Abstract
Objectives
The functional monomer 10-methacryloxydecyl dihydrogen phosphate (10-MDP), recently used in more self-etch adhesives, chemically bonds to hydroxyapatite (HAp) and thus tooth tissue. Although the interfacial interaction of the phosphoric-acid functional group of 10-MDP with HAp-based substrates has well been documented, the effect of the long carbon-chain spacer of 10-MDP on the bonding effectiveness is far from understood.
Methods
We investigated three phosphoric-acid monomers, 2-methacryloyloxyethyl dihydrogen phosphate (2-MEP), 6-methacryloyloxyhexyl dihydrogen phosphate (6-MHP) and 10-MDP, that only differed for the length of the carbon chain, on their chemical interaction potential with HAp and dentin, this correlatively using X-ray diffraction (XRD) and transmission electron microscopy (TEM). Commercial 6-MHP and 10-MDP containing adhesives were investigated as well.
Results
XRD revealed that on HAp only 10-MDP produced monomer-calcium salts in the form of ‘nano-layering’, while on dentin all monomers produced ‘nano-layering’, but with a varying intensity in the order of 10-MDP > 6-MHP > 2-MEP. TEM confirmed that 10-MDP formed the thickest hybrid and adhesive layer. XRD and TEM revealed ‘nano-layering’ for all commercial adhesives on dentin, though less intensively for the 6-MHP containing adhesive than for the 10-MDP ones.
Significance
It is concluded that not only the phosphoric-acid group but also the spacer group, and its length, affect the chemical interaction potential with HAp and dentin. In addition, the relatively strong ‘etching’ effect of 10-MDP forms more stable monomer-Ca salts, or ‘nano-layering’, than the two shorter carbon-chain monomers tested, thereby explaining, at least in part, the better bond durability documented with 10-MDP containing adhesives.
1
Introduction
Of all the ingredients of a dental adhesive, the functional monomer is considered most important; it regulates the interfacial interaction at tooth enamel and dentin . Commonly, functional monomers in self-etch primers/adhesives are acidic esters, originating from the reaction of a bivalent alcohol with methacrylic acid and phosphoric/carboxylic acid derivatives . The diverse commercially available self-etch adhesives contain each one or occasionally two specific functional monomers. The wide variance in effectiveness data, typically recorded for the different adhesives in the worldwide conducted laboratory and clinical studies, should at least in part be attributed to the diversity in kind of functional monomers included in the particular adhesive formulations .
Of the functional monomers contained in self-etch adhesives, 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP) was found to adhere to hydroxyapatite (HAp) and tooth tissue most readily and intensively . According to the adhesion–decalcification (AD) concept, the less soluble the calcium salt of the acidic monomer, the more intense and stable is the molecular adhesion to the HAp-based substrate . Indeed, adhesives containing 10-MDP revealed rather consistently a favorable adhesive performance in many laboratory and clinical studies, in particular also regarding long-term bond durability . Consequently, more manufacturers recently marketed adhesives based on 10-MDP monomer technology, like All-Bond Universal (Bisco, Schaumburg, IL, USA), Clearfil 3S Bond Plus (Kuraray Noritake Dental, Tokyo, Japan), G-aenial Bond (GC, Tokyo, Japan), and Scotchbond Universal (3M ESPE, Seefeld, Germany).
The monomer 10-MDP possesses a phosphoric-acid functional group as main adhesion promoter to interact with HAp, a methacrylate polymerizable group for curing, of which the importance with regard to bond strength and durability should not be underestimated , and finally a 10-carbon chain or decyl group as spacer to separate both other active groups. The latter carbon spacer is known to influence monomer characteristics such as flexibility, solubility, wetting, and the hydrophobicity-hydrophilicity balance . Actually, only a few studies investigated the effect of the spacer of 10-MDP on adhesive performance . There is to our knowledge no paper that searched for the underlying mechanisms by investigating the chemical interaction of different carbon-chain monomers with HAp.
The objective of this study was therefore to investigate the influence of the carbon-chain length of a phosphoric-acid ester monomer on its chemical interaction potential with HAp. This study consisted of three project parts: in Project Part 1, the interaction with HAp of three functional monomers, namely 2-methacryloyloxyethyl dihydrogen phosphate (2-MEP), 6-methacryloyloxyhexyl dihydrogen phosphate (6-MHP) or 10-MDP, all differing only for the length of the carbon-chain spacer, was studied using X-ray Diffraction (XRD); in Project Part 2, XRD was used to assess potential nano-layering at dentin, while transmission electron microscopy (TEM) was used to ultra-morphologically characterize the adhesive interface produced at dentin and so to potentially confirm nano-layering morphologically when detected by XRD; in Project Part 3, finally, the interfacial interaction of one 6-MHP-based and three 10-MDP-based commercial adhesives with dentin was evaluated chemically using XRD and ultra-morphologically using TEM. The overall null-hypothesis tested was that the carbon-chain length of a phosphoric-acid ester functional monomer has no effect on chemical interaction with HAp and dentin.
2
Materials and methods
The different project parts are schematically explained in Fig. 1 .
2.1
Project PART 1 (PP1): chemical interaction of different carbon-chain monomers with HAp analyzed using XRD
2.1.1
Preparation of monomer-coated HAp
2-MEP and 6-MHP-coated HAp particles (referred to as ‘2-MEP_HAp’ and ‘6-MHP_HAp’, respectively) were prepared at room temperature by dispersion of 0.2 g of HAp particles (surface area of 49 m 2 g −1 , mean powder diameter of 19.45 ± 0.49 μm; Pentax, Tokyo, Japan) in 0.1 g of a mixed solution of, respectively, 2-MEP and 6-MHP (Kuraray Noritake Dental), in absolute ethanol and distilled de-ionized water in a 2-MEP or 6-MHP:EtOH:H 2 O composition of 15:45:40 wt% under stirring. The 2-MEP or 6-MHP-coated HAp particles were separated from the mixed solution by centrifugation and decantation, respectively, after 5 min (referred to as ‘2-MEP or 6-MHP_HAp_5min’), 1 h (referred to as ‘2-MEP or 6-MHP_HAp_1h’), and 24 h (referred to as ‘2-MEP or 6-MHP_HAp_24h’). The separated 2-MEP/6-MHP-HAp particles were then washed three times with absolute ethanol, and dried at room temperature in ambient atmosphere. Identical compounds of 10-MDP were prepared similarly in our previous study . Kuraray Noritake Dental confirmed to have provided 10-MDP and 6-MHP in a high purity, while 2-MEP mainly consists of pure monomer and some di-ester monomer. A comparison will be made with commercial adhesives (see below). The level of purity of the three functional monomers in this experiment is thought to be of the same level of that of the monomers contained in the respective commercial adhesives.
2.1.2
XRD
The crystal phases of powder samples were identified by means of an X-ray powder diffractometer (CuKα1 1.5406 Å, RINT2500, Rigaku, Tokyo, Japan), operated under 40 kV acceleration and 200 mA current, and a scanning rate of 0.02° per second for 2 θ / θ scan. The XRD data obtained for 2-MEP and 6-MHP were compared to the respective data obtained before for 10-MDP .
2.2
Project PART 2 (PP2): interaction of different carbon-chain monomers with dentin analyzed using XRD and TEM
2.2.1
Preparation of dentin specimens treated with monomer for XRD (PP2a)
Dentin specimens (10 × 8 × 1 mm) were cut from bovine mandibular front teeth, after which the exposed surfaces were ground using SiC-paper (#600). The same 15:45:40 wt% 2-MEP or 6-MHP/ethanol/water monomer solutions as described above were applied on dentin by lightly rubbing with a micro-brush (Centrix Benda Brush, Centrix, Chelton, CT, USA). After 20 s, the samples were strongly air-dried prior to further chemical analysis using XRD. Identical compounds of 10-MDP were prepared similarly in our previous study .
The surface structure of the dentin specimens treated with the experimental monomer solutions were examined by thin-film X-ray diffraction (TF-XRD) using an X-ray diffractometer (RINT2500, Rigaku; same parameters as mentioned above), with the angle of the incident X-ray beam fixed at 1.0°.
2.2.2
TEM of interfaces produced by experimental adhesive formulations at dentin (PP2b)
Extracted non-carious human third molars (gathered following informed consent approved by the Commission for Medical Ethics of KU Leuven) were used within 1 month of extraction (stored in 0.5% chloramine/water, 4 °C). After removal of the occlusal crown third using an Isomet diamond saw (Isomet 1000, Buehler, Lake Bluff, IL, USA), the exposed dentin was wet-sanded (60 s, #600 SiC-paper) to produce a standard smear layer, after which it was treated with one of the three experimental 15:45:40 wt% 2-MEP or 6-MHP/ethanol/water monomer solutions. After 20 s, the samples were strongly air-dried and then light-cured using an Optilux 500 (Demetron/Kerr, Danbury, CT, USA) light-curing unit (40 s). On top of the adhesive, the flowable composite (Clearfil Protect Liner F, Kuraray Noritake Dental) was applied and again light-cured. After bonding, the resin-bonded dentin specimens were stored for 1 day in distilled water at 37 °C and further processed for TEM following a protocol previously described in detail before . Non-demineralized sections were cut (Ultracut UCT, Leica, Vienna, Austria) to be imaged by TEM (80 kV JEM-1200 EX II TEM, Jeol, Tokyo, Japan).
2.3
Project PART 3 (PP3): chemical interaction of commercial adhesives with dentin analyzed using XRD and TEM
2.3.1
Preparation of dentin specimens treated with a commercial adhesive for XRD (PP3a)
Bovine dentin specimens (10 × 8 × 1 mm) were prepared for XRD like described in PP2. The 6-MHP-containing adhesive, ‘Adper Easy Bond’ (3M ESPE), and the 10-MDP-containing adhesives, ‘All-Bond Universal’ (Bisco), ‘Clearfil S3 Bond (Kuraray Noritake Dental) and ‘ScotchBond Universal’ (3M ESPE), were lightly rubbed on dentin with a micro-brush (Centrix Benda Brush, Centrix). After 20 s, the samples were strongly air-dried, after which XRD analysis was performed, like it was described in PP2.
2.3.2
TEM of interfaces produced by the commercial adhesives at dentin (PP3b)
Another set of extracted non-carious human third molars was prepared in the same manner as described in PP2. The 6-MHP-containing adhesive, ‘Adper Easy Bond’ (3M ESPE), and 10-MDP-containing adhesives, ‘All-Bond Universal’ (Bisco), Clearfil S3 Bond (Kuraray Noritake Dental), and ‘ScotchBond Universal’ (3M ESPE), were applied strictly following the manufacturer’s instructions by lightly rubbing dentin with a micro-brush (Centrix Benda Brush, Centrix). After 20 s, the samples were strongly air-dried and then light-cured using an Optilux 500 (Demetron/Kerr, Danbury, CT, USA) light-curing unit. On top of the adhesive, the flowable composite (Clearfil Protect Liner F, Kuraray Noritake Dental) was applied and again light-cured. After bonding, the resin-bonded dentin specimens were stored for 1 day in distilled water at 37 °C and further processed for TEM (JEM-1200 EX II TEM, and 300 kV TEM JEM-3010, both Jeol) in the same manner as described in PP2.
2
Materials and methods
The different project parts are schematically explained in Fig. 1 .
2.1
Project PART 1 (PP1): chemical interaction of different carbon-chain monomers with HAp analyzed using XRD
2.1.1
Preparation of monomer-coated HAp
2-MEP and 6-MHP-coated HAp particles (referred to as ‘2-MEP_HAp’ and ‘6-MHP_HAp’, respectively) were prepared at room temperature by dispersion of 0.2 g of HAp particles (surface area of 49 m 2 g −1 , mean powder diameter of 19.45 ± 0.49 μm; Pentax, Tokyo, Japan) in 0.1 g of a mixed solution of, respectively, 2-MEP and 6-MHP (Kuraray Noritake Dental), in absolute ethanol and distilled de-ionized water in a 2-MEP or 6-MHP:EtOH:H 2 O composition of 15:45:40 wt% under stirring. The 2-MEP or 6-MHP-coated HAp particles were separated from the mixed solution by centrifugation and decantation, respectively, after 5 min (referred to as ‘2-MEP or 6-MHP_HAp_5min’), 1 h (referred to as ‘2-MEP or 6-MHP_HAp_1h’), and 24 h (referred to as ‘2-MEP or 6-MHP_HAp_24h’). The separated 2-MEP/6-MHP-HAp particles were then washed three times with absolute ethanol, and dried at room temperature in ambient atmosphere. Identical compounds of 10-MDP were prepared similarly in our previous study . Kuraray Noritake Dental confirmed to have provided 10-MDP and 6-MHP in a high purity, while 2-MEP mainly consists of pure monomer and some di-ester monomer. A comparison will be made with commercial adhesives (see below). The level of purity of the three functional monomers in this experiment is thought to be of the same level of that of the monomers contained in the respective commercial adhesives.
2.1.2
XRD
The crystal phases of powder samples were identified by means of an X-ray powder diffractometer (CuKα1 1.5406 Å, RINT2500, Rigaku, Tokyo, Japan), operated under 40 kV acceleration and 200 mA current, and a scanning rate of 0.02° per second for 2 θ / θ scan. The XRD data obtained for 2-MEP and 6-MHP were compared to the respective data obtained before for 10-MDP .
2.2
Project PART 2 (PP2): interaction of different carbon-chain monomers with dentin analyzed using XRD and TEM
2.2.1
Preparation of dentin specimens treated with monomer for XRD (PP2a)
Dentin specimens (10 × 8 × 1 mm) were cut from bovine mandibular front teeth, after which the exposed surfaces were ground using SiC-paper (#600). The same 15:45:40 wt% 2-MEP or 6-MHP/ethanol/water monomer solutions as described above were applied on dentin by lightly rubbing with a micro-brush (Centrix Benda Brush, Centrix, Chelton, CT, USA). After 20 s, the samples were strongly air-dried prior to further chemical analysis using XRD. Identical compounds of 10-MDP were prepared similarly in our previous study .
The surface structure of the dentin specimens treated with the experimental monomer solutions were examined by thin-film X-ray diffraction (TF-XRD) using an X-ray diffractometer (RINT2500, Rigaku; same parameters as mentioned above), with the angle of the incident X-ray beam fixed at 1.0°.
2.2.2
TEM of interfaces produced by experimental adhesive formulations at dentin (PP2b)
Extracted non-carious human third molars (gathered following informed consent approved by the Commission for Medical Ethics of KU Leuven) were used within 1 month of extraction (stored in 0.5% chloramine/water, 4 °C). After removal of the occlusal crown third using an Isomet diamond saw (Isomet 1000, Buehler, Lake Bluff, IL, USA), the exposed dentin was wet-sanded (60 s, #600 SiC-paper) to produce a standard smear layer, after which it was treated with one of the three experimental 15:45:40 wt% 2-MEP or 6-MHP/ethanol/water monomer solutions. After 20 s, the samples were strongly air-dried and then light-cured using an Optilux 500 (Demetron/Kerr, Danbury, CT, USA) light-curing unit (40 s). On top of the adhesive, the flowable composite (Clearfil Protect Liner F, Kuraray Noritake Dental) was applied and again light-cured. After bonding, the resin-bonded dentin specimens were stored for 1 day in distilled water at 37 °C and further processed for TEM following a protocol previously described in detail before . Non-demineralized sections were cut (Ultracut UCT, Leica, Vienna, Austria) to be imaged by TEM (80 kV JEM-1200 EX II TEM, Jeol, Tokyo, Japan).
2.3
Project PART 3 (PP3): chemical interaction of commercial adhesives with dentin analyzed using XRD and TEM
2.3.1
Preparation of dentin specimens treated with a commercial adhesive for XRD (PP3a)
Bovine dentin specimens (10 × 8 × 1 mm) were prepared for XRD like described in PP2. The 6-MHP-containing adhesive, ‘Adper Easy Bond’ (3M ESPE), and the 10-MDP-containing adhesives, ‘All-Bond Universal’ (Bisco), ‘Clearfil S3 Bond (Kuraray Noritake Dental) and ‘ScotchBond Universal’ (3M ESPE), were lightly rubbed on dentin with a micro-brush (Centrix Benda Brush, Centrix). After 20 s, the samples were strongly air-dried, after which XRD analysis was performed, like it was described in PP2.
2.3.2
TEM of interfaces produced by the commercial adhesives at dentin (PP3b)
Another set of extracted non-carious human third molars was prepared in the same manner as described in PP2. The 6-MHP-containing adhesive, ‘Adper Easy Bond’ (3M ESPE), and 10-MDP-containing adhesives, ‘All-Bond Universal’ (Bisco), Clearfil S3 Bond (Kuraray Noritake Dental), and ‘ScotchBond Universal’ (3M ESPE), were applied strictly following the manufacturer’s instructions by lightly rubbing dentin with a micro-brush (Centrix Benda Brush, Centrix). After 20 s, the samples were strongly air-dried and then light-cured using an Optilux 500 (Demetron/Kerr, Danbury, CT, USA) light-curing unit. On top of the adhesive, the flowable composite (Clearfil Protect Liner F, Kuraray Noritake Dental) was applied and again light-cured. After bonding, the resin-bonded dentin specimens were stored for 1 day in distilled water at 37 °C and further processed for TEM (JEM-1200 EX II TEM, and 300 kV TEM JEM-3010, both Jeol) in the same manner as described in PP2.
3
Results
3.1
PP1: chemical interaction of different carbon-chain monomers with HAp analyzed using XRD
All samples showed diffraction peaks representing HAp (range of 2 θ = 10.7°–39.74°) ( Fig. 2 ). Powder-XRD of 2-MEP- and 6-MHP-coated HAp disclosed the formation of dicalcium phoshate dihydrate or DCPD (CaHPO 4 ·2H 2 O), but only after 24 h interaction and very intense for 2-MEP, but not for 6-MHP ( Fig. 2 a and b). The strong peak at 2 θ = 11.8° ( d = 0.75 nm) was accompanied by several peaks at 2 θ = 21.0° ( d = 0.42 nm), 23.0° ( d = 0.39 nm) and 29.4° ( d = 0.30 nm), all to be ascribed to DCPD. Besides peaks representing DCPD, no other peaks that could have represented monomer-Ca salts, were detected in the XRD patterns of 2-MEP- and 6-MHP-coated HAp.
