The study was approved by the ethics committee of the Laboratory Animal Center of the Fourth Military Medical University (Xi'an, China). Rats were supplied by the Laboratory Animal Center of the Fourth Military Medical University, and sacrificed by CO₂ asphyxiation. Rat BMSCs were isolated from the bone marrow of male rats (n=8; age, 6 weeks; weight, 80–100 g), which was obtained by flushing the femoral and tibial medullary cavities with ice-cold low-glucose Dulbecco's modified Eagle's medium (L-DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco). The marrow cell suspension was repeatedly aspirated through a 22-gauge needle and filtered through a 100-µm cell strainer prior to culture (both purchased from Corning, Inc., New York, NY, USA). Marrow cells were plated in 25-cm² tissue culture flasks at a density of 1×10⁶−1×10⁷ cells/ml. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO₂. The media was comprised of L-DMEM supplemented with 10% FBS, 100 IU/ml penicillin, 100 µg/ml streptomycin and 1 µg/ml amphotericin, and was replaced every two days.

When the cell confluence reached 80%, cells were passaged by detachment with 0.02% trypsin (Gibco) and subcultured in a fresh flask at a ratio of 1:2. The cells were cultured in flasks and three passages of monolayer cells were divided into 24-well culture plates. At passage 3, the cells were divided into two groups: The osteogenic induction group, which was further cultured in differentiation medium (L-DMEM containing 10 mM β-glycerophosphate, 100 nm dexamethasone and 50 µg/ml ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA) to induce osteogenic differentiation (31), and the control group, which was cultured in normal medium. To detect the expression of CGRP receptors in the process of differentiation, the cells in the induction group were further divided into four groups, which were then treated with various concentrations of CGRP (0, 10⁻¹², 10⁻¹⁰ and 10⁻⁸ M) to determine the optimal concentration to promote cellular differentiation. In another experiment, cells from the induction group were divided into five groups to determine the effects of CGRP receptor antagonist and sFRP on the differentiation of BMSCs: i) The control group was treated with phosphate-buffered saline; ii) cells were incubated with 20 mM LiCl (Sigma-Aldrich); iii) cells were incubated with CGRP (10⁻⁸ M); iv) cells were incubated with a mixture of CGRP (10⁻⁸ M) and 10⁻⁶ M CGRP receptor antagonist CGRP8-37 (Sigma-Aldrich); v) cells were incubated with a mixture of CGRP (10⁻⁸ M) and 10 µg/ml sFRP (R&D Systems, Inc. Minneapolis, MN, USA).

Alizarin red staining was performed to identify osteoblasts. After three passages of monolayer culture, cells were cultured in differentiation medium for 14 days and then fixed in 4% paraformaldehyde (Sigma-Aldrich) for 30 min, followed by three washes with ice-cold phosphate-buffered saline and staining for 5 min with alizarin red (Sigma-Aldrich). The cells were viewed using a 450 fluorescent inverted phase contrast microscope (Nikon Corporation, Tokyo, Japan).

To assess the expression of various genes in BMSCs in the experimental groups, total RNA was isolated from the cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) and cDNA synthesis was performed using Oligo dT (Invitrogen). The qPCR assay was performed using SYBER Green (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the primers (Shanghai GenePharma Co., Ltd., Shanghai, China) designed with Primer Express software (version 3.0; Applied Biosystems), listed in Table I. The amplification conditions were as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 30 sec. Thermal cycling and fluorescence detection were performed using the StepOnePlus™ Real-Time PCR System (Applied Biosystems). The mRNA levels of alkaline phosphatase, collagen type I, osteocalcin, Runx2, c-myc, cyclin D1, Lef1, Tcf7 and β-catenin mRNA were calculated relative to those of GAPDH using the ΔΔCt method (32). Experiments were performed in triplicate.

Following 7 and 14 days of treatment, the cells in the experimental groups were subjected to protein extraction with lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) and protein extracts were dissolved in sample buffer (Cell Signaling Technology, Inc.) containing 2% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl, 100 mM dithiothreitol (pH 6.80) and 10% glycerol. Protein samples (30 µg) were separated by 10% SDS-polyacrylamide gel (Beyotime Institute of Biotechnology, Haimen, China) electrophoresis and electrotransferred onto a nitrocellulose membrane (EMD Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk in Tris-buffered saline solution (Beyotime Institute of Biotechnology) for 1 h at room temperature, blots were subsequently incubated with polyclonal goat anti-CRL (1:5,000; sc-18007), polyclonal rabbit anti-RAMP1 (1:5,000; sc-11379) and monoclonal mouse anti-β-catenin antibodies (1:5,000; sc-53483) overnight at 4°C (all purchased from Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Following washing for 5 min 3 times with Tris-buffered saline, horseradish peroxidase (HRP)-conjugated polyclonal rabbit anti-mouse IgG (1:5,000; sc-358920), HRP-conjugated polyclonal goat anti-rabbit IgG (1:5,000; sc-2004) and HRP-conjugated polyclonal mouse anti-goat IgG (1:5,000; sc-2345) (all purchased from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were incubated with the membranes for 1 h at room temperature. An Imagestation 2000 MM (Eastman Kodak, Rochester, NY, USA) was then used for capturing images of the blots. Blots were stripped and re-probed with polyclonal goat anti-actin (1:500; sc-1616) and polyclonal rabbit anti-α-tubulin (1:500; sc-5546) (both purchased from Santa Cruz Biotechnology, Inc.) antibodies to demonstrate equal loading and for normalization of the protein content among the groups. Densitometric analysis of the bands was performed using Molecular Imaging Software Version 4.0 (Eastman Kodak). Protein concentration was determined using an Immobilon Western Chemiluminescent HRP Substrate for enhanced chemiluminescence (EMD Millipore, Billerica, MA, USA).

Values are expressed as the mean ± standard error of the mean. Statistical analyses were performed using SPSS software version 13.0 (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance was used to compare mean values between groups. Comparisons among groups were performed using Dunnett's two-tailed t post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Following seven days of primary culture, BMSCs grew in spindle-like, triangular and polygonal shapes (Fig. 1A). At passage 3 and beyond, cell growth was accelerated (Fig. 1B). After seven days of differentiation in the osteogenic medium, the cells grew slowly and were covered with calcium deposits (Fig. 1C). After 14 days of differentiation, alizarin red staining revealed that the cells displayed characteristics of osteoblasts and extracellular matrix mineralization (Fig. 1D).

The expression of CRL and RAMP1 protein in BMSCs on days 7 and 14 of culture in osteogenic medium was assessed by western blot analysis (Fig. 1E and F). While CRL and RAMP1 protein expression was present in the osteogenic induction group as well as in the control group on days 7 and 14, expression levels were approximately two-fold increased in the induction group compared with those in the control group.

The effects of CGRP (10⁻⁸, 10⁻¹⁰ and 10⁻¹² M) on the differentiation of BMSCs were examined by assessing the mRNA expression of the early osteoblastic markers alkaline phosphatase and collagen type I as well as the late osteoblastic marker osteocalcin by using RT-qPCR analysis. Runx2, a transcriptional factor necessary for osteoblast differentiation, was also examined. Compared with the untreated group, CGRP treatment significantly increased the levels of osteoblastic markers at days 7 and 14, with 10⁻⁸ M CGRP exerting a greater effect than the lower concentrations (Fig. 2).

To assess the possible involvement of the Wnt/β-catenin pathway in BMSC differentiation into osteoclasts, the effects of Wnt/β-catenin pathway agonist LiCl, CGRP inhibitor CGRP8-37 and Wnt inhibitor sFRP on the expression of osteoblastic genes in induced BMSCs were examined in the absence or presence of CGRP. RT-qPCR revealed that CGRP (10⁻⁸ M) and LiCl alone significantly increased the expression of osteoblastic genes. Of note, pre-treatment with CGRP8-37 and sFRP significantly inhibited CGRP-induced expression of the osteoblastic markers by induced BMSCs (Fig. 3).

To further assess the involvement of the Wnt/β-catenin signaling pathway, the mRNA expression of signaling molecules of this pathway, including c-myc, cyclin D1, Lef1, Tcf7 and β-catenin, was evaluated by RT-qPCR at days 7 and 14. Incubation of BMSCs with CGRP (10⁻⁸) and LiCl in the osteoinductive medium significantly increased the mRNA expression of c-myc, cyclin D1, Tcf7 and Lef1 on days 7 and 14, as compared with their expression in the control group. This upregulation was significantly inhibited by pre-treatment with CGRP8-37 or sFRP (Fig. 4A–D). However, the expression of β-catenin mRNA was not significantly affected in the experimental groups (Fig. 4E). Therefore, the protein expression of β-catenin in all experimental groups was examined by western blot analysis. The results revealed that CGRP (10⁻⁸ M) and LiCl increased the protein expression of β-catenin on days 7 and 14 compared with that in the control group. Furthermore, pre-treatment with CGRP8-37 or sFRP inhibited the CGRP-induced increase in β-catenin protein expression on days 7 and 14 (Fig. 4F).