The present study was approved by the Ethics Committee of Second Military Medical University (Shanghai, China). All samples were obtained from patients with AS (male:female=11:2, n=13) and patients with aseptic necrosis of the femoral head (control; n=8). All of the patients underwent total hip arthroplasty at Changhai Hospital (Shanghai, China) between January 2017 and December 2017. Patients with AS were diagnosed according to the Modified New York Criteria (1984) (19). The age of patients with AS ranged from 8 to 20 years, with an mean age of 42.67 years. Ligaments were acquired from the femoral neck during surgery. None of the patients had taken anti-osteoporosis drugs, glucocorticoids, anti-tumor necrosis factor-drugs or NSAIDS at least 2 months prior to surgery. All patients agreed to participate and provided written informed consent. Fibroblasts were isolated from ligament tissues using the following method: The ligament tissue was washed twice with Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and finely minced. The minced tissue was incubated with 5 ml DMEM containing 1 mg/ml collagenase type II (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C for 6 h, then with 5 ml 0.25% trypsin at 37°C for 30 min, filtered through a nylon mesh of 75 µm and then washed three times. The single cell suspension was cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml) and streptomycin (100 µg/ml) in a humidified 5% CO₂ atmosphere at 37°C overnight. Subsequently, non-adherent cells were removed, and adherent cells were re-cultured in DMEM plus 10% FBS. Confluent cells were trypsinized with 0.05% trypsin (Thermo Fisher Scientific, Inc.) and re-cultured. All ligament fibroblasts from the third passage were used for all subsequent experiments, as these cells were more purified compared with the first and second passages, and more similar to cells in vivo. Fibroblasts were identified by flow cytometry with CD90-FITC from GeneTex (GeneTex, Inc., Irvine, CA, USA) using a BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Hip tissues in the AS and control groups were lysed in ice-cold lysis buffer [1% Triton X-100, 20 mmol/l Tris-HCl (PH=8.0), 137 mmol/l NaCl, 10% glycerol (v/v), 2 mmol/l EDTA, 1 mmol/l phenylmethysulfonyl fluoride, 10 µg/ml leupeptin, 50 µg/ml trypsin inhibitor, 1 mmol/l sodium orthovanadate] for 15 min. The amount of protein was determined using the BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Protein (100 µg) was loaded onto an 8% SDS-PAGE gel and transferred onto polyvinylidenedifluoride (PVDF) membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Membranes were blocked for 1 h at room temperature in 5% skimmed milk, washed with TBST (150 mM NaCl, 10 mM Tris pH 7.4, 0.1% Tween-20). Primary antibodies used included: Rabbit polyclonal CXCR4 (cat. no. sc-374159; 1:400; Santa Cruz Biotechnology, Inc., Dallas, TX, USA;), polyclonal β-catenin (cat. no. sc-316059; 1:1,000; Santa Cruz Biotechnology, Inc.), polyclonal p-β-catenin (cat. no. sc-316098; 1:500; Santa Cruz Biotechnology, Inc.), or polyclonal GAPDH (cat. no. ab8245; 1:500; Abcam, Cambridge, MA, USA). The antigen-antibody complexes were detected using peroxidase-labeled goat anti-rabbit antibodies (cat. no. ab95071; 1:800; Abcam) in TBS-T containing 2.5% non-fat dry milk for 1 h at room temperature. The results were visualized using the enhanced chemiluminescence Plus detection system (Abcam, Cambridge, MA, USA), and band areas/intensities for all proteins were measured using Image J software (version 1.8.0; National Institutes of Health, Bethesda, MD, USA).

All Immunohistochemistry (IHC) analyses was performed on formalin-fixed and paraffin-embedded samples. Paraffin blocks were sectioned to 4-µm thickness. Then, poly-L-lysine-coated slides were used to promote adhesion of the paraffin-section to the slides. The sections were dewaxed, rehydrated, and antigen-repaired. For dewaxing, the sections were baked in an incubator at 60°C for 2 h, followed by cooling to room temperature and soaking in xylene for 10 min 3 times. For rehydrating, the sections were put into ethanol (I) for 5 min, ethanol (II) for 5 min, 95% ethanol solution for 5 min, 80% ethanol solution for 5 min, 70% ethanol solution for 5 min, 50% ethanol solution for 5 min, and ddH₂O for 5 min successively. The sectioned tissue slides were incubated with the primary polyclonal rabbit antibodies: Polyclonal rabbit CXCR4 (cat. no. sc-374159; 1:400; Santa Cruz Biotechnology, Inc.) for 18 h at 4°C in a humidity chamber. Biotinylated secondary antibody anti-rabbit IgG (cat. no. ab80948; 1:2,000, Abcam) was incubated with the sections for 30 min at 37°C, then incubated with extra-avidin peroxidase for 30 min at 37°C. Images were captured under Nikon Eclipse (E600) fluorescent microscope (Nikon Corporation, Tokyo, Japan) at magnification ×400, and then analyzed using Image-Pro Plus software (version 4.5.1; Media Cybernetics, Rockville, MD, USA).

Flow cytometry was used for fibroblast verification. The CD90 marker was used to identify fibroblasts, as described previously (20–22). In brief, third-generation fibroblasts were collected, and a suspension with a density of 1×10⁵ cells/ml was prepared. Next, 12 ml trypsin/EDTA solution was added to the 75 cm³ cell tubes with 1×10⁶ cells/ml in each tube. A total of 20 µl fluorescein isothiocyanate-labeled mouse anti-human CD90 monoclonal antibody (cat. no. 11-0903-82; 1:1,000; Thermo Fisher Scientific, Inc.) was added, followed by incubation on ice for 20 min in the dark. Tubes without antibodies were used as the negative control. PBS (2 ml) was added to each tube and centrifuged at 400 × g for 6 min at 25°C. Cells were washed twice with 0.5 ml PBS. Flow cytometry was performed using BD FACSCalibur version 4.1 (BD Biosciences). Fibroblasts were plated into chamber slides and fixed with 3% formaldehyde, and then blocked with 5% BSA and incubated with anti-Vimentin (cat. no. ab20346; 1:2,000; Abcam,) overnight at 4°C. Following incubation with the primary antibodies, cells were washed three times in PBS followed by 60 min of incubation at room temperature with anti-rabbit FITC (cat. no. Sfk12258; 1:1,000; Shanghai Shifeng Biological Technology, Co., Ltd., Shanghai, China,). Fluorescence was analyzed using the fluorescence microscopy (Nikon Corporation).

Cell proliferation was assessed using an MTS assay. Aliquots (10 µl) of MTS at a concentration of 0.1 mg/ml from the MTS cell proliferation assay kit (Promega Corporation, Madison, WI, USA) were added to the cell (1×10⁴) cultures. After 4 h at 37°C, the absorbance was measured at 490 nm with a spectrophotometer.

The fibroblasts were digested with 0.25% trypsin and cultured at 37°C in a humidified atmosphere containing 5% CO₂. Following attachment of the cells to the surface of the wells, osteogenic induction medium (OM) containing DMEM/F12 with 100 nM dexamethasone, 50 mg/l ascorbic acid, 10 mM β-sodium glycerophosphate and 10% FBS was added. The medium contained mycillin and was changed every 2–3 days.

Following the induction of osteogenesis, AMD 3100 (10 µM) or SDF-1 (10 ng/ml) was added to the fibroblasts. Cell proliferation was then assessed using the MTS assay as aforementioned.

For in vitro knockdown studies, siRNA targeting rat CXCR4 (Invitrogen; Thermo Fisher Scientific, Inc.) were used. Control cultures were transfected with scramble siRNA (Invitrogen; Thermo Fisher Scientific, Inc.). Fibroblasts were seeded in a 6-cm dish at a density of 5×10⁵ cells/dish and prepared for transfection with 1 µg CXCR4 siRNA or control siRNA. siRNA was added to Opti-MEM with Lipofectamine^® 2000 RNAi MAX (Invitrogen; Thermo Fisher Scientific, Inc.) for transfection according to the manufacturer's protocol. A total of 8 h following incubation, the medium was changed into fresh EBM-2 medium containing 15% FBS. Cells were harvested at 48 h following transfection of siRNA. The sequences of the CXCR4 siRNA used were as follows: 5′-GACUGGUACUUUGGGAAAUTT-3′ and 3′-AUUUCCCAAAGUACCAGUCTT-5′. The scrambled sequence was 5′-GUAGCAGGGCAUGUAUUUATT-3′ and 3′-UAAAUACAUGCCCUGCUACTT-5′, and was designed as a negative control. The efficiencies of CXCR4 siRNA and control siRNA were monitored by western blot analysis, as aforementioned. The fibroblasts were divided into two groups: i) The AS+OM group, containing osteogenesis-induced fibroblasts; and ii) AS+OM + si-CXCR4 group, containing osteogenesis-induced fibroblasts treated with si-CXCR4.

The cells were washed with ice-cold PBS and lysates were prepared in radioimmunoprecipitation assay lysis buffer [50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.25% sodium deoxycholate, 1% NP-40, 100 mg/ml PMSF Protease Inhibitor Cocktail (Roche Diagnostics, Basel, Switzerland)]. Lysates were passed through a 1 ml needle syringe to facilitate the disruption of the cell membranes and centrifuged at 10,000 × g for 15 min at 4°C, and supernatants were collected. Protein concentrations of lysates were determined by PierceH BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Protein (100 µg) was loaded onto a 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes (Merck KGaA). Membranes were then blocked for 1 h at room temperature in 5% skim milk, washed with TBST (150 mM NaCl, 10 mM Tris pH 7.4, 0.1% Tween-20). Primary antibodies used included CXCR4 (cat. no. sc-374159; 1:800; Santa Cruz Biotechnology, Inc.), β-catenin (cat. no. sc-7963; 1:1,000; Santa Cruz Biotechnology, Inc.;), phosphorylated (p)-β-catenin (cat. no. sc-316059; 1:700; Santa Cruz Biotechnology, Inc.), glycogen synthase kinase 3b (cat. no., sc-11757; GSK3b; 1:500; Santa Cruz Biotechnology, Inc.), frizzled-7 (FZD7; cat. no. sc-31061; 1:500; Santa Cruz Biotechnology, Inc.), alkaline phosphatase (ALP; cat. no., sc-214129; 1:1,000; Santa Cruz Biotechnology, Inc.), osteocalcin (OCN; cat. no., sc-139134; 1:1,500; Santa Cruz Biotechnology, Inc.), v-myc avian myelocytomatosis viral oncogene homolog (c-myc; cat. no., sc-261714; 1:1,000; Santa Cruz Biotechnology, Inc.) and cyclin-D1 (cat. no., sc-426371; 1:1,000; Santa Cruz Biotechnology, Inc.). Membranes were washed three times in TBST, incubated with horseradish peroxidase-conjugated secondary antibody (HRP; ab6721; 1:1,000; Abcam) at 1:5,000 in 5% skim milk for 1 h at room temperature, washed and then processed with ECL Plus Western Blotting detection kit (Amersham Biosciences; GE Healthcare, Chicago, IL, USA). The filter was then incubated with the substrate and exposed to X-ray films (Thermo Fisher Scientific, Inc.), and analyzed using Image J software (version 1.8.0; National Institutes of Health, Bethesda, MD, USA).

As described previously (23), cultured cells were stained with 0.1% alizarin red (Thermo Fisher Scientific, Inc.) for 30 min at 37°C. Each well washed twice with PBS, then fixed in 10% formalin for 15 min at room temperature. Then, the wells were rinsed with distilled water, and 1 ml alizarin red S was added to each well (40 mM; pH 4.1). The 24-well plate was then placed on a shaker and incubated at room temperature for 20 min. Then, 4 ml distilled water was added to each well and rinsed for 5 min, and this step was repeated 4 times.

Statistical analysis was performed using SPSS version 18.0 software (SPSS Inc., Chicago, IL, USA). The data are presented as mean ± standard error of the mean, and differences between groups were analyzed by two way analysis of variance. Post-hoc pairwise comparisons were performed using the Bonferroni test. P<0.05 was considered to indicate a statistically significant difference.

The basic clinical data of the two groups are summarized in Tables I and II; there were no significant differences between the two groups in terms of sex and age. The expression of CXCR4 in AS and control ligament tissues was then investigated. Immunohistochemistry (Fig. 1A and B) and western blot analysis (Fig. 1C and D) indicated that the expression of CXCR4 was upregulated in AS tissues compared to control tissues (P<0.01).

Primary fibroblasts were successfully cultured. Following cell sorting for fibroblasts, fibroblasts morphology was determined by vimentin immunocytochemistry (Fig. 2). To determine whether the fibroblasts in patients with AS were different from those in the control group, cell proliferation assays in the two fibroblasts groups were conducted. It was identified that AS fibroblasts exhibited an increased growth rate compared with the control group, as determined by the growth curve.

The role of CXCR4 in the proliferation and osteogenesis of AS fibroblasts was examined by inhibiting CXCR4. Flow cytometric analysis indicated that the inhibition of CXCR4 through the addition of AMD 3100 led to a decrease in the proliferation of AS fibroblasts (Fig. 3). However, the proliferation of AS fibroblasts was significantly increased following the addition of SDF-1 (Fig. 3). Therefore, these data suggest that CXCR4 is involved in the proliferation of AS fibroblasts.

Next, it was identified that the AMD 3100 significantly suppressed the expression of CXCR4, while SDF-1 increased the level of CXCR4 in fibroblasts. Then, the downregulation of β-catenin, GSK3b, FZD7, c-myc, cyclin D1 and the osteogenesis markers ALP and OCN, and the upregulation of p-β-catenin were observed in fibroblasts following the blocking of CXCR4 using AMD 3100 (Fig. 4). Conversely, SDF-1 in fibroblasts led to the opposite results.

Alizarin red staining for calcium revealed significantly decreased levels of mineralization following the addition of AMD 3100 (Fig. 5). By contrast, the amount of mineralization indicated a distinctive increasing trend following the addition of SDF-1 compared with that observed in the AS+OM and AS+OM+AMD 3100 groups.

Alizarin red staining was performed to investigate the mineralization ability of CXCR4. As indicated in Fig. 6A and B, the level of mineralization was markedly decreased following preincubation with CXCR4 siRNA (P<0.05). It was then identified that the si-CXCR4 significantly suppressed the expression of CXCR4, while SDF-1 increased the level of CXCR4 in fibroblasts (Fig. 6C and D). Then, the downregulation of β-catenin, GSK3b, FZD7, c-myc, cyclin D1 and the osteogenesis markers ALP and OCN, and upregulation of phosphorylated β-catenin were observed in fibroblasts following the silencing of CXCR4. Collectively, these data suggested that CXCR4 promoted the functions of osteogenesis and mineralization formation in cultured fibroblasts.