Highlights
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Non-thermal plasmas induce HEMA grafting onto dentin collagen under clinical settings.
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Grafting efficacy of HEMA depends on the treatment time and input power of plasmas.
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A new method to create chemical bond between resin monomers and dentin collagen.
Abstract
Objective
Non-thermal atmospheric plasma (NTAP) brush has been regarded as a promising technique to enhance dental interfacial bonding. However, the principal enhancement mechanisms have not been well identified. In this study, the effect of non-thermal plasmas on grafting of HEMA, a typical dental monomer, onto dentin collagen thin films was investigated.
Methods
Human dentin was sectioned into 10-μm-thick films. After total demineralization in 0.5 M EDTA solution for 30 min, the dentin collagen films were water-rinsed, air-dried, treated with 35 wt% HEMA aqueous solution. The films were then subject to plasma-exposure under a NTAP brush with different time (1–8 min)/input power (5–15 W). For comparison, the dentin collagen films were also treated with the above HEMA solution containing photo-initiators, then subject to light-curing. After plasma-exposure or light-curing, the HEMA-collagen films were rinsed in deionized water, and then examined by FTIR spectroscopy and TEM.
Results
The FITR results indicated that plasma-exposure could induce significant HEMA grafting onto dentin collagen thin films. In contrast, light-curing led to no detectable interaction of HEMA with dentin collagen. Quantitative IR spectral analysis (i.e., 1720/3075 or 749/3075, HEMA/collagen ratios) further suggested that the grafting efficacy of HEMA onto the plasma-exposed collagen thin films strongly depended on the treatment time and input power of plasmas. TEM results indicated that plasma treatment did not alter collagen’s banding structure.
Significance
The current study provides deeper insight into the mechanism of dental adhesion enhancement induced by non-thermal plasmas treatment. The NTAP brush could be a promising method to create chemical bond between resin monomers and dentin collagen.
1
Introduction
A dentin adhesion process using bonding agents generally involves functional hydrophilic monomers to facilitate diffusion of the bonding resin into demineralized dentin. Among such “wetting monomers”, 2-hydroxyethyl methacrylate (HEMA) is one of the most widely used. HEMA has a low molecular weight and ethanol-like hydrophilic portion, which allow it to infiltrate into the network of dentin organic matrix (collagen) and prevent collagen from collapse . This process, meanwhile, provides a method for HEMA (along with other adhesive monomers) to mechanically entangle the collagen matrix after polymerization, thus potentially benefits the resin/dentin bonding . Furthermore, studies also showed that HEMA might exhibit affinity to dentin through some type of chemical or physical interactions with dentinal collagen to promote adhesion.
Despite of extensive use, HEMA presented some obvious problems in dentin bonding applications. For example, HEMA was disclosed to be one of the major components released from adhesive resin, which likely induced cytotoxic effect . The leaching may occur during the setting period of the adhesive resin or later when the resin is degraded . This fact possibly suggests that the affinity of HEMA to dentin (or dentin collagen), if any, might be rather weak. The leaching of HEMA may also be related to the poor polymerization capacity of HEMA , since unpolymerized monomers are easier to be released to surrounding environment with oral fluids. In addition, the release and suboptimal curing of HEMA might lead to enhanced water uptake and hydrolysis in the hybrid layer . All of these factors would consequently undermine the long-term stability of the adhesive/dentin interface.
Non-thermal atmospheric plasma technique has been recently proven to be an “effective” and “clean” approach for materials surface modification . This technique combines exceptional chemical reactivity with a relatively mild, non-destructive character resulting from a cold gas phase. Depending on plasma chemistry and gas composition, the highly reactive plasma species react with, clean, or etch surface materials. Meanwhile, the modification is limited to the outmost layer that allows the bulk properties be kept unchanged . Such great advantages have made non-thermal atmospheric plasmas a promising technique in dentistry such as adhesion enhancement of dental restorations. Recent studies have reported the significant improvement of the adhesive/dentin bonding induced by using non-thermal atmospheric plasma (NTAP) brush. However, the exact mechanism is not well understood. Especially, investigations are needed to obtain deeper insights into plasma-induced interactions of the individual components of adhesive and dentin, such as HEMA and collagen. In this study, dentin collagen thin films were prepared, and the effect of NTAP brush on interaction of HEMA with dentin collagen was investigated. The null hypothesis tested was that plasma treatment would not enhance the interaction of HEMA with dentin collagen.
2
Materials and methods
2.1
Dentin collagen film/HEMA solution preparation
Ten extracted non-carious human third molars were collected after the patients’ informed consent under a protocol approved by the University of Missouri-Kansas City adult health sciences IRB. The teeth were stored in 0.9% w/v phosphate buffered saline (PBS) containing 0.002% sodium azide at 4 °C before use. The occlusal one-third to one-half of the crown was removed using a water-cooled diamond saw (Buehler Ltd, Lake Bluff, IL, USA) until all enamel was removed (as determined by reflective light microscopy). Four additional cuts in the occusal-apical direction were performed to remove all side walls of the enamel. The remaining dentin block was then sectioned into thin film with thickness of 10 μm by means of a tungsten carbide knife mounted on a microtome (SM2500S, Leica, Nussloch, Germany). 15–20 10-μm-thick slices/films were obtained from each tooth. The obtained dentin slices were fully demineralized by immersing into 0.5 mol/L EDTA solution (pH = 7.4) for 30 min to obtain dentin collagen films (The complete demineralization was assured by using FTIR, in which the disappearance of the phosphate peak at 1070 cm −1 was monitored.). The collagen films were washed in deionized water and transferred onto Mylar films (22 mm × 22 mm × 0.25 mm, Fisher Scientific, Pittsburg, PA, USA), air-dried and stored in a desiccator.
The HEMA solutions used for plasma-exposure were either neat HEMA (Acros Organics, Morris Plain, NJ, USA) or 35 wt% HEMA in deionized water. For comparison, a three-component photoinitiator system (0.5 wt% camphorquinone (CQ), 0.5 wt% ethyl 4-dimethylaminobenzoate (EDMAB) and 1.0 wt% diphenyliodonium hexafluorophosphate (DPIHP)) was added to the above solutions to provide light-curing capability. Photoinitiator concentration was calculated based on pure HEMA monomer. These initiator components were all obtained from Sigma–Aldrich (St. Louis, MO, USA).
2.2
Non-thermal atmospheric plasma (NTAP) brush
The non-thermal atmospheric plasma brush ( Fig. 1 ) employed in this study was fabricated by Nanova, Inc (Columbia, MO, USA). The detailed information about this device can be found in previous publications . Compressed argon gas (ultra high purity) was used as the plasma gas supply. A MKS mass flow controller (MKS Instruments Inc., Andover, MA, USA) was introduced to adjust the argon gas flow rate (2000 sccm). A glow discharge by the direct current power source (Model 1556C, Power Designs Inc, Westbury, NY, USA) was ignited between the two electrodes in a walled, Teflon chamber. One of the electrodes was attached to a ballasted resistor which controlled the discharge current. The other electrode was grounded for electrical safety. The formed plasma discharge could be blown out of the chamber to create a brush-shaped non-thermal plasma jet, which was generated in about 2 s after turning on the power. The prepared specimens (HEMA solutions and dentin collagen films) were placed under the plasma nozzle (5–6 mm from bottom of the nozzle) for treatment with varying input power (5, 10 and 15 W) and treatment times (1, 2, 4, 6 and 8 min).
2.3
Plasma-exposure and light-curing of specimens
The prepared HEMA solutions were subjected to plasma-exposure and light-curing. For plasma-exposure, the glass slide substrates were pre-treated by plasma brush for 60 s in order to improve the surface hydrophilicity. A small volume (2 μL) of neat HEMA without photoinitiator was then cast on the glass slide and cured by the plasma brush with different input powers and treatment times. For light-curing, a small volume (2 μL) of HEMA solution with photoinitiator was cast on the diamond crystal top-plate of the ATR accessory (of the FT-IR spectrometer), and covered by a Mylar film. A 40 s-exposure from a conventional dental light polymerization unit (Spectrum Light, DENTSPLY, Milford, DE, USA) emitting 550 mW/cm 2 was applied. The distance from the top of the Mylar film to the distal end of the light guide was kept at ∼2 mm.
The plasma-exposure and light-curing of the prepared dentin collagen films were conducted after they were pre-treated with 35 wt% HEMA aqueous solutions (without and with photoinitiator, respectively), and gently air-dried for 15 s. After air-drying, the collagen films were still wet, but no free, excess liquid could be observed on the film surfaces. The collagen films treated with HEMA without photoinitiator were then plasma-exposed with different input powers and treatment times. The collagen films treated with HEMA containing photoinitiator were covered by Mylar films and light-cured for 40 s at the light intensity of 550 mW/cm 2 . The plasma-exposed and light-cured HEMA and dentin collagen film specimens were then measured by FT-IR spectrometer. After measurements, each of the dentin collagen film specimens was immersed in 30 ml deionized water and placed on a rotational shaker (a speed of 60 r/min) for 5 min. After air drying, the collagen films were examined again using FT-IR spectrometer, or employed for TEM specimen preparation and measurements. As control, some of the dentin collagen films treated with HEMA solutions but without plasma-exposure or light-curing, were also subjected to FT-IR measurements.
2.4
Attenuated total reflectance Fourier transform infrared (ATR/FT-IR) spectroscopy
The FT-IR spectra of the above specimens were collected using a Fourier transform infrared spectrometer equipped with an ATR attachment (Spectrum One, Perkin-Elmer, Waltham, MA, USA) at a resolution of 4 cm −1 and scan time of 32. The specimens were cast or pressed on the crystal top-plate of the ATR accessory. The ATR crystal was diamond with a transmission range between 650 and 4000 cm −1 . At least 3 FT-IR spectra were collected for each specimen group.
2.5
Transmission electron microscopy (TEM)
Collagen film specimens were fixed in 2.5% glutaraldehyde (in sodium cacodylate buffer) for 2 h and post-fixed in 1% osmium tetroxide for 1 h. Then they were dehydrated and embedded in Embed-812 resin. Sectioning was performed with diamond knife (Diatome, Biel, Switzerland) on EM UC7 ultramicrotome (Leica Microsystems, Inc., Buffalo Grove, IL). Sections were stained with phosphotungstic acid. Micrographs were collected at 80 kV accelerating voltage with CM12 TEM (FEI, Hillsboro, OR) equipped with SC1000 ORIUS ® digital camera (Gatan, Pleasanton, CA). At least 3 collagen film specimens of each treatment group were examined with TEM.
2
Materials and methods
2.1
Dentin collagen film/HEMA solution preparation
Ten extracted non-carious human third molars were collected after the patients’ informed consent under a protocol approved by the University of Missouri-Kansas City adult health sciences IRB. The teeth were stored in 0.9% w/v phosphate buffered saline (PBS) containing 0.002% sodium azide at 4 °C before use. The occlusal one-third to one-half of the crown was removed using a water-cooled diamond saw (Buehler Ltd, Lake Bluff, IL, USA) until all enamel was removed (as determined by reflective light microscopy). Four additional cuts in the occusal-apical direction were performed to remove all side walls of the enamel. The remaining dentin block was then sectioned into thin film with thickness of 10 μm by means of a tungsten carbide knife mounted on a microtome (SM2500S, Leica, Nussloch, Germany). 15–20 10-μm-thick slices/films were obtained from each tooth. The obtained dentin slices were fully demineralized by immersing into 0.5 mol/L EDTA solution (pH = 7.4) for 30 min to obtain dentin collagen films (The complete demineralization was assured by using FTIR, in which the disappearance of the phosphate peak at 1070 cm −1 was monitored.). The collagen films were washed in deionized water and transferred onto Mylar films (22 mm × 22 mm × 0.25 mm, Fisher Scientific, Pittsburg, PA, USA), air-dried and stored in a desiccator.
The HEMA solutions used for plasma-exposure were either neat HEMA (Acros Organics, Morris Plain, NJ, USA) or 35 wt% HEMA in deionized water. For comparison, a three-component photoinitiator system (0.5 wt% camphorquinone (CQ), 0.5 wt% ethyl 4-dimethylaminobenzoate (EDMAB) and 1.0 wt% diphenyliodonium hexafluorophosphate (DPIHP)) was added to the above solutions to provide light-curing capability. Photoinitiator concentration was calculated based on pure HEMA monomer. These initiator components were all obtained from Sigma–Aldrich (St. Louis, MO, USA).
2.2
Non-thermal atmospheric plasma (NTAP) brush
The non-thermal atmospheric plasma brush ( Fig. 1 ) employed in this study was fabricated by Nanova, Inc (Columbia, MO, USA). The detailed information about this device can be found in previous publications . Compressed argon gas (ultra high purity) was used as the plasma gas supply. A MKS mass flow controller (MKS Instruments Inc., Andover, MA, USA) was introduced to adjust the argon gas flow rate (2000 sccm). A glow discharge by the direct current power source (Model 1556C, Power Designs Inc, Westbury, NY, USA) was ignited between the two electrodes in a walled, Teflon chamber. One of the electrodes was attached to a ballasted resistor which controlled the discharge current. The other electrode was grounded for electrical safety. The formed plasma discharge could be blown out of the chamber to create a brush-shaped non-thermal plasma jet, which was generated in about 2 s after turning on the power. The prepared specimens (HEMA solutions and dentin collagen films) were placed under the plasma nozzle (5–6 mm from bottom of the nozzle) for treatment with varying input power (5, 10 and 15 W) and treatment times (1, 2, 4, 6 and 8 min).
