All animal procedures were ethically approved by the Ethics Committee of the Guanghua College of Stomatology, Sun Yat-sen University [Guangzhou, China; approval no. ERC-(2014)-21]. Sprague-Dawley rats (Laboratory Animal Center, Sun Yat-sen University) were housed in a specific pathogen-free laboratory animal center at a temperature of 20-26°C, 35 pa higher than the standard atmosphere, light-dark cycle 12/12 h, and fed using standard rodent chow (ad libitum access to food and water). A total of 70 rats (1, 3, 5, 7, 9, 11 and 13 days old; 10 rats for each group, nonsexual selection with 38 female mice and 32 male mice, weight 5-20 g) were obtained from the Laboratory Animal Center and euthanized by cervical dislocation immediately.

Mandibles containing the first molars were dissected, fixed in 4% paraformaldehyde at 4°C for 24 h, decalcified in methanoic acid for 3-7 days and embedded in paraffin. Furthermore, serial 5-mm sections were then cut, collected on 3-aminopropyltriethoxysilane-coated slices and air dried. Mesiodistal sections of mandibular tissues were deparaffinized with two washes in dimethyl benzene for 10 min succeeded by rehydration with serial dilutions (95, 90, 80 and 70%) of ethanol for 5 min respectively. Sections were heated in a water bath (10 min at 95°C) in citrate buffer for antigen retrieval. Following incubation with blocking buffer consisting of 1% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at room temperature for 1 h, the slides were incubated overnight at 4°C with the primary antibody Nkd2 (1:100; cat. no. sc163145; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or with PBS as a negative control. Subsequent to washing using PBS, the slides were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody rabbit anti-goat IgG (1:200; cat. no. A21030; Abbkine Scientific Co., Ltd., Wuhan, China) at room temperature for 1 h and images were obtained using an electron optical microscope (magnification, x200; Zeiss AG, Oberkochen, Germany) as previously described (23). Subsequent to image acquisition, image analysis was performed using ImageJ 1.48 version (National Institutes of Health, Bethesda, MD, USA), as previously described (24). Quantification of the immunoscore of all images was blinded for histopathological diagnosis. A specific brown color was selected as the standard for all positive image scores as previously described (24). The mean gray value of the negative group was used to set the threshold adjustment, and the mean gray value of each image was obtained. The results were evaluated using SPSS software version 16.0 (SPSS, Inc., Chicago, IL, USA) with a paired Student's t-test (each group was compared with the negative group), and the data are expressed as the mean ± standard deviation, with P<0.05 considered to indicate a statistically significant difference.

DFs were gently isolated from the first mandibular molars of Sprague-Dawley rats (5 to 7 days old) and digested with 0.1% collagenase type I (Collagen-I) and 10 U/ml dispase (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 30 min at 37°C to obtain rDFSCs, as previously described (7). To ensure a uniform cell population, the rDFSCs were cultured in α-modified Eagle's medium (α-MEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin for 3 to 4 passages in a 37°C incubator with 5% CO₂. rDFSCs at passages 3-5 were used for all experiments.

rDFSCs were cultured until sub-confluent (>80%) in standard cell culture medium prior to the induction of cementoblast/osteoblast differentiation, and then stimulated with osteoblast induction medium containing 10 mmol β-glycerophosphate (Sigma-Aldrich; Merck KGaA), 0.2 mmol ascorbic acid (Sigma-Aldrich; Merck KGaA) and 100 nmol dexamethasone (Sigma-Aldrich; Merck KGaA). Differentiation of rDFSCs was determined by Alizarin Red staining and the expression of osteoblast markers.

rDFSCs were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 20 min. The cells were blocked with 1% bovine serum albumin at 4°C for 1 h and then incubated overnight at 4°C with an anti-rat Nkd2 antibody (1:100; cat. no. sc163145; Santa Cruz Biotechnology, Inc.). Fluorescein isothiocyanate-conjugated immunoglobulin G secondary antibody (1:1,000; cat. no. E031230; EarthOx, LLC, Millbrae, CA, USA) was added to the cells at 37°C for 2 h as the secondary antibody prior to nuclear staining with 10 µg/ml DAPI at 37°C for 2 min. The cells were finally washed with PBS 3 times and examined under a confocal laser-scanning microscope (magnification, ×400; Zeiss AG).

rDFSCs (passages 3-5) were seeded in 6-well culture plates with a density of 10⁵/ml and 200 ml cell suspension per well, and cultured until sub-confluent (>80%) prior to transfection with 50 nmol/l siRNA targeting the rat Nkd2 gene (si-Nkd2) using Lipofectamine RNAiMax (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 8 h according to the manufacturer's protocol. Cells transfected with non-targeting siRNA (si-NC) or cells incubated without siRNA (mock transfection) were used as negative controls. The oligonucleotides si-Nkd2 (forward, 5′ GCACUCCAGUGUGAUGUCUdTdT 3' and reverse, 3′ dTdTCGUGAGGUCACACUACAGA 5′) and si-NC were synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China).

DFSCs were seeded in 96-well culture plates at a density of 4×10³/ml, 200 µl cell suspension per well and transfected with si-Nkd2 or si-NC for 24, 48 or 72 h. Mock-transfected cells were used as negative controls. CCK-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) reagent was added to each well at 10% of the volume, and the samples were incubated at 37°C in the dark for 2 h. Optical density was measured at 450 nm using a microplate reader (Tecan Group, Ltd., Mannedorf, Switzerland).

DFSCs transfected with si-Nkd2 or si-NC for 72 h were used for cell cycle distribution and apoptosis analyses. For the cell cycle distribution assay, 1×10⁶ treated cells were fixed in 70% pre-chilled ethanol overnight at -20°C. Subsequently, the cells were labeled with Forevergen Cell Cycle kits (Forevergen; Guangzhou Yongnuo Biotechnology Co., Ltd., Guangzhou, China) according to the manufacturer's protocol. The Annexin V/propidium iodide apoptosis assay was performed using the PE Annexin V Apoptosis Detection kit I (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocols. All samples were analyzed using an Accuri C6 Flow Cytometer and CFlow Plus 1.0.264.15 Software (BD Biosciences).

si-Nkd2- or si-NC-transfected rDFSCs were trypsinized with 0.25% Trypsin-EDTA solution (cat. no. 25200-056; Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 1 min and resuspended in serum-free DMEM (Gibco; Thermo Fisher Scientific, Inc.) at a density of 3×10⁵/ml, and 200 ml of the cell suspension was seeded in the upper chamber of a Costar Transwell System (Corning Incorporated, Corning, NY, USA). The lower chamber was filled with 600 ml α-MEM containing 10% FBS. The cells were allowed to migrate through 8-mm pores for 16 h at 37°C and the cells in the upper chamber were removed with a cotton swab. The cells present beneath the membrane were stained with 10 µg/ml DAPI at 37°C for 2 min. Fluorescence microscopy was performed with a electron fluorescence microscope (magnification, x100; Axiovert; Zeiss AG, Oberkochen, Germany). Ten fields per sample were randomly selected for the quantitative analysis of cell migration by counting cell nuclei.

si-Nkd2- or si-NC-transfected rDFSCs seeded at a density of 2×10⁴/ml and 100 µl cell suspension per well in 96-well culture plates were grown in conditioned medium for 7 days. The plates were washed twice, and 55 µl 1% Triton X-100 was added to each well. Subsequent to incubation overnight at 4°C, 30 µl cell lysate per well was subjected to an ALP activity assay using an ALP kit (Nanjing Jiancheng Bioengingeering Institute, Nanjing, China). To normalize enzyme activity, the protein concentration was measured with a BCA protein assay kit (Boster Biological Technology, Pleasanton, CA, USA).

si-Nkd2- or si-NC-transfected rDFSCs were seeded at a density of 2×10⁴/ml and 100 µl cell suspension per well in 96-well culture plates. Subsequent to osteogenic induction for 7 days, si-Nkd2- or si-NC-transfected rDFSCs were fixed with 4% paraformaldehyde at 4°C for 24 h and stained with 1% Alizarin Red S (Sigma-Aldrich; Merck KGaA) at 37°C for 15 min. Following the addition of 10% cetylpyri-dine, the cells were incubated at 37°C for 60 min. The extent of mineralization was detected at 562 nm using a microplate reader (Tecan Group, Ltd.).

Control and induced (0, 1, 2, 3 and 4 weeks), and si-Nkd2- or si-NC-transfected cells (≥10⁶ cells per group) were harvested with radioimmunoprecipitation assay (RIPA) lysis buffer at 0°C for 30 min (10 mmol/l Tris-HCl, 1 mmol/l EDTA, 1% sodium dodecyl sulfate, 1% Nonidet P-40, 1:1,000 proteinase inhibitor cocktail, 50 mmol/l β-glycerophosphate and 50 mmol/l sodium fluoride) to obtain total proteins. Nuclear proteins were extracted using a CelLytic™ NuCLEAR™ Extraction kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. Proteins were quantified using a BCA Protein Assay kit (cat. no. P0012S; Beyotime Institute of Biotechnology, Shanghai, China), and 30 µg protein per lane was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto 0.22-mm nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The nitrocellulose membranes were blocked using 1% bovine serum albumin for 20 min at 37°C. Subsequent to washing with tris-buffered saline (TBS; 10 mM Tris, 154 mM NaCl, pH=7.5 with 0.1% Tween-20) for 5 min at room temperature 3 times, the membranes were incubated overnight at 4°C with goat anti-rat Nkd2 (1:100; cat. no. sc163145; Santa Cruz Biotechnology, Inc.), rabbit anti-rat β-catenin (1:1,000; cat. no. ab6302; Abcam, Cambridge, MA, USA), rabbit anti-rat runt-related transcription factor 2 (Runx2; 1:1,000; cat. no. ab23981; Abcam), rabbit anti-rat osteocalcin (OCN; 1:200; cat. no. AB10911; EMD Millipore), rabbit anti-rat β-actin (1:1,000; cat. no. G3202; Wuhan Saiwei Biotechnology Co., Ltd., Wuhan, China), rabbit anti-rat Cyclin D1 (1:1,000; cat. no. #2978; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-rat c-Myc (1:1,000; cat. no. #5605; Cell Signaling Technology, Inc.), mouse anti-rat GAPDH (1:1,000; cat. no. #5174; Cell Signaling Technology, Inc.) and rabbit anti-rat histone H3 (1:500; cat. no. ab8580; Abcam) antibodies. The membranes were washed with TBS for 5 min at room temperature 3 times. Proteins were detected by incubated with HRP-conjugated secondary antibody rabbit anti-goat immunoglobulin G (IgG; 1:3,000; cat. no. A21030; Abbkine Scientific Co., Ltd.), goat anti-rabbit IgG (1:3,000; cat. no. A0208; Beyotime Institute of Biotechnology) and goat anti-mouse IgG (1:3,000; cat. no. A0216; Beyotime Institute of Biotechnology) at 37°C for 2 h and according to the enhanced chemiluminescence method (EMD Millipore). Densitometric analysis of the protein bands was performed using ImageJ software 1.48 version (National Institutes of Health, Bethesda, MD, USA).

Cells (si-Nkd2- or si-NC-transfected, and control or treated by Wnt3a) at a density of ≥10⁶ per group were harvested with RIPA lysis buffer (10 mmol/l Tris-HCl, 1 mmol/l EDTA, 1% sodium dodecyl sulfate, 1% Nonidet P-40 and 1:1,000 proteinase inhibitor cocktail) for 30 min at 0°C for protein extraction and western blot analysis. Cellular extracts were prepared with Lysis kit-RIPA buffer (ProteinSimple, San Jose, CA, USA). Wes Antibody Diluent2 included in the respective Wes Master kit was used as the blocking buffer and used undiluted. Cellular extracts were blocked with the Wes Antibody Diluent2 for 30 min at room temperature. Immunoreactive proteins were detected using Wes-Rabbit (12-230 kDa) Master kit (cat. no. PS-MK01; ProteinSimple) and Wes-Mouse (12-230 kDa) Master kit (cat. no. PS-MK02; ProteinSimple) by the Wes™ Simple Western System (ProteinSimple) according to the manufacturer's protocol. Rabbit anti-rat Runx2 (1:20; cat. no. ab23981; Abcam), Collagen-I (1:10; cat. no. ab90395; Abcam), ALP (1:10; cat. no. ab83259; Abcam) and β-catenin (1:50; cat. no. ab23981; Abcam) antibodies were used as primary antibodies for detecting Runx2, Collagen-I and ALP, respectively. Cellular extracts were incubated with the primary antibodies for 30 min at room temperature. As the loading control, β-tubulin was detected using a mouse anti-rat β-tubulin monoclonal antibody (1:50; cat. no. AF7011; Affinity Biosciences, Cincinnati, OH, USA). The secondary antibodies were included in the respective Wes Master kit and used undiluted. Cellular extracts were incubated with the secondary antibodies for 30 min at room temperature. Quantitation of the detected proteins and image preparation were performed with Compass Software 2.5.8.0 (ProteinSimple). Parameters: Sample separation time, 25 min; separation voltage, 375 V.

The total RNA of control and induced (0, 1, 2, 3 and 4 weeks), and si-Nkd2- or si-NC-transfected cells was isolated with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. mRNA quantification was performed using an ABI 7300 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Complementary DNA (cDNA) was synthesized with a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) in a total volume of 20 µl. RT was performed at 37°C for 1 h followed by incubation for 10 min at 70°C to inactivate the reverse transcriptase, following which 5 µl of the reaction mixture was incubated with FastStart Universal SYBR Green Master (Roche Diagnostics, Basel, Switzerland) in a total volume of 10 µl. The primers used for qPCR are listed in Table I. All reactions were performed with a hot-start preincubation step of 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C, 60 sec at 60°C and a final 5 min step at 60°C. The amount of template was quantified using the comparative cycle quantification cycle method according to the ABI 7300 Real-Time PCR System manufacturer's protocol. Relative gene expression was calculated with GAPDH as an internal control using the formula 2^−ΔΔCq (25).

Activation of the Wnt/β-catenin signaling pathway was measured using the activity of the transcription factor T-cell factor (TCF)/lymphoid enhancer factor (LEF), a direct downstream modulator of Wnt signaling, as previously described (26). The luciferase assay was performed following the RT protocol described by the manufacturer of TCF/LEF Reporter Assay kit (Qiagen GmbH, Hilden, Germany), which contains a TCF/LEF luciferase reporter construct in addition to a negative control (Cignal negative control; non-inducible firefly luciferase reporter construct) and a positive control (Cignal positive control; constitutively expressed firefly lucif-erase construct). The pGL4 Luciferase Reporter Vectors were used as the plasmid vectors as described by the manufacturer of TCF/LEF Reporter Assay kit. The reporter was a mixture of inducible transcription factor responsive construct and constitutively expressing Renilla luciferase construct (40:1). The inducible transcription factor-responsive construct encodes the firefly luciferase reporter gene under the control of a basal promoter element (TATA box) joined to tandem repeats of a specific Transcriptional Response Element. The negative control was a mixture of non-inducible reporter construct and constitutively expressing Renilla luciferase construct (40:1). The noninducible reporter construct encodes firefly luciferase under the control of a basal promoter element (TATA box), without any additional transcriptional response elements. The positive control was a constitutively expressing GFP construct, pre-mixed with a constitutively expressing firefly luciferase construct, and a constitutively expressing Renilla luciferase construct (40:1:1).

For co-transfection with si-Nkd2, rDFSCs were seeded at a density of 4×10⁵ cells/ml and cultured in α-MEM containing 5% FBS without antibiotics. The constructs were diluted in 25 ml Opti-MEM (Invitrogen; Thermo Fisher Scientific, Inc.), mixed with Lipofectamine RNAiMax reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and incubated at room temperature for 20 min to allow formation of the transfection complex. Subsequently, the mixture was added to cells in 96-well culture plates. After 24 h of transfection at RT, signals were assessed using Dual-Luciferase^® Reporter Assay System (Promega Corporation, Madison, WI, USA). Luciferase activity in control (si-NC-transfected) cells was used for calibration.

si-Nkd2-transfected rDFSCs were seeded in 6-well culture plates at a density of 10⁵/ml and 200 ml cell suspension per well. si-Nkd2-transfected rDFSCs were treated with 100 ng/ml Wnt3a or mock-treated and osteoblast induction medium at 37°C for 7 days. rDFSCs cultured in osteoblast induction medium at 37°C for 7 days served as the control. A simple western size assay was performed to assess the levels of ALP, Runx2 and β-catenin proteins.

All experiments were performed at least 3 times. Unpaired Student's t-test was used to compare the means of two groups. One-way analysis of variance was applied to compare more than two means and Bonferroni's test was performed to compare the means between the groups. The Kruskal-Wallis test was used to assess the variance of heterogeneity. All results were evaluated using SPSS software version 16.0 (SPSS, Inc., Chicago, IL, USA). The data were expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significance difference.

Nkd2 staining was positive on postpartum day 1 in the DF of newborn rats (Fig. 1A). Subsequently, Nkd2 expression peaked on day 3 (Fig. 1B) and became weaker on day 5 (Fig. 1C). Nkd2 staining was not detected on days 7 and 9 (Fig. 1D and E) but was detected again on days 11 (Fig. 1F) and 13 (Fig. 1G). The expression of Nkd2 in the DF at postpartum days 1, 3, 5, 11 and 13 were significantly higher compared with the respective days in the negative control group (Fig. 1H) (P<0.05, P<0.001, P<0.01, P<0.05 and P<0.05, respectively. Fig. 1I). However, at postpartum days 7 and 9, the difference was not significant (P>0.05). In addition, primary rDFSCs were isolated from the first mandibular molars of Sprague-Dawley rats, and the localization of Nkd2 in the cytomembrane, cytoplasm and nucleus of rDFSCs was observed by immunofluorescence and confocal laser-scanning microscopy (Fig. 1J).

Subsequent to culturing for 4 weeks in osteoblast induction medium, RT-qPCR (Fig. 2A) and western blotting (Fig. 2B) revealed that Nkd2 expression was upregulated in rDFSCs following osteogenic induction. This result was consistent with the observed expression of β-catenin, Runx2 and OCN. The expression of Nkd2 mRNA was significantly increased at week 1, 2, 3 and 4 compared with the control group (P<0.01, P<0.001, P<0.001 and P<0.001, respectively), which was the similar to the mRNA expression of β-catenin (P<0.001, P<0.001, P<0.001 and P<0.001, respectively), Runx2 (P<0.001, P<0.001, P<0.001 and P<0.001, respectively) and OCN (P>0.05, P>0.05, P>0.05 and P<0.001, respectively) compared with the control group. The expression of Nkd2 protein was also significantly upregulated on week 3 and 4 compared with the control group (P<0.05 and P<0.01) similar to the protein levels of β-catenin (P<0.001 and P<0.001), Runx2 (P<0.001 and P<0.001) and OCN (P<0.05 and P<0.01).

RT-qPCR and western blotting were employed to investigate the efficiency of si-Nkd2 transfection in rDFSCs. The mRNA and protein levels of Nkd2 were significantly reduced compared with the si-NC transfected group (P<0.05 and P<0.01, respectively; Fig. 3A and B).

Silencing of Nkd2 expression significantly decreased the proliferation of rDFSCs after 72 h of transfection compared with the si-NC group (P<0.05; Fig. 3C). To assess whether si-Nkd2-transfected rDFSCs exhibited decreased proliferation or whether these cells underwent apoptosis, the cell cycle distribution and apoptosis rate were investigated. It was revealed that Nkd2 silencing arrested cells in the G1 phase and significantly reduced their rate of entry into the G2 phase compared with the si-NC group (P<0.001; Fig. 3D). Apoptosis analysis revealed that fewer cells underwent apoptosis due to si-Nkd2 transfection compared with the si-NC group, however this difference was not significant (P>0.05; Fig. 3E). Therefore, si-Nkd2 inhibited cell cycle progression instead of affecting survival. Furthermore, a Transwell assay revealed the significantly increased migration of rDFSCs silenced for Nkd2 compared with the si-NC group (P<0.05; Fig. 3F).

Subsequent to culturing in osteoblast induction medium for 7 days, Alizarin Red staining demonstrated a significant reduction in the formation of mineralized nodules for Nkd2-silenced rDFSCs compared with the si-NC group (P<0.001; Fig. 4A). Furthermore, ALP activity was significantly lower in si-Nkd2-transfected cells compared with in si-NC- and Mock-transfected cells (P<0.05; Fig. 4B). As an indication of osteoblast differentiation, the expression of osteoblast-associated markers were examined using a simple western size assay. Collagen-I and ALP protein levels were significantly lower in si-Nkd2-transfected rDFSCs compared with in si-NC-transfected rDFSCs subsequent to culturing in osteoblast induction medium for 7 days (P<0.05). Runx2 expression was also reduced in Nkd2-silenced rDFSCs, albeit in a non-significant manner (P>0.05; Fig. 4C).

A luciferase activity assay at 72 h after si-Nkd2 transfection revealed that TCF/LEF activity was significantly reduced in Nkd2-silenced rDFSCs with Cignal reporter compared with the si-NC transfected group with the Cignal reporter (P<0.05) or compared with the Cignal positive control (P<0.001; Fig. 5A). Furthermore, western blotting of the nuclear proteins revealed that the expression of β-catenin, cyclin D1 and c-Myc was significantly lower in si-Nkd2-transfected rDFSCs compared with in si-NC-transfected rDFSCs (P<0.05, P<0.01 and P<0.05, respectively; Fig. 5B).

Next, si-Nkd2-transfected rDFSCs were treated with Wnt3a or mock-treated and cultured in osteoblast induction medium for 7 days. According to the results of the simple western size assay, the protein levels of ALP, Runx2 and β-catenin were significantly increased in Wnt3a-treated si-Nkd2-transfected rDFSCs compared with si-Nkd2-transfected rDFSCs (P<0.001, P<0.05 and P<0.05, respectively; Fig. 6).