Bactericidal dental nanocomposites containing 1,2,3-triazolium-functionalized POSS additive prepared through thiol-ene click polymerization

Graphical abstract

Highlights

  • Use of azide-alkyne click reaction to generate triazolium functional POSS.

  • Use of thiol-ene/methacrylate polymerization to prepare dental materials with low shrinkage strain.

  • Lower deterioration of mechanical strength under wet condition by proper selection of biocidal additive.

  • Preparation of dental materials with improved bactericidal activity against Streptococcus mutans .

Abstract

Objective

Deterioration of mechanical strength for the dental composite containing ionic bactericidal compounds restricts the widespread utilization of this class of useful materials. This problem is originated from the reduction of the intermolecular interaction of polymeric network due to plasticization effect of absorbed water molecules penetrated between the chain segments. The main goal of this study is the synthesis of the highly efficient bactericidal additive with low hydrophilicity and consequently the least adverse effect on the final mechanical strength of the dental composite.

Methods

The bactericidal 1, 2, 3-triazolium functional groups were chemically anchored on the surface of hydrophobic POSS nanoparticles (Triazolium-POSS) and incorporated into a dental restorative system composed of a ternary thiol-allyl ether-methacrylate resin and glass fillers. A similar system was also prepared, in which the POSS additive was replaced with quaternized dimethyl aminoethyl methacrylate monomer (DMAEMA-BC). The chemical structure of POSS derivatives was evaluated by 1 HNMR and FTIR spectra. The water uptake of dental composites was evaluated at days 1 and 14 after immersion into water. The bactericidal activity of composite specimens against Streptococcus mutans (ATCC 35668) was determined based on ASTM E 2180 – 07. The flexural properties of samples were investigated through three-point bending assay and the shrinkage-strain of photo-cured resins was measured using the bonded-disk technique. The degree of conversion (DC %) of methacrylate functions was followed by FTIR spectroscopy. MTT assay was performed to investigate the cytocompatibility of samples.

Results

Regardless of the partial increase in water uptake for Triazolium-POSS-containing sample, this parameter was much favor than the composite made from DMAEMA-BC. Therefore, the lower decline in flexural properties was recorded under the wet condition for the former system. Incorporation of Triazolium-POSS had no significant effect on shrinkage strain and cytocompatibility of composite specimen, meanwhile, a higher degree of conversion of methacrylate functional groups was recorded. The Triazolium-POSS-containing nano composite showed significantly higher bactericidal activity against Streptococcus mutans than another studied model system.

Significance

The new derivative of bactericidal POSS nanoparticles decorated with 1, 2, 3-Triazolium moieties is a highly efficient bactericidal compound. If Triazolium-POSS is incorporated into a proper dental resin formulation, it can provide a strong bactericidal activity for dental materials; in the meantime, it leads to minimum deterioration of their mechanical strength due to its low water uptake.

Introduction

Despite the type of restorative materials, secondary caries, the lesion at the margin of a restoration, has been widely considered as the most important and common reason for restoration replacement . This phenomenon is mainly attributed to the bacterial activity at the marginal gap between tooth tissue and restorative materials. Three main strategies are followed for solving this problem. These include: a) decreasing the marginal gap formation by reduction of photopolymerization-induced shrinkage strain and stress and enhancement of fracture toughness of dental restorative composites , b) development of materials with the promising anti-cariogenic properties acting through the release of OH , calcium and fluoride which are able to inhibit bacterial growth, decreasing both superficial colonization and acid production by microorganisms and c) introducing bactericidal agent to dental restorative composites .

The recently developed thiol-ene polymerization system with its special radical mediated step growth mechanism is considered as an important method which can lead to the formation of the polymeric network with reduced shrinkage and improved fracture toughness . However, elimination of crack propagation is not completely achievable and marginal gap formation is an inevitable phenomenon in clinical practice. Therefore, a combination of the new polymerization technique of thiol-ene system and the introduction of bactericidal agents into the backbone of dental restorative composites seems to be an effective approach for reduction of troubles arisen from secondary caries.

Very recently we have reported our findings of the physicomechanical and bactericidal activity of dental materials prepared through ternary thiol-allyl ether-methacrylate system containing quaternary ammonium salts (QAS) methacrylate monomers . The results were also compared with the common restorative materials prepared through conventional radical photopolymerization system with the same reactive bactericidal methacrylate monomers. In addition to the significant reduction of shrinkage stress and strain, much better bactericidal activity was recorded for thiol-allyl rich system. The latter phenomenon was attributed to the higher degree of freedom for chain segments and a consequently higher chance for exposing of reactive QAS moieties on the surface of materials. Unfortunately, the accessible polar QAS moieties presented in the backbone of such materials can also increase the possibility of oral fluid uptake. The plasticization effect of absorbed water molecules may lead to an overall reduction of mechanical strength of restorative materials. Using bactericidal compounds possessing QAS moieties chemically anchored to a hydrophobic core is the strategy we have followed to compensate the overall increase of the system hydrophilicity introduced by QAS groups. To fulfill this goal, application of cage type polyhedral oligomeric silsesquioxane (POSS) nanoparticles decorated with QAS moieties was considered in the present study.

A POSS molecule can be considered as a smallest possible particle of silica. Thus, these materials may be compounded with polymers yielding true nanocomposites with molecular-level dispersion. POSS consists of an inner hydrophobic core of inorganic silicon and oxygen and an outer layer of organic groups . Investigation of literature data showed some interesting reports regarding functionalization of POSS core with bactericidal moieties such as QAS groups . Highly branched structure with the possibility of having up to eight functional groups per molecule while maintaining a compact molecular size was considered as specific characteristics of POSS nanoparticles.

To this end, a new derivative of POSS with 1,2,3-Triazolium functional groups was synthesized and incorporated into a dental restorative system composed of ternary thiol-allyl ether-methacrylate resin and glass fillers. To compare the properties, a similar system was also prepared, in which the POSS additive was replaced with benzyl chloride quaternized 2-(dimethylamino)ethyl methacrylate monomer (DMAEMA-BC). Physico-mechanical, cytocompatibility and antibacterial properties of these composites were evaluated and compared. To the best of our knowledge, no similar reports concerning the application of bactericidal POSS-based additive in thiol-ene type dental composite have been reported yet.

Experimental

Materials

Camphorquinone (CQ), 2-(dimethylamino)ethyl methacrylate (DMAEMA), 1-methylimidazole, phenylacetylene (PhAc), N , N- dimethylformamide (DMF), tetrahydrofuran (THF) and methyl iodide (CH 3 I) were purchased from Merck (Germany). 2,2-Bis-(2-hydroxy-3-methacryloxypropoxy)phenyl propane (Bis-GMA) and triethylene glycol dimethacrylate (TEGDMA) were kindly provided by Evonik (Germany) and used as received. Pentaerythritol tetrakis(3-mercaptopropionate) (PETMP) was supplied by Aldrich (Germany). Urethane tetra-allyl ether monomer (UTAE), as ene monomer was synthesized by condensation reaction of isophorone diisocyanate (IPDI) and trimethylolpropane diallyl ether (DAE) according to the procedure reported in our previous article . DMAEMA-BC was synthesized according to the procedure reported in our previous article . Sodium azide (NaN 3 ) and tetrafluoroboric acid (HBF 4 , 50% w/w aqueous solution) were supplied by Daejung (Korea). Glass filler with the average particle size of 2–5 μm, (SP345; aluminum fluoride: 5–10%, barium fluoride: 5–10%, calcium fluoride: 5–10%, silicon dioxide: 40–70%, zinc oxide: 5–10%) was kindly supplied by Specialty Glass (USA) and octakis-(3-glycidoxypropyl) octasilsesquioxane (Glycidyl-POSS cage mixture, EP0409) was obtained from hybrid plastics (USA). Streptococcus mutants, ATCC 35668 ( Streptococcus mutans ) was purchased from Iranian Research Organization for Science and Technology (Tehran, Iran). Mouse L929 fibroblast cells were also obtained from Pasteur Institute of Iran and used as received.

Methods

Synthesis of ionic liquid catalyst

1-Methylimidazolium tetrafluoroborate ([Hmin] + BF4 ) was synthesized according to the procedure reported in . Briefly, 1-methylimidazole (30.80 g, 0.38 mol) was charged into a 250 ml two-necked round-bottomed flask equipped with a dropping funnel, a condenser, and a magnetic stirrer. The flask was cooled to 0 °C and then, tetrafluoroboric acid (65.90 g, 50% solution in water, 0.38 mol) was dropped slowly over a period of 30 min and the mixture was stirred at 0–5 °C for 2 h. The product as a viscous colorless liquid, which solidified on cooling, was obtained after removing the water using a vacuumed rotary evaporator.

Synthesis of octakis-(3-[(1-azido-2-hydroxy)propoxy]propyl) octasilsesquioxane (N 3 -POSS)

A clickable azide derivative of POSS was synthesized through ring opening reaction of Glycidyl-POSS with NaN 3 in the presence of [Hmin] + BF4 as a catalyst. Glycidyl-POSS (1.00 g, 5.97 mmol epoxy) and ([Hmin] + BF 4 ) (1 g, 5.87 mmol) were placed in 250 ml two-neck round-bottomed flask equipped with a thermometer, a condenser, a magnetic stirrer and an oil bath. A solution of NaN 3 (1.90 g, 29.80 mmol) in THF/DMF (20 ml, 1:2 V/V) was added and the reaction mixture was stirred at 95 °C for 48 h. At the end of the reaction, the solvent was removed by a rotary evaporator under reduced pressure. The product was dissolved in chloroform (20 ml) and transferred to a separatory funnel, washed successively with distilled water (60 ml) and brine (20 wt%, 60 ml) to remove the unreacted NaN 3 and catalyst residue. Then, the organic layer was dried over magnesium sulfate and the product as a pale-yellow and viscous liquid was obtained in 90% yield after removing the solvent.

Synthesis of octakis-(3-[(1-(4-phenyl-1,2,3-triazole)-2-hydroxy)propoxy]propyl) octasilsesquioxane (Triazole-POSS)

Triazole-POSS was prepared through click reaction of N 3 -POSS and phenylacetylene under catalyst free, thermal cycloaddition reaction. N 3 -POSS (1.00 g, 4.70 mmol azide functional groups), phenyl acetylene (1.45 g, 14.20 mmol) and DMF (25 ml) were charged into a 100 ml two-neck round-bottomed flask equipped with a thermometer, a condenser, a magnetic stirrer and oil bath. The reaction mixture was stirred at 95 °C for 48 h. The workup and purification of the product were done according to the procedure described above for the preparation of N 3 -POSS. The pale-brown product was obtained in 95% yield.

Synthesis of octakis-(3-[(1-(4-phenyl-3-methyl-1,2,3-Triazoliumiodide)-2-hydroxy)propoxy]propyl) octasilsesquioxane (Triazolium-POSS)

Triazolium-POSS was synthesized through N -alkylation reaction of 1,2,3-triazole rings of Triazole-POSS with methyl iodide. A 100 ml two-neck round-bottomed flask equipped with a thermometer, a condenser, a magnetic stirrer and oil bath was charged with Triazole-POSS (1.00 g, 3.2 mmol triazole rings), methyl iodide (2.27 g, 16 mmol) and chloroform (20 ml). The reaction mixture was stirred at 38 °C for 48 h. At the end of the reaction, the solvent and excess amount of methyl iodide were removed by a rotary evaporator under reduced pressure. The product as a dark-brown, viscous liquid was obtained in 95% yield.

Characterization

1 H NMR spectra of the synthesized compounds were recorded on an AVANCE 300 MHz spectrometer (Bruker, Germany) using DMSO-d6 and CDCl 3 as solvents. FTIR spectra were obtained using an Equinox 55 Bruker instrument (Bruker, Germany). All spectra were obtained in the air as a function of time with 16 scans at a resolution of 4 cm −1 and a spectral range of 500–4000 cm −1 .

The degree of conversion of methacrylate functions was followed using FTIR spectroscopy as described in Supplementary information S1.

Transmission electron microscopy (TEM) was used to identify of POSS nanoparticles within the composite specimen. Ultrathin sections with a thickness of about 100 nm were cut using a Leica EM UC7 ultramicrotome equipped with a diamond knife and TEM micrographs were obtained with a Philips EM 208 apparatus using an acceleration voltage of 100 kV.

The procedure followed for assessment of cytocompatibility of composites was described in Supplementary information S2. The bactericidal activity of composite specimens against S. mutans (ATCC 35668) was determined based on ASTM E 2180-07 and the related procedure was described in Supplementary information S3.

The method followed for the evaluation of the flexural strength of the samples was described in Supplementary information S4. The viscoelastic properties of the specimens were determined by dynamic mechanical analysis (DMA) as described in Supplementary information S5. The technique applied for measurement of shrinkage-strain was also described in Supplementary information S6.

The procedure followed for evaluation of bulk hydrophilicity of composites was described in Supplementary information S7.

Statistical analysis

Statistical analyses were performed by a PASW statistics program package, version 18 (SPSS Inc., Chicago, IL, USA). Comparison of the obtained data for different samples was performed with One-Way ANOVA with Tukey’s post-hoc test. The significance level was set at p ≤ 0.05.

Experimental

Materials

Camphorquinone (CQ), 2-(dimethylamino)ethyl methacrylate (DMAEMA), 1-methylimidazole, phenylacetylene (PhAc), N , N- dimethylformamide (DMF), tetrahydrofuran (THF) and methyl iodide (CH 3 I) were purchased from Merck (Germany). 2,2-Bis-(2-hydroxy-3-methacryloxypropoxy)phenyl propane (Bis-GMA) and triethylene glycol dimethacrylate (TEGDMA) were kindly provided by Evonik (Germany) and used as received. Pentaerythritol tetrakis(3-mercaptopropionate) (PETMP) was supplied by Aldrich (Germany). Urethane tetra-allyl ether monomer (UTAE), as ene monomer was synthesized by condensation reaction of isophorone diisocyanate (IPDI) and trimethylolpropane diallyl ether (DAE) according to the procedure reported in our previous article . DMAEMA-BC was synthesized according to the procedure reported in our previous article . Sodium azide (NaN 3 ) and tetrafluoroboric acid (HBF 4 , 50% w/w aqueous solution) were supplied by Daejung (Korea). Glass filler with the average particle size of 2–5 μm, (SP345; aluminum fluoride: 5–10%, barium fluoride: 5–10%, calcium fluoride: 5–10%, silicon dioxide: 40–70%, zinc oxide: 5–10%) was kindly supplied by Specialty Glass (USA) and octakis-(3-glycidoxypropyl) octasilsesquioxane (Glycidyl-POSS cage mixture, EP0409) was obtained from hybrid plastics (USA). Streptococcus mutants, ATCC 35668 ( Streptococcus mutans ) was purchased from Iranian Research Organization for Science and Technology (Tehran, Iran). Mouse L929 fibroblast cells were also obtained from Pasteur Institute of Iran and used as received.

Methods

Synthesis of ionic liquid catalyst

1-Methylimidazolium tetrafluoroborate ([Hmin] + BF4 ) was synthesized according to the procedure reported in . Briefly, 1-methylimidazole (30.80 g, 0.38 mol) was charged into a 250 ml two-necked round-bottomed flask equipped with a dropping funnel, a condenser, and a magnetic stirrer. The flask was cooled to 0 °C and then, tetrafluoroboric acid (65.90 g, 50% solution in water, 0.38 mol) was dropped slowly over a period of 30 min and the mixture was stirred at 0–5 °C for 2 h. The product as a viscous colorless liquid, which solidified on cooling, was obtained after removing the water using a vacuumed rotary evaporator.

Synthesis of octakis-(3-[(1-azido-2-hydroxy)propoxy]propyl) octasilsesquioxane (N 3 -POSS)

A clickable azide derivative of POSS was synthesized through ring opening reaction of Glycidyl-POSS with NaN 3 in the presence of [Hmin] + BF4 as a catalyst. Glycidyl-POSS (1.00 g, 5.97 mmol epoxy) and ([Hmin] + BF 4 ) (1 g, 5.87 mmol) were placed in 250 ml two-neck round-bottomed flask equipped with a thermometer, a condenser, a magnetic stirrer and an oil bath. A solution of NaN 3 (1.90 g, 29.80 mmol) in THF/DMF (20 ml, 1:2 V/V) was added and the reaction mixture was stirred at 95 °C for 48 h. At the end of the reaction, the solvent was removed by a rotary evaporator under reduced pressure. The product was dissolved in chloroform (20 ml) and transferred to a separatory funnel, washed successively with distilled water (60 ml) and brine (20 wt%, 60 ml) to remove the unreacted NaN 3 and catalyst residue. Then, the organic layer was dried over magnesium sulfate and the product as a pale-yellow and viscous liquid was obtained in 90% yield after removing the solvent.

Synthesis of octakis-(3-[(1-(4-phenyl-1,2,3-triazole)-2-hydroxy)propoxy]propyl) octasilsesquioxane (Triazole-POSS)

Triazole-POSS was prepared through click reaction of N 3 -POSS and phenylacetylene under catalyst free, thermal cycloaddition reaction. N 3 -POSS (1.00 g, 4.70 mmol azide functional groups), phenyl acetylene (1.45 g, 14.20 mmol) and DMF (25 ml) were charged into a 100 ml two-neck round-bottomed flask equipped with a thermometer, a condenser, a magnetic stirrer and oil bath. The reaction mixture was stirred at 95 °C for 48 h. The workup and purification of the product were done according to the procedure described above for the preparation of N 3 -POSS. The pale-brown product was obtained in 95% yield.

Synthesis of octakis-(3-[(1-(4-phenyl-3-methyl-1,2,3-Triazoliumiodide)-2-hydroxy)propoxy]propyl) octasilsesquioxane (Triazolium-POSS)

Triazolium-POSS was synthesized through N -alkylation reaction of 1,2,3-triazole rings of Triazole-POSS with methyl iodide. A 100 ml two-neck round-bottomed flask equipped with a thermometer, a condenser, a magnetic stirrer and oil bath was charged with Triazole-POSS (1.00 g, 3.2 mmol triazole rings), methyl iodide (2.27 g, 16 mmol) and chloroform (20 ml). The reaction mixture was stirred at 38 °C for 48 h. At the end of the reaction, the solvent and excess amount of methyl iodide were removed by a rotary evaporator under reduced pressure. The product as a dark-brown, viscous liquid was obtained in 95% yield.

Characterization

1 H NMR spectra of the synthesized compounds were recorded on an AVANCE 300 MHz spectrometer (Bruker, Germany) using DMSO-d6 and CDCl 3 as solvents. FTIR spectra were obtained using an Equinox 55 Bruker instrument (Bruker, Germany). All spectra were obtained in the air as a function of time with 16 scans at a resolution of 4 cm −1 and a spectral range of 500–4000 cm −1 .

The degree of conversion of methacrylate functions was followed using FTIR spectroscopy as described in Supplementary information S1.

Transmission electron microscopy (TEM) was used to identify of POSS nanoparticles within the composite specimen. Ultrathin sections with a thickness of about 100 nm were cut using a Leica EM UC7 ultramicrotome equipped with a diamond knife and TEM micrographs were obtained with a Philips EM 208 apparatus using an acceleration voltage of 100 kV.

The procedure followed for assessment of cytocompatibility of composites was described in Supplementary information S2. The bactericidal activity of composite specimens against S. mutans (ATCC 35668) was determined based on ASTM E 2180-07 and the related procedure was described in Supplementary information S3.

The method followed for the evaluation of the flexural strength of the samples was described in Supplementary information S4. The viscoelastic properties of the specimens were determined by dynamic mechanical analysis (DMA) as described in Supplementary information S5. The technique applied for measurement of shrinkage-strain was also described in Supplementary information S6.

The procedure followed for evaluation of bulk hydrophilicity of composites was described in Supplementary information S7.

Statistical analysis

Statistical analyses were performed by a PASW statistics program package, version 18 (SPSS Inc., Chicago, IL, USA). Comparison of the obtained data for different samples was performed with One-Way ANOVA with Tukey’s post-hoc test. The significance level was set at p ≤ 0.05.

Results

Synthesis and spectroscopic characterization

Scheme 1 represents the chemical routes followed for the preparation of 1,2,3-triazolium-functionalized POSS (Triazolium-POSS).

Scheme 1
Synthetic route followed for the preparation of Triazolium-POSS.

At first, a clickable derivative of POSS was prepared from Glycidyl-POSS via ring opening reaction with NaN 3 . The FT-IR and 1 H NMR spectra of N 3 -POSS are depicted in Fig. 1 . The peaks related to the vibration of epoxy rings at 851 and 907 cm −1 present in the structure of Glycidyl-POSS (Fig. 1S) are completely disappeared at FTIR spectrum of N 3 -POSS ( Fig. 1 b) while new peaks at 2100 and 3470 cm −1 related to azide and hydroxyl groups appear. The other peaks related to POSS structure are remained intact after functionalization with an azide group. The 1 H NMR spectrum of N 3 -POSS is also in accordance with the proposed structure of this material ( Fig. 1 a). The signals related to methylene protons of the epoxy ring at 2.60 and 2.80 ppm are completely disappeared (Fig. 1S). The peak related to methylene group bearing azide function is detected at 3.20 ppm and the proton of methine group attached to hydroxyl function appears at 3.90 ppm.

Nov 22, 2017 | Posted by in Dental Materials | Comments Off on Bactericidal dental nanocomposites containing 1,2,3-triazolium-functionalized POSS additive prepared through thiol-ene click polymerization
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