Chemicals and cell culture
All reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA) unless otherwise noted. TEGDMA and HEMA were obtained from Aldrich Chemical Company (Deisenhofen, Germany). Anti-phospho-ERK, −JNK, and -p38 polyclonal antibodies, in addition to horseradish peroxidase-conjugated secondary antibodies, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Under the approval of the Institutional Review Board of Seoul National University Dental Hospital, human dental pulp cells were extracted from incisors that had been removed from patients for orthodontic purposes. After swabbing teeth with 70% ethanol and phosphate-buffered saline (PBS) (pH 7.4), teeth were cut aseptically at the apex. Dental pulp was obtained from the pulp chamber and immersed in minimal essential medium (MEM) containing 20% fetal bovine serum (FBS) and antibiotic solution (100 U/mL of penicillin-G and 100 mg/mL of streptomycin). MEM and FBS were obtained from GIBCO-BRL (Carlsbad, CA, USA). The pulp was minced into several pieces and incubated at 37°C in a humidified atmosphere (5% CO2/95% air), with medium exchanges every 3 days. After 20 days, pulp cells were collected by treatment with trypsin solution and maintained in fresh medium. Cells were used in their fifth passage after confirming their alkaline phosphatase (ALP) activity in differentiation medium (MEM containing 50 mg/mL L-ascorbic acid, 10−8 M dexamethasone, and 2 mM β-glycerolphosphate) (9).
Cytotoxicity test
HDPCs were incubated in 96-well plates until confluent and then treated with various concentrations of HEMA and TEGDMA. After treatment of resin monomers in growth medium which contained 10% FBS for 24 hrs, cell viability was measured using the WST-8 method as previously described [18]. Briefly, 10% WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] (Dojindo Laboratories, Kumamoto, Japan) was added to culture medium without phenol red, and cells were incubated for 1 h. The absorbances were detected at a test wavelength of 450 nm and a reference wavelength of 600 nm using an automated microplate reader (Sunrise, TECAN, Salzburg, Austria). All experiments were performed in triplicate.
ALP activity assay
The effects of resin monomers on HDPC ALP activity were evaluated using a 4-nitrophenyl phosphate-based colorimetric assay, as described previously [19]. HDPCs were exposed to 0.5 mM TEGDMA and 2 mM HEMA for 24 hrs in growth medium, and the cells were then incubated in growth medium or differentiation medium for 6 days before ALP activity assessment. Each well was washed with PBS, and then cells were incubated in a mixture of 140 μl of alkaline buffer solution (67 mM 4-nitrophenyl phosphate; Fluka, Buchs, Switzerland) and 10 μl of 1.5 mM MgCl2 solution for 30 min at 37°C. The reaction was stopped by the addition of 0.5 mM NaOH, and absorbances were measured at 405 nm. ALP activity is expressed as enzyme activity units per microgram of protein. Protein quantitation of each well was performed with a bicinchoninic acid (BCA) protein assay kit (iNtRON Biotechnology; Sungnam, Korea). To observe the effects of resin monomers after washout of the monomers from cell cultures, the treated cells were re-incubated in fresh medium for certain periods before initiation of differentiation.
Gene expression analysis by real-time polymerase chain reaction
To investigate the effects of resin monomers on the differentiation of HDPCs, the mRNA levels of dentin sialophosphoprotein (DSPP), osteocalcin (OCN), and osteopontin (OPN) were measured by real-time polymerase chain reaction (RT-PCR). After treatment with resin monomers for 24 hrs, cells were washed and maintained in differentiation medium for 12 days. HDPCs treated with 0.2 mM H2O2 were used as a control for oxidative stress. Total RNA was obtained with the WelPrep Total RNA Isolation Reagent (Welgene Inc, Daegu, Korea), and cDNA was prepared with a Power cDNA Synthesis kit (iNtRON Biotechnology). Real-time PCR was performed in a mixture of 10 μl SYBR Premix Ex Taq (Takara Bio, Otsu, Japan), 0.4 μl ROX Reference Dye II (Takara Bio), cDNA, and primers on an ABI PRISM 7500 Sequence Detection System Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The following primers were used: DSPP, forward 5′-GCATTCAGGGACAAGTAAGCA-3′, reverse 5′-CTTGGACAACAGCGACATCCT-3′; OCN, forward 5′-GTGACGAGTTGGCTGACC-3′, reverse 5′-CAAGGGGAAGAGGAAAGAAGG-3′; OPN, forward 5′-CAGACGAGGACATCACCTCA-3′, reverse 5′-TGGCTGTGGGTTTCAGCA-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward 5′-GTCGGAGTCAACGGATTTGG-3′, reverse 5′-GGGTGGAATCA ATTGGAACATG-3′. The PCR thermocycling conditions were: 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing at 60°C for 30 sec. DSPP, OCN, and OPN expression levels were calculated based on their threshold cycle (CT) values and are expressed as relative mRNA expression ratios normalized to a reference gene (GAPDH).
Protein expression analysis by western blotting
The expression of phospho-ERK, JNK, and p38 in resin-treated cells was observed by western blotting. HDPCs were incubated in 6-well plates until confluent, and then cells were washed and treated with resin monomers for 1, 3, and 6 hrs. HDPCs treated with 0.2 mM H2O2 were used as a positive control. Proteins were extracted in cold NP-40 lysis buffer [50 mM Tris–HCl (pH 7.6); 150 mM NaCl; 10% glycerol; 1% NP-40; 1 mM phenylmethylsulfonyl fluoride; and 1 μg/mL each of leupeptin, aprotinin, and pepstatin] for 15 min at 4°C and lysates were then spun by centrifugation at 14,000 × g for 10 min at 4°C. The total protein concentrations of the lysates were measured using a Pro-Measure kit (iNtRON Biotechnology, Seoul, Korea). Equal amounts of protein (30 μg) were subjected to SDS-polyacrylamide gel electrophoresis on 10% gels and then transferred to polyvinylidene difluoride (PVDF) transfer membranes (Hybond-P; Amersham Biosciences, Bucks, England). After blocking [6% (w/v) dried low-fat milk and 0.1% (v/v) Tween 20 in PBS (PBST)], the blots were incubated with anti-phospho-ERK, −JNK, and -p38 polyclonal antibodies in PBST for 1 hr followed by two washes (15 min each) in PBST. The blots were then probed with goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase. Immunoreactive bands were visualized using a chemiluminescence kit (WEST-ZOL plus Western Blot Detection System; iNtRON Biotechnology, Seoul, Korea). Chemiluminescence was detected with a MicroChemi Bio-image analyzer (DNR, Jerusalem, Israel).
Statistical analysis
Data are expressed as means ± SDs of three or more experiments. The significance of differences between control and treated groups was analyzed using the paired Student’s t-test.