Synthesis and characterization of a novel resin monomer with low viscosity

Graphical abstract

Abstract

Objective

In this study, we designed and synthesized a novel macromolecule (tetramethyl bisphenol F acrylate, TMBPF-Ac) with low viscosity, excellent mechanical properties, and good biocompatibility. It could be used as a monomer for dental resin composites, which could reduce the risk of human exposure to bisphenol A derivatives in the oral environment. In addition, the monomer could be used without diluent, thereby avoiding the negative effect of a diluent

Methods

TMBPF-Ac was synthesized by a multistep condensation reaction. Its structure was confirmed by 1 H NMR spectra. Different resin mixtures were prepared, and then a number of performance and cytotoxicity tests were performed on these specimens.

Results

1 H NMR spectra showed that the structure of TMBPF-Ac was in accordance with the design. The viscosity of TMBPF-Ac was obviously lower than that of bisphenol-A diglycidyl methacrylate. The three kinds of resins used in this study were in line with ISO 4049:2009 and ISO 10993-5:2009. TMBPF-Ac-based resin had better physical and biological properties.

Introduction

Dental resin composites, as a kind of direct and indirect bonding repair material for dental restoration, play an indispensable role in clinical work. Their advantages of terrific aesthetic effect, simple operation procedure, excellent user experience, and high biological safety made them widely used in various oral clinical areas such as filling material, binder, indirect prosthetic restoration, overcoating of metal restoration, and post core restoration. Nowadays, commercial resin composites usually consist of dimethacrylate-based resin matrix, photo-initiator system, and coupling agent-treated inorganic fillers. Bisphenol-A diglycidyl methacrylate (bis-GMA), which is also known as Bowen’s resin, was first synthesized by Bowen in 1965 and used as a monomer for resin composite. From then on, Bowen’s resin has been widely used in clinical work. At present, more than 70% of the commercial resin composites use bis-GMA as monomer. Bis-GMA (Mw = 512 g mol −1 ) has high viscosity (η = 600–1000 Pa·s) , which increases the difficulty of practical application. Therefore, the use of diluent co-monomers became necessary to allow the introduction of inorganic fillers.

The routine co-monomer used was triethylene glycol dimethacrylate (TEGDMA). The low molecular weight (Mw = 286 g mol −1 ) and low viscosity (η = 0.05 Pa·s) of TEGDMA reduced the viscosity of the mixture and significantly increased the degree of conversion of polymer (DC) . However, the introduction of TEGDMA had clinically undesirable effects on the increase of polymerization shrinkage and water absorption and the decrease of mechanical properties. TEGDMA is a cytotoxic monomer, and a certain amount of residual unreacted monomer will release freely from the resin-based materials after the polymerization reaction, resulting in contamination of the pulp cells . Even a sublethal dose of TEGDMA can damage the function of the cells considerably, changing their dynamic balance, tissue repair, and the modulation pathway of mineralization. Some reports have also demonstrated that TEGDMA could inhibit the mineralization of dental pulp cells and the formation of the reparative dentin . For these reasons, to reduce the application of TEGDMA, some monomers with low viscosity and high molecular weight replacing TEGDMA partially or totally were presented in several commercial formulations such as ethoxylated bisphenol-A-dimethacrylate (bis-EMA, Mw = 540 gmol −1 , η = 3.00 Pa·s) .

Recently, people’s knowledge about environmental hormones, which are commonly known as endocrine disrupting chemicals, has gradually deepened. Environmental hormones are chemical substances that interfere with incretion in organisms, and after entering the body, they can combine with hormone acceptors or affect the quantity of endogenous hormones; interfere with hormone synthesis, function, and removal; and cause endocrine disorders. Their mechanism is similar to that of estrogen . In daily life, the most likely environmental hormone to be contacted by humans is bisphenol A (BPA), which could induce body feminization, sperm count decrease, genital dysplasia, and body function abnormality . Currently acknowledged resin monomers are bis-GMA and bis-EMA, which are synthesized from BPA. In addition, BPA and its derivatives can be released from the resin composites and be detected . Although the quantity of BPA and its derivatives is in trace amounts and is safe to the human body, they still have the risk of being absorbed into the blood and accumulating in the body . To reduce human exposure to BPA, designing and synthesizing a novel structure as an alternative resin monomer for composite materials without BPA is necessary.

In this study, a novel monomer tetramethyl bisphenol F acrylate (TMBPF-Ac) was designed and synthesized. The aim of the study was to compare the comprehensive properties and cytotoxicity of TMBPF-Ac-based resins with those of bis-GMA-based resins. TMBPF-Ac-based resins were expected to present excellent properties: lower viscosity, low polymerization shrinkage, high degree of double bond conversion, high mechanical properties, and good biocompatibility. TMBPF-Ac resins without TEGDMA still exhibited prominent properties. They could not only reduce exposure to BPA but also avoid the negative impact from diluents. The novel resin monomer could conform to the fundamental requirements for applications as resin matrix for composite materials.

Materials and methods

Materials

TMBPF epoxy resin (TMBPF-ER) monomer was prepared as described in a previous work . 1,3-bis [2(3,4-epoxycyclohex-1-yl) ethyl] tetra-methyldisiloxane (Siepoxy) was purchased from Fluorochem Ltd. (UK). Bis-GMA (99%), TEGDMA (98%), 2-(N, N-dimethylamino) ethyl-methacrylate (DMAEMA; 99%), and triphenyl-phosphine (99%) were purchased from Shanghai No. 1 Chemical Reagent Co. Ltd. (China). Phenol (99%) and hydroquinone (99%) were obtained from Shantou Xilong Chemical Factory Co. Ltd. (China). Camphorquinone (CQ; 98%) was obtained from Alfa Aesar Co. (USA). Ethylene oxide (99.9%) was purchased from Sigma-Aldrich (USA). Acrylic acid (75%) was purchased from Tianjin Guangfu Fine Chemical Industry Research Institute (China). Tetrabutyl ammonium bromide (99%) was obtained from Zhenjiang Jingrun high purity chemical Reagent Co. Ltd (China). Epichlorohydrin (AR) and toluene (AR) were purchased from Beijing Chemical Reagent Co. Ltd. (China). Fetal bovine serum was obtained from Tianjin Haoyang Biological Technology Co. Ltd. (China). Trypsin and Dulbecco’s modified Eagle medium (DMEM) were obtained from Gibco (Grand Island, NY, USA).

Synthesis of monomer TMBPF-Ac

In a 100-mL three-necked flask equipped with a mechanical stirrer, nitrogen inlet, and condenser, 3.680 g (10.00 mmol) of TMBPF-ER, 0.050 g (0.19 mmol) of triphenyl-phosphine, and 0.005 g (0.05 mmol) of hydroquinone were added. The system was heated to 70 °C, and then 1.40 mL (20.40 mmol) of acrylic acid was added. The mixture was heated to 105 °C and kept at the same temperature for 12 h. The reaction was terminated by adding 60 mL of ethyl acetate, and the mixture was then transferred into a separating funnel; washed with 1 M HCl thrice, 1 M NaOH thrice, and deionized water thrice; and dried with an appropriate amount of anhydrous MgSO 4 for 30 min. The mixture was filtered and distilled under reduced pressure. The resulting faint yellow viscous liquid was dried under vacuum at 25 °C for 24 h to remove any solvents, thus producing TMBPF-Ac.

Preparation of resin mixture

Taking TMBPF-Ac/TEGDMA resin as example, the typical preparation procedure of the resin curing mixture was as follows. TMBPF-Ac/TEGDMA = 7:3 (w/w) was mixed with 1.0 wt.% CQ and 1.0 wt.% DMAEMA under mechanical stirring. The preparation of bis-GMA/TEGDMA and TMBPF-Ac resins without TEGDMA was similar to that of TMBPF-Ac/TEGDMA resin.

Measurements

The 1 H NMR spectra

The 1 H NMR spectra were recorded on a Bruker 510 instrument using DMSO-d 6 as the solvent and tetramethyl saline as the reference.

The viscosity

The viscosity of each resin (n = 3) was measured in pascal seconds (Pa·s) using a rheometer (AR2000, TA Instruments Co.; USA) (cone-and-plate geometry: 40 mm, 2° cone; truncation gap: 61) . For each test run, a unidirectional steady shear rate sweep was performed from 0.1 to 1000 reciprocal seconds with 10 points per decade. Each sample was tested in triplicate. Sample temperature was maintained at 25 °C using the Peltier plate on the rheometer.

Water contact angle

Disc-shaped specimens (d = 15.0 mm, h = 1.0 mm, n = 5) were prepared for each group, which were light cured for 40 s on both the top and bottom surfaces, stored at 37 ± 1 °C for 24 h, polished with 500-grit silica carbide paper, and sonicated for 5 min in distilled water. The specimens were then tested according to the ISO 4049:2009 . The measurement of water contact angle (WSA) was performed with a Drop Shape Analysis System DSA10-MK2 (Kruess, Germany) at ambient temperature with a 1-μL double-distilled water droplet as the indicator. The water contact angle was measured for 10 s after drop placement. Three positions for each specimen were measured and the average was obtained.

Double bond conversion

The degree of double bond conversion (DC) was determined by FT-IR spectrometer (VERTEX70, Brooke Inc.; GER) with an attenuated total reflectance accessory. Small amount of each specimen was coated on KBr sheet, and the sheet was then put into a sample pool. First, the sample was scanned without being irradiated. Then, the sample was irradiated for 60 s with a visible light-curing unit (MiNi LED F02616 = 420–480 nm, I ≈ 1250 mW cm −2 , SATELEC. Inc. FRN.). To determine the percentage of reacted double bonds, the absorbance intensities of the methacrylate double bonds (C C), with absorbance peak at 1636 cm −1 , were decreased after being irradiated and those of the carbonyl (C O), with peak at 1720 cm −1 , were used as the standard. The ratios of the absorbance intensities were calculated before and after polymerization . The DC was calculated using Eq. (1) :

D C = [ 1 − ( A C = C / A C = O ) 0 ( A C = O / A C = O ) t ] × 100 %

where A C C and A C O are the absorbance intensities of methacrylate double bonds (C C) at 1636 cm −1 and carbonyl (C O) at 1720 cm −1 , respectively, and ( A C C / A C O ) 0 and ( A C C / A C O ) t are the normalized absorbance intensities of the functional groups before and after being irradiated, respectively.

Polymerization shrinkage

The densities of the specimens were measured to determine the polymerization shrinkage according to the Archimedes’ principle and ISO 17304:2013 . The method determines the densities of the unpolymerized and polymerized samples by measuring their weights in air and water at room temperature. Analytical balance (AG 204 Mettler Toledo) equipped with a density kit was used. First, the mass of the holder was weighed in air and water, and the density of the holder (D h ) was calculated as shown in Eq. (2) :

D h = M h a ( M h a − M h w ) × D w

where M ha is the mass of the holder in air, M hw is the mass of the holder in water; D w is the density of water at the exactly measured temperature.

Second, the entire mass of the specimen and the holder were weighed in air and water, and the density was directly calculated according to Eq. (3) :

D s = ( M a − M h a ) [ M a − M w − ( M h a × D w / D h ) ] × D w

where D s is the density of the sample, M a is the mass of the sample and holder in air, M w is the mass of the sample and holder in water, D h is the density of the holder.

Finally, the percentage of volume shrinkage ( VS ) after polymerization was calculated from the densities according to Eq. (4) :

V S = ( D t − D 0 ) D t × 100 %

where D 0 and D t are the densities of the sample before and after curing, respectively.

Water sorption and solubility

Disc-shaped specimens (d = 15.0 mm, h = 1.0 mm, n = 5) were prepared for each group according to ISO 4049:2009 . These specimens were kept in one of the two desiccators maintained at 37 ± 2 °C for 22 h. The specimens were then transferred into another desiccator maintained at 23 ± 1 °C for 2 h and weighed to an accuracy of 0.1 mg. M 1 was obtained when the mass loss of each specimen was not more than 0.1 mg in any 24-h period. After the final drying, the diameter of each specimen was measured to an accuracy of 0.01 mm, and the mean diameter was calculated. The thickness of the specimens was measured to an accuracy of 0.01 mm at the center of the specimen and at four equally spaced points on the circumference. The volume ( V ; mm 3 ) of the specimens was calculated from the averages of the diameter and the thickness. Each specimen was immersed in 15 mL of deionized water at 37 ± 1 °C for 7 days and then washed with deionized water. The surface water was blotted away until the specimens were free from visible moisture. Finally, the specimens were waved in the air for 15 s and weighed after 60 s to obtain the water sorption mass ( M 2 ). Then the specimens were kept in desiccators as described above until a constant mass ( M 3 ) was obtained. The water sorption value ( W SP ) and the water solubility value ( W SL ) were calculated as

W S P = ( W 2 − W 3 ) V
W S L = ( M 1 − M 2 ) V

Flexural strength, flexural modulus, indentation hardness, and indentation modulus

Bar-shaped specimens (a = 25.0 mm, b = 2.0 mm, h = 2.0 mm, n = 8) were prepared for each group according to ISO 4049:2009 . Each specimen was light cured for 40 s in four overlapping irradiation zones, on both the top and bottom surfaces. The specimens were then wet finished with 500-grit silica carbide paper and stored in distilled water at 37 °C for 24 h. Three-point bending test (span 20.0 mm) of the specimens was performed using a material testing machine (AG-X plus, SHIMADZU Corporation; Japan) with a cross-head speed of 1.0 mm min −1 . Flexural strength ( FS ; MPa) and flexural modulus ( FM ; GPa) were calculated from the data obtained from the initial linear portion of the load–displacement curve according to the following equations:

F S = 3 F L 2 b h 2
F M = F L 3 × 1000 − 3 4 b h 3 d

where F is the load (N) corresponding to the displacement of the cross-head, L (mm) is the distance between the supports, b (mm) is the width of the specimen, and h (mm) is the height of the specimen.

Disc-shaped specimens (d = 5.0 mm, h = 1.0 mm, n = 3) were prepared for each group as described earlier. These specimens were subjected to indentation hardness ( H IT ) and indentation modulus ( E IT ) tests with Nano Indenter G200 (TriboIndenter, Hysitron Inc, Minneapolis, MN; USA). Each specimen was sandwiched between Mylar strips and glass slides to ensure consistent thickness and smooth surfaces and to avoid air inhibition. Loss tangent was obtained by applying a 500-μN static load using a Berkovich indenter, followed by a 25-μN dynamic loading at 10 Hz with an amplitude of 1.5 nm. Twenty indentations were made on each specimen. The test started 10 min after photoactivation (500 Mw cm −2 × 32 s) and required 30 min to run. Because we found no difference in the loss tangent values as a function of time, we used the mean value of the 20 indentations to represent the loss tangent value of the specimens. H IT and E IT were calculated as follows:

H I T = P max A ( h c ( max ) )
E I T = ( 1 − V s 2 ) [ 2 S A π − ( 1 − V i 2 ) E i ] − 1

where P max is the maximum load (μN), h c ( max) is the maximum contact depth (nm), A is the contact area (nm 2 ), ν is the Poisson’s ratio, S is the elastic contact toughness, and the subscripts s and i correspond to the sample and the indenter, respectively.

Cytotoxicity

The cytotoxicity of the resins prepared in this study was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay following ISO 10993-5:2009 . The MTT assay reduces the yellow tetrazolium bromide to a purple formazan product with mitochondrial dehydrogenases in active cells. Disc-shaped specimens (d = 5.0 mm, h = 1.0 mm, n = 5) were prepared for each group as previously described. The specimens were first sterilized using ethylene oxide (10.5 psi, 54.4 °C, 12 h) and were then individually immersed in 12.0 mL of DMEM supplemented with 10% FBS (Hyclone, Logan, UT; USA) at 37 °C in incubators (Thermo Fisher Scientific; USA) for 24 h. The obtained extracts were prepared at 100.0% 50.0% concentrations . L929 mouse fibroblasts (Chinese Academy of Sciences cell library cell bank of Chinese Academy of Sciences) were cultured in DMEM supplemented with 10% FBS and then were seeded at the cell density of 3 × 10 3 /well in 96-well tissue culture microtiter plates (Corning; USA) with 100 mL of DMEM. After 4 h of incubation (37 °C; > 90% humidity; 5% CO 2 /air), the medium was removed and replaced with 100 μL of the previously prepared extracts into each well in quadruplicate . Control columns of four wells were prepared with a medium without cells (blank) and a medium with cells but without the extract (100% survival; negative control). Moreover, 0.3 vol% phenol solutions were used as positive control. Following incubation for 72 h, 20 μL of CellTiter 96 AQueous One Solution Reagent (Promega Corporation, Madison, WI; USA) was added to each well. After 4 h of incubation, 100 μL of solubilization/stop mix was added to each sample and allowed to react for 2 h (37 °C; > 90% humidity; 5% CO 2 /air). The morphology and growth of the cells were observed under an inverted microscope (4000 B DMI, Microsystems Leica; GER) at 24, 72, and 120 h. The incorporated dye was measured by reading the absorbance at 490 nm in a microplate reader (Elx800, BioTek instruments, Inc; USA) against a blank column . The relative growth rate ( RGR ) of the cells was calculated according to Eq. (11) :

R G R % = ( O D e − O D b ) ( O D n − O D b ) × 100 %

where OD e is the OD of the experimental group, OD n is the OD of the negative control group, and OD b is the OD of the blank. The cytotoxicity grade (CG) of each group was presented as follows: RGR ≥100%, CG = 0; 99% ≥RGR ≥ 75%, CG = 1; 74% ≥RGR ≥ 50%, CG = 2; 49% ≥RGR ≥25%, CG = 3; 24% ≥ RGR ≥1%, CG = 4. RGR = 0, CG = 5.

Statistical analysis

All the results were statistically analyzed using analysis of variance (ANOVA) at the p < 0.05 significance level. Subsequent multiple comparisons were made using the Duncan’s multiple range test for significant differences between multiple sets of sample analysis.

Materials and methods

Materials

TMBPF epoxy resin (TMBPF-ER) monomer was prepared as described in a previous work . 1,3-bis [2(3,4-epoxycyclohex-1-yl) ethyl] tetra-methyldisiloxane (Siepoxy) was purchased from Fluorochem Ltd. (UK). Bis-GMA (99%), TEGDMA (98%), 2-(N, N-dimethylamino) ethyl-methacrylate (DMAEMA; 99%), and triphenyl-phosphine (99%) were purchased from Shanghai No. 1 Chemical Reagent Co. Ltd. (China). Phenol (99%) and hydroquinone (99%) were obtained from Shantou Xilong Chemical Factory Co. Ltd. (China). Camphorquinone (CQ; 98%) was obtained from Alfa Aesar Co. (USA). Ethylene oxide (99.9%) was purchased from Sigma-Aldrich (USA). Acrylic acid (75%) was purchased from Tianjin Guangfu Fine Chemical Industry Research Institute (China). Tetrabutyl ammonium bromide (99%) was obtained from Zhenjiang Jingrun high purity chemical Reagent Co. Ltd (China). Epichlorohydrin (AR) and toluene (AR) were purchased from Beijing Chemical Reagent Co. Ltd. (China). Fetal bovine serum was obtained from Tianjin Haoyang Biological Technology Co. Ltd. (China). Trypsin and Dulbecco’s modified Eagle medium (DMEM) were obtained from Gibco (Grand Island, NY, USA).

Synthesis of monomer TMBPF-Ac

In a 100-mL three-necked flask equipped with a mechanical stirrer, nitrogen inlet, and condenser, 3.680 g (10.00 mmol) of TMBPF-ER, 0.050 g (0.19 mmol) of triphenyl-phosphine, and 0.005 g (0.05 mmol) of hydroquinone were added. The system was heated to 70 °C, and then 1.40 mL (20.40 mmol) of acrylic acid was added. The mixture was heated to 105 °C and kept at the same temperature for 12 h. The reaction was terminated by adding 60 mL of ethyl acetate, and the mixture was then transferred into a separating funnel; washed with 1 M HCl thrice, 1 M NaOH thrice, and deionized water thrice; and dried with an appropriate amount of anhydrous MgSO 4 for 30 min. The mixture was filtered and distilled under reduced pressure. The resulting faint yellow viscous liquid was dried under vacuum at 25 °C for 24 h to remove any solvents, thus producing TMBPF-Ac.

Preparation of resin mixture

Taking TMBPF-Ac/TEGDMA resin as example, the typical preparation procedure of the resin curing mixture was as follows. TMBPF-Ac/TEGDMA = 7:3 (w/w) was mixed with 1.0 wt.% CQ and 1.0 wt.% DMAEMA under mechanical stirring. The preparation of bis-GMA/TEGDMA and TMBPF-Ac resins without TEGDMA was similar to that of TMBPF-Ac/TEGDMA resin.

Measurements

The 1 H NMR spectra

The 1 H NMR spectra were recorded on a Bruker 510 instrument using DMSO-d 6 as the solvent and tetramethyl saline as the reference.

The viscosity

The viscosity of each resin (n = 3) was measured in pascal seconds (Pa·s) using a rheometer (AR2000, TA Instruments Co.; USA) (cone-and-plate geometry: 40 mm, 2° cone; truncation gap: 61) . For each test run, a unidirectional steady shear rate sweep was performed from 0.1 to 1000 reciprocal seconds with 10 points per decade. Each sample was tested in triplicate. Sample temperature was maintained at 25 °C using the Peltier plate on the rheometer.

Water contact angle

Disc-shaped specimens (d = 15.0 mm, h = 1.0 mm, n = 5) were prepared for each group, which were light cured for 40 s on both the top and bottom surfaces, stored at 37 ± 1 °C for 24 h, polished with 500-grit silica carbide paper, and sonicated for 5 min in distilled water. The specimens were then tested according to the ISO 4049:2009 . The measurement of water contact angle (WSA) was performed with a Drop Shape Analysis System DSA10-MK2 (Kruess, Germany) at ambient temperature with a 1-μL double-distilled water droplet as the indicator. The water contact angle was measured for 10 s after drop placement. Three positions for each specimen were measured and the average was obtained.

Double bond conversion

The degree of double bond conversion (DC) was determined by FT-IR spectrometer (VERTEX70, Brooke Inc.; GER) with an attenuated total reflectance accessory. Small amount of each specimen was coated on KBr sheet, and the sheet was then put into a sample pool. First, the sample was scanned without being irradiated. Then, the sample was irradiated for 60 s with a visible light-curing unit (MiNi LED F02616 = 420–480 nm, I ≈ 1250 mW cm −2 , SATELEC. Inc. FRN.). To determine the percentage of reacted double bonds, the absorbance intensities of the methacrylate double bonds (C C), with absorbance peak at 1636 cm −1 , were decreased after being irradiated and those of the carbonyl (C O), with peak at 1720 cm −1 , were used as the standard. The ratios of the absorbance intensities were calculated before and after polymerization . The DC was calculated using Eq. (1) :

D C = [ 1 − ( A C = C / A C = O ) 0 ( A C = O / A C = O ) t ] × 100 %
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Jun 19, 2018 | Posted by in General Dentistry | Comments Off on Synthesis and characterization of a novel resin monomer with low viscosity
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