Extracellular vesicles derived from bone marrow mesenchymal stem cells loaded on magnetic nanoparticles delay the progression of diabetic osteoporosis via delivery of miR-150-5p

Establishment of rat models of DO

Seventy male SD rats (7–8 weeks old; 180 ± 20 g) were housed with 60–70% relative humidity at 25 ± 2 °C under a 12-h light/dark cycle. They were free access to food and water. Following 7 days of acclimatization, 6 rats were injected with normal saline and used as control and 64 rats were randomly selected for DO modeling by intraperitoneal injection with streptozotocin (STZ; 60 mg/kg, Sigma-Aldrich, St Louis, MO) (Wang-Fischer and Garyantes 2018; Cao et al. 2021; An et al. 2019). The study, approved by the Animal Ethics Committee of the First Affiliated Hospital of Bengbu Medical College (approval no.: 2021–245), was conducted according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

After 72 h, random blood glucose > 16.7 mmol/L was indicative of successful modeling. Bone mineral density (BMD) and bone mineral content (BMC) were assessed every 4 weeks using dual-energy X-ray absorptiometry (DXA; Hologic, Marlborough, MA). The significant statistical difference in BMD and BMC between the two groups denoted successful establishment of DO (Supplementary Fig. 1).

After 8 weeks, 55 rats were successfully modeled, among which, 6 rats were randomly selected and treated with GMNPE-EVs (n = 3) or EVs (n = 3); the remaining 48 rats were treated with lentiviral vector harboring mimic negative control (NC), inhibitor NC, miR-150-5p mimic, or miR-150-5p inhibitor, PBS alone, or co-treated with GMNPE-EV-mimic NC + lentiviral vector harboring sh-NC, GMNPE-EV-miR-150-5p mimic + lentiviral vector harboring shRNA (sh)-NC, and GMNPE-EV-miR-150-5p mimic + lentiviral vector harboring sh-β-catenin (n = 6 for rats upon each treatment). GMNPE (10 mg/kg body weight in 400 μL PBS), EVs (100 μL), and lentiviral vector (100 μL) were injected into rats via tail vein, and N52 neodymium magnet was then used to provide external magnetic field on rat femur, with the flowing GMNPE collected to the femur. Lentivirus was purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). After 8 weeks, femoral tissues were collected for subsequent micro-CT and other tests.

Isolation and culture of primary BMSCs and osteoblasts

BMSCs were isolated as previously described (Huang et al. 2021). The bilateral tibia and femur of rats were collected, and the bone marrow was washed with DMEM/F12 to obtain a mixed cell suspension. The suspension was subsequently centrifuged (1000 r/min, 5 min) and a precipitate containing BMSCs was harvested, then re-suspended in DMEM/F12 and cultured with 5% CO2 at 37 °C. For the characterization of BMSCs (Duan et al. 2020; Dominici et al. 2006), alizarin red S staining was performed to test the calcium deposition at the 3rd week of osteogenic differentiation induction, so as to examine the osteogenesis of BMSCs, and oil red O staining was applied to observe the lipid droplets at the 3rd week of adipogenic differentiation induction to assess the adipogenesis of BMSCs. Cell morphology and growth were observed and identified under an inverted microscope (Supplementary Fig. 2).

The osteoblasts were isolated from trabecular bone fragments by sequential enzymatic digestion (Huang et al. 2021; Zhao et al. 2010). The morphology of cells was finally observed with an inverted microscope (Supplementary Fig. 3).

Cell transfection

Upon attaining about 75% confluence, cells were transfected with miR-150-5p mimic, mimic NC, miR-150-5p inhibitor, inhibitor NC, oe-NC, oe-MMP14, sh-NC, sh-MMP14#1, sh-MMP14#2, sh-MMP14#3, sh-β-catenin#1, sh-β-catenin#2, and sh-β-catenin#3 alone or in combination (Supplementary Table 1).

The gene overexpression plasmid pCMV6-AC-GFP was purchased from Shanghai Yaji Biotechnology Co., Ltd. (YC-13849RJ), and the shRNAs from Thermo Fisher Scientific.

RT-qPCR

Total RNA was extracted with TRIzol method and reverse-transcribed into cDNA with PolyA Tailing Reverse Transcription Kit (B532451, Sangon; for miRNA detection) or RevertAid RT Reverse Transcription Kit (K1691, Thermo Fisher Scientific; for mRNA detection). RT-qPCR was followed using Applied Biosystems QuantStudio System (4,489,084, Thermo Fisher Scientific). As normalized to U6 or GAPDH, the fold changes were calculated using the 2−ΔΔCt method (Livak and Schmittgen 2001) (Supplementary Table 2).

Immunoblotting

Total protein was extracted, separated, and transferred onto PVDF membranes. The membrane was blocked using 5% skimmed milk powder and underwent incubation with primary antibodies to CD63 (1:1000, rabbit, PA5-92,370, Thermo Fisher Scientific), HSP70 (1:1000, mouse, ab2787, Abcam), TSG101 (1:1000, rabbit, ab125011, Abcam), GM130 (1:500, rabbit, PA5-95,727, Thermo Fisher Scientific), MMP14 (1:1000, rabbit, MA5-32,076, Thermo Fisher Scientific), β-catenin (1:5000, rabbit, ab32572, Abcam), Wnt1 (1:1000, rabbit, PA5-85,217, Thermo Fisher Scientific), RUNX2 (mouse, ab76956, Abcam), BSP (1:1000, rabbit, PA5-114,915, Thermo Fisher Scientific), OPN (1 µg/mL, rabbit, ab63856, Abcam), OCN (1–10 µg/mL, mouse, MA1-20,786, Thermo Fisher Scientific), and β-actin (1:5000, mouse, ab8226, Abcam). The membrane was re-probed with HRP-labeled secondary antibody IgG (goat anti-rabbit, ab205718, 1:20,000, Abcam) or goat anti-mouse (ab6789, 1:5000, Abcam). Following ECL development, quantification was conducted with the ImageJ 1.48 software, with β-actin as the normalization.

ELISA

Using rat ELISA kit of tartrate-resistant acid phosphatase (TRAP) (BS-E11304R2, Boshen Biotechnology Co., Ltd., Jiangsu, China) and rat ELISA kit of C-terminal telopeptide of type I collagen (CTX-I) (XY-SJH-DS1027, Xuanya Biotechnology Co., Ltd., Shanghai, China), we detected levels of CTX-I and TRAP5b in the serum of rats.

Isolation and identification of EVs from BMSCs

The EVs were isolated from BMSCs by differential ultracentrifugation-based method as previously described (Liao et al. 2019).

The obtained EVs were then observed under a TEM (Hitachi H-7650, BAHENS, Shanghai, China) (Zhou et al. 2018). Nanoparticle Tracking Analyzer ZetaView_Particle Metrix (DKSH, China) was adopted to detect size distribution of EVs (Kooijmans et al. 2013). Furthermore, immunoblotting was conducted to examine the EV surface marker proteins (CD63, HSP70, TSG101, and GM130).

Co-culture of BMSC-EVs with osteoblasts and uptake of EVs by osteoblasts

BMSCs were transiently transfected with Cy3-labeled miR-150-5p, and then co-cultured with osteoblasts using a Transwell co-culture system (Guan et al. 2020; Zhu et al. 2019). After nucleus staining with Hoechst 33,342, uptake of the labeled EVs by osteoblasts was visualized by a confocal laser scanning microscope (ZEISS LSM 800).

Detection of osteoblast viability, mineralized nodules, and ALP activity

For viability assay of osteoblasts, CCK-8 kit (10 μL, C0038, Beyotime, Shanghai, China) was applied to assess cell viability (Ge et al. 2019). Osteoblasts were fixed and then stained with 1% alizarin red or ALP staining solution to observe the number of mineralized nodules or ALP activity respectively (Li et al. 2021).

Synthesis and characterization of nanoparticles

Various chemical substances were fixed on the surface of MNPs (102,138, XFNANO, Nanjing, Jiangsu, China) to produce GMNPs or RhB-labeled GMNPs (Supplementary Table 3). The Fe3O4@SiO2-PEG-CHO (GMNPs or RhB-labeled GMNPs) was obtained according to previous report (Liu et al. 2020).

The intermediate products and final products were analyzed by SEM (S-4800, Hitachi, Shanghai Fulai Optical Technology Co., Ltd., Shanghai, China) with energy-dispersive spectrometer (IQLAAHGABMFAAWMACL, Thermo Fisher Scientific) and Fourier transform infrared spectrometer (912A0770, Thermo Fisher Scientific). DLS (Nano ZS90, Malvern) was applied for size and size distribution measurement.

Preparation and biocompatibility of GMNPE

GMNPE was generated with GMNPs (Fe3O4@SiO2-PEG-CHO) bound by the rabbit antibody to CD63 (PA5-92,370, Thermo Fisher Scientific). In short, 1 mg/mL GMNP solution was purified by magnetic separation, and incubated with anti-CD63 antibody overnight at 4 °C to obtain GMNPE. Fluorescent GMNPE was prepared using the same method, with the aforementioned GMNPs replaced by RhB-labeled GMNPs.

In order to assess drug carrying capacity of GMNPE, thermogravimetric analysis (TGA; STA 6000, PerkinElmer) was conducted for analyzing MNPs, GMNPs, and GMNPE. The adsorption capacity and stability of HSA were measured with FITC-labeled HSA by fluorescence spectroscopy.

In vitro identification of GMNPE-EV binding and targeting efficiency

Purified EVs were labeled with PKH67 (green) (MINI67, Sigma). After magnetic separation, GMNPE-EVs were obtained, with the morphology observed by a high-resolution TEM (200 kV, CM200, Philips). Furthermore, RhB and PKH67 were separately used to label GMNPE and EVs to confirm this binding (Liu et al. 2020).

Immunoblotting was applied for confirmation of magnetic separation of serum EVs. Samples were probed with CD63 (rabbit, PA5-92,370, Thermo Fisher Scientific) and then with IgG (goat anti-rabbit, ab205718, 1:20,000, Abcam).

Immunofluorescence

GMNPBSA and GMNPE were fixed in 4% paraformaldehyde, blocked, and probed with rabbit antibody to CD63 (PA5-92,370, Thermo Fisher Scientific). The cells were re-probed with goat anti-rabbit (#60,839, 1:50, Cell Signaling Technologies, Beverly, MA) conjugated to Alexa Fluor® 555. Finally, images were captured under an 80i fluorescence microscope.

Uptake of GMNPE-EVs by osteoblasts

GMNPE-EVs (RhB-labeled GMNPE, PKH67-labeled EVs) were incubated with osteoblasts for 3 h at 37 °C and fixed with PFA. Cytoskeleton staining was performed to observe the localization of GMNPE-EVs in the cells, and the nuclei were counterstained with Hoechst 33,342.

Flow cytometry

FITC-labeled GMNPE or PKH67-labeled GMNPE-EVs containing different ratios of anti-CD63 were co-cultured with osteoblasts in PBS at 4 °C for 30 min. These samples were analyzed using a FC500 flow cytometer (Morey Biosciences, Inc., Shanghai, China). The FlowJo v.10 (Tree Star) software was applied for data processing.

Dual-luciferase reporter assay

3′-UTR sequence of MMP14 with predicted miR-150-5p binding sites was cloned into pGL3-basic vector (6107, Jiran Biotechnology Co., Ltd. Shanghai, China) to generate luciferase reporter vector pGL3-basic MMP14-3′-UTR-WT and pGL3-basic MMP14-3′-UTR-MUT. The reporter vectors underwent co-transfection with mimic NC or miR-150-5p mimic into HEK-293 T cells (Biobw, Beijing, China). Luciferase activity, as normalized to renilla luciferase, was determined using Dual-Luciferase Reporter Assay System (Shenzhen TOP Biotechnology Co., Ltd., Shenzhen, Guangdong, China).

Immunofluorescence detection of GMNPE-EV uptake in rat femoral tissues

GMNPE-EVs (RhB-labeled GMNPE, PKH67-labeled EVs) were injected into DO rats via tail vein. An N52 neodymium magnet was used to provide an external magnetic field in the rat femur, and the flowing GMNPE nanoparticles were collected to the femur. Next, the rats were euthanized and the femoral, brain, liver, and kidney tissues were removed to prepare longitudinal frozen sections, which were subjected to immunofluorescence staining with OCN (10 µg/mL, mouse antibody, 33–5400, Thermo Fisher Scientific) and 80i fluorescence microscopy.

Immunohistochemistry and hematoxylin–eosin staining

The femoral specimens were decalcified with 18% EDTA and cut into 5-μm-thick sections. The sections were immunostained with OCN (10 µg/mL, mouse, 33–5400, Thermo Fisher Scientific) and type I collagen (1:100, rabbit, 600–406-103, Thermo Fisher Scientific), and observed with a microscope (Olympus CX31, TUSEM, Shanghai, China). HE staining was used for histological observation as previously described (Liu et al. 2021b).

Histomorphometric analysis

On days 10 and 3 before euthanasia, the rats were intraperitoneally injected with 0.1% calcein (10 mg/kg body weight, C0875, Sigma-Aldrich) dissolved in PBS. After euthanasia, the femurs were removed from the rats. The samples were fixed, dehydrated, embedded in methyl methacrylate, and cut into 60-μm-thick sections. The double labeling of calcein was observed under a fluorescence microscope and the Image-Pro Plus 6 software was applied to measure mineral apposition rate (MAR) of trabecular bones.

Micro-CT scanning

Femoral tissues were scanned with mCT-40 micro-CT system (Scanco Medical, Switzerland) to analyze femoral tissue growth (Zuo et al. 2019). Image reconstruction was performed by the NRecon software (Bruker, Kontich, Belgium), and data were analyzed using the CTAn program (Bruker). The following parameters were determined: trabecular thickness (Tb.Th), bone volume/tissue volume (BV/TV), trabecular number (Tb.N), and trabecular separation (Tb.Sp).

Statistical analysis

Unpaired t-test, one-way ANOVA and two-way ANOVA, or repeated measures ANOVA with Tukey’s tests were utilized to calculate statistical significance for two-group, multi-group, and time-based data. All results processed using the SPSS 21.0 software were presented as mean ± SD. p < 0.05 suggests statistically significant difference.

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