All the experiments in the present research were approved by the Third Hospital of Hebei Medical University. The macrophage cell line RAW264.7 was incubated in α-MEM culture medium containing 10% fetal cattle serum (FBS) and 1% penicillin-streptomycin. For induction of M2 macrophage differentiation, RAW264.7 cells were cultured with 20 ng/ml Interleukin-4 (IL-4) for 24 h. Ten BALB/c male mice were obtained from the animal experimental center of Hebei Medical University and were used to separate BMSCs. Briefly, primary BMSCs were isolated from bone marrow and subsequently cultured in DMEM complete medium. For induction of osteogenesis, BMSCs were grown in osteogenic medium containing 10 mM β-glycerophosphoric acid, 10 nmol/L dexamethasone, and 50 μg/ml ascorbic acid. Adipogenesis was induced by 10 mg/ml insulin, 500 mmol/L methyl-isobutylxanthine, and 1 μmol/L dexamethasone. All cells were cultured at 37°C with 5% CO₂.

After RAW264.7 cells were cultured with 20 ng/ml Interleukin-4 (IL-4) for 24 h, the positive surface markers CD206 (BioLegend, San Diego, CA, USA) was analyzed by flow cytometry analysis.

After M2 differentiation of macrophages was induced, the MinuteTM efficient exosome precipitation reagent purchased from Inent Biotechnologies Company was used to obtain the exosomes according to the instructions. In short, the culture medium of M2 macrophages was harvested after culture in serum-free medium for 15 h. Then, cells were removed from the samples by low-speed centrifugation (5 min, 1,000 g), and the culture medium was obtained for further separation. The collected media was subsequently incubated with exosome precipitant overnight at 4°C. Next, the samples were centrifuged for 1 h at 10,000 g, and the supernatant was removed. The isolated exosomes were harvested and stored at −80°C for future use.

For further identification, the morphology and diameter of exosomes were characterized by transmission electron microscopy (Hitachi HT7700 TEM, Tokyo, Japan). Twenty microliter exosome samples were aspirated with a pipette gun and placed in carbon membrane copper mesh for 5 min. Then, the excess liquid was aspirated with filter paper. The sample was subsequently stained with 2% phosphotungstic acid for 2 min. The excess liquid was absorbed with filter paper. Finally, images of exosomes were collected by TEM. The number and size of exosomes were characterized by nanoparticle tracking analysis (NTA) using NanoSight NS-300. In addition, CD63 and CD81, exosomal specific surface proteins, were detected by western blotting.

According to the instructions, exosomes were labeled with PKH26 (Sigma-Aldrich, Germany). First, 30 μl of exosomes was diluted in 1 ml of diluent C and 6 μl of PKH26 dye for 5 min. Then, 10% bovine serum albumin (BSA) was added to neutralize the excess dye, followed by washing in PBS for 60 min. Finally, BMSCs were treated with the labeled exosomes and analyzed by a fluorescence microscope.

Cells were transfected with 30 nM miR-690 inhibitor or 30 nM inhibitor negative control (inhibitor NC) using Lipofectamine 3000 (Thermo Fisher Scientific, USA) based on the manufacturer’s protocol. Briefly, miR-690 inhibitor or inhibitor NC was mixed with Lipofectamine 3000 for 20 min separately before being cultured with cells. Cells were collected for further miRNA analysis at 48 h post-transfection. The miR-690 inhibitor and inhibitor negative control were all obtained from Zhongshi Tongtru (Tianjin, China). The sequences were as follows: miR-690 inhibitor 5’-UUUGGUUGUGAGCCUAGCCUUU-3’ and miR-690 inhibitor NC 5’-CAGUACUUUUGUGUAGUACAA-3’.

BMSCs were transfected with Si-IRS-1 or control plasmid (Genechem, Shanghai, China) using Lipofectamine 3000 after reaching 80% confluence, in accordance with manufacturer’s instructions.

A-R staining was performed after 2 weeks of osteoblast induction. In short, BMSCs were fixed with 4% paraformaldehyde (Solarbio, China) for 10 min after being washed twice with PBS. Then, 1% A-R staining solution was incubated for half an hour. Finally, red mineralized nodules were observed. In addition, the absorbance at 570 nm was subsequently measured via a microplate spectrophotometer (BioTek Instruments, San Jose, CA, USA).

Lipid droplet formation was detected by oil red O staining after culture in adipogenic medium for 2 weeks. Briefly, BMSCs were washed twice with PBS followed by fixation with 4% paraformaldehyde for 10 min. Then, oil red O staining solution was added to stain the cells for half an hour. Finally, the formation of lipid droplets was captured by a microscope. Moreover, the absorbance at 520 nm was subsequently analyzed.

For the analysis of mRNA, total RNA was extracted using TRIzol^® reagent (Tiangen, Beijing, China) before reverse transcription into cDNA via a RevertAid™ First Strand cDNA synthesis kit (Thermo, Waltham, USA) based on the instructions. Then, RT-PCR analysis was performed according to the protocols for Tiangen SuperReal PreMix Plus.

For the analysis of miRNA, microRNA assay kits were purchased from Zhongshi Tongtru (Tianjin, China) and used following the manufacturer’s instructions.

Table 1 shows the PCR primer sequence, which is designed by primer software.

RIPA lysis buffer containing 1% protease inhibitor (Zhongshi Tongtru, Tianjin, China) was used to obtain total protein from the samples. Then, 20 μg protein was separated via SDS-PAGE and transferred to PVDF membranes before being blocked with 5% milk for 1 h. The PVDF membranes were stained at 4°C overnight with antibodies specific for IRS-1 (1:1,000, #2382, Cell Signaling, Beverly MA,USA), TAZ (1:1,000, #72804, Cell Signaling, Beverly MA,USA), OCN (1:1,000, ab274873, Abcam, Cambridge, England), RUNX2 (1:1,000, ab236639, Abcam, Cambridge, England), CEBPβ (1:1,000, ab32358, Abcam, Cambridge, England), PPARγ (1:1,000, ab272718, Abcam, Cambridge, England), and GAPDH (1:1,000, ab8248, Abcam, Cambridge, England). Finally, blots were stained with fluorescence secondary antibodies and detected with the Odyssey Infrared Imaging System (Li‐COR Biosciences).

Experimental data were obtained from at least three replicates and are shown as the mean ± standard deviation. Student’s t-tests and one-way ANOVA with Tukey’s post hoc test were used to compare the data between different groups as appropriate. p < 0.05 was considered statistically significant.

Compared to PBS-treated macrophages, IL-4-treated macrophages showed significant upregulation of CD206 ( Figure 1A ), suggesting that the RAW264.7 cells were successfully induced to M2 polarization after stimulation with IL-4.

Exosomes were first harvested from the supernatants of M2 macrophages via exosome separation reagent. TEM, NTA, and western blotting analyses were performed to identify the collected M2D exosomes. Typical round or cup-shaped exosomal structures were revealed by TEM ( Figure 1B ). Western blotting analysis further provided evidence that the isolated particles were positive for exosomal surface markers, including CD63 and CD81 ( Figure 1C ). NTA analysis revealed that the diameters of these exosomes ranged from 50 to 150 nm ( Figure 1D ).Therefore, these above analyses confirmed the successful collection of M2D-Exos.

Subsequently, to verify whether the M2D-Exos could be endocytosed by BMSCs, we labeled the M2D-Exos with PKH26 and further cocultured them with BMSCs. As shown in Figure 1E , PKH26-labeled exosomes were localized in the BMSC region, which exhibited efficient internalization of the M2D-Exos by BMSCs.

To further analyze the effect of M2D-Exo function on the osteogenesis of BMSCs, we performed western blotting, RT-PCR, and Alizarin red staining. The expression of the osteogenic differentiation-related proteins RUNX2 and osteocalcin (OCN) was upregulated after culture with M2D-Exos ( Figures 2A, B ). Consistent with this finding, significantly higher mRNA levels of RUNX2 and OCN were detected in the M2D-Exo group than in the control group (treated with PBS) ( Figure 2C ). In addition, the Alizarin red staining results revealed that the mineral deposition staining in the M2D-Exo-treated BMSCs was significantly larger than that in the control group ( Figures 2D, E ). The above findings indicate that M2D-Exos could increase the osteogenic differentiation of BMSCs.

For further elucidation of the function of M2D-Exos in the adipogenesis of BMSCs, adipogenic differentiation-related genes, and proteins as well as the formation of lipid droplets were analyzed. The expression of CCAAT/enhancer binding protein β (C/EBPβ) and PPARγ was downregulated after treatment with M2D-Exos ( Figures 3A, B ). Similarly, the RT-PCR results showed lower mRNA levels of C/EBPβ and PPARγ in the M2D-Exo group compare to PBS-treated BMSCs ( Figure 3C ). In addition, oil red O staining revealed reduced formation of lipid droplets in the M2D-Exo-treated cells ( Figures 3D, E ), indicating an inhibitory effect of M2D-Exos on adipogenesis.

To explore the mechanism involved in the regulation of the differentiation of BMSCs by M2D-Exos, we then detected the expression of miR-690, IRS-1, and TAZ. Western blotting analysis showed that TAZ and IRS-1 expression during osteogenesis was significantly increased in the M2D-Exo-induced BMSCs ( Figures 4A, B ). Moreover, M2D-Exo intervention led to an increased level of miR-690 during osteogenic differentiation ( Figure 4C ). During adipogenic differentiation, we also detected the expression of miR-690 and the IRS-1 and TAZ proteins. The results were consistent with those for osteogenic differentiation. M2D-Exo administration resulted in an increase in the levels of miR-690, IRS-1, and TAZ ( Figures 4D–F ). We then knocked down IRS-1 expression using si-IRS-1 plasmids ( Figures 4G, H ). Western blot analyses suggested that Si-IRS-1 significantly blocked the upregulation of TAZ expression after the intervention of M2D-Exos ( Figures 4I, J ). The above results indicated that M2D-Exos might up-regulate miR-690 of BMSCs and increase the expression of IRS-1 and TAZ to regulate the balance between adipogenesis and osteogenesis.

To investigate whether miR-690 mediates the M2D-Exo-derived regulation of adipogenesis and osteogenesis, we first detected the expression of miR-690 in BMSCs after the intervene of M2D-Exos. The level of miR-690 was significantly increased after treated with M2D-Exos ( Figure 5A ), showing that M2D-Exos might transmit miR-690 to BMSCs. We then transfected a miR-690 inhibitor and inhibitor control into M2 macrophages. Compared with the inhibitor control, the miR-690 inhibitor significantly inhibited the level of miR-690 in M2D-Exos ( Figure 5B ). In addition, BMSCs were cocultured with miR-690 inhibitor-treated exosomes or miR-690 inhibitor control-treated exosomes, and the miR-690 inhibitor-treated exosome group showed a lower level of miR-690 than the inhibitor control group ( Figures 5C, D ). Furthermore, the miR-690 inhibitor attenuated the M2D-Exo-induced increases in IRS-1 and TAZ levels and the subsequent upregulation of osteogenic marker gene (RUNX2 and OCN) expression in osteogenic medium ( Figure 5E ). Similarly, the miR-690 inhibitor-treated exosomes inhibited the mRNA expression of IRS-1 and TAZ and increased the mRNA expression of C/EBPβ and PPARγ during adipogenic differentiation ( Figure 5F ). Furthermore, the results of Alizarin Red staining and oil red O staining were consistent with the real-time RT-PCR analyses and verified the mechanism by which M2D-Exos facilitated osteogenic differentiation and inhibited adipogenic differentiation by miR-690 ( Figures 5G–J ).