MSCs were isolated from the long bone (femur and tibia) of 12 six-week-old male balb/c mice (Orient Bio, Inc., Seongnam, Korea) following euthanization in a CO₂ chamber. The bone marrow was flushed out using needles and α-minimal essential medium (α-MEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) without fetal bovine serum (FBS), and unless otherwise stated, the cells were maintained in α-MEM containing 10% FBS medium at 37°C with 5% CO₂ for 7 days. The medium was replaced at 4 day intervals.

Modified α-MEM containing FBS was purchased from Gibco; Thermo Fisher Scientific, Inc. Smad1/5/8 (cat. no. sc-6031), phosphorylated (p)-Smad1/5/8 (cat. no. 9511S) and β-actin (cat. no. sc-47778) antibodies, and the horseradish peroxidase (HRP)-conjugated rabbit anti-rabbit immunoglobulin G secondary antibodies (cat. no. sc-2004) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and Cell Signaling Technology, Inc. (Beverly, MA, USA). The anti-actin antibody, MSM, Alizarin red S, ascorbic acid phosphate, β-glycerophosphate disodium salt hydrate, L-glutamine and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide were obtained from Sigma-Aldrich (St. Louis, MO, USA). Tri-RNA reagent was purchased from Favogen Biotech Corp. (Kaohsiung, Taiwan). The kit used for reverse transcription-polymerase chain reaction (RT-PCR) analysis was obtained from Bioline, Ltd. (London, UK). The ALP, BSP, OCN, Runx2 and 18S primers for use in RT-PCR were purchased from Bioneer (Daejeon, Korea), and enhanced chemiluminescence (ECL) and detection kits were purchased from GE Healthcare (Piscataway, NJ, USA). The Coomassie Protein Assay kit and Restore Western Blot Stripping Buffer were purchased from Pierce Biotechnology, Inc. (Rockford, IL, USA).

The MSCs were cultured in the osteogenic medium with MSM (20 mM) and BMP-2 (100 ng/ml) during the initiation of osteoblast differentiation. The cells were lysed in whole lysis buffer, containing 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, and protease and phosphatase inhibitors. Lysates were incubated on ice for 40 min and then centrifuged at 15,000 × g for 10 min at 4℃. The supernatant was collected and the protein concentrations were determined using a Coomassie protein assay. An equivalent quantity (20 µg) of protein extract from each sample was electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were blocked for 1 h with 5% bovine serum albumin (Merck Millipore, Darmstadt, Germany) in 20 mM Tris-HCl (pH 7.6) 137 mM NaCl and 0.16 Tween 20 (T-TBS), and incubated overnight at 4°C with primary antibodies (Smad1/5/8, p-Smad1/5/8 or β-actin; dilution, 1:1,000). The membranes were then washed three times in T-TBS and incubated with the corresponding anti-rabbit or anti-mouse immunoglobulin G HRP-conjugated secondary antibodies (1:2,000) in T-TBS with 0.5% bovine serum albumin for 1 h under agitation at room temperature. Following incubation, the membranes were washed three times in T-TBS, following which the membranes were developed using the ECL PLUS kit and analyzed using an LAS 4000 machine (Fujifilm, Tokyo, Japan).

The bone marrow MSCs were cultured in osteogenic medium, and the mRNA expression levels were measured following treatment with MSM (20 mM) and BMP-2 (100 ng/ml) at day 5 for ALP, day 14 for BSP and day 21 for OCN. Total RNA was prepared using Tri-RNA reagent, and cDNA was synthesized using the SensiFAST™ cDNA Synthesis kit (Bioline, Ltd.), according to the manufacturer's protocol. The PCR analysis was performed on aliquots of cDNA to detect Runx2 (301 bp), ALP (454 bp), BSP (81 bp) and OCN (113 bp). The PCR primer sequences were as follows: Runx2, sense 5′-TCACTACCAGCCACCGAGAC-3′ and antisense 5′-ACGCCATAGTCCCTCCTTT-3′; ALP, sense 5′-TGGAGCTTCAGAAGCTCAACACCA-3′ and antisense 5′-ATCTCGTTGTCTGAGTACCAGTCC-3′; BSP, sense 5′-CAGAGGAGGCAAGAGTCACT-3′ and antisense 5′-CTGTCTGGGTGCCAACACTG-3′; OCN, sense 5′-AAGCAGGAGGGCAATAAGGT-3′ and antisense 5′-CAAGCAGGGTTAAGCTCACA-3′. For control purposes, the levels of 18S mRNA were measured using the following primer: Sense 5′-CGGCTACCACATCCAAGGAA-3′ and antisense 5′-CCGGCGTCCCCTCTTAATC-3′. The PCR reaction was performed as follows: 25 cycles at 94°C for 30 sec, 56°C for 30 sec and 72°C for 45 sec. Following amplification, the PCR products were analyzed on a 1.5% agarose gel stained with ethidium bromide. mRNA levels were semi-quantified using Multigauge software (version 3.1; Fujifilm Corporation, Tokyo, Japan) with 18S as the reference gene.

Alizarin Red staining and von Kossa staining of the MSC cultures were performed 21 days following the MSM (20 mM), BMP-2 (100 ng/ml) and combination treatments to evaluate the effects of MSM and BMP-2 on the matrix mineralization of MSCs. The cells were washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde in PBS for 15 min, and rinsed with distilled water three times. For Alizarin Red S staining, 40 mM Alizarin Red S (pH 4.2) stain was added to the plates and incubated for 10 min at room temperature; the plates were then rinsed three times with distilled water and washed with PBS to reduce non-specific staining. For the von Kossa staining, the fixed cells were stained with freshly prepared 5% silver nitrate under ultraviolet light for 1 h to detect phosphate deposits. Background color was removed using 5% sodium thiosulfate, and the cells were rinsed three times with distilled water. The cells were observed using phase contrast microscopy (Olympus DP71; Olympus Corporation, Tokyo, Japan), and images were captured using DPC controller software and a Nikon digital camera (Nikon, Tokyo, Japan).

Immunofluorescence microscopy of the MSC cultures was performed 5 days following the MSM (20 mM), BMP-2 (100 ng/ml), and combination treatments to evaluate the effects of MSM and BMP-2 on Smad1/5/8. The cells were permeabilized with Triton X-100 (0.1%) for 15 min on ice, and blocked with 1% horse serum (Thermo Fisher Scientific, Inc.) in PBS for 1 h, followed by incubation in a closed humid chamber with the p-Smad1/5/8 antibody in wash buffer (1% horse serum and 0.1% triton X-100 in PBS), and subsequent incubation with the Alexa Fluor 594 (rabbit) secondary antibody, (Invitrogen; Thermo Fisher Scientific, Inc.) in wash buffer. For nuclear staining, the cells were incubated with TO-PRO3 (Molecular Probes; Thermo Fisher Scientific, Inc.) in wash buffer for 5 min and rinsed with wash buffer. The slides were then observed under a fluorescence microscope (LSM710; Carl Zeiss, Inc., Oberkochen, Germany).

The results of the experiments are expressed as the mean ± standard error of the mean. Statistical analysis was performed using one-way analysis of variance on the SAS program (version 9.3; SAS Institute, Inc., Cary, NC, USA). P<0.05 was considered to indicate a statistically significant difference.

The present study examined the combination effects of MSM and BMP-2 on the differentiation of primary bone marrow MSCs. The cells were cultured with the combination of MSM and BMP-2 for 21 days, following which alterations in morphology were observed (Fig. 1A). Alizarin Red staining was used to visualize the precipitated calcium incorporated into the matrix; it stains intracellular calcium, calcium-binding proteins and proteoglycans, and is thus useful for evaluating the early phases of differentiation. Extracellular calcium deposition by mature osteoblasts was confirmed using von Kossa staining, which detects the phosphate of the calcium phosphate from the secreted matrix. The cells undergoing osteoblast differentiation and mineralization stained positive, enabling comparison in the third week of differentiation. As shown in Fig. 1B, compared with the cells treated with BMP-2 alone, the combination of MSM and BMP-2 increased the area of mineralization, visualized by the Alizarin Red staining for calcium. The relative concentration of mineralization with respect to the control is shown in Fig. 1C. Similar results were verified by the von Kossa staining (Fig. 1D). The percentage of calcification relative to the control is shown in Fig. 1E.

The expression levels of various proteins involved in BMP signaling were assessed using western blot analysis. It was hypothesized that treatment with a combination of MSM and BMP-2 leads to a more marked increase in the expression levels of Smad1/5/8 in MSCs, compared with treatment with BMP-2 alone. As shown in Fig. 2, the combination treatment increased the expression of p-Smad1/5/8 more markedly, compared with the MSCs treated with either BMP-2 or MSM alone. This result confirmed that the combination of MSM and BMP-2 involved the BMP signaling pathways in the MSCs.

The present study analyzed the mRNA expression levels of Runx2, ALP, BSP and OCN in the different treatment groups. Runx2 is a key transcription factor associated with osteoblast differentiation. During the differentiation process, the expression of the early-stage osteogenic differentiation marker, ALP, middle-stage markers, BSP and OPN, and late-stage marker, OCN, increased. However, RT-PCR analysis of the expression of OPN showed no change, compared with the control. The mRNA levels of Runx2 and osteogenic-specific markers in the MSCs were increased following combined treatment with MSM and BMP-2, compared with the levels following the individual treatments (Fig. 3).

Phosphorylation of the Smad1/5/8 protein enhances the expression of osteoblastic marker genes. The present study performed p-Smad1/5/8 localization analysis using immunofluorescence microscopy in the MSCs. The cells were cultured with a combination of MSM and BMP-2 for 5 days, as shown in Fig. 4, and this combination resulted in the upregulation of p-Smad1/5/8 localization from the cytosol to the nucleus.