2.1 Cell cultures

The human DPSCs were derived from impacted fully developed healthy third molars, which were surgically removed from healthy young patients in the clinic with the indication of ectopia (18–23 years of age, n = 7), and healthy premolars extraction from orthodontic patients (13–14, years of age, n = 2). Informed consent was obtained from each patient, and the study was approved by the institutional Medical Ethics. Pulp tissue was isolated from the crown and digested in phosphate-buffered saline (PBS) containing 3 mg/ml collagenase type I and 4 mg/ml dispase for 1 h at 37°C. Single-cell suspensions were obtained by passing the digested tissues through a 70-mm cell strainer (BD Falcon). Cell suspensions of dental pulp were then seeded into 10-cm culture dishes and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco-BRL; Life Technologies Inc.), 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in 5% CO₂. Cells were passaged at the ratio of 1:3 when they reached 85%–90% confluence. Cells between the third to fifth passages were used in the following experiments.

2.2 Odontogenic differentiation

Third-passage DPSCs (2 × 10⁴ cells/dish) were seeded in 35-mm culture dishes (Costar) and cultured with odontogenic differentiation medium (ODI) consisting of α-MEM supplemented with 10% FBS, 50 mg/ml α-ascorbic acid, 10 mmol/L β-glycerophosphate, 10 nmol/L dexamethasone (Sigma-Aldrich) and 0.3 mg/ml glutamine, 100 units/ml penicillin G, 100 mg/ml streptomycin, respectively, for 0, 3, 7, and 14 days with or without TNF-α (1, 10, 50, 100 ng/ml), replacing the medium every 2 days. TNF-α was added every 3 days. At the indicated times, the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min before staining with Alizarin red S (Sigma) and alkaline phosphatase (JianCheng) at room temperature.

DPSCs were plated at a density of 2 × 10⁴ cells/cm² and cultured in an odontogenic medium with or without TNF-α (10 ng/ml) for 3 days. For inactivation of ERK signaling pathway, DPSCs were supplemented with 50 mM PD98059 (PD, a potent and selective inhibitor of MEK1, which is a kinase upstream of ERK; Sigma) for 3 days.

2.3 Alizarin red S and ALP staining

Third-passage DPSCs were plated to six-well plates (Falcon) and cultured in different dishes supplemented with ODI. DPSCs were fixed with 4% PFA for 1 h and washed with PBS. Cells were then stained with 40 mmol/L alizarin red S (pH 4.2) for 10 min under conditions of gentle agitation. After decoloration and air-drying, culture plates were evaluated by light microscopy using an inverted microscope. To quantify the red dye, the stain was solubilized by shaking with 10% cetylpyridinum chloride for 20 min and the absorbance was measured at a wavelength of 570 nm. The alkaline phosphatase (JianCheng) staining was used to estimate the degree of extracellular matrix calcification. DPSCs were subjected to alkaline phosphatase (ALP) staining using the ALP assay kit according to the manufacturer's instructions.

2.4 Western blot analysis

Western blot analysis was performed according to a previous study (Tsugawa et al., 2003). In brief, cells were washed twice with ice-cold PBS and lysed in buffer containing 50 mM Tris (PBST), 150 mM NaCl, 2% sodium dodecyl sulfate (SDS), and a protease inhibitor mixture. After centrifugation at 12,000 rpm for 12 min, protein concentrations were determined using the Bradford Microassay (Bio-Rad). The resulting supernatant (50 mg of protein) was subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were then transferred onto polyvinylidene difluoride membranes (Amersham Biosciences, Amersham, UK). The samples were incubated with the primary antibodies (1:400) overnight at 4°C and the HRP-conjugated secondary antibodies (1:1000) for 2 h. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control for the cytoplasmic and nuclear proteins. Concomitantly, the primary antibodies (Santa Cruz) used in this study were as follows: GAPDH (anti-mouse), DMP-1 (anti-mouse), DSPP (anti-mouse), ALP (anti-mouse), RICK (anti-mouse), IκBα (anti-rabbit), p-IκBα (anti-mouse), ERK (anti-mouse), p-ERK (anti-mouse), JNK (anti-mouse), p-JNK (anti-mouse), p38 (anti-mouse), p-P38 (anti-mouse). Western blot band area/intensity was measured as previously described (Huang et al., 2019). Bands area/intensity for all proteins was measured by Image J software (Wayne Rasband, National Institutes of Health).

2.5 Immunofluorescent staining

DPSCs were fixed with 4% PFA for 1 h, washed with PBST, and blocked for 30 min in PBST supplemented with 10% FBS. Cells were then incubated with one of the following primary antibodies (1:100; Santa Cruz) in the same solution overnight at 4°C Vimentin (anti-mouse), p65 (anti-rabbit), STRO-1 (anti-mouse), and CK-14 (anti-mouse). Cells were washed and incubated in secondary antibodies for 2 h at room temperature. Nuclei were stained with DAPI (1:800; Sigma). The cells were finally examined by a Leica fluorescence microscope (Germany).

2.6 RICK RNA silencing

The sequences of the sense and antisense strands of the human RICK siRNA were as follows: 5′-AGAAUAUCCUAUUGGACAATT-3′ (sense), 5′-UUGUCCAAUAGGAUAUUCUTT-3′ (antisense). The siRNA was commercially synthesized (Genepharma). For controls, scrambled RNA oligonucleotides were used. For each well, 33.3 nM of each were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After incubation for 6 h, the medium was replaced with DMEM containing 10% FBS. Transfected cells were used for the subsequent experiments 48 h after transfection.

2.7 Reverse-transcription polymerase chain reaction (RT-PCR) analysis

Total RNA was extracted from DPSCs using the TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. RNA (2 μg) was reverse-transcribed into first-strand cDNA (Gibco BRL). PCR amplification was performed using the following primer sets: β-actin, F, 5ʹ-CGTTGACATCCGTAAAAGAC-3ʹ; R, 5ʹ-TGGAAGGTGGACAGTGAG-3ʹ. RICK, F, 5ʹ-CCATCCCGTACCACAAGCTC-3ʹ; R, 5ʹ-GCAGGATGCGGAATCTCAAT-3ʹ. p65, F, 5ʹ-CAAGCGAGGAGGGGACGTG-3ʹ; R, 5ʹ-CCCCCAGAGCCTCCACCC-3ʹ. DSPP, F, 5ʹ-GGAGCCACAAACAGAAGCAACA-3ʹ; R, 5ʹ-TGGACAACAGCGACATCCTCAT-3ʹ. DMP-1, F, 5ʹ-CAGGAAGAGGTGGTGAGTGAGT-3ʹ; R, 5ʹ-TGGATTCGCTGTCTGCTTGCT-3ʹ. GAPDH, F, 5ʹ-CCAGAACATCATCCCTGCCTCT-3ʹ; R, 5ʹ-GACGCCTGCTTCACCACCTT-3ʹ. PCR reactions were carried out using an ABI Prism 7900HT Sequence Detection System (Applied Biosystems) with the following program: 95°C for 15 min, 94°C for 15 s, and 57°C for 30 s for 40 cycles. The mRNA levels of target genes were calculated after normalization to β-actin mRNA.

2.8 Statistical analysis

Data are shown as mean ± SEM from at least three independent experiments, each performed with triplicate samples. The differences between control and the experimental groups were determined by using one-way analysis of variance (ANOVA), multi-way ANOVA and Student's t test for post hoc comparisons. All statistical analyses were performed using SPSS 20.0 software. *p < .05 was considered statistically significant.

3.1 TNF-α influences the odontogenic differentiation of DSPCs

DPSCs were treated with different concentrations of TNF-α in ODI to examine the reparative responses of DPSCs to inflammatory stimuli. After 7 days of incubation, the activity of ALP was inhibited by TNF-α at high concentrations (50, 100 ng/ml) while the odontogenic potential of DPSCs was promoted at low concentrations (1, 10 ng/ml), especially at 10 ng/ml (Figure 1a,b; *p < .05). The expression of the mineralization-associated proteins DMP-1 and DSPP were tested during the 7 days of incubation. Western blot analysis showed that high concentrations (50, 100 ng/ml) of TNF-α downregulated the expressions of DMP-1 and DSPP in DPSCs compared with the control group (0 ng/ml TNF-α), while in low concentrations (1, 10 ng/ml) of TNF-α the expressions of DMP-1 and DSPP increased significantly, especially in 10 ng/ml (Figure 1c,d; *p < .05). RT-PCR analysis showed that low concentrations (1, 10 ng/ml) of TNF-α upregulated the expressions of DMP-1 and DSPP in DPSCs compared with the control group (0 ng/ml TNF-α), especially in 10 ng/ml (Figure 1e,f; *p < .05).

To investigate the effect of TNF-α on odontogenic differentiation, DPSCs were stimulated with TNF-α (10 ng/ml) for 0, 3, 7, and 14 days. ALP staining showed that TNF-α accelerated odontogenic differentiation of DPSCs time-dependently (Figure 2a,b; *p < .05). The results of Alizarin red S staining also indicated that TNF-α (10 ng/ml) promoted the formation of mineralized nodules (Figure 2c,d; *p < .05). The western blot analysis also showed that TNF-α (10 ng/ml) promoted the expressions of DMP-1 and DSPP at 3 days of odontogenic differentiation (Figure 2e–g; *p < .05).

3.2 The expression of RICK increased in the odontogenic differentiation of DPSCs

The expression of RICK has also been tested to investigate the effect during odontogenic differentiation. TNF-α (10 ng/ml) was added into the medium for 3 days. Western blot analysis indicated that the level of RICK protein was higher after TNF-α treatment (Figure 3; *p < .05).

3.3 ERK/p-ERK signaling pathway was activated during the odontogenic differentiation of DPSCs after being treated with TNF-α

DPSCs were cultured with ODI or ODI + TNF-α (10 ng/ml) for 3 days. We found that p-ERK increased significantly in the TNF-α treated group. The JNK and p38 pathways were not activated in any of the groups (Figure 4a,b; *p < .05). To get a better understanding of the ERK pathway in the differentiation of DPSCs, we tested the phosphorylation of ERK at 0, 3, 7, and 14 days. The phosphorylation of ERK increased significantly after the TNF-α treatment from Day 3 (Figure 4c,d; *p < .05). Furthermore, we investigated whether phosphorylation of IκBα (p-IκBα) participated in the process. Western blot analysis showed that the phosphorylation of IκBα did not change significantly in the differentiation of DPSCs (Figure 4e,f; *p < .05) and the expression of p65 and phosphorylation of p65 did not change during the differentiation of DPSCs (Figure 4g–j; *p < .05). Moreover, immunocytochemical staining found that TNF-α treatment did not change the level of p65 in the nucleus (Figure 4k; *p < .05). These results showed that ERK/p-ERK signaling pathway was activated during the odontogenic differentiation of DPSCs, while the NF-κB signaling pathway was not.

3.4 Inhibition of the ERK pathway suppressed the odontogenic differentiation of DPSCs treated with TNF-α

To test the role of the ERK signaling pathway in odontoblastic differentiation of DPSCs, the cells were treated with the ERK signaling pathway inhibitor PD98059. The DPSCs were cultured with ODI + TNF-α for 3 days, and then PD98059 was added to the experimental group. The results showed that, in the presence of PD98059, the phosphorylation of ERK was inhibited and the expression level of DMP-1 and DSPP was reduced (Figure 5a–c; *p < .05).

3.5 Inhibition of RICK reduced the odontogenic differentiation of DPSCs

SiRNAs have been used to knock down the expression of RICK. The silencing effect of siRNA was verified by western blot and RT-PCR analyses (Figure 6a–d; *p < .05). All DPSCs were cultured in an odontogenic differentiated medium for 3 days with TNF-α (10 ng/ml). The expression of ALP, DMP-1, and DSPP were downregulated significantly when RICK was inhibited (Figure 6e,f; *p < .05). Alizarin red S staining also indicated that mineralized nodules were reduced significantly when RICK was inhibited (Figure 6g,h). Furthermore, western blot has also shown that the phosphorylation of ERK was significantly lower in the group of siRICK than that in the control group (Figure 6i,j; *p < .05).