Normal impacted third molar tooth germs were collected from patients (n=10; 16–18 years old) undergoing orthodontic treatment in the West China Hospital of Stomatology (Chengdu, China), following the provision of written informed consent. All the experiments were performed in accordance with the ethical protocol approved by the Ethics Committee of Sichuan University (Chengdu, China). The DFCs and DPCs were isolated and cultured, as previously reported (15,16). In brief, the DFCs and DPCs were isolated from the dental follicle and dental pulp, and incubated in α-minimum essential medium (α-MEM; Gibco; Thermo Fisher scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher scientific, Inc.), 100 U/ml penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 100 mg/ml streptomycin (Sigma-Aldrich) in humid conditions at 37°C of 5% CO₂ in air. The medium was renewed every three days.

hDFCs and hDPCs were seeded at a density of 3×10³ into each well of a 24-well plate overnight, respectively. The cells were then washed with phosphate-buffered saline (PBS) three times, fixed with 4% cold polyoxymethylene (Sigma-Aldrich) for 15 min and permeabilized by 0.5% Triton (Sigma-Aldrich) for 10 min at room temperature. Following rinsing three times with PBS, the cells were blocked with 1% bovine serum albumin (Shanghai Zhangyun Chemical Co., Ltd., Shanghai, China) diluted in PBS for 30 min at 37°C. The cells were then treated with primary antibodies for 2 h in a humidified environment at 37°C. Following being washed three times with PBS, the cells were treated with secondary antibodies for 1 h in a humidified environment at 37°C. Following three rinses with PBS, the nuclei were counterstained with 100 ng/ml DAPI blue (Beyotime Institute of Biotechnology, Shanghai, China) for 3 min in the dark. The primary antibodies used were as follows: Anti-vimentin (mouse IgG; 1:200; OMA1-06001; Thermo Fisher Scientific, Inc.), anti-CK14 (mouse IgG; 1:200; MAB3232; EMD Millipore, Billerica, MA, USA) and anti-Stro-1 (monoclonal mouse IgG; 1:100; FAB1038F; R&D Systems, Minneapolis, MN, USA). Secondary antibodies were conjugated to Alexa FluoR 488 goat anti-mouse (A11001) or Alexa FluoR 555 goat anti-mouse (1:500; A21422; Invitrogen; Thermo Fisher Scientific, Inc.). Images were captured and analyzed under a confocal imaging system (Olympus FV1000; Olympus Corporation, Tokyo, Japan).

To characterize the immunophenotype of the hDFCs and hDPCs, flow cytometric analysis was used to measure the expression of mesenchymal stem cell surface markers. Cells were trypsinized (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) and incubated at 37°C with phycoerthyn (PE)-conjugated cluster of differentiation (CD)29 integrin β1 (555443); fluorescein isothiocyanate (FITC)-conjugated CD31 (555445), which is also known as platelet-endothelial cell adhesion molecule-1; FITC-conjugated CD34 (555821), which is also known as hematopoietic progenitor cell antigen; FITC-conjugated CD44 (555478), which is also known as hyaluronate/lymphocyte homing associated cell adhesion molecule; PE-conjugated CD90 (Thy-1/Thy-1.1) (555596); and PE-conjugated CD166 (559263), which is also known as activated leucocyte cell adhesion molecule and is a mesenchymal stem cell marker. All antibodies were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Flow cytometry was performed using a Beckman Coulter Cytomics FC 500 MPL system (Beckman Coulter, Fullerton, CA, USA).

According to previous studies, a total of 5×10⁴ hDFCs and hDPCs were seeded into each well of a 6-well plate at 37°C overnight, respectively. At 50% cell density, 1 ml α-MEM (without FBS), containing 1.5 µl of green-fluorescence protein-labeled kif3a short hairpin (sh) RNA lentivirus (NeuronBiotech Co., Ltd., Shanghai, China) and 0.5 µl polybrene (Sigma-Aldrich), were added into each well. Following incubation for 6 h at 37°C, the supernatant was removed and replaced with 2 ml fresh α-MEM supplemented with 10% FBS. After 3 days at 37°C, the infected cells were observed and images were captured under a fluorescence microscope (Leica Optical, Leica Microsystems GmbH, Wetzlar, Germany) (17).

For osteoblastic induction, a total of 1×10⁵ hDFCs and hDPCs at passage three were seeded into each well of a 6-well plate overnight respectively. These cells were then cultured at 37°C in osteoblastic inductive medium (α-MEM supplemented with 10% FBS, 10 mmol/l β-glycerophosphate (Sigma-Aldrich), 0.2 mmol/l ascorbic acid (Sigma-Aldrich), 10 nmol/l 1,25-dihydroxyvitamin D3 and 100 nmol/l dexamethasone (Sigma-Aldrich) for 3–30 days, depending on the subsequent experiments. The inductive medium was replaced every three days.

As previously described (18), following fixation with 4% paraformaldehyde (Sigma-Aldrich) for 30 min at room temperature, the cultures were incubated in 0.1% Alizarin Red solution (Sigma-Aldrich) in Tris HCl (pH 8.3; 0.5–1 ml per well; Kelong Chemical Co., Ltd., Chengdu, China) in a 6-well plate for 30 min at room temperature.

The control and kif3a-knockdown hDFCs and hDPCs were seeded (3×10³) into each well of a 24-well plate overnight, respectively. The methods used were the same as described above. The primary antibodies used were anti-acetylized α-tubulin (mouse IgG; 1:500; ab24610; Abcam). Secondary antibodies were conjugated to Alexa FluoR 555 (goat anti-mouse; 1:500; Invitrogen; Thermo Fisher Scientific, Inc.). Images were captured and analyzed under a confocal imaging system (Olympus FV1000; Olympus Corporation).

The control and kif3a-knockdown hDFCs and hDPCs were harvested from each well of a 6-well plate following 3 days osteoblastic induction in preparation for RNA isolation. Total RNA was obtained using RNAiso™ Plus (Takara Bio Inc., Otsu, Japan). cDNAs were synthesized by reverse transcription using the extracted RNA with PrimeScript^® Perfect Real Time reagent kit (Takara Bio., Inc.). The relative expression levels of genes were quantified using qPCR with SYBR^® Premix Ex Taq™ (Perfect Real Time; Takara Bio, Inc.) in a 10 µl total reaction volume using an ABI Prism 7300 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The qPCR conditions were as follows: Initial cycle was 95°C for 30 sec, 40 cycles at 95°C for 5 sec and 60°C for 30 sec; the final cycle was 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec. Dissociation curves were used to confirm primers specificity. D-glyceraldehyde-3-phosphate (GAPDH) was used as an internal reference, and relative mRNA levels were quantified using the 2^−ΔΔCq method (19). The primer sequences used in the present study for GAPDH, dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP-1), osteocalcin (OCN), Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteopontin (OPN), axis inhibition protein 2 (Axin2) and β-catenin, lymphoid enhancer-binding factor 1 (Lef1) are listed in Table I. All experiments were performed three times.

Control and kif3a-knockdown hDFCs and hDPCs were harvested from each well of a 6-well plate following 1 week of osteoblastic induction prior to protein extraction. Cells were rinsed three times in PBS prior to being lysed using cell lysis buffer, containing 0.5% 100 mmol/l phenylmethanesulfonyl fluoride, 0.1% protease inhibitor and 0.5% phosphatase inhibitor, on ice. Proteins in the supernatant were measured by modified Bradford Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). An equal quantity of total protein (50 mg) was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (Invitrogen; Thermo Fisher Scientific, Inc.). Following blocking with non-fat milk in PBS with Tween 20 (PBST) for 1 h at room temperature, the membranes were incubated with primary antibodies at 4°C overnight. The membranes were subsequently washed three times with PBST for 10 min and incubated with secondary antibodies at room temperature for 1 h, respectively. Membranes were visualized with a chemiluminescent horseradish peroxidase (HRP) substrate (EMD Millipore) using a ChemiDoc XRS system (Bio-Rad Laboratories, Inc.). The primary antibodies used for western blot analysis were as follows: Anti-β-actin (1:500; ab8227), anti-ALP (1:500; ab67228), anti-DMP1 (1:500; ab103203), anti-kif3a (1:500; ab133587), anti-bone sialoprotein (BSP; 1:200; ab52128), anti-OPN (1:1,000; ab8448), anti-Runx2 (1:500; ab76956), anti-phosphorylated glycogen synthase kinase 3β (GSK3β; 1:1,000; ab32391; Abcam), anti-Lef1 (1:500; ab85052) and anti-active β-catenin (1:4,000; 8814; Cell Signaling Technology, Inc., Danvers, MA, USA). The secondary antibodies were HRP-conjugated AffiniPure goat anti-mouse IgG (H+L; 1:10,000; ZB-2305) and HRP-conjugated AffiniPure goat anti-rabbit IgG (H+L; 1:10,000, ZB-2301; ZSGB-Bio, Beijing, China). All experiments were performed three times.

The data in the present study were collected from three different samples of the same cell sample in triplicate. Values are presented as the mean ± standard deviation. Statistical significance was evaluated using one-way analysis of variance using SPSS software (Version 10.0; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The present study identified the characterization of hDFCs and hDPCs as dental mesenchymal stem cells using cellular immunofluorescent detection. The two cell types were positive for the vimentin and Stro-1 mesenchymal stem cells markers, but negative for the CK-14 epithelial cell marker (Fig. 1). The immunophenotypic characterization was performed using flow cytometry. The DFCs and DPCs were positive for the CD29, CD44, CD90 and CD166 mesenchymal stem cell markers. However, the two types of cell were negative for the leucocyte precursor markers, CD31 and CD34, suggesting a lack of cells of hematopoietic and angiogenic lineages (Fig. 2). These data indicated that the DPC and DFC cell lineage were pure mesenchymal cells and maintained stem cell-like properties.

The present study then investigated the existence of primary cilia in the hDFCs and hDPCs. The primary cilia were immunostained using acetylated α-tubulin antibody and observed under a laser scanning confocal microscope, as described previously (14) (Fig. 3A). The expression of kif3a was then determined using western blot analysis, and the target bands were detected with purified anti peptide antibodies in the two cell cultures (Fig. 3B) Lentiviruses expressing four different shRNAs specific for kif3a were transfected into the cells, as described above, and the shRNA with the most marked effect was selected. The efficiency of kif3a suppression was determined using immunoblot analysis (Fig. 3C). Following kif3a knockdown, the number of primary cilia were substantially reduced in the two cell cultures (Fig. 3D–G).

To examine the association between the loss of primary cilia caused by kif3a knockdown and osteoblastic differentiation in hDFCs and hDPCs, the present study performed Alizarin Red staining following treatment with osteoblastic inductive medium. Following 10 days induction, the kif3a-knockdown cells formed fewer mineralized nodules, compared with the cells in the control group, in the hDFCs and hDPCs. This difference became more significant following 30 days induction (Fig. 4A and B).

The present study then assessed the expression levels of osteoblastic genes using RT-qPCR (Fig. 4C and D), and protein expression levels using western blot analysis (Fig. 4E). The results showed that the osteoblastic genes and proteins, including ALP, BSP, DMP1, OCN and Runx2, were significantly downregulated following kif3a knockdown in the hDFC and hDPC cells.

To examine the mechanisms by which the loss of kif3a causes alterations in hDFC and hDPC functions, the present study examined whether the Wnt signaling pathway was involved in this suppression, which has a close association with tooth development (20,21). Following treatment with osteoblastic medium and recombinant Wnt3a for 6 days, the mRNA and protein expression levels of osteoblastic markers of hDPCs and hDFCs, were assessed, respectively. Significant increases in the mRNA (Fig. 5A and B) and protein (Fig. 5C) expression levels of osteoblastic markers were found in the Wnt3a-treated wild-type cells, compared with the control cells. By contrast, there was no significant increase in the kif3a-knockdown cells (Fig. 5A–C). In accordance with these results, the kif3a-knockdown cells formed fewer mineralized nodules, compared with the control group cells, in the hDFCs and hDPCs following culture in osteoblastic medium for 14 days and treatment with recombinant Wnt3a (Fig. 5D and E). This confirmed that the loss of kif3a suppressed osteoblastic differentiation in the hDFCs and hDPCs by the inhibition of Wnt signaling.

The present study further examined whether the loss of primary cilia of the hDFCs and hDPCs following inhibition of kif3a leads to interference in the canonical Wnt pathway. Using RT-qPCR and western blot analyses, it was found that the expression levels of canonical Wnt pathway factors, including Axin2, β-catenin and Lef1, were significantly lower in the kif3a-knockdown cells, compared with the control cells, at the transcriptional (Fig. 6A and B) and translational (Fig. 6C) levels following treatment in osteoblastic medium for 6 days. However, the addition of 100 ng/ml recombinant human Wnt3a protein to the osteoblastic hDFC and hDPC cultures resulted in a significant increase in Wnt signaling activity, at the mRNA (Fig. 6D and E) and protein (Fig. 6F) expression levels, in the control cells. By contrast, there was no significant increase in the kif3a-knockdown cells (Fig. 6D–F), suggesting that the loss of kif3a impaired the Wnt signaling response in the hDFCs and hDPCs. This result confirmed that kif3a, at least part, regulated hDFC and hDPC osteoblastic differentiation by the Wnt signaling pathway.