Bone marrow was obtained from the iliac crest marrow aspirates of 3 healthy donors (one 34-year old male, two females at 19 and 38-year's old) undergoing iliac crest bone transplantation at Jingzhou Central Hospital, Tongji Medical College of Huazhong University of Science and Technology (Jingzhou, China) between January and July 2018. The procedure was performed following approval from the Ethics Committee of Huazhong University of Science and Technology and after obtaining donors' informed consent. The collected bone marrow was treated with heparin anticoagulant and then diluted in α-minimal essential medium (α-MEM; Gibco; Thermo Fisher Scientific, Inc.) (30). hBMSCs were then isolated by density gradient centrifugation (400 × g for 20 min at room temperature) with Ficoll-Paque (GE Healthcare) and plastic adherence, purified by discarding suspended cells through exchanging medium, and then grown in α-MEM supplemented with 20% fetal bovine serum (Atlanta Biologicals; Bio-Techne Corporation), 100 U/ml penicillin (Invitrogen; Thermo Fisher Scientific, Inc.), 100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.), and 2 mM L-glutamine (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were then cultured at 37°C with 5% humidified CO₂. The hBMSCs were passaged and maintained at low density. Adherent cells reaching 80% confluence were harvested by using 0.25% trypsin at 37°C for 3 min. Then, 1×10⁵ cells were replated, and the remainder was used for the analysis of gene expression.

hBMSCs were plated in 6-well plates in standard growth medium. At 80% confluence, the medium was replaced with osteogenic medium consisting of α-MEM + 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin supplemented with 10 mM β-glycerophosphate (Calbiochem; Merck KGaA), 50 µg/ml L-ascorbic acid (Wako Chemicals GmbH), 10 nM dexamethasone (Sigma-Aldrich; Merck KGaA), and 10 nM calcitriol (Sigma-Aldrich; Merck KGaA). The medium was replaced every 3 days. The control cells were grown in standard growth medium. Cell pellets were harvested for RNA isolation at different time points following induction.

hBMSCs were cultured in 24-well plates at 2×10⁴ cells/cm². At 80% confluence, cells were induced to differentiate into osteoblasts using StemPro™ Osteogenesis Differentiation kit (Gibco; Thermo Fisher Scientific, Inc.) supplemented with KR-12-a6 (synthesized and purified by GL Biochem (Shanghai) Ltd.) at 0, 20, 30, 40, 60, or 80 µg/ml at 37°C for 7 days. For ALP staining, hBMSCs in each group were washed twice with PBS at the end of the 7-day KR-12-a6-treatment period, fixed with 4% paraformaldehyde for 30 s, and then stained using the ALP staining kit (Maokang Biotechnology, Shanghai) according to the manufacturer's instructions. Images were acquired by an inverted phase contrast microscope (CKX41, Olympus) under ×100 magnification. At least five fields per sample were randomly selected and observed. To quantitatively analyze ALP activity, hBMSCs after the 7-day KR-12-a6-treatment period were washed twice with PBS and lysed with Triton X-100 (1%) for 15 min. Then, the activity was measured by Alkaline Phosphatase Assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. The final estimation was based on the absorbance at 405 nm measured by a spectrophotometer.

Assessment of ex vivo mineralization was performed by employing alizarin red staining. hBMSCs at the density of 5×10⁴ cells/well in 6-well plates underwent osteoblast differentiation in medium supplemented with KR-12-a6 at 0, 20, 30, 40, 60, or 80 µg/ml at 37°C for 21 days. Then cells were washed in PBS, fixed in 70% ethanol at −20°C for 1 h, and rinsed in dH₂O. The cultures were stained with 40 mM alizarin red (Sigma-Aldrich; Merck KGaA) at pH 4.2 for 10 min at room temperature with rotation. Cells were then rinsed twice with dH₂O, followed by washing three times with PBS to reduce nonspecific staining. Images were acquired by an inverted phase contrast microscope (CKX41, Olympus Corporation) under ×100 magnification. At least five fields per sample were randomly selected and observed. For further quantitative analysis, a 10% chlorinated 16 alkyl pyridine solution of sodium phosphate (pH 7.0) was added into each sample to dissolve the dye, and the absorbance was measured at 620 nm by a spectrophotometer.

hBMSCs at the density of 5×10⁴ cells/well in 12-well plates were treated with KR-12-a6 at 0, 20, 30, and 40 µg/ml and then cultured at 37°C for 3, 7, and 14 days. On day 3, 7, and 14 post-KR-12-a6 treatment, hBMSCs were harvested in Buffer RLT using an RNeasy Mini kit (Qiagen, Inc.). Total RNA was prepared according to standard protocols. Complementary DNAs (cDNAs) were synthesized from 0.5 µg total RNA using a QuantiTect Reverse Transcription kit (Qiagen, Inc.) according to the manufacturer's protocols. qPCR was conducted using a QuantiTect SYBR Green PCR kit (Qiagen, Inc.) and an ABI PRISM 7900HT SDS instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was performed as follows: 2 min activation at 95°C, then 40 cycles of 15 sec denaturation at 95°C and 15 sec annealing at 62°C, and a final 20-sec extension at 68°C. All reactions were in a final volume of 20 µl consisting of 2X master mix (10 µl), forward and reverse primers (2 µl), cDNA, and RNase H₂O, and each sample was run in triplicate. The specificity of PCR products was confirmed at the end of each run via melting curve analysis. All signals were normalized to β-actin, and the quantification cycle was determined. RT-qPCR was quantified by 2^−∆∆Cq method (31). Oligonucleotide primers used for RT-qPCR are presented in Table I. β-actin was used as the housekeeping gene to normalize gene expression levels.

hBMSCs at the density of 5×10⁴ cells/well in 12-well plates were treated with KR-12-a6 at 0, 20, 30, and 40 µg/ml and then cultured at 37°C for 7 days. On day 7 post-KR-12-a6 treatment, hBMSCs were harvested and lysed in immunoprecipitation lysis buffer (10 mM Tris, 0.15 M NaCl, 1% NP-40, and 10% glycerol, pH 7.4, at 22°C) containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich; Merck KGaA). Cell lysates were then centrifuged at 15,000 × g for 45 min at 4°C. The supernatants were collected, and the protein concentration was determined by BCA assay with a Bio-Rad Model 680 Plate Reader. Then, 40 µg/sample of protein was separated via 7.5% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk for 1 h at 4°C, and then incubated with primary antibodies targeting phosphorylated (p)-Smad1/5 (cat. no. 9516S, Cell Signaling Technology, 1:500 dilution), Smad1/5 (cat. no. sc-6201, Santa Cruz Biotechnology, 1:200 dilution), or β-actin (cat. no. sc-1616, Santa Cruz Biotechnology, 1:200 dilution) overnight at 4°C. The following day, membranes were washed and incubated with bovine anti-rabbit IgG-HRP secondary antibody (cat. no. sc-2370, Santa Cruz Biotechnology, 1:1,000 dilution) or bovine anti-goat IgG-HRP secondary antibody (cat. no. sc-2350, Santa Cruz Biotechnology, 1:1,000 dilution) for 1 h at room temperature, and immunoreactive bands were detected and visualized by ECL reagent (EMD Millipore) and a FluorChem FC Imaging system (Alpha Innotech). The protein expression was then quantified by AlphaEase FC StandAlone software (version 6.0.0.14, Alpha Innotech).

hBMSCs at the density of 5×10⁴ cells/well in 12-well plates were pre-treated with 10 µM LDN-212854 (Selleck Chemicals, Shanghai) or DMSO (vehicle control) at 37°C for 14 h, then washed 3 times with plain culture medium. hBMSCs continued to be cultured in medium containing 1 µM LDN-212854 alone or 1 µM LDN-212854 in combination with 40 µg/ml KR-12-a6 at 37°C for 7 days. After washing for times with PBS, hBMSCs were harvested and subjected to western blot analysis.

Each experiment was repeated at least three times, data are expressed as the mean ± standard deviation. One-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used to determine statistical significance. All statistical analyses were performed using SPSS 19.0 (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

To determine the effects of KR-12-a6 on the osteogenic differentiation of hBMSCs, ALP staining and alizarin red staining were performed. With increasing concentrations of KR-12-a6, the intensity of ALP (Fig. 1) and alizarin red staining (Fig. 2) also increased. KR-12-a6 at 40 µg/ml exhibited the strongest effects on both ALP (Fig. 1) and alizarin red staining (Fig. 2), whereas KR-12-a6 at 60 and 80 µg/ml did not notably increase the staining intensity compared with KR-12-a6 at 40 µg/ml.

The mRNA expression levels of osteoblastic differentiation-associated genes, including RUNX2 (encoding runt-related transcription factor 2; Fig. 3A), ALP (Fig. 3B), COL1A1 (encoding type 1 collagen alpha 1 chain; Fig. 3C), BSP (encoding bone sialoprotein; Fig. 3D), BMP2 (encoding bone morphogenic protein 2; Fig. 3E), OSX (encoding osterix; Fig. 3F), OCN (encoding osteocalcin; Fig. 3G) and OPN (encoding osteopontin; Fig. 3H), were determined via RT-qPCR analysis following treatment of hBMSCs with KR-12-a6 for 3, 7 or 14 days. The mRNA levels of RUNX2 and ALP increased in a dose-dependent manner as early as 3 days post-KR-12-a6 treatment. The mRNA expression of COL1A1, BSP and BMP2 was significantly upregulated from day 7 post-KR-12-a6 treatment compared with the control. In contrast, the mRNA levels of OSX, OCN and OPN were only significantly upregulated at day 14 following KR-12-a6 stimulation.

As a significant elevation of BMP2 mRNA was observed in Fig. 3E, it was next investigated as to whether BMP/SMAD signaling was involved in KR-12-a6-induced hBMSC osteogenic differentiation. The activation of SMAD signaling was examined via western blotting following KR-12-a6-induced hBMSC osteogenesis. The results showed that KR-12-a6 promoted the phosphorylation of Smad1/5 in a dose-dependent manner following 7 days of KR-12-a6 treatment (Fig. 4A and B) and exhibited the maximum activation at 40 µg/ml. These results suggested that KR-12-a6 activated BMP/SMAD signaling in a dose-dependent manner.

To further elucidate the role of BMP/SMAD signaling in osteoblast differentiation, LDN-212854, a novel BMP inhibitor that exhibits greater selectivity for BMP compared with the TGF-β type I receptors, was used to suppress BMP/SMAD signaling. Western blotting was performed to observe the changes of several Smad proteins after 7 days of KR-12-a6 treatment with or without LDN-212854 (Fig. 5). The results showed that KR-12-a6 at 40 µg/ml significantly promoted the phosphorylation of Smad1/5 in hBMSCs, which was consistent with the results in Fig. 4. However, the use of LDN-212854 significantly reduced Smad1/5 phosphorylation in KR-12-a6-treated hBMSCs (Fig. 5). RT-qPCR analysis was then performed to further examine the effects of LDN-212854 (Fig. 6). The results showed that LDN-212854, which inhibited BMP/SMAD signaling, significantly suppressed the mRNA expression of several osteoblastic differentiation-associated genes, including RUNX2 (Fig. 6A), ALP (Fig. 6B), COL1A1 (Fig. 6C), BSP (Fig. 6D), BMP2 (Fig. 6E), OSX (Fig. 6F), OCN (Fig. 6G), and OPN (Fig. 6H) in hBMSCs at day 7 post-KR-12-a6 treatment. Collectively, these results indicated that BMP/SMAD signaling exerts a positive role in KR-12-a6-induced hBMSC osteogenesis.