Specimens collection

Glucocorticoid-induced osteonecrosis of femoral head (ARCO stage II) samples were collected from a patient who received bone grafting in Zhongshan Hospital, Fudan University (Shanghai, China). The patient with ONFH had a medical history of the steroid administration owing to systemic lupus erythematosus (SLE). This study was approved by the Ethical Committee of the Zhongshan Hospital (B2019-135R) and informed content was obtained from the patient.

Reagents

Fetal bovine serum (FBS), penicillin and streptomycin were provided by Gibco Life Technologies (Grand Island, NY, USA). α-Modified Eagle’s Medium (α-MEM) was purchased from Hyclone (Waltham, MA, USA). Endothelial Cell Medium (ECM) was purchased from ScienCell (Carlsbad, CA, USA). Osteogenic differentiation medium of MC3T3-E1 Cell was purchased from Cyagen (Santa Clara, CA, USA). Matrigel was purchased from Corning (New York, USA). Phalloidin was provided by Beyotime (Shanghai, China). Cy3- conjugated goat anti-rabbit secondary antibody (BA1032) and 4’, 6-Diamidino-2-phenylindole (DAPI) (AR1177) were purchased from Boster (Wuhan, China). Mouse SOST Elisa Kit (ELK 6089) was purchased from ELK Biotech (Wuhan, China). Dexamethasone (CAS No. 50-02-2) and other reagents were of the highest commercial grade and were purchased from Sigma Chemical (St. Louis, MO, USA).

Cell culture

Murine osteocyte (Ocy454) was provided by Prof. Pajevic (Massachusetts General Hospital and Harvard Medical School). Murine Osteoblast cell line (MC3T3-E1) was purchased from ATCC. Murine arterial vascular endothelial cells were isolated from previous study (Wang et al. 2016). Ocy454 and MC3T3-E1 were cultured in α-MEM contained 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. Vascular endothelial cells were cultured in ECM contained 5% FBS, 5 ml endothelial cell growth supplement, 100 U/mL penicillin and 100 mg/mL streptomycin. Ocy454, MC3T3-E1 and vascular endothelial cells were maintained in humid incubator with 5% CO2 at 37 °C. The medium was replaced every 2 days and passaged upon 80%–90% confluency. To investigate the role of sclerostin in GA-ONFH, Trans-well chamber system was used for co-culture experiment. MC3T3-E1 or vascular endothelial cells were cultured in the lower chamber with corresponding medium. Ocy454 or SOST-silencing Ocy454 were seated in the upper wells with α-MEM containing dexamethasone.

Cell viability assay

A cell counting kit (CCK, Dojindo, Japan) was used to analyze cell viability in the co-culture experiment. Different groups of MC3T3-E1 or vascular endothelial cells were seeded at density 1 × 104 cells/well. To measure cell viability, 10 μL CCK-8 solution and 90 μL medium were added to each well, and then, the plates were incubated in the dark at 37 °C for 1.5 h. The absorbance values of the supernatants were recorded in 450 nm.

ELISA assay

In co-culture system, MC3T3-E1 or vascular endothelial cells were passaged at 80% confluence, cells were washed, and fresh medium was added. After 24 h, the supernatant was collected, and Sclerostin levels were measured using mouse SOST Elisa kit as per the instruction manual.

Alizarin red (ALR) staining

MC3T3-E1 were seeded onto 24-well plates at a density of 1 × 105 cells/well and incubated for 21 days in an osteogenic differentiation medium. Upon completion of osteogenic differentiation, cells were washed with PBS twice and fixed in 4% paraformaldehyde for 30 min at room temperature. After fixation, cells were treated with 1 mL alizarin red staining solution for 3–5 min and gently rinsed by tri-distilled water before analyzed under a light microscope.

Tube formation assay

In tube formation assay, 200ul of Matrigel was coated in the lower chamber and the vascular endothelial cells were seeded on the Matrigel at density of 5 × 104 cells/well. After the plate was placed into an incubator for 1 h, tube formation was monitored using a microscope 6 h after inoculation.

Total RNA extraction and quantitative real-time RT-PCR

Total RNA was extracted by total RNA extraction kit in accordance with the manufacturer’s instructions, OMEGA. The purity and concentration of the RNA were determined by a spectrophotometer (Thermo Fisher Scientific, USA). Complementary DNA (cDNA) was synthesized from total RNA and amplified with SYBR Green Master Mix in an ABI PRISM 7500 PCR Sequence Detection System (Applied Biosystems, Foster City, CA, USA) according to following condition: 30 s of denaturation followed by 40 cycles of 94 °C for 5 s and 60 °C for 35 s. The melting curve was generated to test for primer dimer formation and false priming for each reaction. Relative expression of gene-specific products was analyzed using the comparative Ct (2 − ΔΔCt) method and normalized to the reference gene β-actin.

Western blotting analysis

The total proteins were obtained from different groups of MC3T3-E1 or vascular endothelial cells by RIPA lysis buffer containing 1% proteinase inhibitor and 1% phosphatase inhibitors cocktail for 30 min on ice at the indicated time points. The concentration of protein was measured using the BCA protein assay kit (Boster, Wuhan, China). Then, 20 μg of protein was separated resolved on 10% SDS-PAGE gels and transferred to PVDF membrane (Millipore), blocked with 5% BSA in TBS-T (0.1% Tween-20) and incubated with primary antibody (2% BSA in TBS-T) overnight at 4 °C. Subsequently, the membrane was washed with TBS-T and incubated the corresponding secondary antibodies for 2 h at room temperature. Finally, the protein bands were visualized through Western ECL Substrate Kit (Yseasen, Shanghai, China) on Tanon imaging system and grayscale was analyzed with ImageJ software. Antibodies against phospho-GSK-3β (8213), GSK-3β (12,456), β-catenin (9562), non-phospho (Active)-β-catenin (8814), Connexin 43 (3512) were bought from Cell Signaling Technology (Beverly, MA, USA). Antibodies against ALP (ab229126), Runx2 (ab192256), Sclerostin (ab63097), VEGF (ab1316) were bought from Abcam (Cambridge, UK). Antibodies against VEGF and β-actin were bought from Proteintech Group (Wuhan, China).

Small interfering RNA (siRNA) assays

In line with the manufacturer’s protocol, osteocyte (Ocy454) was transfected with 20 μM siRNAs by using siRNA Transfection Reagent (Ribbio, Guangzhou, China). After 2 days of cultivation, transfected osteocytes were harvested for subsequent experiment. siRNAs used for siRNA assays: si-1 CTGAGAACAACCAGACCAT; si-2 ATCCCTATGACGCCAAAGA; si-3 ACACCCGCTTCCTGACAGA.

Animal model

In our study, all experimental procedures on the animals were in accord with the Ethics Committee on Animal Experimentation of Zhongshan Hospital, Fudan University (Shanghai, China). All rats were kept in the animal care facility of Zhongshan Hospital. The living environment of mice were maintained at 25 °C with 12:12 light/dark cycle, and mice were fed with normal chow and water. SOST-Knockout (SOST −/−) rats were generated using the CRISPR/Cas9 system as previously decribed (Bäck et al. 2019; Ma et al. 2014). We designed two single guide RNA (sgRNA) targeting exons 2 in SOST gene (NM_030584.1). The mixture of transcribed Cas9 and sgRNA was injected into Sprague–Dawley rat monocytic embryos. We obtained SOST heterozygous rats (F0) and the heterozygous rats were hybridized wide-type rat to obtain next generation of SOST heterozygous rats (F1). After genomic DNA sequencing, the F1 were inbred to produce F2 generation rats, and then the homozygous were identified by the same methods. As shown in Fig. 6c, 3-month-old wide-type rat (n = 12) and SOST-KO rats (n = 12) were injected 20 μg/kg lipopolysaccharide (LPS) intravenously for two consecutive days. One day after last injection of LPS, methylprednisolone (MPS 60 mg/kg) was injected intramuscularly in rats 3 times a week for 4 weeks. After 4 weeks, all rats were sacrificed with 4 ml 10% chloral hydrate. Before injection of LPS, the blood samples were collected and then we collected blood samples every 2 weeks. All collected samples were tested for β-CTX, CTX-I, CTX-II, OCN and 25(OH)D3 using ELISA kits by following the user manual.

Micro-computed tomography (μ-CT) scanning and analysis

The bone tissue was scanned by Sanco viva CT40 instrument (Scanco, Brüttisellen, Switzerland) under the scanning conditions of 100 kV and 98μA. The thickness of the tomographic image was 10.5 μm. The results of bone structure measurement were analyzed according to previous literature (Huang et al. 2017a; Huang et al. 2018).

Histological and immunohistochemical staining

All samples were collected and decalcificated in 10% tetrasodium-EDTA aqueous solution at 4 °C for 1 months. Decalcified tissue was embedded by paraffin and sectioned for hematoxylin and eosin (H&E staining) and immunohistochemical staining. In immunohistochemical staining, sections were deparaffinized, antigen retrieved, blocked and incubated with primary antibodies of ALP and VEGF and corresponding biotinylated secondary antibodies. Then sections were stained with DAB and counterstained with haematoxylin.

Statistical analysis

The experiments were at least performed three times. All data were presented as mean ± standard deviation (SD). Statistical analyses were performed using Graph Pad Prism software and SPSS 18.0 (IBM, Armonk, USA). For differences among treatments, Student’s t-test was used for the comparisons between two groups and data involving more than two groups were analyzed by one-way ANOVA followed by Tukey post hoc test. P values less than 0.05 were considered statistically significant.