Culturing and identification of BMSCs
As previously described [21], BMSCs were flushed from the femurs and tibias of 2-week-old SD rats and subjected to density gradient centrifugation to obtain mononuclear cells. The cells were then cultured in α-MEM supplemented with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin (Gibco, USA) at 37°C with 5% CO2. The medium was changed every 2 days, and the cells were trypsinized and subcultured (1:3 per passage). Cells at the third passage were used for all experiments.
To identify the multi-lineage differentiation potential, the BMSCs were induced to differentiate by switching to osteogenic, adipogenic, or chondrogenic differentiation medium. Flow cytometry was used to confirm the surface markers of BMSCs.
Extraction and identification of BMSC-Exos
The extraction and identification of exosomes have been described in our previous studies [20, 22]. Briefly, the culture medium of BMSCs was centrifuged at 10,000 g for 20 min. The supernatant was collected, and filtered through a 0.22-µm sterile filter (Millipore, USA). Then, the liquid was centrifuged at 100,000 g for 70 min (HITACHI Limited, Japan), and BMSC-Exos were obtained. The supernatant was centrifuged again at 100,000 g for 70 min to enrich the BMSC-Exos. The BMSC-Exos were then carefully resuspended in phosphate buffered saline (PBS) and the particle size distribution was examined with nanoparticle tracking analysis (NTA). The morphology of the EVs was observed with an 80 KV transmission electron microscope (TEM, HT7700, HITACHI, Japan), and exosome-specific proteins CD63, CD81, and TSG101 were detected by western blotting.
Exosome labeling and uptake assay
According to the instructions, BMSC-Exos were labeled with Dil (Sigma-Aldrich, USA), and the excess Dil dye was removed by supercentrifugation. MG-63 cells and the labeled exosomes were incubated for 4 h, before being fixed with 4% paraformaldehyde for 10 min at room temperature. After fixation, the nuclei were stained with Hoechst 33342 dye (Beyotime, China), and the cytoskeleton was stained with Phalloidin (Invitrogen, USA) for 15 min at room temperature. The internalization of BMSC-Exos was observed by laser confocal microscopy (Olympus, Japan).
Cell viability assay
To determine the effect of exosomes on cell viability, MG-63 cells were seeded at a density of 2000 cells/well (three replicates per group) in a 96-well plate and the MEM standard culture medium (containing 10% exosome-depleted FBS) supplemented with different concentrations of exosomes, 20 µg/ml, 40 µg/ml, and 80 µg/ml, or an equal volume of PBS for 24 h, 48 h, or 72 h. Then, 10 µL CCK-8 solution (sigma, USA) was added to each well and the plate was incubated at 37°C for 2 h. Finally, the absorbance of each well was measured at 450 nm under a microplate reader (Bio-Tek, USA).
Cell alkaline phosphatase activity and mineralization assay
As described previously [20], MG-63 cells were seeded at a density of 3 × 105 cells/well in a 12-well plate. The role of exosomes was tested by supplementing osteogenic induction medium (OIM; Cyagen Biosciences Inc., China) during cell culture, and additional BMSC-Exos (80 µg/ml) or an equal volume of PBS were added to each well. After 7-day induction, staining for alkaline phosphatase (ALP) was conducted by a BCIP/NBT ALP Color Development Kit (Beyotime, China). After induction for 14 d, the cells were stained with 1% Alizarin red S (Solarbio, China). For quantitative mineralization measurement, the stained cells were eluted with 10% cetylpyridinium chloride (Sigma, USA) at room temperature for 20 min and the absorbance value was measured at 570 nm. Images of the cells were captured under a Leica microscope.
Flow cytometric assay
MG-63 cells were seeded at a density of 2000 cells per well in a 96-well plate. The MEM standard culture medium (containing 10% exosome-depleted FBS) was changed into medium containing 1% FBS, and the cells were cultured for another 24 h. Then, the cells were treated with 80 µg/ml exosomes derived from BMSCs or PBS for 12, 24, 48, or 72 h, harvested, resuspended in a propidium iodide–RNase solution, and incubated for 2 h in the dark. Finally, the cells were detected by flow cytometry, and the cell cycle distribution was analyzed using the ModFitLT DNA analysis program (Becton Dickinson, San Jose, CA, USA).
Western blotting
The procedures for western blotting were described in our previous reports [20, 22]. The following primary antibodies were used for western blotting: ERα (1:500 dilution, Abcam), ERK (1:1000 dilution, Cell Signaling Technology), p-ERK (1:1000 dilution, Cell Signaling Technology), and β-actin (1:5000 dilution, Abcam). The secondary antibody was obtained from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. (Beijing, China). Briefly, the cells were homogenized at 13,000 rpm for 5 min at 4°C, and the protein was obtained. The protein (20 µg) was subjected to SDS-PAGE, and electro-transferred to PVDF membrane (Whatman, Maidstone, Kent, UK). The membranes were incubated with different primary antibodies and then secondary antibodies and then filmed using a chemiluminescent imaging system (Fusion SL2, Vilber Lourmat, Marne-la-Vallée Cedex, France).
Animal model
All animal care and experiments were approved by the Animal Research Ethics Committee of Beijing Jishuitan Hospital. Female Sprague-Dawley (SD) rats (10 weeks old, weighing 230–250 g) were purchased from SPF Beijing Biotechnology Co. Ltd. (Beijing, China). All of the rats were housed at a 12/12-h light/dark cycle, with a controlled temperature (23–25°C) and steady humidity (40–60%). Based on our previous study [20], the osteoporosis (OP) model was established by bilateral removal of rat ovaries. All rats were randomly divided into three groups as follows: (1) sham group (removal of a similar volume adipose tissue around the ovary); (2) OVX group (bilateral ovaries were removed and treated with PBS at 8 weeks after surgery); and (3) OVX + BMSC-Exos group (bilateral ovaries were removed and treated with BMSC-Exos at 2 weeks after surgery).
ELISA for inflammatory factors
The blood samples were obtained by cutting the rat tail, transferring the blood samples to a 2-ml tube, and centrifuged at 2600 rpm for 10 min to obtain the serum samples for ELISA. The concentration of serum samples was evaluated by measuring the levels of OCN and CTX-I according to the procedures of the ELISA kit (CUSABIO, Wuhan, China).
Microcomputed tomography (micro-CT) and histological analyses
The femurs were removed and fixed in 4% paraformaldehyde for 72 h. A Skyscan 1172 (Belgium) was used to scan the femurs at a resolution of 9 µm per pixel. Bone parameters, including bone mineral density (BMD), bone volume/tissue volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp), were analyzed to evaluate the bone mass and microstructure.
After fixation, the femurs were decalcified in 10% EDTA solution (Sigma) for approximately 1 month, before dehydrating, paraffin embedding, and sectioning into 4-µm slices. H&E staining was used to show the bone trabecular structure. Immunohistochemistry was performed to determine the ERα expression in vivo among different groups and evaluate the function of exosomes in terms of ERα expression. The sections were dehydrated, the antigens were extracted, blocked, and treated with primary ERα (1:200, Abcam) antibody at 4°C overnight. Then, DAB solution was added after 30 min of incubation with secondary antibody at room temperature. Finally, the images were observed using an optical microscope.
Statistical analyses
Quantitative data are presented as the mean ± standard deviation. SPSS (version 21.0, SPSS Inc.; Chicago, Ill) was used to perform all statistical analyses. The Student’s t-test was used to compare the differences between the two groups. The differences among three or more groups were examined by one-way analysis of variance (ANOVA), and the subsequent between-group differences were confirmed by Tukey’s test. A P-value < 0.05 was considered significant.