Bacterial strains, growth conditions, and chemical reagents
The S. aureus strain used in this study was ATCC®BAA-1717™ (USA300-HOU-MR). The SaeR-knockdown strain (kd-SaeR)58 and bioluminescent strain Xen2959 were constructed following established protocols. All S. aureus strains were cultured in brain heart infusion (BHI) broth or on BHI agar plates supplemented with chloramphenicol (10 µg/mL). Escherichia coli (E. coli) strains were grown in Luria–Bertani (LB) broth at 37 °C, with kanamycin (50 µg/mL) added when appropriate. Unless otherwise specified, all the cultures were grown at 37 °C in a shaking incubator. The strains involved are listed in Supplementary Table 4. BI, a natural product with a purity of 99.43%, was purchased from Letian Mei Biotech Co., Ltd. (Chengdu, China), and a quality inspection report is provided in Supplementary Fig. 3. The general reagents used were obtained from Sangon Biotech (Shanghai, China).
Expression and purification of recombinant SaeR and its mutants
The primers used were designed on the basis of the genome sequence of S. aureus MW2 (NC-003923) published by the National Center for Biotechnology Information (NCBI). For recombinant 6-His-tagged SaeR protein expression, the full-length SaeR gene was amplified from USA300 genomic DNA. The PCR product was digested with BamHI and XhoI and inserted into the pET28a plasmid, which was then transformed into E. coli DH5α. The recombinant plasmid pET28a-saeR was further transformed into E. coli BL21 (DE3) for expression of the recombinant BL21-pET28a-saeR strain, followed by overnight culture at 37 °C. Site-directed mutagenesis of K121A-saeR, K156A-saeR, W182A-saeR, and R199A-saeR was performed via a site-directed mutagenesis kit (TianGen®, China), and the mutations were verified via DNA sequencing. The mutant plasmids were subsequently transformed into E. coli BL21 (DE3) and cultured overnight at 37 °C.
The cultures were diluted 1:100 into 1000 mL of LB medium containing kanamycin (50 µg/mL) and grown until the OD600 nm reached 0.6–0.8. Isopropyl β-d-1-thiogalactopyranoside was added to a final concentration of 0.1 mmol/L to induce protein expression at 16 °C for 16 h. The bacteria were harvested via centrifugation (4000 rpm, 30 min), washed twice with phosphate-buffered saline (PBS), and resuspended in 25 mL of non-denaturing lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl). The cells were lysed on ice via an ultrasonic homogenizer (Xiaomei ultrasonic instrument Co., Ltd., China. 200 W; 5 s pulses with 10 s intervals for 2–3 h), followed by centrifugation at 12,000 rpm for 1 h at 4 °C. The supernatant was added to Ni-NTA His-tag purification agarose (MedChemExpress, China), which had been preequilibrated with a nondenaturing binding buffer. The contaminating proteins were washed with a buffer containing 50 mmol/L imidazole, and the target protein was eluted with 400 mmol/L imidazole. SDS‒PAGE was performed to verify protein purity, and the purified SaeR protein was stored at −80 °C for further experiments.
Virtual screening with discovery studio
The 3D structure of SaeR (PDB ID: 4QWQ) was obtained from the Protein Data Bank (www.rcsb.org). For virtual screening, a small-molecule library was assembled using data from the ZINC website (https://zinc.docking.org/) and additional compounds from PubChem (https://pubchem.ncbi.nlm.nih.gov/). The binding site for SaeR was predicted on the basis of the shape of the receptor via Discovery Studio60. CDOCKER, a CHARMM-based docking tool, was used for rigid receptor docking. Ligand conformations were first generated via high-temperature molecular dynamics with various random seeds, and the final conformations were minimized via a rigid receptor model. For each conformation, the CHARMM energy and interaction energy was calculated61, and the top-ranked conformations on the basis of binding energy were selected for further evaluation.
Thermal shift assay
TSA was conducted as previously described, and SYPRO Orange dye (Thermo Fisher Scientific, China) was used to monitor the interaction between SaeR and small molecules. A 100× SYPRO Orange solution was mixed with SaeR protein (final concentration of 2 μM) on ice. Then, 6 μL of the mixture was combined with 6 μL of the small-molecule mixture and 8 μL of buffer (150 mM NaCl, 10 mM HEPES, pH 7.5), and the samples were added to a 96-well PCR plate. Fluorescence was measured via the IQ5 Real-Time PCR Detection System (Bio-Rad, USA) as the temperature increased from 25 °C to 95 °C at a rate of 1 °C/min, with Ex/Em wavelengths of 490 nm/530 nm. Wells containing the same buffer composition but without small molecules were used as controls.
MIC assay and growth curve analysis
The MIC, defined as the lowest drug concentration that inhibits bacterial growth (OD600 nm < 0.01), was determined according to the Clinical and Laboratory Standards Institute (CLSI) guidelines62. A 96-well plate containing S. aureus (1 × 105 CFU) and various concentrations of BI (1‒256 µg/mL) was incubated at 37 °C for 18 h. To plot the growth curve, S. aureus was incubated with 128 μg/mL BI at 37 °C, and the OD600 values were measured at various time points.
Resistance induction assay
The resistance of S. aureus to BI was assessed via a twofold dilution method. Briefly, 180 μL of S. aureus (1 × 105 CFU/mL) was incubated with 20 μL of BI (5.12 mg/mL) or Oxacillin (5.12 mg/mL) in a 96-well plate. The subsequent wells contained serial dilutions of BI and Oxacillin at concentrations of 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μg/mL. The plate was incubated at 37 °C for 24 h, after which the MIC was determined. Following the determination of the MIC, 50 μL of bacterial suspension from wells containing sub-MIC concentrations (1/2 MIC) of BI or Oxacillin was transferred to 10 mL of BHI medium and incubated at 37 °C with shaking for 8 h. This process was repeated for 20 generations, with MIC measurements taken every 5 generations to monitor the development of bacterial resistance.
Cytotoxicity assay
The ATDC5 cell line, derived from mouse teratocarcinoma cells, is a continuously cultured line widely used as an in vitro model for chondrocyte research. The cytotoxic effects of BI on ATDC5 cells were evaluated via the MTT assay. ATDC5 cells were seeded at a density of 2 × 10⁴ cells/well in a 96-well plate and incubated at 37 °C in a 5% CO2 incubator for 24 h. The cells were then treated with various concentrations of BI (8‒128 µg/mL) for 24 h. After the medium was removed, 5 mg/mL MTT solution was added to each well, and the mixture was incubated for 4 h. The formazan crystals were dissolved in 100 µL of DMSO, and the absorbance was measured at 490 nm.
Western blot analysis
S. aureus was cultured to an OD600 of 0.3, after which various concentrations of BI (8‒128 µg/mL) were added, and the cultures were grown until the late logarithmic phase (OD600 of 2.5). The bacteria were harvested, and the total protein was extracted via lysis buffer. Protein concentrations were quantified via a BCA protein assay kit (Beyotime, China). Equal amounts of protein (20 μg) were separated by SDS‒PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% nonfat milk and incubated overnight at 4 °C with an anti-Hla antibody (1:2000, Sigma, USA) or anti-PVL polyclonal antibody (1:2000, Abcam, England). After washing, the membranes were incubated with an HRP-conjugated secondary antibody (1:5000, Abcam, England) for 1 h at room temperature (RT). Visualization was achieved via an enhanced chemiluminescence kit (Servicebio, China), and images were captured via a chemiluminescence detection system.
Hemolysis assay
S. aureus was cultured to an OD600 of 0.3, and BI (8–128 µg/mL) was added. The cultures were incubated until the late logarithmic phase (OD600 of 2.5) and centrifuged (5500 rpm, 4 °C, 1 min), and the supernatant was filtered for further analysis. For the hemolysis assay, 100 µL of supernatant was mixed with 25 µL of defibrinated rabbit blood and 775 µL of PBS in a 1 mL reaction volume. Triton X-100 served as the negative control. The samples were incubated at 37 °C until complete hemolysis was observed in the untreated group. After centrifugation (5500 rpm, RT, 1 min), the OD543 nm was measured via a microplate reader.
Adhesion of S. aureus to immobilized fibrinogen
To assess the effect of BI on S. aureus adherence to fibronectin, a 96-well plate was coated with bovine fibronectin (50 µg/mL; Source Leaf, China) overnight at 4 °C. After the fibronectin solution was removed, the wells were blocked with 3% bovine serum albumin (BSA; Sigma, USA) for 2 h to prevent nonspecific binding. S. aureus was grown to the logarithmic phase in the presence of various concentrations of BI (8–128 µg/mL). The 96-well plate was then washed and incubated with BI-treated S. aureus (5 × 10³ CFU/well) at 37 °C for 1 h. After incubation, the wells were washed with PBS, and the adherent cells were fixed with 4% paraformaldehyde for 30 min. The wells were then stained with 0.1% crystal violet for 20 min and washed, and the absorbance was measured at 600 nm via a microplate reader.
RNA extraction and real-time quantitative reverse transcription PCR (RT‒qPCR)
To examine the expression of virulence factors in S. aureus USA300, the bacteria were coincubated with BI (64 µg/mL) or the kd-saeR strain at 37 °C and 200 rpm for 24 h. Total RNA was extracted via TRIzol reagent, and reverse transcription was performed via a commercial kit (Servicebio, China). The synthesized cDNA was stored at −80 °C. RT‒qPCR was carried out via SYBR Green mix (Servicebio, China) to quantify the expression levels of target genes. The cycling conditions were as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 15 s, 60 °C for 10 s, and 72 °C for 30 s. Primers for key virulence genes were designed on the basis of the nucleotide sequences provided in Supplementary Table 5. The relative RNA levels of each gene were calculated via the 2-ΔΔCt method, with gyrb as the internal control.
Fluorescence Staining of Intracellular bacteria
The kd-SaeR strain was cultured on BHI agar plates supplemented with 10 μg/mL chloramphenicol. Wild-type S. aureus strains were incubated with BI (32 and 64 μg/mL) at 37 °C for 24 h, with DMSO (0.1%) added to the control groups. The bacterial cells were labeled with 5(6)-carboxyfluorescein N-hydroxysuccinimide ester (MedChemExpress, China). ATDC5 cells were cultured in DMEM/high-glucose medium (Servicebio, China) and seeded at a density of 1 × 105 cells/well in a 24-well plate. After 24 h of incubation, ATDC5 cells were infected with S. aureus at a multiplicity of infection (MOI) of 5 for 1 h. The extracellular bacteria were then killed by treatment with 50 μg/mL gentamicin for 1 h, after which the cells were washed three times with sterile PBS (pH 7.4). The cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with PBS containing 0.1% Triton X-100 for 10 min, and stained with TRITC-conjugated phalloidin (Yeasen, China) and DAPI (Invitrogen, USA). Confocal laser scanning microscopy (Leica TCS DMI8, Germany) was used to capture images.
Quantification of internalized bacteria via CFU counting
ATDC5 cells were seeded at a density of 1 × 105 cells/well in a 24-well plate and incubated for 24 h. The cells were infected with the S. aureus kd-saeR strain or BI-treated/untreated S. aureus strains (1 × 105 CFU/mL) for 1 h. After 1 h, the cells were treated with gentamicin (50 µg/mL) for 1 h to kill the extracellular bacteria. The cells were washed three times with sterile PBS and lysed with 0.1% Triton X-100 to release intracellular bacteria. Serial dilutions of cell lysates were plated on blood agar and incubated overnight at 37 °C, after which the CFU counts were determined. The relative number of internalized bacteria was calculated by dividing the CFU by the total number of ATDC5 cells.
Neutrophil isolation and transwell migration assay
Peripheral blood samples were collected from the rats, and neutrophils were isolated via a rat neutrophil isolation kit (Tianjin Haoyang Huake Biotechnology, China) according to the manufacturer’s instructions. The isolated neutrophils were resuspended in Hank’s balanced salt solution (HBSS) containing 1% BSA. S. aureus USA300 was incubated with various concentrations of BI (16–64 µg/mL) at 37 °C and 220 rpm for 4 h. The supernatant was collected and heat-inactivated at 60 °C for 30 min. A 24-well plate with 3.0 µm transwell inserts was used to evaluate neutrophil migration. Heat-inactivated S. aureus (3 × 108 cells/insert) was added to the inserts, and 600 µL of HBSS containing 1% BSA was added to the lower chamber. Neutrophils (1.5 × 104 cells) were added to the upper chamber and incubated at 37 °C for 1 h. After incubation, nonmigrating neutrophils were removed, and the remaining cells were stained with Alexa Fluor 647-conjugated Ly6G. The cells were washed three times with sterile PBS. The fluorescence was measured at 535 nm and 488 nm.
Infection of ATDC5 cells and LDH assay
ATDC5 cells were seeded at a density of 2 × 104 cells/well in a 24-well plate and incubated for 12 h. The culture medium was replaced with a fresh RPMI 1640 medium containing 10% serum. S. aureus USA300 was added at an MOI of 50, and the cells were treated with various concentrations of BI (32 and 64 µg/mL) for 6 h. Cell viability was assessed via calcein-AM/PI staining to distinguish between live and dead ATDC5 cells, and images were captured via fluorescence microscopy. The control group consisted of untreated cells, and ImageJ software (ImageJ 1.52a) was used to quantify the ratio of live to dead cells. To measure LDH release, the supernatants were collected after 6 h of incubation, and LDH levels were measured via an LDH assay kit (Beyotime, China).
Biofilm formation and development assays
S. aureus was cultured in a 96-well plate (1 × 106 CFU/mL) with or without BI treatment (128 µg/mL) at 37 °C overnight. After 24 h, the biofilms were gently washed three times with sterile PBS, fixed with 100 µL of methanol for 15 min, and stained with 0.1% crystal violet ethanol solution for 15 min. After the plate was washed, images were captured, and biofilm formation was quantified by adding 33% acetic acid to each well and measuring the absorbance at 570 nm.
The impact of BI on various stages of biofilm formation was analyzed as previously described63, with slight modifications. More specifically, rabbit plasma-coated 96-well plates were inoculated with S. aureus. The control wells contained no compounds. BI was added sequentially to the wells at various times to a final concentration of 128 µg/mL. After coincubation for 24 h, the biofilms were stained with 1% crystal violet, and the absorbance was measured at 570 nm.
Cell motility assay
To assess sliding motility, plates containing 1% tryptone, 0.25% NaCl, and 0.3% agar were prepared. A 15-µL aliquot of a standard S. aureus cell suspension (1 × 10⁸ CFU/mL) was inoculated at the center of the plates, either in the absence or presence of BI (64 µg/mL). After 24 h of incubation at 37 °C, the motility of S. aureus was observed.
Triton X-100-induced autolysis assay
Overnight cultures of S. aureus, either treated or not treated with BI (64 µg/mL), were centrifuged to collect the bacterial pellets. The pellets were washed and resuspended in 0.05 M Tris-HCl buffer (pH 7.0) containing 0.05% Triton X-100. The suspensions were incubated at 30 °C with shaking at 150 rpm. The OD600 nm was measured hourly to assess autolysis.
CLSM and SEM for biofilm observation
Glass coverslips were coated with lyophilized rabbit plasma at 4 °C overnight in a 24-well plate. S. aureus suspensions (1 × 105 CFU/mL) containing varying concentrations of BI (64 and 128 µg/mL) were added, and biofilms were allowed to form by incubating the plates at 37 °C for 24 h. The samples were gently washed with precooled PBS and fixed with glutaraldehyde for 15 min. FITC (0.001%) was added, and the mixture was incubated at 37 °C with shaking for 5 min. The samples were then stained in the dark at 4 °C for 15 min to 1 h. After washing with PBS, the coverslips were mounted on slides, and biofilm formation was observed via a confocal laser scanning microscope (Leica TCS DMI8, Germany). Live bacteria were visualized as green fluorescence.
For SEM, glass coverslips coated with rabbit plasma were treated similarly to the CLSM setup. After incubation with S. aureus and BI, the samples were washed with sterile PBS to remove nonadherent bacteria and fixed overnight with glutaraldehyde at 4 °C. The samples were dehydrated in a series of ethanol concentrations, freeze-dried, and sputter-coated with platinum before the biofilm structure was imaged via SEM (HITACHI Regulus 8100, Japan).
Quantification of eDNA, protein, and PIA in biofilms
S. aureus was cultured overnight at 37 °C and diluted in BHI to 1 × 106 CFU/mL. Bacterial suspensions (1 mL) were added to a 6-well plate, either treated or not treated with BI (64 µg/mL), and incubated at 37 °C for 24 h. The biofilms were collected by washing with PBS, filtered through 0.22 µm filters, and suspended in PBS. The concentrations of eDNA and proteins in the biofilm matrix were quantified via a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, USA). The PIA content was evaluated by color changes on Congo red agar plates.
Electrophoretic mobility shift assay
Purified SaeR (20 μM) and SaeSC (1 μM) were mixed in phosphorylation buffer (10 mM Tris-HCl (pH 7.4), 50 mM KCl, 5 mM MgCl2, and 10% glycerol), followed by the addition of 1 mM ATP and incubation at RT for 5 min. EMSA was performed with 80 ng of fluorescein (FAM)-labeled DNA probes mixed with varying amounts of SaeR in 25 µL of gel mobility shift buffer (10 mM Tris-HCl, pH 7.4; 50 mM KCl; 5 mM MgCl2; 10% glycerol; and 3 μg/mL sheared salmon sperm DNA). After 30 min of incubation at RT, the samples were subjected to electrophoresis on an 8% polyacrylamide gel (100 V for prerun, 85 V for 30 min of separation), and the gels were imaged.
Cellular thermal shift assay
SaeR proteins were expressed via the same method, specifically through E. coli BL21(DE3) pET28a-SaeR64. At the outset, the bacterial lysates were centrifuged to collect the supernatants. One portion of the supernatant was treated with BI (32 μg/mL), and the other portion was treated with DMSO as a control. After incubation at 37 °C for 1 h, the supernatants were centrifuged and distributed into PCR tubes. The samples were heated at specific temperatures for 5 min, followed by immersion in a quick 3-minute ice bath. The supernatant was collected by centrifugation and mixed with protein loading buffer. The samples were subjected to 12% SDS‒PAGE for separation. Protein band intensities related to stability were quantified via ImageJ software.
Fluorescence quenching assay
The binding constant (KA) of BI to SaeR and the SaeR mutant proteins was determined via fluorescence quenching. Purified SaeR (4 µM) was mixed with increasing concentrations of BI (0–174.92 µmol/L). Fluorescence measurements were taken at an excitation wavelength of 280 nm, with the emission spectra recorded between 280 and 400 nm via a microplate reader (Thermo Fisher Scientific, China). The fluorescence quenching data were plotted as the relative fluorescence intensity (RFI = F/F0×100) against the BI concentration. A Stern‒Volmer plot was used to calculate the KA value through linear regression.
SPR analysis
SPR analysis was performed via a Biacore 1 K system (Cytiva, Sweden). A CM5 sensor chip was installed according to standard operating procedures, and PBS (pH 7.4) was used as the running buffer at a flow rate of 150 µL/min. SaeR protein was diluted in sodium acetate and immobilized on the chip surface. After functionalization with NHS, EDC, and ethylenediamine, different concentrations of BI (0.19–100 μmol/L) were injected at a flow rate of 30 µL/min. The association and dissociation times were set at 120 s and 180 s, respectively. The data were analyzed via a one-to-one binding model.
Molecular docking
Molecular docking of BI (PubChem CID: 5488822) with SaeR was performed via AutoDock Vina 1.1.2. The three-dimensional structure of SaeR (PDB ID: 4QWQ) was downloaded from the Protein Data Bank (www.rcsb.org), and the 3D structure of BI was drawn via ChemBioDraw Ultra 14.0. The input files for docking were prepared via AutoDockTools 1.5.6, and the ligand structure was prepared by merging nonpolar hydrogen atoms and defining rotatable bonds. The docking grid for SaeR was defined at center_x: −18.047, center_y: −4.024, and center_z: 11.259, with dimensions of size_x: 45.11, size_y: 50.75, and size_z: 50.75. Exhaustiveness was set to 20 to improve accuracy. Default parameters were used unless otherwise stated.
Site-directed mutagenesis
On the basis of the binding sites predicted via molecular docking, site-directed mutagenesis of key amino acid residues in SaeR was performed via a site-directed mutagenesis kit, with pET28a-saeR serving as the template. The mutations converted specific amino acids to alanine (Ala) (the primer sequences are listed in Supplementary Table 5). The mutated proteins were expressed and purified following standard induction protocols. Fluorescence quenching experiments were conducted to assess the specific binding of BI to the SaeR protein.
G. mellonella infection model
G. mellonella larvae (220–260 mg) were used to assess the in vivo toxicity and therapeutic potential of BI. The larvae were randomly divided into four groups (n = 5): a control group treated with PBS and two groups treated with BI (20 or 50 mg/kg). The control group received PBS (containing 0.1% DMSO) or BI via a 10 μL Hamilton syringe into the last right proleg of each larva. The survival rate and degree of melanization were monitored and recorded daily for 5 days.
To evaluate the therapeutic potential of BI against systemic S. aureus infection, G. mellonella were divided into infection groups (WT), kd-saeR strain infection groups (kd-saeR), and groups treated with BI (20 or 50 mg/kg) or vancomycin (50 mg/kg). Each group contained 10 larvae (n = 10). The infection groups received 5 × 10⁸ CFU/mL S. aureus USA300 or the kd-saeR strain in 10 μL, which was injected into the last right proleg of each larva. One hour post infection, treatment interventions began. The larvae were kept at a constant temperature of 37 °C throughout the experiment. Survival rates were monitored every 12 h over a 120-hour period. For bacterial load assessment, larvae were harvested at 48 h post infection. The larvae were surface sterilized, homogenized in sterile PBS, serially diluted, and plated on BHI agar. The plates were incubated at 37 °C for 24 h, and CFUs were counted. The sensitivity threshold for detection in this assay was 100 CFU/mL of homogenized larva.
MRSA-induced rat model of septic arthritis
All animal experiments were conducted in accordance with protocols approved by the Animal Care & Welfare Committee of Changchun University of Chinese Medicine (Approval No: 2024635). Male Sprague–Dawley (SD) rats (6–8 weeks old) (Liaoning Changsheng Biotechnology Co., Ltd., China) were anesthetized via an intraperitoneal injection of 1% pentobarbital. The fur around the knee joints was shaved, and the samples were gently washed with warm water. The skin around the joints was disinfected via povidone-iodine and alcohol swabs.
After the joints were disinfected, a 10 μL Hamilton syringe was used to inject 4 × 106 CFU/10 μL of MRSA or kd-saeR strain into the joint cavity beneath the patella. Vancomycin (50 mg/kg) or BI (50 mg/kg) was administered subcutaneously once daily for 6 days. The control animals received the same volume of PBS. The rats were kept on a warming pad and monitored until they could move freely. All the animals were housed in ventilated cages with a 12-hour light‒dark cycle at 22 ± 3 °C and were given free access to food and water.
The severity of arthritis was assessed via clinical scoring via macroscopic inspection of the knee joints, assigning a score of 0–4 for each limb (0 = normal, 1 = periarticular erythema, 2 = articular erythema, and edema, 3 = functional impairment with difficulty in locomotion and joint extension, 4 = purulent process with abscess formation). After 7 days of observation, the rats were euthanized via CO2 in a chamber filled at a low rate (30% of the chamber volume per minute). Knee joint diameters were measured, and representative images of the knees were taken.
For real-time monitoring of knee joint infection, bioluminescence imaging was performed using the luminescent strain S. aureus Xen29 or SaeR-knockdown of Xen29 (knockdown). A 10 μL Hamilton syringe was used to inject 4 × 106 CFU/10 μL S. aureus Xen29 into the joint cavity beneath the patella. Bioluminescence was measured daily for 7 days to track infection progression.
After 7 days, the rats were euthanized, and the knee joints were collected. The joints were fixed in 4% paraformaldehyde, decalcified with 12% EDTA, embedded in paraffin, and sectioned at a thickness of 4 μm. H&E staining was performed to assess joint tissue integrity. Images of the stained sections were captured under a light microscope.
To evaluate the bacterial load in the synovial fluid, the knee joints were flushed with 10 μL of PBS via a Hamilton syringe, and the fluid was transferred into sterile microcentrifuge tubes. The mixture was plated on BHI agar containing chloramphenicol (10 μg/mL) and incubated for 24–48 h, followed by colony counting.
Micro-CT analysis
Knee joint tissues were fixed in 10% buffered formalin for 5 days and then transferred to 70% ethanol for storage at 4 °C. Micro-CT scans of the distal femur and proximal tibia were performed via the NEMO Micro-CT system (NMC-200, Pingseng Scientific, China) with a voltage of 80 kV, a current of 0.06 mA, and a scan time of 200 s. The acquired images were reconstructed into three-dimensional models via Recon software. Coronal and sagittal views were analyzed for the smoothness of the cartilage surface and the degree of subchondral bone sclerosis. The BV/TV and TMD of the femoral and tibial condyles were calculated via Cruiser CT software.
Statistical analysis
All the statistical analyses were conducted via GraphPad Prism 8.0 (GraphPad Software). Two-tailed Student’s t tests were used to assess significant differences between two groups, whereas one-way or two-way analysis of variance was used for comparisons among multiple groups. Log-rank tests were performed for survival analyses. Differences were considered statistically significant at P < 0.05. The error bars represent the standard error of the mean. The experiments were repeated in triplicate when necessary.
