Patients
The specimens were obtained from the Orthopedic Department of the hospital between March 2018 and November 2018. A total of 12 participants were enrolled, six of whom had glucocorticoid-induced ONFH. These six patients formed the ONFH group while the other six had femoral neck fractures, and these formed the control group. The inclusion criteria were (1) male or female patients between 40 and 70 years old; (2) diagnosis of glucocorticoid-induced ONFH or femoral neck fracture; (3) indications for total hip arthroplasty. The exclusion criteria included (1) alcohol-induced or trauma-induced ONFH and (2) preoperative diagnosis of either HIV, hepatitis B or C infections. The ONFH group comprised 3 women and 3 men (average age of 51.7±5.2 years) at the time of surgery, while the control group comprised five women and one man (average age of 65.3±2.9 years) at the time of surgery (Table 1). Femoral heads were removed during total hip arthroplasty, and a band saw was used to cut all the specimens into half at the coronary level. Half of the femoral heads were fixed in 10 % formalin for examination while the remaining half was used for the isolation and culture of BMECs.
Hematoxylin‐eosin(HE) staining
The collected tissues were fixed in 10 % formalin after which they were decalcified in 10% EDTA solution for six weeks. The samples were subsequently dehydrated in a graded series of ethanol, embedded in paraffin, sliced into 5μm thick sections. After staining with HE, a light microscope was used to examine the level of necrosis of the bone and marrow tissues. The existence of empty lacuna was used to indicate the level of osteonecrosis [17]. The average number of lacunae from three fields was calculated for each group.
Isolation and culture of BMECs
The BMECs of patients undergoing hip arthroplasty were obtained as described previously [18]. Briefly, cancellous bones were harvested from the subchondral region of the femoral head. The bone debris was digested with 0.2% type I collagenase and 0.25% Trypsin–EDTA for 5 minutes. The reaction was stopped with the addition of Dulbecco's modified Eagle's medium (DMEM, Gibco, USA). The cell lysates were filtered with 70‐μmol/L cell strainer and centrifugated at 1500 rpm for 6 min. The supernatant was removed and the cells were bathed in endothelial cell medium (ECM, ScienCell, USA) containing 5% fetal bovine serum (FBS), 5 ml recombinant human VEGF and antibiotics in a 37°C humidified incubator with 5% CO2. A fresh medium was added the next day, and at intervals of three days subsequently. The cells were passaged when they reached 90% confluence.
Examination of cell proliferation and viability
The proliferation of BMECs was assessed using the CCK-8 kit following the instructions on the kit. After BMCEs incubation in the 96 wells, 100 μL of ECM and 10 μL of CCK-8 solution were added to each well for a 2-h incubation period. The OD of each well was determined using a microplate reader at a wavelength of 450 nm.
The neutral red uptake assay was used to assess cell viability, as detailed before [19]. BMECs were added to 96-well plate (2×105 cells/well) and allowed to adhere. Next, a fresh FBS-free medium was added and then the cells were treated with neutral red dye (in a fresh medium) for 2 h. Subsequently, a neutral red lysis buffer was used to remove the neutral red stain, and the OD was assessed at 570 nm.
TUNEL assay
Apoptosis of BMECs was measured with TUNEL staining using the In Situ Cell Death Detection Kit (Roche). Cultured BMECs were collected in a six-well plate, 12 h post preparation. The cells were fixed with freshly prepared 4% paraformaldehyde for 1 h, incubated with 3% H2O2 and 0.1% Triton X-100 for 10 min, and washed thrice with phosphate-buffered saline (PBS) during each step. Cells were subsequently stained with DAPI according to the In Situ Cell Death Detection Kit manual. Images were observed and captured using a fluorescence microscope (Olympus, Tokyo, Japan). The number of apoptotic cells was quantified by counting TUNEL-positive cells by three independent researchers.
Tube formation assay
A 96-well plate was coated with 100 μL of Matrigel (BD, USA) and then allowed to solidify and polymerize at 37 °C for 1 h. The Matrigel was subsequently overlaid with 100 μL of a suspension of BMECs (2×105 cells/well). Tube formation by BMECs was observed under a microscope after 6 h of incubation and quantified by NIH Image J.
Transwell assay
Corning transwell chambers (Corning, 8μm, USA) were used for the transwell assay. For the migration assay, 5×105 BMECs suspended in 200 μL serum-free medium were added in the upper chambers, and 500 μL ECM containing FBS were added in the lower chamber. Following incubation for 12 h, the upper chambers were washed with PBS, and the cells on the upper surface were wiped off using a cotton swab. Subsequently, cells on the bottom surface of the membrane were fixed in 4% paraformaldehyde and stained with 1% crystal violet. The number of migrated cells was assessed and counted under an optical microscope.
Western blot analysis
Cellular total protein was extracted using RIPA lysis buffer and then quantified using the BCA kit (Beyotime, China). After that, proteins were denatured by heating at 95℃ for 5 min. A 30 μg sample of proteins were resolved by 10% SDS-PAGE and transferred to a PVFD membrane. The membrane was bathed in fat-free milk to block non-specific binding for 1 h followed by incubating overnight at 4°C with primary antibodies against caspase-3 (Abcam, 1:1000), cleaved caspase-3 (Abcam, 1:1000), Bax (Abcam, 1:1000), Bcl‑2 (Abcam,1:500), and β-actin (Abcam,1:3000). Subsequently, they were incubated with their corresponding secondary antibodies. The immunoblots were visualized with Electrochemiluminescence Plus Reagent (Invitrogen), and the expression was quantified with Image Lab 3.0 software.
Statistical analysis
All data were expressed as the mean ± standard deviation (SD). Data analysis was done using one-way analysis of variance (ANOVA) and the Student’s t-test using SPSS version 21.0 (SPSS Inc., Chicago, IL, USA). A value of P < 0.05 was considered statistically significant.