Functional monomer impurity affects adhesive performance

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Functional monomer impurity affects adhesive performance Kumiko Yoshihara a , Noriyuki Nagaoka b , Takumi Okihara c , Manabu Kuroboshi c , Satoshi Hayakawa d , Yukinori Maruo e , Goro Nishigawa e , Jan De Munck f , Yasuhiro Yoshida g , Bart Van Meerbeek f,∗ a

Center for Innovative Clinical Medicine, Okayama University Hospital, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan b Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan c Division of Chemical and Biological Technology, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan d Biomaterials Laboratory Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan e Department of Occlusion and Removable Prosthodontics, Okayama University, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan f BIOMAT, Department of Oral Health Research, KU Leuven (University of Leuven) & Dentistry, University Hospitals Leuven, Kapucijnenvoer 7 blok a bus 7001, B-3000 Leuven, Belgium g Department of Biomaterials and Bioengineering, Graduate School of Dental Medicine, Hokkaido University, Kita 13, Nishi 7, Kita-ku, Sapporo, Hokkaido 060-8586, Japan

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Article history:

Objective. The functional monomer 10-MDP has been considered as one of the best perform-

Received 30 May 2015

ing functional monomers for dental adhesives. Different adhesives containing 10-MDP are

Received in revised form 9 July 2015

commercially available, among which many so-called ‘universal’ adhesives. We hypothesize

Accepted 25 September 2015

that the quality of the functional monomer 10-MDP in terms of purity may affect bonding performance. Methods. We therefore characterized three different 10-MDP versions (10-MDP KN provided

Keywords:

by Kuraray Noritake; 10-MDP PCM provided by PCM; 10-MDP DMI provided by DMI) using

Functional monomer

NMR, and analyzed their ability to form 10-MDP Ca salts on dentin using XRD. The ‘imme-

NMR

diate’ and ‘aged’ micro-tensile bond strength (␮TBS) to dentin of three experimental 10-MDP

TEM

primers was measured. The resultant interfacial adhesive-dentin ultra-structure was char-

Impurity

acterized using TEM.

Adhesion

Results. NMR disclosed impurities and the presence of 10-MDP dimer in 10-MDP PCM and 10-

Dentin

MDP DMI. 10-MDP PCM and 10-MDP DMI appeared also sensitive to hydrolysis. 10-MDP KN,

∗

Corresponding author. Tel.: +32 16 33 75 87; fax: +32 16 33 27 52. E-mail addresses: [email protected] (K. Yoshihara), [email protected] (N. Nagaoka), [email protected] (T. Okihara), [email protected] (M. Kuroboshi), [email protected] (S. Hayakawa), [email protected] (Y. Maruo), [email protected] (G. Nishigawa), [email protected] (J. De Munck), [email protected] (Y. Yoshida), [email protected] (B. Van Meerbeek). http://dx.doi.org/10.1016/j.dental.2015.09.019 0109-5641/© 2015 Published by Elsevier Ltd on behalf of Academy of Dental Materials. All rights reserved.

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on the contrary, contained less impurities and dimer, and did not undergo hydrolysis. XRD revealed more intense 10-MDP Ca salt deposition on dentin induced by 10-MDP KN. The adhesive based on the experimental 10-MDP KN primer resulted in a significantly higher ‘immediate’ bond strength that remained stable upon aging; the ␮TBS of the experimental 10-MDP PCM and 10-MDP DMI adhesives significantly dropped upon aging. TEM revealed thicker hybridization and more intense nano-layering for 10-MDP KN. Significance. It was concluded that primer impurities and the presence of 10-MDP dimer affected not only hybridization, but also reduced the formation of 10-MDP Ca salts and nano-layering. 10-MDP in a high purity grade is essential to achieve durable bonding. © 2015 Published by Elsevier Ltd on behalf of Academy of Dental Materials. All rights reserved.

1.

Introduction

Phosphate functional monomers are designed to chemically interact with dentinal hydroxyapatite, being thought of importance for bond durability. Among many functional monomers, 10-MDP was most shown to improve bonding effectiveness in laboratory and clinical research [1–6]. In our previous research, 10-MDP’s chemical interaction with hydroxyapatite (HAp) of tooth tissue was proven using diverse chemical analytical tools, such as XRD, XPS and NMR [7–9]. Furthermore, 10-MDP was documented to self-assemble into so-called ‘nano-layering’, a process driven by the deposition of 10-MDP Ca salts with low solubility [8,10–12]. Each nano-layering consists of two sublayers of parallel oriented 10MDP monomers in opposite direction. 10-MDP’s methacrylate group is directed inwards, enabling mutual co-polymerization between two opposed monomers. Its functional phosphate group is directed outwards, capturing Ca released from dentin thanks to the etching effect of 10-MDP and so coupling adjacent nano-layers. Further in-depth research into the process of nano-layering is warranted to assess the relevance and actual contribution of this compact 3D structure to the stability of the adhesive interface. The functional monomer 10-MDP was originally synthesized by Kuraray (today Kuraray Noritake, Tokyo, Japan) and has been introduced in 1981 (following ‘Clearfil SE Bond’ Technical Information from Kuraray Noritake). The actual 10-MDP patent expired in 2011, and has led other companies to develop and launch 10-MDP adhesives, many of them have been referred to as so-called ‘universal’ adhesives, such as Adhese Universal (Ivoclar Vivadent, Schaan, Liechtenstein), All-Bond Universal (Bisco, Schaumburg, IL, USA), Clearfil Universal Bond (Kuraray Noritake), Futurabond U (Voco, Cuxhaven, Germany; although the MSDS document does not specify what the ‘acidic adhesive monomer’ is), G-Premio Bond (GC, Tokyo, Japan) and Scotchbond Universal (3M ESPE, Seefeld, Germany). Besides indicated for direct and indirect restorative procedures, universal adhesives allow the dentist to opt for either an etch-and-rinse (E&R) or self-etch (SE) application protocol. Bonding effectiveness of the different commercial 10-MDPbased adhesives was found to vary [13,14]. Self-evidently, difference in composition is the most plausible reason. Bond strength was shown to depend on the actual concentration of 10-MDP [12]. The interfacial interaction potential of 10-MDP with HAp was found to be inhibited by other monomers like

HEMA [15]. 10-MDP’s performance may also degrade with time, as the monomer is sensitive to hydrolytic degradation [16]. To date, the effect of 10-MDP’s purity on bonding effectiveness is unknown, although several chemical companies (PCM, Krefeld, Germany; DMI, San Diego, CA) commercially offer the functional monomer to be used for dental purposes. Different purities are reported in the accompanied technical documentation. Moreover, patent literature not clearly describes the synthesis process and actual purification process [17,18]. We therefore investigated in this study three different 10-MDP versions, provided by three different companies, on their purity and their chemical interaction potential as well as bonding effectiveness to dentin. The null hypothesis tested was that the adhesive performance of experimental primers varying for the 10-MDP version was not different when they were used as part of a 2-step SE adhesive protocol.

2.

Materials and methods

Three different 10-MDP monomers were selected, as they were provided by DMI (further referred to as ‘10-MDP DMI’), by Kuraray Noritake (referred to as ‘10-MDP KN’), and by PCM (referred to as ‘10-MDP PCM’). The purity grade of 10-MDP DMI was reported by DMI to be 90%, versus 83% reported by PCM for 10-MDP PCM. The purity grade of 10-MDP KN was not released by Kuraray Noritake, but was informed to be higher than that of the other 10-MDP’s tested and to be the same as that of 10-MDP included as functional monomer in the commercial adhesive Clearfil SE Bond (Kuraray Noritake). Three respective experimental primers were prepared to consist of a 15:45:40 wt% solution of 10-MDP/ethanolD6/water-D2 and were stored for 1 month at 37 ◦ C. The pH of the experimental primers was 2.66 for 10-MDP DMI, 2.03 for 10-MDP KN, and 2.73 for 10-MDP PCM.

2.1.

Nuclear magnetic resonance spectrocopy (NMR)

NMR is a research technique that exploits the magnetic properties of atomic nuclei and can provide detailed information about the electronic structure of a molecule. In order to investigate the molecular structure of 10-MDP and potential impurities, we analyzed the three 10-MDP experimental primers using 1 H (with respect to hydrogen), 13 C (with respect to carbon) and 31 P (with respect to phosphorus) NMR spectroscopy. The three experimental primers were disposed

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in 5-mm diameter NMR glass tubes (528PP, Wilmad glass, Vineland, NJ). An NMR spectrometer (JNM-ECS 400, JEOL, Tokyo, Japan) was employed to acquire 1 H NMR spectra with a 30 s relaxation delay between pulses to allow complete relaxation, 31 P NMR and 13 C NMR spectra, both with a 2 s delay between pulses. 1 H NMR and 13 C NMR spectra were referenced externally to neat chloroform-d3 (ı = 7.27 ppm); 31 P NMR spectra were referenced externally to neat 85% aq. phosphoric acid (ı = 0.00 ppm).

2.2.

X-ray diffraction analysis (XRD)

XRD is a tool used for identifying the atomic and molecular structure of crystals or materials with a highly ordered microstructure. Fifteen dentin specimens (10 mm × 8 mm × 1 mm) were cut from fifteen bovine mandibular front teeth (n = 5 for each experimental group), after which the exposed surfaces were ground using SiC paper (#600). The 15:45:40 wt% 10-MDP/ethanol/water monomer solutions were applied on dentin as well as on glass plates 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 being further chemically analyzed using XRD. The surface structures of the dentin specimens and glass plates treated with the experimental monomer solutions were examined by thin-film X-ray diffraction (TF-XRD) using an X˚ RINT2500, Rigaku, Tokyo, ray diffractometer (Cu K␣1 1.5406 A, Japan), operated under 40 kV acceleration and 200 mA current, and a scanning rate of 0.02◦ /s, with the angle of the incident X-ray beam fixed at 1.0◦ .

2.3.

Energy dispersive X-ray spectroscopy (EDS)

EDS is an analytical technique used for elemental analysis. Since crystals precipitated upon mixing 10-MDP DMI with ethanol, the chemical element composition of solely 10MDP DMI was determined using EDS (Noran Voyager III M3100, NORAN Instruments, Middleton, Wisconsin, USA), connected to a scanning electron microscope (SEM; DS-720, Topcon Corp., Tokyo, Japan), after having dissolved 10-MDP DMI in ethanol and the precipitate separated.

2.4.

Micro-tensile bond strength (TBS) to dentin

Three respective experimental primers were prepared to consist of 15 wt% 10-MDP dissolved in 45 wt% ethanol and 40 wt% water, to which 1 wt% camphorquinone (CQ)/ethyl-4 dimethylaminobenzoate (amine) (Sigma–Aldrich, St. Louis, MO) was added to achieve a final concentration of 14.7 wt% 10-MDP, 44.1 wt% ethanol, 39.2 wt% water, 1 wt% CQ and 1 wt% amine. Mid-coronal dentin of fifteen extracted human third molars (n = 5 for each experimental group; the teeth were gathered following informed consent approved by the Commission for Medical Ethics of KU Leuven under the file number S57622) was exposed using a diamond saw (Buehler, Illinois, USA) and next polished with #600 SiC paper. The experimental primer was applied for 20 s followed by gently air-drying, after which the bonding agent of Clearfil SE Bond (Kuraray Noritake) was applied, and subsequently air-thinned

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and light-cured (BluePhase G2, Ivoclar Vivadent). Finally, a micro-hybrid composite (Clearfil AP-X, Kuraray Noritake) was applied to a thickness of 2 mm, after which the specimens were stored for 24 h in water at 37 ◦ C (‘immediate’). The teeth were cut into 1-mm2 stick-shaped micro-specimens with the aid of a 0.3 mm diamond cut-off wheel (Struers, Ballerup, Denmark) mounted in an Accutom-50 cutting machine (Struers, Ballerup, Denmark). Half of the specimens were additionally thermocycled (30 s immersion, alternatively, in a 5 and 55 ◦ C water bath) during 100,000 cycles before being subjected to ␮TBS (‘aged’). The micro-specimens were fixed to a BIOMAT jig [19] with the aid of cyanoacrylate-based glue (Model Repair II Blue, Dentsply-Sankin, Ohtawara, Japan), and stressed at a crosshead speed of 1 mm/min until failure using a universal testing device (LRX, Lloyd Instruments, Hampshire, UK) equipped with a load cell of 100 N. The ␮TBS was expressed in MPa, as derived from dividing the imposed force (N) at the time of fracture by the bond area of the individual specimen (mm2 ). The occurrence of failure prior to actual testing was included in the calculation of the mean ␮TBS as 0 MPa, with an explicit note of the number of pre-testing failures (ptf). The data were statistically evaluated by two-way ANOVA and Tukey HSD test (˛ = 0.05). The statistical analysis was performed using SPSS (IBM, Armonk, NY, USA).

2.5. Interfacial characterization using transmission electron microscopy (TEM) Extracted non-carious human third molars (gathered following informed consent approved by the Commission for Medical Ethics of KU Leuven under the file number S57622) 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 for 20 s with one of the three experimental 15:45:40 wt% 10-MDP/ethanol/water monomer solutions to which 1 wt% CQ/amine was added. On top of the adhesive, the flowable composite (Clearfil Protect Liner F, Kuraray Noritake) was applied and light-cured for 10 s using an Optilux 500 (Demetron/Kerr, Danbury, CT, USA) light-curing unit. 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 [20]. 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).

3.

Results

3.1.

NMR

31 P

NMR of all 10-MDP samples showed a strong singlet peak at 0.4 ppm, which must be assigned to the actual 10-MDP monomer (Fig. 1a). In addition to this peak, a small peak was detected at −14.5 ppm for 10-MDP PCM and 10-MDP DMI, which was assigned to 10-MDP dimer (Fig. 1a).

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Fig. 1 – Chemical characterization of 15 wt% 10-MDP/ethanol solution containing one of the three 10-MDP versions using 31 P NMR in (a), 13 C NMR after 24 h water storage in (b) and after 1 Mo storage in (c), and using 1 H NMR in (d). (a) 31 P NMR of all 10-MDP samples showing a strong peak at 0.4 ppm, which must be assigned to the 10-MDP monomer. In addition to this peak, a small peak was detected at −14.5 ppm for 10-MDP DMI and 10-MDP PCM, which was assigned to 10-MDP dimer. (b) 13 C NMR of 10-MDP PCM revealed a strong (4) and a weak (d) peak around 170 ppm. (c) After 1 Mo water storage, 10-MDP DMI also revealed the weaker ‘d’ peak, representing methacrylic acid and indicating that some hydrolysis of the 10-MDP monomer had occurred. (d) Using 1 H NMR, 10-MDP PCM showed additional peaks around 3.2 ppm. Two pairs of distinct peaks were detected in the range from 3.9 ppm to 4.2 ppm for 10-MDP KN, which were assigned to the CH2 spacer chain of 10-MDP. These peaks were much sharper, finer and intenser than those recorded for the other two 10-MDP’s. The peaks revealed for 10-MDP DMI were also distorted.

13 C NMR analysis after 24 h of 10-MDP PCM revealed two peaks around 170 ppm (Fig. 1b). The small peak refers to the carboxyl group (‘d’) of methacrylic acid produced by hydrolysis of the 10-MDP monomer. This ‘d’ peak was also detected after 1 Mo for 10-MDP PCM, but also for 10-MDP DMI (Fig. 1c); the peak was not detected for 10-MDP KN after 24 h (Fig. 1b), nor after 1 Mo (Fig. 1c). In particular for 10-MDP PCM, several other, yet unidentified peaks were detected at 8.4 and 46.6 ppm, which were not detected for 10-MDP DMI and 10-MDP KN (Fig. 1b and c). Using 1 H NMR, the peak shapes from 3.8 to 4.1 ppm that must be assigned to the CH2 groups of the 10-MDP molecule, were sharper and more intense for 10-MDP KN as compared to those detected for the two other 10-MDP’s (Fig. 1d). 10-MDP DMI showed distorted peak shapes from

3.8 to 4.1 ppm; 10-MDP PCM showed additional peaks around 3.2 ppm (Fig. 1d).

3.2.

XRD

Application of the three 10-MDP’s on dentin (D) resulted in three distinct peaks at 2 = 2.52◦ (d = 3.51 nm), 2 = 4.84◦ (d = 1.82 nm) and 2 = 7.16◦ (d = 1.23 nm); they should be assigned to 10-MDP Ca salts (Fig. 2a). The peak intensities of these three 10-MDP characteristic peak differed in the order of 10-MDP KN D > 10-MDP PCM D = 10-MDP DMI D. 10MDP KN showed an additional peak at 2 = 11.8◦ (d = 0.75 nm), which should be attributed to DCPD (Fig. 2a). When applied on a glass plate, 10-MDP KN GP and 10MDP PCM GP did not show any peaks (Fig. 2b). 10-MDP DMI GP

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Fig. 2 – XRD patterns of 10-MDP applied on dentin (D) in (a) and on a glass plate (GP) in (b), and EDS analysis of the salts of 10-MDP DMI in (c). (a) When the three 10-MDP’s were applied on dentin, three characteristic peaks were detected at 2 = 2.52◦ (d = 3.51 nm), 2 = 4.84◦ (d = 1.82 nm) and 2 = 7.16◦ (d = 1.23 nm); they were assigned to 10-MDP Ca salts. The peak intensity differed in the order of 10-MDP KN D > 10-MDP PCM D = 10-MDP DMI D. 10-MDP KN revealed an additional peak at 2 = 11.8◦ (d = 0.75 nm), which should be attributed to DCPD. (b) When the three 10-MDP’s were applied on a glass plate, only 10-MDP DMI GP revealed the abovementioned three Ca-salt characteristic peaks. (c) EDS analysis of 10-MDP DMI revealed four distinct peaks, representing respectively the elements of carbon, oxygen, sodium, and phosphate and thus indicating that the 10-MDP DMI solution contained 10-MDP sodium salts.

however showed three peaks at 2 = 2.52◦ (d = 3.51 nm), 2 = 4.84◦ (d = 1.82 nm) and 2 = 7.16◦ (d = 1.23 nm).

3.3.

EDS

When the three 10-MDP’s were dissolved in ethanol, only 10MDP DMI formed salts. EDS analysis of the 10-MDP DMI salts revealed that it consisted of carbon, oxide, sodium and phosphate (Fig. 2c).

3.4.

experimental 10-MDP KN primer, an about 1.0-␮m thick and HAp-rich hybrid layer was formed (Fig. 4a). High magnification of the adhesive layer immediately above the hybrid layer clearly revealed intense nano-layering (Fig. 4b and c). The hybrid layer produced by the 10-MDP PCM and 10-MDP DMI versions was about 0.5-␮m thick and also rich in HAp (Fig. 4d and g). High magnification of the adhesive layer again revealed nano-layering (Fig. 4e, f, h, and i), although less intense than that observed for 10-MDP KN.

TBS

4. The 10-MDP version as well as the aging imposed by longterm thermo-cycling was found to have affected the ␮TBS (two-way ANOVA, both p < 0.0001). Not all 10-MDP did however degrade in a similar way as the interaction between the two variables (10-MDP and aging) was found to be significant as well (p = 0.0155). The ‘immediate’ ␮TBS to dentin of the 2-step SE adhesive, including the experimental 10-MDP KN primer, was significantly higher than that of the 10-MDP PCM and 10-MDP DMI versions. The ␮TBS of 10-MDP KN did not significantly decrease after 100,000 thermocycles. On the contrary, the ‘aged’ ␮TBS of 10-MDP PCM and 10-MDP DMI significantly decreased after long-term thermo-cycling (Fig. 3). Also, while no pre-testing failures (ptf’s) were recorded for 10-MDP KN, respectively 5 ptf’s (out of 22 micro-specimens) and 7 ptf’s (out of 21 micro-specimens) were recorded for the ‘aged’ 10MDP PCM and 10-MDP DMI specimens.

3.5.

TEM

TEM releaved tight interfaces at dentin for all experimental 10-MDP adhesives. For the 2-step SE adhesive, including the

Discussion

Functional monomers as part of self-etch adhesives are designed to interact with HAp using the functional group at one end, a phosphate group in case of 10-MDP, and at the same time to co-polymerize through the methacrylate group at the other end with the other resin monomers [21]. The spacer of 10-MDP keeps the two functional groups separated by a relatively long hydrophobic chain. The functional monomer in ‘mild’ SE adhesives (self-)etches HAp of dentin, releasing Ca, in an equilibrium with ionic interaction with Ca of HAp, thereby forming 10-MDP Ca bonds. Upon sufficient release of Ca from dentin, the 10-MDP monomers self-assemble into nano-layers [11]. Each nano-layer consists of two rows of parallel oriented 10-MDP monomers, with the monomers in each row directed in opposite direction. The methacrylate ends of two opposed 10-MDP monomers can co-polymerize, while the released Ca can bridge/link two adjacent nano-layers by Ca–P salt formation. The obtained compact structure is thought to contribute to the bond stability, while this assumption needs to be confirmed and potential measures to intensify nanolayering should be explored.

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Fig. 3 – Graph presenting the micro-tensile bond strength (␮TBS) of the experimental adhesives with the SE primers based on the three respective 10-MDP functional monomers, this after 24 h water storage (‘immediate’ ␮TBS) and after long-term 100,000 times thermo-cycling (‘aged’ ␮TBS). The numbers above each box express the mean ␮TBS (in MPa) with the corresponding standard deviation (mean ± SD). Underneath between parenthesis, the number of pre-testing failures (ptf’s) per total number of micro-specimens tested is mentioned. Different small letters underneath refer to statistically significant differences between the experimental groups. 10-MDP KN revealed a significantly higher bond strength than 10-MDP PCM and 10-MDP DMI. The ␮TBS of 10-MDP KN did not decrease upon aging, while the ␮TBS measured for 10-MDP PCM and 10-MDP DMI significantly decreased after long-term thermo-cycling. The boxes represent the spreading of the data between the first and third quartile. The closed dot and the horizontal line in each box represent the mean and median, respectively. The whiskers denote the range of variance; outliers are indicated by the open dot.

In this study, we investigated the effect of the quality or more specifically the purity of 10-MDP as one of the best performing functional monomers for a SE adhesive routine. Two commercially available 10-MDP’s (10-MDP PCM, 10-MDP DMI) and one 10-MDP version (10-MDP KN) provided by Kuraray Noritake and used in the different Clearfil adhesive generations were tested. The experimental adhesive containing the latter 10-MDP KN monomer revealed the significantly highest ‘immediate’ and ‘aged’ ␮TBS. Moreover, aging using longterm thermo-cycling did not significantly reduce its ␮TBS, as opposed to the significant decrease in ␮TBS upon aging for 10MDP PCM and 10-MDP DMI. Hence, the null hypothesis that the three 10-MDP versions did not differ in bonding performance was rejected. In order to identify the reasons for this difference in ‘immediate’ and ‘aged’ bond strength, the three 10-MDP versions were chemically characterized using NMR. 31 P NMR revealed that both 10-MDP PCM and 10-MDP DMI contain 10MDP dimer. Steric hindrance of the two (remaining) OH groups of the dimer can be expected to result in a lower flexibility and thus lower chemical reactivity of the dimer with HAp than the single 10-MDP monomer. This is supported by TEM that revealed a thicker hybrid layer with abundant HAp for the experimental 10-MDP KN adhesive than for the other two 10-MDP versions. This higher etching efficacy of 10-MDP KN may also explain its higher immediate bond strength as compared to that of the other 10-MDP versions. The difference in etching capacity also appeared from the pH of the prepared experimental 10-MDP primers, being most acidic with a pH of 2.03 for 10-MDP KN, a pH of 2.66 for 10-MDP DMI and 2.73 for 10-MDP PCM.

As mentioned above, previous research demonstrated that upon interaction of 10-MDP with Ca of HAp, 10MDP ionically bonded to HAp and even self-assembled in nano-layering. XRD confirmed 10-MDP Ca salt formation and nano-layering for all three 10-MDP versions upon 20-s interaction with dentin. Although in principle XRD cannot be considered quantitative in terms of peak intensity, our previous studies revealed a clear correlation between the intensity of the three nano-layering characteristic XRD peaks and amount of nano-layering [11]. Much more intense nanolayering was produced by 10-MDP KN than by the other 10-MDP monomers. High-magnification TEM corroborated the XRD data. Ultra-structurally, more intense nano-layering was observed with 10-MDP KN, clearly extending from the hybrid layer into the adhesive resin. Although nanolayering was also detected using the experimental adhesives containing 10-MDP PCM and 10-MDP DMI, the amount of nano-layering was clearly much smaller. Also this difference in nano-layering can be explained by the existence of dimers, sterically hindering the formation of nanolayering. Remarkable was the detection by XRD of the three nano-layering characteristic peaks when the experimental 10-MDP DMI primer was applied on a (Ca-free) glass plate. EDS confirmed the presence of 10-MDP Na salts. When 10MDP DMI was dissolved in ethanol, the 10-MDP Na salts remained. In water, the salts must have dissolved. These sodium ions might represent impurities that consume 10MDP, by which it is less able to interact with Ca of HAp. This sodium in 10-MDP DMI may have contributed to the lower ‘immediate’ bond strength.

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Fig. 4 – TEM photomicrographs illustrating the adhesive–dentin interface produced by the experimental 10-MDP KN primer in (a–c), the experimental 10-MDP PCM primer in (d–f) and the 10-MDP DMI primer in (g–i), when they were applied on dentin for 20 s and followed by the application of a low-viscosity resin. All experimental adhesives revealed tight interfaces with dentin (no de-bonding). TEM of 10-MDP KN revealed a 1.0-␮m thick and HAp-rich hybrid layer (a). High magnification of the adhesive layer clearly revealed intense nano-layering (b and c). TEM of 10-MDP PCM and 10-MDP DMI revealed a 0.5-␮m thick and HAp-rich hybrid layer (d and g). High magnification of the adhesive layer clearly revealed nano-layering (e, f, h, and i) but in a lower degree than that observed for 10-MDP KN. PL: primer layer; D: dentin; HL: hybrid layer; LVR: low-viscosity (composite) resin; NL: nano-layering.

1H

NMR revealed distinct, sharp and fine peaks at around 4.0 ppm for 10-MDP KN, which should be assigned to the 10 CH2 spacer chain. The peak sharpness and intensity are a clear indication of high purity. On the contrary, broader and less intense peaks at around 4.0 ppm were detected for 10-MDP PCM; even distorted peaks at around 4.0 ppm were detected for 10-MDP DMI. 10-MDP PCM presented with

several peaks at 1.3, 1.7 and around 3.2 ppm. The latter peaks might also represent the CH2 chain, which may have become shorter by hydrolysis or may represent monomers with a shorter CH2 chain that remained from the synthesis process. All these additional peaks indicate that both 10-MDP DMI and 10-MDP PCM contain impurities. In addition, the peaks at 10 and 45 ppm disclosed by 31 C NMR must also be assigned to

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impurities. As mentioned before, the purity grade of 10-MDP released by the manufacturer was 83% for 10-MDP PCM and 90% for 10-MDP DMI. The purity of 10-MDP KN was not disclosed, but thought to be higher than that of the other two 10-MDP’s (confirmed by personal communication with Kuraray Noritake). Most impurities could not be identified; they may represent residual solvents and/or by-products. The bond strength must have been affected by the impurities, because the existence of impurities reduced the concentration of pure 10-MDP and some impurities may even have inhibited bonding directly. 31 C NMR revealed a peak at 170 ppm for 10-MDP PCM. This peak was assigned to methacrylic acid, indicating that some 10-MDP may have been hydrolysed [22]. 10-MDP DMI also showed this peak after 1 Mo storage. When 10-MDP undergoes hydrolysis, methacrylic acid and 10-hydroxydecyl dihydrogen phosphate are formed [23]. 10-Hydroxydecyl dihydrogen phosphate can still react with HAp, but cannot polymerize and thus prevents other 10-MDP monomers from interaction, thereby not contributing to the bond strength. Aida et al. [22] reported degradation of a HEMA and 10-MDP containing self-etch primer, as revealed by 13 C NMR. HEMA appeared more sensitive for hydrolysis than 10-MDP; HEMA’s degradation strongly affected bond durability [22]. On the other hand, in our study, no signs of hydrolysis were observed for 10-MDP KN. The sensitivity of the monomer for hydrolysis depends on the monomer structure and the solvent. 10-MDP PCM and 10-MDP DMI may contain residual solvent and/or by-products that may promote hydrolysis. Very likely, the degradation of 10-MDP by hydrolysis recorded in this study for 10-MDP PCM and 10-MDP DMI must have resulted in the significantly decreased bond strength upon long-term thermo-cycling. It is concluded that the three 10-MDP’s studied in this study clearly revealed a different purity. Differences in the ultrastructure of the resultant hybrid layers were observed for the three 10-MDP versions. Both the impurities and the presence of dimers affected the etching efficacy of HAp, the intensity of nano-layering and the ‘immediate’ bond strength. Furthermore, these impurities may have promoted monomer hydrolysis and so have affected bond durability as well. Hence, the purity of 10-MDP present in commercial dental primers, adhesives and cements can be expected to influence bonding performance. Studies investigating the purity of monomers in commercial dental adhesive materials are needed.

Conflict of interest The authors declare no conflict of interest.

Acknowledgements The current research was supported by a Grant-in-Aid for Research Activity Start-up (26893156). We thank the respective manufacturers, Kuraray Noritake (Tokyo, Japan), PCM (Krefeld, Germany) and Designer Molecule (DMI, San Diego, CA) for providing the different 10-MDP monomers.

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