Sprague Dawley rats (n=10; age, 6–8 days old; male:female, 1:1; weight, 20–30 g) were purchased from the Experimental Animal Center of Chongqing Medical University (Chongqing, China). The rats were housed in cages under a 12-h light/dark cycle with free access to food and water, at a temperature of 22–25°C and 50–60% humidity; the rats were monitored every day. Rats were sacrificed with 35 mg/kg sodium pentobarbital (1%) followed by decapitation then soaked in 20% ethanol for 10 min; respiratory arrest was confirmed following decapitation. Dental sac tissues were obtained from the first and second mandibular molars, and rDFCs were obtained as previously described (6). rDFCs were cultured in DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich; Merck KGaA), and maintained at 37°C in a humidified atmosphere containing 5% CO₂.

Flow cytometric analysis and multi-lineage differentiation were performed for the characterization of mesenchymal stem cells, as previously described (6). The identification of rDFCs and the determination of appropriate concentrations of TNF-α (10 ng/ml; GenScript) and DKK1 (0.1 and 0.4 µg/ml; Sino Biological, Beijing China) were determined based on previously published studies (6,8).

At 80% confluence (passage 3), cells were digested by 0.25% trypsin, re-suspended and re-cultured at 37°C in a humidified atmosphere containing 5% CO₂ for 24 h. AdBMP9 (MOI=100) and polybrene (1 µm/ml) were added to the adherent culture according to previously described methods (6,14). Following the addition of AdBMP9, cells were incubated for 4 h, then 2 ml complete medium was added followed by incubation for 24 h at 37°C in an atmosphere containing 5% CO₂. The green fluorescence was observed under a fluorescence microscope (magnification, ×40) AdGFP(MOI=100) was used as the vector control. Cells were subsequently collected for further experimentation. Ad-BMP9 and Ad-GFP were provided by Dr Tong-Chuan He (Molecular Oncology Laboratory, University of Chicago Medical Centre, USA).

A total of 5×10⁴ cells/well were plated into 24-well plates and cultured in a conventional osteogenic medium for 7 days at 37°C (Beyotime Institute of Biotechnology). After transfection with AdBMP9, TNF-α (10 ng/ml, GenScript) and DKK1 (0.1 or 0.4 µg/ml; Sino Biological Inc.) were added into the medium and cultured at 37°C in an atmosphere containing 5% CO₂. The medium was refreshed every three days. At 7 days post-culture, cells were fixed with 4% paraformaldehyde at room temperature for 2 h and washed with distilled, deionized water. Stained cells were observed under a light microscope (magnification, ×100). ALP staining was performed using an ALP staining kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol.

Cells (5×10⁴ cells/well) were plated into 24-well plates and cultured in a conventional osteogenic medium at 37°C for 14 days. TNF-α (10 ng/ml) and DKK1 (0.1 or 0.4 µg/ml) were added into the medium and cultured at 37°C in an atmosphere containing 5% CO₂. The medium was refreshed every three days. Following 14 days of culture, mineralized matrix nodules were stained for calcium precipitation using Alizarin Red S. Briefly, cells were fixed with 4% paraformaldehyde at 20°C for 15 min and then washed with distilled, deionized water. Subsequently, the cells were stained with Alizarin Red S (Beyotime Institute of Biotechnology) at room temperature for 30 min, followed by three washes with deionized water. Deionized water was added into each well to prevent cells from drying before they were observed under a light microscope (magnification, ×40).

Total RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using the iScript™cDNA Synthesis kit (Bio-Rad Laboratories, Inc.) according to the manufacturer's protocol. Reverse transcription was performed using the following temperature protocol: 85°C for 2 min and 37°C for 30 min. Subsequently, qPCR was performed using SYBR-Green Master mix (Thermo Fisher Scientific, Inc.). The sequences of the primers used for qPCR are presented in Table I. Primers used for qPCR amplification are presented in Table I, and β-actin was used as the internal control. The following thermocycling conditions were used for qPCR: 94°C for 2 min; followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 64°C for 30 sec, and extension 72°C for 30 sec. Relative gene expression was calculated using the 2^−ΔΔCq method (26) and normalized to the internal reference gene β-actin.

Cells were washed in precooled PBS twice and total protein was extracted from cells using RIPA lysis buffer (Pierce; Thermo Fisher Scientific, Inc.), containing protease inhibitor, phosphatase inhibitor and 1 mM PMSF. The cells were incubated on ice for 30 min and the lysate was subsequently collected in a 1.5 ml tube and centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was transferred into 1.5 ml new tubes, followed by the addition of 5X loading buffer (volume 1:4), which was then boiled for 5–8 min and stored at −20°C until use. Total protein was quantified using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology) and proteins (30 µg) were separated via 8% SDS-PAGE. The separated proteins were subsequently transferred via the wet method (6,8,14) to PVDF membranes and blocked with PBS containing 0.2% Tween-20 and 5% non-fat milk at 4°C for 2 h. The membranes were then incubated overnight at 4°C with the following primary antibodies overnight at 4°C: Anti-BMP9 (cat. no. ab35088; 1:1,000; Abcam), anti-β-catenin (cat. no. ab6302; 1:1,000; Abcam), anti-phosphorylated (p)-glycogen synthase kinase 3β (GSK3β; cat. no. ab131097; 1:1,000; Abcam), anti-GSK3β (cat. no. ab93926; 1:1,000; Abcam), anti-calcium/calmodulin dependent protein kinase II (CaMKII; cat. no. ab52476; 1:1,000; Abcam), anti-nemo like kinase (NLK; cat. no. ab26050; 1:1,000; Abcam) and anti-β-actin (cat. no. 4970; 1:1,000; Cell Signaling Technology, Inc.). Following the primary antibody incubation, the membranes were incubated at room temperature for 2 h with a HRP-conjugated goat anti-rabbit (cat. no. ab205718; 1:3,000; Abcam) and anti-mouse (cat. no. 58802; 1:3,000; Cell Signaling Technology, Inc.). Protein bands were visualized using an ECL chemiluminescent substrate kit (Beyotime Institute of Biotechnology) and densitometric analysis was performed using ImageJ software (version 1.52; National Institutes of Health).

The present study was approved by the Institutional Animal Experimental Ethics Committee of Chongqing Medical University (Chongqing, China). A total of 20 4-week-old Sprague Dawley rats (male; weight, 200–250 g) were purchased from the Experimental Animal Center of Chongqing Medical University. The rats were housed in cages under a 12-h light/dark cycle with free access to food and water, at 22–25°C and 50–60% humidity. Animal health and behavior was monitored every day. The rats were randomly divided into 4 groups (n=5 per group): i) AdBMP9 group; ii) AdBMP9 + 0.1 µg/ml DKK1; iii) AdBMP9 + 0.4 µg/ml DKK1; and iv) AdBMP9 + TNF-α and received implants respectively as detailed below. All cells had been previously co-cultured for 7 days with hydroxyapatite (40 mg/ml; Biomaterials Engineering Research Center of Sichuan University), a scaffold material, at 37°C (14).

Rats were anaesthetized using an intraperitoneal injection of 35 mg/kg sodium pentobarbital (1%). The periapical alveolar bone of the first and second molars was exposed by surgery and an osseous defect (2×3×3 mm) was created. Cells got 7-day-co-cultured with hydroxyapatite. The defect of was filled with rDFCs-hydroxyapatite in accordance with the grouping of each rat (4×10⁶ cells; 20 mg; injection site, periapical defect; age of rats at injection, 8 weeks) then covered with a collagen membrane (5×5 mm; Bopei Biotech Co., Ltd). The wound was closed with interrupted sutures. The 4th week following surgery was chosen as the humane endpoint; although the rats were in a good mental state and exhibited good locomotor activity, the scientific goal of the research has been achieved, thus, it was of little significance to continue the experiment. The rats were transferred from the rearing room into the testing room at ≥30 min prior to the initiation of the experiment. Subsequently, rats were anesthetized with an intraperitoneal injection of 35 mg/kg sodium pentobarbital (1%) and sacrificed by decapitation; respiratory arrest and death were confirmed.

The alveolar bones were extracted and fixed in 4% paraformaldehyde for 48 h at room temperature. Subsequently, sections (1 mm³) were stained with hematoxylin for 5 min at room temperature and eosin for 3 min at room temperature. Stained sections were observed under a light microscope (magnification, ×100).

Data are presented as the mean ± SD of ≥3 independent experiments. Statistical analysis was performed using SPSS 21.0 software (IBM Corp.). Data from the different groups were compared and analyzed using a one-way ANOVA, followed by a Bonferroni's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

rDFCs are polygonal or fusiform in morphology (6,8,14). rDFCs were transfected with AdBMP9 or AdGFP and green fluorescence was detected at 24 h post-transfection (Fig. 1A). In addition, following AdBMP9 transfection for 24 h, the expression levels of BMP9 were upregulated compared with the AdGFP and blank groups (Fig. 1B). To determine the effect of TNF-α on AdBMP9-induced osteogenic differentiation, rDFCs were transfected with AdBMP9 prior to treatment with 10 ng/ml TNF-α, which was selected as the stimulatory concentration for use in the experiments (8). Cells were collected 7- and 14-days post-transfection and analyzed via ALP staining and Alizarin Red S staining, respectively. The results revealed that TNF-α treatment not only the reduced osteogenic medium-induced osteogenesis, but it also downregulated BMP9-induced osteogenic differentiation (Fig. 2A). The expression levels of bone-related genes, ALP, collagen type 1 α1 (COLI), and the late osteogenesis marker osteocalcin (OCN), were further analyzed, in addition to the osteogenic differentiation-related transcription factor, RUNX2. RT-qPCR analysis discovered that the expression levels of these genes were significantly upregulated in the rDFCs + AdBMP9 group compared with the rDFCs group; however, these expression levels were significantly downregulated in the rDFCs + AdBMP9 + TNF-α group (Fig. 2B-E). These results indicated that AdBMP9 may promote the osteogenic differentiation of rDFCs, which may be suppressed by TNF-α.

Wnt signaling has been discovered to be crucial for BMP9-induced osteogenic differentiation (8,24). AdGFP was used in preliminary western blotting experiments as the vector control as a comparison to the effects of AdBMP9 (Fig. 3A and C). After TNF-α stimulation, the expression levels of β-catenin and p-GSK3β in the TNF-α and BMP9 + TNF-α groups were upregulated compared with the control and BMP9 groups, respectively (Fig. 3B). The expression levels of CaMKII and NLK were also downregulated following TNF-α stimulation in the TNF-α and BMP9 + TNF-α groups compared with the control and BMP9 groups, respectively (Fig. 3D). CaMKII and NLK are parts of the non-canonical Wnt signaling pathway (21,23) Thus, these results suggested that TNF-α may inhibit the non-canonical Wnt signaling pathway. Cyclin D1 and c-Myc are downstream genes in the canonical Wnt signaling pathway (27,28). The RT-qPCR results demonstrated that the expression levels of cyclin D1 and c-Myc were significantly upregulated in the TNF-α stimulation group compared with the non-TNF-α stimulation group (Fig. 3E), indicating that the canonical Wnt signaling pathway of rDFCs may be activated by TNF-α.

DKK1 is an inhibitor of the Wnt/β-catenin signaling pathway (8,23). The present study selected 0.1 and 0.4 µg/ml DKK1 as the stimulatory concentrations to treat the rDFCs with. Following the treatment with the higher concentration of DKK1 or TNF-α, AdBMP9-induced osteogenic differentiation was reduced compared with the AdBMP9 group (Fig. 4A); however, the addition of low concentrations of DKK1 promoted osteogenesis. The RT-qPCR analysis revealed a similar result; the expression levels of RUNX2, OCN and COLI were significantly upregulated following the treatment with lose dose DKK1 in AdBMP9-transduced rDFCs compared with the rDFCs + AdBMP9 group (Fig. 4E). However, TNF-α treatment and high dose DKK1 treatment significantly downregulated the expression levels of these genes compared with the rDFCs + AdBMP9 group (Fig. 4E). In addition, the expression levels of β-catenin, p-GSK3β and cyclin D1 were downregulated following the treatment with 0.1 and 0.4 µg/ml DKK1 compared with the rDFC + AdBMP9 group (Fig. 4B and D). Thus, these findings suggested that DKK1 may suppress the canonical Wnt/β-catenin signaling pathway. Furthermore, following the treatment with 0.4 µg/ml DKK1, the expression levels of CaMKII and NLK were upregulated compared with the rDFCs + AdBMP9 group, which suggested the partial enhancement of the non-canonical Wnt signaling pathway (Fig. 4C and D). Conversely, low dose DKK1 (0.1 µg/ml) downregulated the expression levels of CaMKII and NLK compared with the rDFCs + AdBMp9 group, which suggested the inhibition of the non-canonical Wnt signaling pathway. The abovementioned results suggested that high concentrations of DKK1 may activate non-canonical Wnt signaling and suppress canonical Wnt signaling, whereas low concentrations of DKK1 may suppress both canonical and non-canonical Wnt signaling in AdBMP9-transduced rDFCs.

Furthermore, the effect of restoring a bone defect was evaluated in a Sprague Dawley rat model. H&E staining demonstrated that TNF-α and high dose DKK1 treatment promoted a small amount of bone formation from AdBMP9-transduced rDFCs. Conversely, a mass of newly formed bone and numerous blood vessels were observed in Sprague Dawley rats that were treated with AdBMP9-transduced rDFCs and low dose DKK1 (Fig. 4F).

These results suggested that high concentrations of DKK1 and TNF-α may act via the non-canonical and canonical Wnt signaling pathways to downregulate bone formation, whereas low concentrations of DKK-1 may promote BMP-9-induced osteogenic differentiation via inhibition of the non-canonical and canonical Wnt signaling pathways.