Isolation of primary porcine chondrocytes and human OA chondrocytes
Porcine chondrocytes were isolated from full-depth cartilage obtained from porcine metacarpophalangeal joints through a process of sequential enzyme digestion using pronase and collagenase as previously described. Isolated porcine chondrocytes were cultured in Dulbecco's modified Eagle medium (DMEM, Gibco, 11054020) supplemented with 15% fetal bovine serum (Gibco, 10099141), 1% penicillin-streptomycin (Gibco, 15140122), 10 mM HEPES (Sigma, H0887), 4 mM l-glutamine (Corning, 25005CI) and 0.15 mg/ml l-ascorbic acid (Sigma, A4544).
Human articular cartilage samples were obtained from three male and seven female donors (age 60-81 years, mean 70 years) who underwent total knee arthroplasty after obtaining written informed consent, as approved by the Institutional Review Board of the Third Affiliated Hospital of Guangzhou Medical University (no. 2023-075). Human OA chondrocytes were isolated through enzymatic digestion in a manner similar to porcine chondrocytes. Isolated human chondrocytes were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were seeded and cultured at 37 °C with 5% CO for 24 h without passaging, and then subjected to the following experiments.
EPA (Sigma, E2011) was prepared in a complex with defatted bovine serum albumin (BSA, Sigma, A8806) as previously described. In brief, 300 µg/ml EPA-BSA complex (20 mM HEPES, 140 mM NaCl, 4.5 mM KCl, 1 mM MgCl, 2.5 mM CaCl, 11 mM glucose and 3.5 mg BSA, pH 7.4) was incubated at 37 °C for 16 h. EPA-BSA complex was then added to cells to achieve a final EPA-BSA concentrations of 10, 30, 50, 100 and 200 µg/ml. Porcine chondrocytes were treated with EPA for 8 h and then stimulated with 10 ng/ml IL-1α (PeproTech, 20001A) for 24 h, while human OA chondrocytes were treated with EPA for 8 h.
Cell viability was assessed using Cell Counting Kit-8 (CCK-8, GlpBio, GK10001). Freshly isolated porcine chondrocytes were seeded in a 96-well plate at a density of 20,000 cells per well in 200 μl of culture medium. Then, chondrocytes were cultured overnight and treated with or without various concentrations of EPA (10, 30, 50, 100 and 200 µg/ml) for 48 h. Chondrocytes were then treated with 20 μl of CCK-8 solution for 4 h at 37 °C in an incubator. Absorbance was measured at 450 nm using a Bio-Rad Microplate Reader.
DNA fragmentation, a hallmark of late apoptosis, was detected using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay (Beyotime, C1086). In brief, after EPA treatment, porcine chondrocytes cultured on coverslips were fixed with 4% paraformaldehyde for 15 min at room temperature, followed by permeabilization with 0.1% Triton X-100 for 10 min. Cells were incubated with TUNEL reaction mixture for 1 h at 37 °C in the dark. Cell nuclei were stained with 1 μg/ml DAPI for 5 min. Fluorescent images were captured using a Zeiss LSM980 Confocal Laser Microscope System. TUNEL-positive cells (green fluorescence) and total nuclei (blue) were counted using ImageJ. Apoptotic rate was expressed as the percentage of TUNEL-positive cells relative to total nuclei.
Total RNA was extracted from the cells using TRIzol reagent (Invitrogen, 15596026). Reverse transcription of total RNA and cDNA amplification were performed as previously described. The gene expression levels were calculated using the 2 method. The data were presented as fold changes relative to GAPDH. The primer sequences used in the study are listed in Table 1.
Cells were lysed using RIPA buffer (Beyotime, P0013B) for 30 min on ice. Cell extracts were obtained by centrifugation at 12,000g at 4 °C for 15 min, and the supernatant was collected and subjected to protein quantification as described previously. Approximately 20 μg proteins was subjected to SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad). After blocking with 5% skim milk, the membranes were incubated with primary antibodies, including MMP3 (Biorbyt, orb11062), COL2 (Abcam, ab34712), CD44 (Abcam, ab157107), NF-κB p65 (CST, 6956S), p-NF-κB p65 (CST, 3033S), p38 MAPK (CST, 8690T), p-p38 MAPK (CST, 4511T), JNK (Proteintech, 66210-1-Ig), p-JNK (80024-1-RR), c-fos (CST, 2250T), p-c-fos (CST, 5348T), c-Jun (Biorbyt, orb338983), p-c-Jun (CST, 3270T), PTD-p65-P1 peptide (GlpBio, GC38520) and GAPDH (Abcam, ab8245) overnight at 4 °C. After incubation with secondary antibodies, protein bands were visualized with chemiluminescence (Tanon) and quantified using ImageJ. Protein expression levels were normalized to the internal control GAPDH, and relative expression was calculated by dividing the target protein intensity by the GAPDH intensity.
Cells were cultured on glass coverslips at 2 × 10 per well in 24-well plates. Cells were fixed with 4% formaldehyde solution (Thermo Fisher) for 10 min, permeabilized with 0.2% Triton X-100 (BioFroxx, 1139ML100) for 5 min and blocked with 1% BSA (Sigma, A8806) for 1 h. The cells were incubated with phalloidin (Invitrogen, A34055) at room temperature for 1 h. Cells were then incubated with CD44 primary antibodies (Abcam, Ab157107) overnight at 4 °C and secondary antibodies (Invitrogen, A11055) for 1 h at room temperature. Next, the cells were incubated with DAPI-containing fluorescence mounting medium (SouthernBiotech, 010020) and examined by Zeiss LSM980 Confocal Laser Microscope System. Cell area, circularity and fluorescence intensity were quantified using ImageJ analysis software. Circularity was used to describe the roundness of the cell, with lower values indicating either an elongated shape or an increase in cell protrusions. Cell circularity is mathematically defined as 4πA/P, where A represents the area of the cell and P denotes its perimeter. Phalloidin-stained images were converted to 8-bit grayscale and thresholded to define cell boundaries. Individual cells were selected using the 'Wand Tool' to automatically trace cell contours. The 'Analyze Particles' tool measured A and P, with circularity values ranging from 0 (elongated) to 1 (perfect circle). For fluorescence intensity quantification, raw images were converted to 8-bit grayscale. Individual cell boundaries were delineated with the freehand selection tool, and mean fluorescence intensity was measured for individual cells.
Atomic force microscopy (AFM; JPK NanoWizard II) analysis was performed as previously described. In brief, the AFM was mounted on an inverted optical microscope (Nikon eclipse Ti-U, Nikon) and operated in contact mode with a loading rate of 0.4 μm/s and set point in 0.3 V. A silicon nitride cantilever tip (CSG01, NT-MDT) was driven to approach the cell surface and held for 30 s, and the end of the tip was cut off using focused ion-beam milling (FEI Quanta 200 3D FIB/SEM) to form a flat-ended cylindrical tip with a diameter of 3 μm before used. The tip was then unloaded at the same loading rate, and the data were collected simultaneously during the entire indentation. Quantitative analysis of acquired data was performed using the JPK data processing software from the AFM manufacturer. To estimate the viscoelastic of the cells, the indentation curves were fitted using the rate-jump method. The Young modulus was calculated using the following formula:
where h and P are the indentation depth and indentation force, respectively; E and ν are the sample's intrinsic Young's modulus and Poisson's ratio, respectively; and a is the radius of the cylindrical end of the tip. and are jumps in the rates of P and h, respectively, across the unloading point.
Total RNA was extracted using TRIzol reagent (Invitrogen) from porcine chondrocytes in different treatment groups. RNA samples were sent to Gene Denovo Biotechnology, for conducting RNA sequencing (RNA-seq) analysis on the Illumina HiSeq2500 platform. The reference genome used in this study was obtained from the Ensembl database (version: Ensembl 96, species: Sus scrofa, genome assembly: Sscrofa11.1). The data were accessed via the Ensembl website (https://www.ensembl.org). The mapped reads of each sample were assembled using StringTie v1.3.1 in a reference-based approach. For each transcription region, a fragment per kilobase of transcript per million mapped reads (FPKM) value was calculated to quantify its expression abundance and variations, using RSEM software. Transcripts with log|fold change| >1 and false discovery rate (FDR) <0.05 were considered differentially expressed. Volcano plot generation, Venn diagram analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using the Omicsmart online platform (https://www.omicsmart.com) based on the screened-out DEGs. The original RNA-seq data generated in this study have been deposited in the NCBI SRA database under BioProject accession number PRJNA1236871 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1236871/). For the analysis of the published database on OA human knee cartilage tissue and healthy controls, KEGG pathway analysis was performed using the Omicsmart online platform, based on DEGs obtained from GSE113825 (obtained from supplementary table 4 in ref. ) and GSE114007 (obtained from supplementary table 1 in ref. ), respectively.
The following CD44 ligands, including A6 peptide (MCE, HY-P2230), low-molecular-weight hyaluronan (LMWHA, Sigma, 75046) and high-molecular-weight hyaluronan (HMWHA, Sigma, 53747), were used in this study. In most experiments, cells were treated with EPA for 8 h and then stimulated with 10 μM A6 peptide or 1 mg/ml LMWHA or 1 mg/ml HMWHA for 30 min. In some experiments, cells were treated with 10 μM A6 peptide and 1 mg/ml HMWHA or 1 mg/ml LMWHA and 1 mg/ml HMWHA for 30 min. In other experiments, cells were stimulated with 10 ng/ml IL-1α for 24 h and then treated with 1 mg/ml HMWHA for 30 min.
Porcine cartilage explants were obtained from the metacarpophalangeal joints of pigs. In brief, cylindrical cartilage explants (3 mm in diameter and 1 mm in thickness) were collected using a biopsy punch (Miltex). The explants were washed with phosphate-buffered saline (PBS) and incubated in the DMEM containing 1% penicillin-streptomycin solution for 48 h. The explants were then transferred to a 24-well plate and cultured in the medium supplemented with 30 µg/ml EPA and 10 ng/ml IL-1α. The method for preparation of human OA cartilage explants was similar to the preparing porcine cartilage explants. Human OA cartilage explants were treated only with 30 µg/ml EPA.
Porcine cartilage explants were cultured in medium supplemented with 30 µg/ml EPA and 10 ng/ml IL-1α for 10 days. The culture medium was changed and collected every other day. Dimethylmethylene blue (Sigma, 341088) assay was used to determine the sGAG release in the culture medium due to breakdown of cartilage ECM. Different concentrations of chondroitin sulfate (Sigma, C9819) were used to plot a standard curve.
The Young's modulus of porcine cartilage explants was measured using the nanoindentation (Piuma Chiaro, Optics11). A spherical nanoindentation probe with a radius of 9 μm and a stiffness of 262.7 N/m was used. Explants were attached to 6-cm dishes containing PBS at room temperature and indented at a loading rate of 2 μm/s. The tip was held in this indentation depth for 10 s. After the holding, the tip was unloaded at the same loading rate. The indentation curves were fitted using the rate-jump method. The Young's modulus of explants was calculated as previously described in the 'Atomic force microscopy' section.
EPA emulsion was prepared through ultrasonic emulsification. EPA (Sigma, ≥99%, 10417-94-4) and 1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly (ethylene glycol) (DSPE-PEG) (molecular weight 2,000 Da, Aladdin, 147867-65-0) were co-dissolved in dichloromethane (GR, Macklin) at a fixed mass ratio of 5:1. Then, 100 μl of EPA/DSPE-PEG solution was added dropwise to 5 ml aqueous dispersion phase under agitation. Next, the mixed suspension was emulsified by tip sonication (ultrasonic cell disruptor, SCIENTZ, power: 100%; mode: 2 min working time with 2 s ON and 5 s OFF intervals) in an ice bath. Subsequently, the organic solvent was completely removed using a rotary evaporator (IKA) at 40 °C under reduced pressure, stepped down from 550 mbar to 10 mbar, yielding the EPA emulsion. The concentration of EPA emulations can be adjusted to 2-50 mg/ml with desired size distribution and colloidal stability. The EPA emulsion was filtered through a 0.22-μm filter membrane and stored under aseptic conditions at 4 °C. Before use, the EPA emulsion (50 mg/ml) and clinical-grade hyaluronic acid (HA) injections (molecular weight 600-1,170 kDa; ARTZ Dispo, Seikagaku Corporation) were mixed at a volume ratio of 1:9 to obtain EPA emulsion-integrated HA injections (EPA 5 mg/ml).
The size and size distribution of EPA emulsions were characterized by dynamic light scattering analysis (Nicomp 380 N3000, PSS). Measurements were performed after dilution, using 488-nm incident light and at a measuring angle of 90° at 25 °C.
The rheological properties of EPA emulsion-integrated HA injections were studied using a stress-controlled rheometer (Kinexus, Malvern). The dynamic viscosity of HA injections with and without EPA emulsion was measured with 20-mm cone and plate (1° cone angle) at shear rates from 0.01 to 1,000 s. Similarly, dynamic viscoelasticity was measured over an angular frequency range of 0.1 to 100 Hz at a fixed shear strain of 1%.
All animal experiments were approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University (no. S2020-140). This study was reported in accordance with the ARRIVE guidelines 2.0. Sixty Male C57BL/6J mice (aged 10-12 weeks, weighing 30 ± 2 g) were obtained from Guangdong Medical Laboratory Animal Center (Guangzhou, China). The mice were housed with an average of six per cage and had free access to food and water. Anterior cruciate ligament transection (ACLT) surgeries were performed to induce posttraumatic OA as previously described. In brief, mice were anesthetized by inhalation of isoflurane, and ACLTs were performed by transecting the anterior cruciate ligament in the right knee joints using a surgical microscope. Sham surgeries were performed using the same approach, but without ACLT. The mice were randomly divided into five groups (12 mice per group): sham, ACLT injected with PBS (ACLT), ACLT injected with HA solution (HA), ACLT injected with EPA emulsion (EPA) and ACLT injected with EPA emulsion-integrated HA injection (EPA-HA). Random numbers were generated in Excel using the RAND function. Intraarticular injections were initiated 14 days after ACLT surgery. A total of four weekly injections (10 μl per dose) of PBS, EPA emulsion, HA or EPA emulsion-integrated HA solution were administered into the knee joint cavity at 2, 3, 4 or 5 weeks after surgery. Four or 8 weeks after surgeries, six mice in each group (n = 6) were euthanized, and their knee joints were collected for histological and immunohistochemical analysis. None of the mice exhibited wound infection, knee joint infection or mortality. Sample size was calculated using the resource equation approach, which indicated that an n of six is sufficient for our histological studies.
Mouse knee joints were scanned using the Skyscan 1172 micro-computed tomography (micro-CT) scanner (Skyscan 1172, Bruker). Sagittal views of the knee joint were reconstructed using NRecon software (v2.0.4.0, Skyscan) and then visualized using Dataviewer software (v1.5.6.2, Bruker). The subchondral bone of the medial tibial plateau was identified as the region of interest and quantified using CT Analyzer software (v1.13, Skyscan) for trabecular bone volume per tissue volume (BV/TV) and trabecular bone pattern factor (Tb.Pf).
Samples from mouse, porcine and human OA cartilage were fixed in 4% paraformaldehyde for 24 h, decalcified in 0.5 M ethylenediaminetetraacetic acid (EDTA) for 3 weeks, embedded in paraffin and sectioned at a thickness of 6 μm. Tissue slides were stained with hematoxylin and eosin (H&E) to evaluate cartilage thickness or Safranin O/Fast Green to assess cartilage degradation using the Osteoarthritis Research Society International (OARSI) score system. The boundaries of the hyaline cartilage (HC) and calcified cartilage (CC) regions were identified using the tidemark and subchondral bone lines in HE cartilage sections. HC was defined as the nonmineralized cartilage layer above the tidemark, while CC comprised the mineralized tissue between the tidemark and subchondral bone line. Cartilage thickness is defined as the sum of HC and CC. Sagittal sections were divided into anterior, middle and posterior regions. Three measurements per region (nine points per section) were averaged across six nonconsecutive sections per joint to minimize regional bias. For immunohistochemistry, the sections were blocked with 3% BSA for 30 min at room temperature and incubated with CD44 primary antibodies overnight at 4 °C. After washing, the sections were incubated with secondary antibodies and visualized using 3,3'-diaminobenzidine-peroxidase substrate solution.
All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software). Data are presented as mean ± standard deviation (s.d.). Statistical analysis was performed using unpaired Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's multiple-comparisons test. All tests were two-tailed. Differences were considered statistically significant at P < 0.05.