin reactant residues was then determined.

Determination of production amount of MDP-Ca salts

The peak intensity for MDP-Ca salts was determined by totaling the relative intensity ratio of the simulated peak assigned to each MDP-Ca salt in each experimental group. The amount of MDP that had been consumed yielding several types of MDP-Ca salts was determined by assuming that the peak intensity for MDP-Ca salts was 2.641 when the 116.1 mg MDP in 1.0000 g of the EX adhesive had completely yielded MDP-Ca salts as described previously .

Determination of production rate of MDP-Ca salts

The regression line between the amount of MDP added in the EX adhesive and the consumption amount of MDP for yielding MDP-Ca salts was determined using a least-square method. The slope of the regression line was determined as the production rate of MDP-Ca salts. The regression slopes were compared between the enamel and dentin by one-way ANOVA and Scheffé’s multiple comparison tests. The level of statistical significance was set at 0.05.

Observation of XRD patterns

The XRD patterns of enamel and dentin reactant residues were recorded using an X-ray diffractometer (RINT2000, Rigaku Co., Tokyo, Japan). The experimental conditions were as follows: 50 kV accelerating voltage, 300 mA current, CuKα wavelength of 0.1542 nm, 1.0°/min scan speed, 1/2° divergence slit, 0.73 mm scattering slit, 0.3 mm receiving slit, 0.02° step width, 20.0 rpm rotation rate, 1.6–70° scanning range (2 θ degree) and a graphite crystal monochromator.

Statistical analysis

The amount of MDP-Ca salts produced was analyzed by one-way analysis of variance and Tukey-Kramer post-hoc test and a standard t-test with Bonferroni correction. In addition, the amount of DCPD produced was also analyzed using the same procedure. The level of statistical significance was set at 0.05.

Results

31 P NMR analyses of enamel and dentin reactant residues of EX adhesives and curve-fitting of corresponding 31 P NMR spectra

Fig. 1 shows the typical 31 P NMR spectra of enamel (a) and dentin (b) reactant residues of EX adhesives containing different amounts of MDP, respectively. In the 31 P NMR spectrum of each enamel or dentin reactant residue, the observed 31 P NMR spectrum is represented with the black line. The 31 P NMR peaks detected in both NMR spectra were assigned and listed in Table 1 .

Nov 22, 2017 | Posted by in Dental Materials | Comments Off on Layering mechanism of MDP-Ca salt produced in demineralization of enamel and dentin apatite
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