Animals
All animal care and experimental procedures were approved by the Ethical Committee for Animal Research of Affiliated Zhongshan Hospital of Dalian University. Male 3-month-old healthy adult New Zealand white rabbits weighing 3-4 kg were purchased from Dalian Medical University, Liaoning, China. The rabbits were kept in specialized animal centers at Affiliated Zhongshan Hospital of Dalian University and were acclimated for 4 days before initiation of studies.
Animal Model and grouping
NONFH rabbit models were generated by previously well-validated protocols[26]. All rabbits were first randomly divided into 2 groups. The rabbits in the experimental (GC) group (n = 8) were intravenously injected with 10 μg/kg lipopolysaccharide (Sigma, USA). After 24 h, the experimental (GC) group was intramuscularly injected with 20 mg/kg of methylprednisolone acetate (Pfizer Manufacturing Belgium, USA) 3 times every 24 h. The control group (n = 8), the normal group, which was given the same volumes of saline. miR-451-5p group (n = 3): random three of the control group rabbits received AgomiR-451-5p (25 nmol, Genepharma Co. Lt., China) at a dose of 1 mL by intravenous injection one week after first injection of saline. miR-133b-3p group (n = 3): random three of the experimental (GC) group rabbits received agomiR-133b-3p (25 nmol, Genepharma Co. Lt., China) at a dose of 1 mL by intravenous injection one week after first injection of methylprednisolone acetate during modeling procedure. All rabbits were sacrificed four weeks later, and the organs and bone tissue of the lower extremities of the rabbits in the four groups were obtained. For protein extraction, tissue samples were homogenized in liquid nitrogen and then dissolved in ice-cold protein buffer. For RNA isolation, tissues samples were snap-frozen in liquid nitrogen.
Cell isolation and culture
BMMSCs were isolated from the femoral head of healthy rabbits by bone marrow aspiration as described[27]. The cells were culture in Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient medium (DME/F12, Hyclone, GE, USA) with 10% fetal bovine serum (FBS, HyClone; GE, USA) and 1% antibiotic antimycotic solution and changed every two days. Vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) were isolated from rabbit thoracic aortas by digestion with different enzymes as described [28, 29]. VECs were maintained in endothelial cell medium (ECM, ScienCell Research Laboratories, USA) and cultured in culture flasks that were coated with gelatin, and the medium was changed every 1-2 days. VSMCs were cultured in the same medium as what was used for BMMSCs, and the medium was changed every day. The cells were separately identified by flow cytometry and immunohistochemistry. All cells were incubated at 37°C with 5% CO2. Cells from passages 3-5 were used in experiments.
Characterization of rabbit cells
BMMSCs and VECs were identified by flow cytometry. Details on the experimental procedures are provided in Supplementary material. Characterization of VSMCs was performed by alpha-smooth muscle actin (α-SMA) -labeled VSMCs immunohistochemistry analysis. Detailed procedures are also provided in supplementary material.
Micro-CT Assay
Micro-CT (Siemens, Inveon Micro-CT, Berlin, Germany) images were obtained to evaluate trabecular bone structure of the femoral head. The scanning protocol was 80 kV and 500 μA, with an effective pixel size of 15.48 μm. Based on the CT images, a volume of interest (VOI) was selected from these regions for three-dimensional reconstruction and values of bone volume/total volume (BV/TV), bone surface area/bone volume, trabecular thickness (Tb.Th, mm) and trabecular spacing (Tb.Sp, mm) were compared.
Histological and immunohistochemistry (IHC) analysis
The expression of PAI-1 was observed by IHC analysis. Paraffin sections were dewaxed by routine methods, and antigen were retrieved. After that, 10% bovine serum albumin (BSA, HyClone; GE, USA) was incubated with the slides in a humid box at room temperature for 60 mins, then primary rabbit anti-PAI-1 antibody (1:500; Abcam, USA) was incubated with the slides overnight at 4°C. After washing three times in PBS, the sections were incubated with poly-HRP goat anti-rabbit IgG at 37°C for 30 mins. Then, the sections were stained with a DAB dye solution and restained with hematoxylin. In addition, hematoxylin and eosin (H&E) staining and Masson’s trichrome staining were conducted to evaluate the histological morphology. The sections were observed under an optical microscope. The number of empty lacunae in five random regions in each section (5 sections in each group) was counted and the percentage of empty lacunae was defined as the ratio of empty lacuna number to the total lacuna count.
Alkaline phosphatase (ALP) staining
After three days of glucocorticoid stimulation, cell culture medium was removed; then, cells were determined by ALP kits (Beyotime Biotechnology, China) according to the manufacturer's user guide. All cells were observed under a microscope.
Oil Red O staining
After three days of glucocorticoid stimulation, cell culture medium was removed. Cells were washed with PBS and fixed with 4% paraformaldehyde solution at room temperature for 30 mins. Then the freshly prepared Oil Red O working solution (Sangon Biotech, China) was added into culture flask and incubated with the cells at room temperature for 60 mins. The staining solution was removed and cells were washed with PBS 3 times. All cells were observed under a microscope.
Isolation and Purification of Exosome
BMMSCs were seeded at 5×105 cells per 25 cm2 culture flask in 5 mL of culture medium, and were reached confluence 5 days later. The cells were cultured in medium supplemented with 10% serum without FBS-derived exosomes and methylprednisolone (0 or 5 μg/mL). Over the next 72 h, the culture medium was collected. The exosomes were collected from the 40 mL of medium and were subsequently purified via an exosome concentration kit (Liaoning Rengen Biosciences, China) according to the manufacturer's information. The samples were stored at -80°C.
Co-culturing BMMSCs exosomes with vascular cells
VECs and VSMCs were seeded at a density of 105 cells/well in a 24-well plate in 1 mL culture medium before co-culture treatment. After overnight plating, cells were treated with BMMSCs (GC treated or not) exosome for 72 hours. The same dose of exosomes (20 μL) were added to each group.
Nanoparticle Tracking Analysis (NTA)
Particle sizes of exosomes were measured by dynamic light scattering (DLS) (Zetaview, Particle Metrix, Germany).
Transmission Electron Microscope (TEM)
Exosomes were dropped on a copper grid for 3 mins. Excess liquid was removed with filter paper and dried at room temperature. Next, 3% ammonium molybdate negative staining solution (Solarbio Science & Technology, China) was added to the copper grid for 3 mins. After removing excess staining solution with filter paper, the grid was examined, and TEM images were recorded (JEM-2100, JEOL, Japan).
Enzyme-linked immunosorbent assay (ELISA)
The concentration of PAI-1 protein was determined by rabbit PAI-1 ELISA kit (Shanghai Lengton Biotechnology, China) according to the manufacturer's instructions.
Quantitative real-time polymerase chain reaction (qRT‑PCR)
All Cells were lysed with RNAiso Plus (Takara Biomedical Technology, Japan) and reverse transcribed using a PrimeScript™ RT reagent kit with gDNA Eraser (Takara Biomedical Technology, Japan) following the manufacturer’s instructions. The generated cDNA was amplified using TB GreenTM Premix Ex TaqTMⅡ (Takara Biomedical Technology, Japan). The primers (SERPINE1, ocu-miR-133b-3p, ocu-miR-451-5p, U6 and GAPDH) (see Table S1 in the online-only supplementary material) were synthesized by TAKARA BIO (Takara Biomedical Technology, Japan). Target mRNA expression levels were normalized to GAPDH, using the 2-△△Ct method, the result are analysis results are presented as fold change.
Western blotting
Western blotting was conducted as described[30]. Details on the western blotting experimental procedures are provided in supplementary material. All the antibodies mentioned were as follows: PAI-1 (1:1000) (OmnimAbs, USA), GAPDH (1:1000) (Novusbio, USA).
MicroRNA sequencing
Extracting and purifying total RNA from cell supernatants was performed via SeraMirTM exosome RNA amplification kit (System Biosciences, USA). The purified total RNA was analyzed via Novogene’s genomics platform via illumina HiSeqTM2500/MiSeq for gene clustering and sequencing. Other detailed procedures are provided in the supplementary material.
Transfection of miR-133b-3p Mimics and Inhibitor
miR-133b-3p mimics (AgomiR), inhibitor (AntagomiR), stable negative control and inhibitor N.C. were synthesized by Genepharma Co. Lt. (China) (Table S2). They were transfected into vascular endothelial cells at a final concentration of 40 nM using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. All cells were collected for qRT-PCR and western blotting 48 h after transfection.
Statistical analyses
Every experiment was repeated at least three times. All data was analyzed by using SPSS version 23.0 (IBM Corp., Armonk, USA). Measurement data were expressed as the mean ± standard error of mean (s.e.m). Comparisons between groups were analyzed by t-tests or ANOVA. A P-value of < 0.05 was considered statistically significant.