Mouse chondrocyte cells (CP-M087) were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). Cells were grown routinely in Dulbecco's modified Eagle's medium/F-12 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and cultured for 24 h before refreshing the medium in a 37°C humidified atmosphere containing 5% CO2. Knockdown of Ihh in chondrocyte cells was achieved by transfection with 5 µl (10⁹ U) lentivirus containing Ihh shRNA (Ihh-shRNA; Shanghai GenePharma Co., Ltd., Shanghai, China) for 48 h using Lipofectamine 3000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cells transfected with empty lentivirus were used as a negative control (NC), and untreated cells were used as blank control. Cells (5×10⁵ cells/ml) were seeded in 6-well clusters (1 ml) or 96-well plates (0.2 ml) and transfected for 48 h. Transfected cells were used in further assays or RNA/protein extraction.

Total RNA was extracted from cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc.) was used to reverse transcribe the mRNA to cDNA according to the manufacturer's protocol. Briefly, 0.5 ng of the template RNA, 1 µl oligo(dT)18 primer and 12 µl RNase free water were mixed, and incubated at 65°C for 5 min, then cooled down on ice. The mixture was added to Reaction Buffer (4 µl), RiboLock RNase inhibitor (1 µl), 10 mM dNTP mix (2 µl) and RevertAid Reverse Transcriptase (1 µl), and incubated at 42°C for 1 h. Finally, the mixture was heated to 70°C for 5 min to obtain the cDNA. Ihh, parathyroid hormone-related protein (PTHrP), transforming growth factor (TGF)-β, osteocalcin (OCN), runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), mothers against decapentaplegic homolog 2 (Smad2), Smad3, collagen type X α1 chain (COL10A) and receptor activator of nuclear factor-κB ligand (RANKL) mRNA expression was measured using SYBR Fast qPCR Mix (cat. no. RR430A; Takara Biotechnology Co., Ltd., Dalian, China) under the following conditions: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 10 sec, and then for melting curves (one cycle of 95°C for 5 sec and 60°C for 60 sec, followed by cooling to 50°C in 30 sec) β-actin was used as an endogenous control. Primers sequences used for qPCR were as follows: Ihh, forward 5′-CTCAGCCTGCTCTCACTACG-3′ and reverse 5′-AAGCACATCCAACCCACCTC-3′; PTHrP, forward 5′-CGAGGTTCAAAGGTTTGCCTC-3′ and reverse 5′-GGCCAGAGAAGCCTGTTACC-3′; TGF-β, forward 5′-AGGGCTACCATGCCAACTTC-3′ and reverse 5′-TGACACAGAGATCCGCAGTC-3′; OCN, forward 5′-TCCTTTGGGGTTTGGCCTAC-3′ and reverse 5′-CTTGGACACAAAGGCTGCAC-3′; RUNX2, forward 5′-CGCCTCACAAACAACCACAG-3′ and reverse 5′-TCACTGTGCTGAAGAGGCTG-3′; OPG, forward 5′-CTGGAACCCCAGAGCGAAAT-3′ and reverse 5′-GCGTTTACTTTGGTGCCAGG-3′; Smad2, forward 5′-GTTCCTTTCCTCCTCCGCTC-3′ and reverse 5′-AGTCTCTTCACAACTGGCGG-3′; Smad3, forward 5′-TTCACTGGTGCTGGGGTTAG-3′ and reverse 5′-GGTAGGGATTCACGCAGACC-3′; COL10A, forward 5′-CTCCCAGCACGCAGAATC-3′ and reverse: 5′-TTCCCTACAGCTGATGGTCC-3′; RANKL, forward 5′-GGAGTTGGCCGCAGACAAGA-3′ and reverse 5′-TGATGTGCTGTGATCCAACGA-3′; and β-actin, forward 5′-GCAGGAGTATGACGAGTCCG-3′ and reverse 5′-AACAACGCATCTCATATTTGGAA-3′. Data were processed using the 2^−ΔΔCq method (12).

Cell proliferation rates were measured using CCK-8 (Beyotime Institute of Biotechnology, Haimen, China). A total of 0.5×10⁴ cells were seeded in 96-well plates for 24 h, transfected with the indicated lentivirus, and further incubated for 24, 48 and 72 h at 37°C, respectively. A total of 10 µl CCK-8 reagents were added to each well at 1 h prior to the endpoint of incubation (23, 47 and 71 h). The optical density of each well was subsequently determined using a microplate reader at a wavelength of 490 nm.

An Annexin V Apoptosis Detection kit (Thermo Fisher Scientific, Inc.) was used to assess apoptosis. Following incubation for 24, 48 or 72 h at 37°C, the cells were trypsinized for 2 min at 37°C, collected by centrifugation (960 × g) for 5 min at room temperature and resuspended in 2 ml medium. Approximately 2×10⁵ cells were harvested, washed twice with cold PBS and resuspended in 500 µl binding buffer provided in the kit. A total of 10 µl Annexin V-fluorescein isothiocyanate and 10 µl PI was added to the solution and mixed well. Following incubation for 15 min at 37°C, cells were analyzed using a flow cytometer (BD Biosciences, San Jose, CA, USA).

Cell cycle analysis was determined by flow cytometry using a Cell Cycle and Apoptosis Analysis kit (cat no. C1052, Beyotime Institute of Biotechnology) and analyzed the data using Flowjo (7.6 version; Flowjo LLC, Ashland, OR, USA). Briefly, ~1×10⁴ cells were seeded in each well of a 6-well plate. Following 48 h transfection with Ihh-shRNA or empty lentivirus, cells were harvested and fixed in 70% ice-cold ethanol for 24 h, followed by staining with PI for 10 min at room temperature. The different phases of the cell cycle were analyzed using a FACSCalibur flow cytometer (BD Biosciences).

Alkaline Phosphatase Assay Kit (cat no. P0321, Beyotime Institute of Biotechnology) was used to detect ALP activity according to the manufacturer's protocol. Black cobaltous sulfide staining indicated a positive signal. Cells were counterstained with nuclear fast red for 5 min at room temperature to label the nucleus and cytoplasm of cells. Images were captured using an inverted microscope (magnification, ×100; Nikon Eclipse TC 100; Nikon Corporation, Tokyo, Japan).

Von Kossa staining (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to determine the extent of minerals deposited on chondrocyte cells. Silver nitrate solution was added to the cells and the plate was exposed to ultraviolet light for 30 min. Cells were subsequently washed with PBS and the reaction was stopped with the addition of 500 µl of 5% sodium thiosulfate (Sigma-Aldrich; Merck KGaA). The positive mineral depositions were stained in black. Cells were counterstained with nuclear fast red for 5 min at room temperature to label the nucleus and cytoplasm. Images were captured using an inverted microscope (magnification, ×100; Nikon Eclipse TC 100; Nikon Corporation).

Immunoblotting was performed to detect the expression of Ihh, PTHrP, TGF-β, OCN, Runx2, OPG, Smad2, Smad3, COL10A and RANKL in chondrocyte cells. Cultured or transfected cells were lysed in radioimmunoprecipitation assay buffer (Wuhan Boster Biological Technology, Ltd., Wuhan, China) with 1% phenylmethylsulfonyl fluoride. The concentration of protein was determined using the BCA Protein Assay kit (cat no. P0011; Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's protocol. A total of 60 µg/lane of proteins were separated by 12% SDS-PAGE and transferred onto polyvinyl difluoride membranes. The membranes were blocked in non-fat milk for 1 h at 37°C. Membranes were incubated at 4°C overnight with the following primary antibodies: Anti-Ihh, (cat. no. SAB1403965; dilution, 1:1,000); anti-PTHrP (cat. no. SAB5300029; dilution, 1:1,000); anti-TGF-β (cat. no. SAB4502958; dilution, 1:2,000); anti-OCN (cat. no. SAB1306277; dilution, 1:2,000; all from Sigma Aldrich; Merck KGaA); anti-RUNX2 (cat. no. 12556; dilution, 1:1,000); anti-OPG (cat. no. 4816; dilution, 1:1,000); anti-Smad2 (cat. no. 5339; dilution, 1:1,000), anti-Smad3 (cat. no. 9523; dilution, 1:1,000; all from Cell Signaling Technology, Inc., Danvers, MA, USA); anti-COL10A (cat. no. ab58632; dilution, 1:1,000); anti-RANKL (cat. no. ab9957; dilution, 1:1,000) and anti-GAPDH (cat. no. ab9485; dilution, 1:3,000; all from Abcam, Cambridge, UK). The blots were subsequently incubated with goat anti-rabbit IgG (cat. no. ab7090) and goat anti-mouse (cat. no. ab97040; both 1:5,000; Abcam) horseradish peroxidase-conjugated secondary antibodies for 1 h at 37°C. Signals were visualized using enhanced chemiluminescence substrates (EMD Millipore, Billerica, MA, USA). The densitometry of bands was analyzed using Quantity One software (version 4.6.9; Bio-Rad Laboratories, Inc., Hercules, CA, USA). GAPDH was used as an endogenous protein for normalization.

All data are expressed as the mean ± standard deviation of three independent experiments. Statistical analyses were performed using SPSS v. 17.0 (SPSS, Inc., Chicago, IL, USA). Differences between groups were analyzed using Student's t-test or one-way analysis of variance with Tukey's post hoc test dependent on the conditions. P<0.05 was considered to indicate a statistically significant difference.

To investigate the biological role of Ihh in chondrocyte cell growth, chondrocytes transfected with Ihh shRNA were used in the present study. The results of RT-qPCR revealed that Ihh expression was significantly reduced in transfected cells compared with NC cells (P<0.001; Fig. 1A). Ihh downregulation significantly inhibited cell growth (P<0.05; Fig. 1B) and significantly increased the apoptosis rate compared with the NC group (P<0.001; Fig. 2A). In addition, Ihh knockdown significantly reduced the percentage of cells in S phase (P<0.05) and increased the percentage of cells in G1 phase compared with the NC group (P<0.01), indicating cell cycle arrest at G1 to S phase (Fig. 2B).

ALP is an enzyme that is required for bone mineralization (13). ALP activity was assessed in the present study and was markedly decreased in Ihh knockdown cells compared with the NC group (Fig. 3A). The calcification and mineralization of bone matrix are essential for the strength and rigidity of the spinal skeletal system (14). To estimate mineralization and calcification, von Kossa staining was performed following Ihh knockdown. Representative images of von Kossa staining were obtained by bright field microscopy (Fig. 3B). Compared with the NC cells, mineral deposition was markedly decreased in the Ihh-shRNA group.

The effects of Ihh on TGF-β/Smads and OPG/RANKL signaling were also investigated. The mRNA expression of COL10A, RANKL, Samd2 and Smad3 was significantly upregulated in the Ihh-shRNA group compared with the NC cells (P<0.001; Fig. 4), whereas the levels of OCN, OPG, PTHrP, Runx2 and TGF-β were significantly downregulated in the Ihh-shRNA compared with the NC group (P<0.001; Fig. 4). Protein expression of these genes was further assessed by western blot analysis (Fig. 5). The protein expression of COL10A, RANKL, Samd2 and Smad3 were upregulated, whereas levels of OCN, OPG, PTHrP, Runx2 and TGF-β were significantly downregulated in Ihh knockdown cells compared with the NC group (P<0.05; Fig. 5). These findings suggest that the inhibitory effects of Ihh knockdown on bone differentiation and generation are achieved via the TGF-β/Smads and OPG/RANKL signaling pathway.