Reagents and cell culture

Hyaluronic acid sodium salt (CAS number 9067-32-7) was purchased from Nacalai Tesque Co. (Kyoto, Japan). This reagent was slowly dissolved in double-distilled water to a final concentration of 10 mg/mL (1 %). HA was further diluted in culture medium to required concentrations prior to use in cell culture experiments. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS) was purchased from Nichirei Bioscience (Tokyo, Japan).

Human DPSCs were obtained from AllCells LLC (Emeryville, CA, USA). Cell cultures were maintained in DMEM supplemented with 10 % FBS and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37 °C in a humidified atmosphere containing 5 % CO2. The passage numbers were limited at 2–5 to avoid cell deterioration.

Immunofluorescence

Monolayers of DPSCs were cultured with DMEM containing 10 % FBS for 48 h in four-well covered glass chamber slides. After two washes with phosphate-buffered saline (PBS) containing 1 % bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA), cell surface Fc receptors were blocked with immunoglobulin G (IgG) (Santa Cruz Biotechnology Inc., Dallas, TX, USA) on ice for 15 min. The cells were then stained for 30 min at 37 °C with a 1:100 dilution of a fluorescein isothiocyanate (FITC)-conjugated anti-CD44 monoclonal antibody (BD Biosciences, Franklin Lakes, NJ, USA) or an isotype-matched FITC-conjugated IgG control antibody (BD Biosciences). After washing, the cells were analyzed using an ECLIPSE TS100-F microscope equipped with an Intensilight C-HGFIE illumination system (Nikon Co., Ltd., Tochigi, Japan). Digital images were processed with NIS Elements BR3.2 imaging software (Nikon Co., Ltd.) and Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA, USA).

Evaluation of cell growth using the MTT assay

DPSCs were seeded into 96-well microtiter plates at a density of 1 × 103 cells/well and allowed to adhere for 24 h. Cell viability was assessed on a daily basis by addition of 5 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) using a Cell Proliferation Kit I (Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s instructions. The number of viable cells was assessed by measuring the absorbance of the produced formazan crystals at 595 nm with a MultiSkan JX microplate reader and Ascent software (Thermo Labsystems, Vantaa, Finland). The measurement was performed once per day for 5 days. Cell growth was calculated relative to the value on the first day, which was set at 100 %.

Quantification of alkaline phosphatase

DPSCs were seeded into 24-well plates at a density of 5 × 104 cells/well, and incubated with HA (1–20 μg/mL) for 1 week. The cells were then harvested as cell lysates using a SensoLyte® FDP Alkaline Phosphatase Assay Kit (AnaSpec, San Jose, CA, USA), which uses a fluorogenic assay to determine alkaline phosphatase (ALP) activity. The assay was performed according to the manufacturer’s instructions, and fluorescence signals were measured with SpectraFluor plus XFluor4 software (Tecan Japan Co., Ltd., Kawasaki, Japan).

Flow cytometry analysis

For analysis of CD44-positive cell surface antigen expression, untreated and HA-treated DPSCs were harvested by trypsinization, washed with PBS, centrifuged into cell pellets and resuspended in fluorescence-activated cell sorting (FACS) buffer (PBS containing 0.5 % bovine serum albumin). The cells were stained for 30 min at 4 °C with a FITC-conjugated anti-human CD44 antibody (BD Biosciences, San Jose, CA, USA) or an isotype-matched FITC-conjugated IgG control antibody (BD Biosciences). Flow cytometry was performed using an EPICS Altra flow cytometer (Beckman Coulter, Brea, CA, USA) and the data were analyzed using Expo-3 v1.2B software (Beckman Coulter).

For cell cycle analysis, the cell cycle distribution of cells was assayed after 48 h by using flow cytometry to measure the DNA content of nuclei labeled with PI according to the manufacturer’s instructions (BD Pharmingen, BD BioSciences). Data acquisition and analysis were performed using an EC800 flow cytometer (Sony Biotechnology, Tokyo, Japan) with EC800 analysis software (Sony Biotechnology).

Immunoblot analysis

Whole-cell extracts from DPSCs were obtained using a lysis buffer (10× RIPA buffer; Cell Signaling Technology, Beverly, MA, USA) supplemented with 1 mM phenylmethanesulfonyl fluoride plus one tablet of protease inhibitor cocktail (Complete, ethylenediaminetetraacetic acid (EDTA)-free; Roche Diagnostics GmbH, Mannheim, Germany). Aliquots of cell lysates (50 μg protein) were separated by 8 % or 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membranes. The membranes were probed with primary antibodies, comprising anti-CD44 mouse monoclonal antibody, anti-phospho-Akt rabbit monoclonal antibody, anti-Akt rabbit monoclonal antibody, anti-phospho-GSK3β rabbit monoclonal antibody, anti-phospho-Smad1 rabbit monoclonal antibody, anti-Smad1 rabbit monoclonal antibody, anti-phospho-β-catenin (Ser552) rabbit monoclonal antibody, anti-phospho-β-catenin (Ser675) rabbit monoclonal antibody, anti-β-catenin rabbit monoclonal antibody, anti-phospho-p44/42 MAPK rabbit monoclonal antibody (all from Cell Signaling Technology, Danvers, MA, USA), anti-dentin sialophosphoprotein (DSPP) mouse monoclonal antibody (Santa Cruz Biotechnology Inc.), anti-dentin matrix protein-1 (DMP-1) rabbit polyclonal antibody, anti-ALP rabbit monoclonal antibody (both from Abcam PLC, Cambridge, UK), and anti-beta-actin antibody (Sigma-Aldrich) at the dilutions recommended by the manufacturers. Signals were detected using corresponding peroxidase-conjugated secondary antibodies (anti-rabbit IgG antibody or anti-mouse IgG antibody; Cell Signaling Technology), and signal bands were visualized by chemoluminescence (Clarity™ Western ECL substrate; Bio-Rad, Hercules, CA, USA). The membranes and images were developed with a ChemoDoc™ Imaging System (Bio-Rad).

Real-time polymerase chain reaction

Total RNA was purified using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and 600 ng of total RNA was used for reverse transcription with an iScript™ Advanced cDNA Synthesis Kit (Bio-Rad). For real-time polymerase chain reaction analysis, 1 μL of cDNA sample at 1:20 dilution, 1 μL each of forward and reverse primers (final, 500 nM), 7 μL of nuclease-free water, and 10 μL of SsoAdvanced SYBR Green Supermix (Bio-Rad) were used. The following primers were used: DMP-1 (GenBank ID: NM_004407.3) forward primer 5′-CCTGAGGATGAGAACAGCTCCA-3′ and reverse primer 5′-GATCTGCTGCTGTCTTGAGAGTCAC-3′; DSPP (GenBank ID: NM_014208.3) forward primer 5′-CCAGAGCAAGTCTGGTAACGGTAA-3′ and reverse primer 5′-GTCACTGCCTTCACTGTCACTGTC-3′; bone morphogenetic protein (BMP)-2 (GenBank ID: NM_001200.2) forward primer 5′-GGAACGGACATTCGGTCCT-3′ and reverse primer 5′-GGAAGCAGCAACGCTAGAAG-3′; BMP-4 (GenBank ID: NM_001202.3) forward primer 5′-TCACTGCAACCGTTCAGAGGTC-3′ and reverse primer 5′-CCAATCTTGAACAAACTTGCTGGA-3′; and GAPDH (GenBank ID: NM_002046.5) forward primer 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse primer 5′-TGGTGAAGACGCCAGTGGA-3′. Reaction conditions were one 5-min cycle at 95 °C, followed by 45 cycles of 95 °C for 10 s and 72 °C for 10 s. The reactions and relative quantification analyses were performed using a LightCycler 480 instrument (Roche Diagnostics, Indianapolis, IN, USA).

Signal blocking assays

DMH-1, a Smad1/5 inhibitor and SCH772984, a novel ERK1/2-specific inhibitor were purchased from Selleckchem.com (http://www.selleckchem.com/). LY294002, an Akt inhibitor, was purchased from Cell Signaling Technology. An anti-CD44 monoclonal antibody (Clone A3D8; Sigma-Aldrich) was used in neutralization assays.

DPSCs were pretreated with the inhibitors or CD44-blocking antibody for 30 min before stimulation with HA (10 μg/mL). After 30 min of stimulation, the cells were harvested to investigate the inhibition of phosphorylation for several signaling molecules. After 24 h, the odontoblastic differentiation markers DMP-1 and DSPP were evaluated by immunoblotting.

Statistical analysis

Data are presented as the mean ± SD and evaluated using one-way analysis of variance followed by Dunnett’s multiple comparison. Values of P < 0.05 were accepted as statistically significant.