Evaluation of cell responses toward adhesives with different photoinitiating systems

Abstract

Objectives

The photoinitiator diphenyl-(2,4,6-trimethylbenzoyl)phosphine oxide (TPO) is more reactive than a camphorquinone/amine (CQ) system, and TPO-based adhesives obtained a higher degree of conversion (DC) with fewer leached monomers. The hypothesis tested here is that a TPO-based adhesive is less toxic than a CQ-based adhesive.

Methods

A CQ-based adhesive (SBU-CQ) (Scotchbond Universal, 3 M ESPE) and its experimental counterpart with TPO (SBU-TPO) were tested for cytotoxicity in human pulp-derived cells (tHPC). Oxidative stress was analyzed by the generation of reactive oxygen species (ROS) and by the expression of antioxidant enzymes. A dentin barrier test (DBT) was used to evaluate cell viability in simulated clinical circumstances.

Results

Unpolymerized SBU-TPO was significantly more toxic than SBU-CQ after a 24 h exposure, and TPO alone (EC 50 = 0.06 mM) was more cytotoxic than CQ (EC 50 = 0.88 mM), EDMAB (EC 50 = 0.68 mM) or CQ/EDMAB (EC 50 = 0.50 mM). Cultures preincubated with BSO ( l -buthionine sulfoximine), an inhibitor of glutathione synthesis, indicated a minor role of glutathione in cytotoxic responses toward the adhesives. Although the generation of ROS was not detected, a differential expression of enzymatic antioxidants revealed that cells exposed to unpolymerized SBU-TPO or SBU-CQ are subject to oxidative stress. Polymerized SBU-TPO was more cytotoxic than SBU-CQ under specific experimental conditions only, but no cytotoxicity was detected in a DBT with a 200 μm dentin barrier.

Significance

Not only DC and monomer-release determine the biocompatibility of adhesives, but also the cytotoxicity of the (photo-)initiator should be taken into account. Addition of TPO rendered a universal adhesive more toxic compared to CQ; however, this effect could be annulled by a thin dentin barrier.

Introduction

During the past decade, most developments in the field of dental adhesive technology have been based on the simplification of multi-step systems. In line with this, so-called ‘universal adhesives’, which recently have been introduced onto the market, represent one further step in simplification. Typically, universal adhesive systems can be used for bonding not only to enamel and dentin, but also to ceramics, metal and composites. Universal adhesives are actually not new, but new is that the latest generation of universal adhesives come as one-component, one-bottle systems .

Even though application of adhesives on exposed pulp tissue is nowadays advised against , the biocompatibility of adhesives remains very important. There is ample evidence that adhesive ingredients such as monomers and additives may be toxic for pulp cells as they were shown to seriously disrupt vital cell functions .

The dentin substrate should be regarded as a permeable substrate, through which ingredients may permeate to the pulp. Self-evidently, the thickness of the remaining dentin after cavity preparation plays an important role , and a remaining dentin thickness of 300 μm is considered critical to maintain pulp health . Permeation of monomers can occur during the application of the unpolymerized adhesive, but also after polymerization ingredients may be released . In this regard, the degree of polymerization, often also called ‘degree of conversion (DC)’ is important. The higher the DC, the lower is the release of unpolymerized monomers .

The monomers in methacrylate-based adhesives polymerize thanks to a radical polymerization reaction, for which purpose photoinitiators are added in small amounts to the composition of adhesives . Conventionally, the co-initiator camphorquinone/teriary amine is added to adhesives, but a major drawback of this photoinitiator system is its intense yellow color . Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO) is an alternative photoinitiator belonging to the group of acylphosphine oxides, whose initiating system is based on photofragmentation . In contrast to the camphorquinone/co-initiating system, which is characterized by a broad absorption spectrum with peak absorption around 468 nm, the absorption spectrum of TPO is situated more toward the UV spectrum (380–425 nm). Several studies showed that methacrylate composites obtained similar or higher degree of conversion when TPO was used as photoinitiator. It was also shown that TPO is more reactive than camphorquinone . Significantly fewer monomers eluted from a TPO-based methacrylate resin compared to a CQ-based material in ethanol-based extraction solutions .

This specific finding is also of particular relevance considering biological effects of these two dentin adhesives. It has been clearly established that resin monomers disrupt the redox homeostasis in cells of the oral cavity through the generation of elevated levels of reactive oxygen species (ROS). As an adaptive response, cells modify the expression of enzymatic antioxidants like superoxide dismutase (SOD1), which eliminates superoxide anions, and glutathione peroxidase (GPx1/2) or catalase, which reduce increasing levels of hydrogen peroxide (H 2 O 2 ) to water. In addition, an increased expression of the stress-responsive haem oxygenase (HO-1) supports antioxidant defense by the generation of the antioxidant bilirubin. Remarkably, the expression of these cytoprotective enzymes depends on the availability of glutathione (GSH), a non-enzymatic antioxidant . Moreover, monomer-induced oxidative burden exceeding the cells antioxidant capacities to regain balanced intracellular redox homeostasis finally leads to cell death via apoptosis through the intrinsic mitochondrial pathway .

The objective of this study was to use these parameters for a detailed analysis of oxidative stress related cellular responses toward a CQ/amine or TPO based universal adhesive. To this end, cytotoxicity, generation of ROS and expression of enzymatic antioxidants were analyzed, and the raw photoinitiators were evaluated as well. It could be hypothesized that the TPO-based adhesive is biologically less active as it releases fewer monomers, but the initiator itself is a leachable compound whose biological activity should also be taken into account. The null hypothesis tested in the current investigation was that the TPO adhesive would be less cytotoxic than the CQ/amine-based adhesive.

Materials and methods

All chemicals and reagents have been listed in Table 1 .

Table 1
Chemicals and reagents used.
Productname CAS no. (if available) Company City, country
MEMα Gibco Life Technologies Karlsruhe, Germany
Fetal bovine serum Gibco Life Technologies Karlsruhe, Germany
Penicillin/streptomycin Gibco Life Technologies Karlsruhe, Germany
Phosphate buffered saline (PBS) Gibco Life Technologies Karlsruhe, Germany
CMF-PBS (calcium- and magnesium-free) Gibco Life Technologies Karlsruhe, Germany
PBS-EDTA Gibco Life Technologies Karlsruhe, Germany
Genetecin Gibco Life Technologies Karlsruhe, Germany
RPMI 1640 containing l -glutamine Pan Biotech Aidenbach, Germany
NaHCO 3 (2.0 g/l) 144-55-8 Pan Biotech Aidenbach, Germany
2-hydroxyethyl methacrylate (HEMA) 868-779 Merck KGaA Darmstadt, Germany
DMSO (dehydrated, SeccoSolv) 67-68-5 Merck KGaA Darmstadt, Germany
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 57360-69-7 Sigma Aldrich Steinheim, Germany
l -Buthionine sulfoximine (BSO) 83730-53-4 Sigma Aldrich Steinheim, Germany
Accutase PAA Cölbe, Germany
2′-7′ dichlorofluorescin (H 2 DCF) 4091-99-0 MoBiTec Goettingen, Germany
BCA protein assay Sigma Aldrich Steinheim, Germany
Polyclonal antibodies anti-catalase (H-300, sc-50508) Santa Cruz Biotechnology Santa Cruz, CA, USA
Polyclonal antibodies anti-catalase (H-300, sc-50508) Santa Cruz Biotechnology Santa Cruz, CA, USA
Polyclonal antibodies anti-haem oxygenase-1 (HO-1, M-19, sc-1797) Santa Cruz Biotechnology Santa Cruz, CA, USA
Monoclonal antibodies: anti-Cu-Zn superoxide dismutase (SOD-1, B-1, sc-271014) Santa Cruz Biotechnology Santa Cruz, CA, USA
Monoclonal antibodies: anti-glutathione peroxidase 1/2(GPx1/2, D-12, sc-133152) Santa Cruz Biotechnology Santa Cruz, CA, USA
Monoclonal antibodies: anti-glutathione peroxidase 1/2(GPx1/2, D-12, sc-133152) Santa Cruz Biotechnology Santa Cruz, CA, USA
Anti-rabbit IgG horseradish peroxidase linked antibodies (n° 7074) Cell Signaling NEB Frankfurt, Germany
Goat anti-mouse IgG with horseradish peroxidase conjugate Bio-Rad Laboratories Munich, Germany
Amersham hyperfilm enhanced chemiluminiscence GE Healthcare Munich, Germany
Protease inhibitor cocktail (complete mini) Roche Diagnostics Mannheim, Germany
Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (clone 6C5) Millipore Schwalbach, Germany
CHEMICON re-blot plus mild antibody stripping solution Millipore Schwalbach, Germany

Adhesives tested

One commercial camphorquinone-based adhesive (Scotchbond Universal, 3 M ESPE, Seefeld, Germany) and its experimental counterpart were included in this study, which were also used in the study by Pongprueksa et al. . Their compositions can be found in Table 2 . Both adhesives were identical in composition, except that they contained a different photoinitiator. Whereas the commercial adhesive contained camphorquinone and ethyl 4-(dimethylamino)benzoate (EDMAB) as co-initiator, the non-commercialized experimental version contained diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO). In the remainder of the text, they will be referred to as SBU-CQ and SBU-TPO, respectively.

Table 2
Composition of the adhesives tested.
Adhesive Composition Photoinitiator
SBU-CQ Bis-GMA 15–25 wt% (cas: 1565-94-2)
HEMA 15–25 wt% (cas: 868-77-9)
Water 10–15 wt% (cas: 7732-18-5)
Ethanol 10–15 wt% (cas: 64-17-5)
Silanized silica 5–15 wt% (cas: 122334-95-6)
DMDMA 5–15 wt% (cas: 6701-13-9)
2-Proprionic acid, 2-methyl-,reaction products with 1,10-decanediol and phosphorus oxide (P 2 O 5 ) 1–10 wt% (cas: 1207736-18-2)
copolymer of acrylic and itaconic acid 1–5 wt% (cas: 25948-33-8)
Ethyl 4-dimethylaminobenzoate <2 wt% (cas: 10287-53-3)
Butanone <0.5% (cas: 78-93-3)
(Dimethylamino)ethyl methacrylate <2 wt% (cas: 2867-47-2)
Camphorquinone ∼2 wt% (cas: 10373-78-1)
EDMAB ∼2 wt% (cas: 10287-53-3)
SBU-TPO idem TPO ∼2 wt% (cas: 75980-60-8)
Abbreviations : Bis-GMA: bisphenol A diglycidyl methacrylate; CQ: camphorquinone; EDMAB: ethyl 4-(dimethylamino)benzoate; HEMA: 2-hydroxyethyl methacrylate; DMDMA: 1,10 decamethylene dimethacrylate, TPO: diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide.

Dissolved unpolymerized adhesive (i), 24h-extracts of polymerized adhesive (ii) and the raw photoinitators (iii) were used for further testing.

  • (i)

    Unpolymerized adhesives : the uncured adhesives were dissolved in pure ethanol (0.5 g/ml; w/v) at room temperature and stock solutions were prepared in culture medium at a concentration of 10 mg/ml following ISO standards . Serial dilutions in cell culture medium were prepared. In a pilot study, it was found that the ethanol in the tested concentrations was not toxic for the cells used in following experiments.

  • (ii)

    Extracts of polymerized adhesives : Polymerized adhesive disks were prepared in a standardized teflon mold (diameter 5 mm and heigth 0.5 mm). After applying the uncured adhesive in the mold and gently air-blowing for 5 s (as per manufacturer’s instructions), the adhesive was covered by a glass plate to prevent incomplete polymerization due to oxygen inhibition, and light-cured for 20 s with a Bluephase C8 (Ivoclar-Vivadent, Schaan, Liechtenstein; output = 1220 mW/cm 2 ). One minute after light curing, the disks were weighed to monitor variations in weight and each disk was immersed in either 150, 200 or 300 μl MEMα medium per well in a 48-well plate following ISO standards . After 24 h, the media were collected for further testing.

  • (iii)

    Photoinitiator : The photoinitiators CQ (cas: 10373-78-1) and TPO (cas: 75980-60-8), and the co-initiator EDMAB (cas: 10287-53-3) were purchased from Sigma-Aldrich (Sigma-Aldrich Chemie, Steinheim, Germany). Stock solutions of 100 mM in ethanol were prepared for further testing.

Cell culture

Clonal SV 40 large T-antigen transfected human pulp-derived cells (tHPC) were cultivated using a minimal essential medium (MEMα) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (100 U/ml penicillin, 100 μg/ml Streptomycin), and 0.2% geneticin (0.1 mg/ml) at 37 °C and 5% CO 2 . The medium was refreshed twice per week.

RAW264.7 macrophages (ATCC TIB71) were cultivated in RPMI 1640 medium containing l -glutamine, sodium-pyruvate and 2.0 g/l NaHCO 3 supplemented with 10% fetal bovine serum (FBS), and penicillin-streptomycin at 37 °C and 5% CO 2 . These cells were only used for Western blotting, which are considered ‘golden standard cells’ , to confirm the results obtained with the pulp-derived cells.

Cytotoxicity testing

To test the influence of the unpolymerized adhesives, the extracts of the polymerized adhesives and the raw photoinitiators on cell viability, the MTT assay, which is based on the reduction of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), was used . To assess the role of oxidative stress on cytotoxicity, BSO, an inhibitor of glutathione synthesis, was added while testing the unpolymerized adhesives . Briefly, to assess the cytotoxicity of the unpolymerized adhesives, tHPCs were seeded in 96-well plates at a density of 1 × 10 4 cells/well in 100 μl and cultivated for 4 h at 37 °C and 5% CO 2 . Then 100 μl of 50 μM BSO or medium was added to beforehand determined wells, to pre-incubate half of the cell cultures with BSO for at least 20 h. The next day, the cells that were pre-incubated in medium were exposed to 200 μl of diluted unpolymerized adhesives and those pre-incubated to BSO were exposed to unpolymerized adhesives in the presence of 50 μM BSO. Either after 1 h or 24 h, the supernatant was removed, and the cells were incubated with 0.5 mg/ml MTT in PBS solution for 1 h before absorbance reading at 540 nm using a spectrophotometer (Infinite F200, TECAN, Männedorf, Zurich, Switzerland) after complete solubilization of the formazan crystals in DMSO.

To evaluate the cytotoxicity of the extracts of the polymerized adhesives and the raw photoinitators, tHPCs were seeded in 96-well plates in 200 μl culture medium and cultivated at 37 °C and 5% CO 2 . After 24 h, the medium was removed and replaced by the undiluted or diluted extracts (in cell culture medium), or by different concentrations of the photoinitators originally prepared as a stock solution in ethanol and further diluted in cell culture medium. After 24 h, cell viability was determined using MTT as described above. The effect of ethanol concentrations was tested in separate experiments. While ethanol concentrations present in extracts and dilutions of unpolymerized materials had no influence on cell viability, a very small effect on viability was detected for the highest concentrations with photo-initiators. This effect was taken into account for each individual photo-initiator solution while calculating the final viability. HEMA (6 and 8 mM) was included here as a positive control substance .

Each experiment was performed in duplicate or triplicate as specified in the figure legends and four replicate cell cultures were analyzed in each experiment. The optical density readings obtained from treated cell cultures were expressed as the percentage of untreated cells. The half-maximum-effect concentrations (EC 50 ) were calculated by plotting the viability results onto a dose-effect sigmoidal curve (Table Curve 2D, Version 5.01, Systat Software, San Jose, CA, USA). Differences between the EC 50 values were statistically analyzed using the Tukey interval method (SPSS 22.0, SPSS, Chicago, IL, USA).

Cellular reactive oxygen species (ROS) generation and expression of antioxidant enzymes

The generation of ROS in tHPCs was measured following a previously described protocol based on the intracellular oxidation of 2′–7′ dichlorofluorescin (H 2 DCF) to 2′–7′dichlorofluorescein (DCF) . tHPCs seeded in six-well plates at a density of 2 × 10 5 were exposed to different concentrations (0.1, 0.25, 0.5 and 1 mg/ml) of unpolymerized adhesives prepared as described above. 2-hydroxyethyl methacrylate (HEMA, 6 and 8 mM) was applied on the cells as positive control, whereas cell culture medium only was applied as negative control. DCF fluorescence was determined by flow cytometry (Becton Dickinson FACSCanto, San Jose, CA, USA) at an excitation wave length of 495 nm and an emission wave length of 530 nm. Main fluorescence intensities were obtained by histogram statistics using the FACSDiva™ 5.0.2 software. Individual values of fluorescence intensities were normalized to fluorescence detected in untreated control cultures (=1.0). At least four independent experiments were performed, individual values were summarized as medians (with 25–75% quartiles), and differences between medians were statistically analyzed with the Mann–Whitney U test ( α = 0.05) (SPSS 22.0).

The expression of several antioxidant enzymes in tHPCs and RAW264.7 cells after exposure to the unpolymerized adhesives was determined by Western blot analysis following a previously described protocol . Briefly, the cells were seeded at a density of 1.5 × 10 6 in petri-dishes and cultivated for 24 h. Subsequently, they were exposed to 0.25 mg/ml or 0.5 mg/ml SBU-CQ or 0.1 mg/ml or 0.25 mg/ml SBU-TPO for 24 h. These concentrations were selected based on the results of cell viability measurements. After exposure, cells were detached, and collected through centrifugation and lysed as described before . The supernatant was collected by centrifugation, and the amount of proteins present in the cell lysates was determined by BCA protein assay (Sigma) using bovine serum albumin as a standard. Subsequently, Western blot analysis was performed as described before . Proteins (10 μg/lane) were separated by electrophoresis on a sodium dodecyl sulfate polyacrylamide gel and transferred to a nitrocellulose membrane (blot), which was incubated with primary antibodies specific for the detection of SOD-1, GPx1/2, catalase or HO-1. These primary antibodies were detected by horseradish peroxidase-conjugated secondary antibodies visualized by enhanced chemiluminescence (ECL). Finally, the nitrocellulose membranes were stripped using an antibody stripping solution and the enzymes were reprobed with an anti-GAPDH antibody as described previously . All details have been described before .

Dentin barrier test

Three dimensional cultures of tHPCs were cultivated according to a previously described protocol on polyamide meshes . Bovine incisors were cut to obtain dentin disks (200 ± 20 μm), and the pulpal side of the disks was etched with 50% citric acid to remove the smear layer. They were then autoclaved and applied in a cell culture perfusion chamber (Minucells and Minutissue GmbH, Bad Abbach, Germany) to obtain two compartments. After an incubation time of 14 days ± 2 days, the three-dimensional tHPC cultures were introduced into the lower ‘pulpal’ compartment in direct contact with the pulpal side of the dentin disk. This pulpal compartment was perfused with perfusion medium (MEMα with 5.96 g/l HEPES and 20% FBS) at a rate of 0.3 ml/h and left for 24 h before starting the experiment. The next day, perfusion was briefly stopped and SBU-CQ and SBU-TPO were applied onto the dentin disks at the cavity side following the instructions of the manufacturer. After light-curing the adhesives, a flowable composite (Tetric EvoFlow, Ivoclar-Vivadent, Schaan, Liechtenstein) was applied and light-cured for 20 s. As positive control, an experimental light-curing glass ionomer was prepared ( Table 3 ) and as negative control, a polyvinysiloxane impression material (President Regular, Coltene-Whaledent AG, Altstätten, Switzerland) (100% cell viability) was used. The perfusion rate through the pulpal compartment was subsequently increased to 2 ml/h. After an exposure time of 24 h, cell viability in the three-dimensional tHPC cultures was assessed by the MTT assay as described above. Each experiment was performed with five replicates and carried out at least two times. Medians (with 25–75% percentiles) were calculated from individual values, and differences between medians were statistically analyzed with the Mann–Whitney U test ( α = 0.05) (SPSS 22.0).

Table 3
Composition of the experimental light-curing glass ionomer.
Ingredient Manufacturer Final wt% after mixing (%)
Powder 1 Glass particles Schott (GM 35429) 66.3
Diphenyliodoniumchlorid Sigma Aldrich (cas 10287-53-3) 2.0
Powder 2 Polyacrylic acid Sigma Aldrich (cas 9003-01-4) 11.7
Liquid CQ Sigma Aldrich (cas 10373-78-1) 0.05
EDMAB Merck (cas 10287-53-3) 0.05
HEMA Merck (cas 868-77-9) 15.0
Water 4.9
Abbreviations : CQ: camphorquinone; EDMAB: ethyl 4-(dimethylamino)benzoate; HEMA: 2-hydroxyethyl methacrylate.

Exposed three-dimensional cell cultures were also analyzed by confocal laser microscopy (LSM 510 Meta, Zeiss, Jena, Germany) and managed with a LSM Image VisArt software after dying with the a viability/cytotoxicity kit for mammalian cells (Invitrogen, Karlsruhe, Germany). Images were obtained using 10× magnification and further adjusted with an electronic zoom, and image analysis was performed using Zeiss LSM Image Browser software.

Materials and methods

All chemicals and reagents have been listed in Table 1 .

Table 1
Chemicals and reagents used.
Productname CAS no. (if available) Company City, country
MEMα Gibco Life Technologies Karlsruhe, Germany
Fetal bovine serum Gibco Life Technologies Karlsruhe, Germany
Penicillin/streptomycin Gibco Life Technologies Karlsruhe, Germany
Phosphate buffered saline (PBS) Gibco Life Technologies Karlsruhe, Germany
CMF-PBS (calcium- and magnesium-free) Gibco Life Technologies Karlsruhe, Germany
PBS-EDTA Gibco Life Technologies Karlsruhe, Germany
Genetecin Gibco Life Technologies Karlsruhe, Germany
RPMI 1640 containing l -glutamine Pan Biotech Aidenbach, Germany
NaHCO 3 (2.0 g/l) 144-55-8 Pan Biotech Aidenbach, Germany
2-hydroxyethyl methacrylate (HEMA) 868-779 Merck KGaA Darmstadt, Germany
DMSO (dehydrated, SeccoSolv) 67-68-5 Merck KGaA Darmstadt, Germany
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 57360-69-7 Sigma Aldrich Steinheim, Germany
l -Buthionine sulfoximine (BSO) 83730-53-4 Sigma Aldrich Steinheim, Germany
Accutase PAA Cölbe, Germany
2′-7′ dichlorofluorescin (H 2 DCF) 4091-99-0 MoBiTec Goettingen, Germany
BCA protein assay Sigma Aldrich Steinheim, Germany
Polyclonal antibodies anti-catalase (H-300, sc-50508) Santa Cruz Biotechnology Santa Cruz, CA, USA
Polyclonal antibodies anti-catalase (H-300, sc-50508) Santa Cruz Biotechnology Santa Cruz, CA, USA
Polyclonal antibodies anti-haem oxygenase-1 (HO-1, M-19, sc-1797) Santa Cruz Biotechnology Santa Cruz, CA, USA
Monoclonal antibodies: anti-Cu-Zn superoxide dismutase (SOD-1, B-1, sc-271014) Santa Cruz Biotechnology Santa Cruz, CA, USA
Monoclonal antibodies: anti-glutathione peroxidase 1/2(GPx1/2, D-12, sc-133152) Santa Cruz Biotechnology Santa Cruz, CA, USA
Monoclonal antibodies: anti-glutathione peroxidase 1/2(GPx1/2, D-12, sc-133152) Santa Cruz Biotechnology Santa Cruz, CA, USA
Anti-rabbit IgG horseradish peroxidase linked antibodies (n° 7074) Cell Signaling NEB Frankfurt, Germany
Goat anti-mouse IgG with horseradish peroxidase conjugate Bio-Rad Laboratories Munich, Germany
Amersham hyperfilm enhanced chemiluminiscence GE Healthcare Munich, Germany
Protease inhibitor cocktail (complete mini) Roche Diagnostics Mannheim, Germany
Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (clone 6C5) Millipore Schwalbach, Germany
CHEMICON re-blot plus mild antibody stripping solution Millipore Schwalbach, Germany
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Nov 23, 2017 | Posted by in Dental Materials | Comments Off on Evaluation of cell responses toward adhesives with different photoinitiating systems
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