Amphiregulin (AREG) (262-AR-100) was obtained from R&D Systems (R&D, CA, USA). The specific inhibitors of ERK kinase, p38 kinase, JNK kinase, and AKT kinase are U0126, SB203580, SP600125, and LY294002, respectively, were all obtained from Cell Signaling Technology (Danvers, MA, USA). AREG lentivirus kits were purchased from Hanbio Biotechnology (Hanbio, China).
Freshly extracted sound teeth were collected from patients (18–22-years-old), under the approval of the Ethics Committee of the Fourth Military Medical University (FMMU), Xi’an, China with informed consent. The hDPSCs were isolated, cultured, and characterized as previously described [19, 20]. Briefly, the pulp tissue was dissected and digested with 3 mg/mL type I collagenase and 4 mg/mL dispase (Sigma-Aldrich, St Louis, MO, USA) for 45–60 min at 37℃. Single-cell suspensions were cultured in 60-mm culture dishes and maintained in a-minimum essential medium (α-MEM; Invitrogen, Carlsbad, CA, USA) with 10% (v/v) fetal bovine serum (Gibco-BRL, Grand Island, NY, USA), 100 units/mL penicillin-G, and 100 mg/mL streptomycin (Invitrogen) in a humidified atmosphere with 5% CO2 at 37℃. Single-cell clones of DPSCs were isolated and passaged as previously described . DPSCs were grown in a 5% CO2 incubator at 37 °C, and cultures at passages 3–5 were used for all studies.
Multi-lineage differentiation in vitro
Cells were cultured in 6-well plates in osteo/odontogenic induction medium containing 50 mg/mL ascorbic acid, 10 mM β-glycerophosphate, and 10 nM dexamethasone (all from Sigma-Aldrich) with 10% FBS for 14 days. The cells were washed twice in PBS and fixed with 4% polyoxymethylene (Sigma-Aldrich, St Louis, MO, USA) for 15 min. The cells were then stained with Alizarin Red S (Sigma-Aldrich, St Louis, MO, USA). For adipogenic induction, the cells were cultured in adipogenic induction medium containing 1 mM IBMX, 0.4 mM indomethacin, 0.2 μM dexamethasone, 0.02 mg/mL insulin, 100 μg/mL streptomycin, 100 U/mL penicillin, and 10% FBS in α-MEM medium) for 21 days. Cultures were stained with 0.3% (w/v) Oil-Red O (Sigma-Aldrich)/60% isopropanol reagent for 60 min. Finally, cultures were washed three times in water prior to analysis.
Flow cytometric analysis of stem cell surface markers
DPSCs (2 × 105 cells) were digested by trypsinization and harvested. Cells (1 × 106/100 µL) were resuspended and incubated with fluorescein isothiocyanate (FITC)-coupled anti-human monoclonal antibodies against CD105-FITC, CD34-PE, CD45-PE, CD90-PE, CD29-PE, and CD146-PE (1: 100 dilution; BD Biosciences, San Jose, CA, USA) in PBS with 3% FBS for 1 h in the dark at room temperature. Finally, the samples were analyzed on a flow cytometer (FACS Calibur; BD Biosciences, Franklin Lakes, NJ, USA) using CellQuest PROTM software (BD Biosciences).
Flow cytometric analysis of proliferation and apoptosis in hDPSCs
Third-passage DPSCs (3 × 106 cells/dish) were inoculated in 60 mm dishes. The experimental groups were treated with 0.01 μg/mL and 0.1 μg/mL AREG for 16 h, respectively. The control group received no AREG treatment. After removal from the dishes, cells were washed three times in pre-cooled phosphate-buffered saline (PBS) and were fixed in 75% ice-cold ethanol. A flow cytometer (BD Biosciences, San Jose, CA, USA) was used to monitor the changes in the G0/G1, S, and G2/M phases of the cell cycle and to calculate the cell proliferation index (PI = G2/M + S). For the measurement of apoptosis, the AREG-treated DPSCs were harvested and washed as above, and 500 μl of cells was diluted with 1 × Annexin V Binding Buffer working solution according to the instructions of the apoptosis kit (KeyGENBioTECH, China). Then, 5 μl Annexin V-APC and 5μL 7-ADD staining solution were added to the cells and the number of apoptotic cells was detected by FCM.
Cell counting kit-8 assay (CCK-8)
Five thousand DPSCs per well were seeded and cultured in 96-well plates with a range of concentrations of AREG (0-1 μg/mL). In a separate experiment, DPSCs were incubated with 20 µL CCK-8 reagents (KeyGENBioTECH, China) at 37 °C for 2 h for 1, 3, 5, and 7 days. Finally, absorbances at 450 nm were read in a microplate reader (ModelELX 808; Bio-Tek, Winooski, VT, USA).
Alizarin Red S staining and quantitation
DPSCs (4 × 105 cells/well) were seeded into 6-well plates and cultured in either control medium or osteo/odontogenic induction medium with AREG for 14 days. At specified times, the cells were fixed in 1 ml of 4% paraformaldehyde for 30 min at room temperature. After three washes with distilled water, 0.1 g/ml Alizarin Red S (ARS) (Sigma-Aldrich) was used to stain the cultures for 10 min at room temperature. The unbound stain was removed by washing with deionized water until the discarded liquid appeared colorless. Five hundred microliters of water were added to each dish to ensure the cells remained hydrated. Cells were observed under an Olympus inverted microscope (Tokyo, Japan). For quantitation, alizarin red S stain dissolved in 10% cetylpyridinium chloride (CPC) (Sigma-Aldrich) was added to the cells and the absorbance at 450 nm was read in the microplate reader.
In vivo studies
All animal surgical procedures were approved by the Animal Care Committee and the Institutional Review Board (IRB) for Human Subjects Research of the Fourth Military Medical University. The high-stiffness three-dimensional (3D) nanofibrous gelatin (NF-gelatin) scaffolds were a kind gift from Prof. Tiejun Qu . Initially, the NF-gelatin scaffolds were placed in 70% alcohol for half an hour and then washed three times in sterile PBS to remove residual ethanol. Before the seeding of human DPSCs (5 × 105), the scaffolds were soaked in α-MEM containing 10% FBS. The cell-scaffold composites were cultured in α-MEM supplemented with 10% FBS for 24 h on an orbital shaker (Orbi-shaker™, Benchmark, USA) in an incubator with 5% CO2 at 37℃. Subsequently, the cell-scaffold composites were stimulated with 0.1 μg/mL AREG in osteo/odontogenic induction medium for 7 days. Controls received no AREG exposure. The medium was changed every other day. After 7 days, the cell-scaffold composites were implanted subcutaneously on the dorsal surfaces of immune-compromised nude mice (nu/nu, 6–8 weeks old). After 4 weeks, the mice were euthanized by an anesthetic overdose and the tissue growths were then harvested. Tissue samples were immediately fixed in 4% paraformaldehyde overnight. The samples (n = 4 in each group) were scanned and analyzed using a micro-CT (eXplore Locus SP micro-CT; GE Healthcare, USA) as previously described  and the 3D micro-architectural properties of specimens were evaluated using analysis software (MicroView; GE Healthcare). After decalcification in 17% ethylenediamine tetra-acetic acid, hematoxylin–eosin (H&E), Masson’s trichrome, and von Kossa staining were used for histological observation.
Overexpression and knockdown of AREG in DPSCs
AREG-green fluorescent protein lentivirus kits and their respective control kits were purchased from Hanbio (China). DPSCs were transfected with AREG lentivirus according to the manufacturer's instructions. In brief, third-passage DPSCs were inoculated in 12-well plates at 0.5 × 105/mL and infected for 4 h, after which 0.5 mL fresh complete medium was added. After a further 24-h infection period, the medium was replaced with 1 mL fresh medium and after a further 72 h, the transfection rate was examined under a fluorescence microscope (DMI8, Leica, German). Puromycin was used to select stably transfected cells with the puromycin concentration determined in a preliminary experiment. For simplicity, AREG-overexpressing and AREG-silenced cells were referred to as AREG (+) and AREG (−), respectively. The effects on the oriented differentiation of DPSCs were examined by ARS and Western blot analysis as above and described above and below.
Western blot analysis
DPSCs were cultured in serum-free medium for 24 h and were treated with 0.1 µg/mL AREG for 0, 30, 60, and 90 min in the presence or absence of the specific inhibitors U0126 (25 µM), SP600125 (25 μM), SB203580 (25 μM), and LY294002 (10 μM). Protein extraction from the cells, SDS-PAGE, and blotting was performed as previously described . After incubating with secondary antibody (1:4000; Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h, the protein bands were imaged by using an enhanced chemiluminescence system (Amersham, Piscataway, NJ, USA).
All experiments were repeated separately in triplicate or quintuplicate. Data are expressed as means ± SD. For statistical processing, we used SPSS software 16.0 (version 16.0; SPSS, Chicago, IL, USA). Inter-group differences were compared by the ONE test. P < 0.05 was considered to be statistically significant.