Cell tradition
Mesenchymal stem cells (MSCs) had been remoted and cultured from bone marrow utilizing a beforehand established methodology. In easy phrases, bone marrow stromal cells (BMSCs) had been remoted from the femurs of 4-week-old male Sprague-Dawley rats (90–100 g) after which cultured in α-MEM (Minimal Important Medium) supplemented with 1% penicillin-streptomycin (Gibco, NY, USA) and 10% fetal bovine serum (FBS, Gibco, USA) [50]. The cells had been incubated at 37 °C in a 5% CO2 surroundings. Passage 3–4 BMSCs had been chosen for subsequent experiments. Chondrocytes had been obtained from rat knee joints. The small items of cartilage tissue (roughly 1mm3 in dimension) had been subjected to digestion utilizing 0.2% sort II collagenase ( Sigma-Aldrich, USA) at a temperature of 37 °C for a period of 6 h [51]. Following centrifugation at 1500 rpm for five min, the cells had been suspended once more in DMEM/F-12 and positioned in tradition inside DMEM/F-12 medium. This medium was enriched with 1% penicillin-streptomycin and 10% fetal bovine serum, all beneath equivalent situations as these for BMSCs. Passage 1–3 chondrocytes had been used for mobile experiments. Raw264.7 cells (Beyotime, Shanghai) had been cultured in DMEM (Dulbecco’s Modified Eagle Medium) containing 10% FBS and 1% penicillin-streptomycin [52]. For the remedy of BMSCs, fucoidan (Santa Cruz, USA) was used for pretreatment (Fig. S1A) [53]. After 48 h of pretreatment, the BMSCs had been collected for exosome extraction.
MSCs-Exo and F-MSCs-Exo preparation and characterization
MSCs-derived exosomes (MSCs-Exo) and Fucoidan-preconditioned MSCs-derived exosomes (F-MSCs-Exo) had been remoted from the supernatant utilizing an ultracentrifugation-based methodology [54]. In brief, the supernatant was collected and centrifuged at 300 and 2000 g for 10 min every, respectively, to take away lifeless cells and cell particles. Subsequently, a further centrifugation step at 10,000 g for 30 min was carried out to eradicate apoptotic our bodies. Afterward, ultracentrifugation at 100,000 g for 30 min utilizing Ultraclear™ tubes (Beckman Coulter, USA) was carried out to take away residual proteins. Lastly, the purified samples had been resuspended in PBS following centrifugation at 100,000 g for 90 min and saved at -80 °C for additional experiments. The overall protein focus within the exosomes was decided utilizing the bicinchoninic acid assay (BCA) protein detection package (Beyotime, China). The scale distribution and zeta potential of the exosomes had been measured utilizing a Zetasizer Nano ZS90 (Malvern, UK) by dynamic gentle scattering (DLS). Moreover, the morphology of the exosomes was examined utilizing transmission electron microscopy (TEM, Hitachi, Japan). Moreover, Western blotting was employed to determine the expression of the marker proteins CD9, CD63, CD81, TSG101, and Calnexin (ProteinTech, USA) within the exosomes.
Mobile uptake assay
Exosomes had been labeled utilizing the inexperienced fluorescent dye PKH67 (Sigma-Aldrich, USA), whereas the mobile cytoskeleton was labeled with the pink fluorescent dye TRITC Phalloidin (Thermo Fisher Scientific, USA). The co-culture of exosomes (10 μg/ml) with rat chondrocytes befell in a serum-free medium at 5% CO2 and 37 °C for 12 h. Subsequently, the cells had been mounted with 4% paraformaldehyde. DAPI (Beyotime, China) was used for nuclear staining. Lastly, the uptake of exosomes was visualized and captured utilizing a fluorescence microscope (Zeiss, Germany).
Cell viability assay
The consequences of MSCs-Exo and F-MSCs-Exo on cell viability had been analyzed utilizing the CCK-8 methodology. Rat chondrocytes (8*103 cells per nicely) had been seeded into the wells of a 96-well plate and allowed to incubate for 12 h. Subsequently, the cells had been handled with various concentrations (0, 1, 5, 10 μg/ml) of MSCs-Exo, in addition to with 10 μg/ml of each MSCs-Exo and F-MSCs-Exo [39]. After 24, 48, and 72 h of remedy, 10 μl of CCK-8 answer (Meilunbio, China) was added to every nicely, adopted by a 4-hour incubation at 37 °C [55]. The absorbance was measured at 450 nm utilizing a microplate reader (Leica Microsystems, Germany) to evaluate cell viability.
Cell toluidine blue, alcian blue, safranin O staining
Rat chondrocytes had been seeded right into a 24-well plate and allowed to develop till they reached 75% confluency. Afterward, the cells had been gently washed 3 times with PBS and stuck with 4% paraformaldehyde for 15 min, adopted by one other spherical of PBS washing. Subsequently, fastidiously add the respective staining options, together with toluidine blue, Alcian blue, and safranin O (solarbio, China), to the wells, and allow them to incubate undisturbed at room temperature for 30 min. Lastly, take away the surplus dye by washing with PBS, and observe the stained cells beneath an odd gentle microscope.
Western blotting
For protein extraction, chondrocytes from the first to third passages had been utilized. Briefly, the cells had been lysed in RIPA lysis buffer (Beyotime, China) containing protease and phosphatase inhibitors. The lysate was maintained on ice for 15 min, after which it underwent centrifugation at 12,000 rpm for 30 min at a temperature of 4 °C. This process was carried out to assemble the resultant supernatant. Utilizing the BCA methodology, the protein focus was established. Following this, 15 μg of protein was segregated on an 8–12% SDS-PAGE gel and subsequently transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% non-fat milk at room temperature for two h after which incubated in a single day at 4 °C with the first antibody. After washing, the membrane was incubated with an HRP-conjugated secondary antibody at 37 °C for two h. Lastly, protein imaging and quantification had been carried out utilizing the ChemiDoc™ XRS + and Picture Lab 3.0 imaging system (Bio-Rad, USA).
The experiment utilized the next main antibodies: COX2 (27308-1-AP), INOS (18985-1-AP), Collagen II (28459-1-AP), Aggrecan (13880-1-AP), MMP-13 (18165-1-AP), ADAMTS4 (11865-1-AP), all of which had been bought from Proteintech Group. Moreover, the antibodies ATG7 (ab133528), LC3 (ab192890), beclin1 (ab207612), P62 (ab109012), and TRAF6 (ab40675) had been acquired from Abcam. The antibodies PI3K (AF6241), AKT (AF0836), mTOR (AF6308), P-PI3K (AF3242), P-AKT (AF0016), P-mTOR (AF3308), GAPDH (AF7021), and β-actin (AF7018) had been bought from Affinity Biosciences. Goat anti-rabbit and anti-mouse IgG-HRP antibodies had been additionally procured from Affinity Biosciences.
Enzyme-linked immunosorbent assay (ELISA)
The cell tradition supernatant is collected and saved at -20 °C for subsequent ELISA testing. In every nicely, the concentrations of IL-6, TNF-α, Collagen II, Aggrecan, MMP-13, and ADAMTS4 had been quantified utilizing ELISA kits [56]. Concisly, the cell tradition supernatant was launched onto a plate, adopted by the introduction of a main antibody to ascertain binding with the antigen current within the pattern. Subsequently, an enzyme-labeled secondary antibody was administered onto the plate, resulting in the formation of a posh. Lastly, a substrate was added to provoke the enzyme-catalyzed colour response. The absorbance was measured utilizing a spectrophotometer (Leica Microsystems, Germany) to calculate the precise protein or molecule’s focus.
Actual-time quantitative reverse transcription PCR (RT-qPCR)
Whole mRNA was extracted from rat chondrocytes utilizing TRIzol reagent (Sangon, China), and the RNA content material was quantified utilizing the Nanodrop 2000 spectrophotometer. cDNA synthesis was carried out utilizing the cDNA synthesis package (Takara, Japan). RT-qPCR was carried out with the SYBR Inexperienced detection reagent (Takara, Japan) on the LightCycler® 96 real-time PCR system (Roche, USA). The relative expression ranges of mRNA had been decided utilizing the two − ΔΔCq methodology. The primer sequences for mRNA could be present in Desk S1.
Immunofluorescence
Briefly, the cells had been subjected to a collection of steps as follows: first, they had been washed with PBS after which mounted with 4% formaldehyde for 15 min. After one other spherical of washing, the cells had been permeabilized utilizing 0.2% Triton X-100 for 15 min at room temperature. Subsequently, the cells had been handled with 10% goat serum for 30 min at 37 °C to dam non-specific binding. Subsequent, the cells had been incubated in a single day with a panel of main antibodies, particularly Collagen II (1:200), MMP-13 (1:200), LC3 (1:200), and TRAF6 (1:200). On the next day, the cells had been uncovered to secondary antibodies conjugated with Alexa®488 (1:400) for 1 h. Following one other wash, the samples had been stained with 4’,6-diamidino-2-phenylindole (DAPI) for 60 s to visualise the cell nuclei [57]. Lastly, the samples had been visualized beneath a fluorescence microscope (Carl Zeiss, Germany), and the ImageJ software program was employed for quantitative evaluation in every specified area.
Circulation cytometry evaluation
Raw264.7 cells had been cultured in a 6-well plate till they reached 80-90% confluency. Upon reaching the specified confluency, the cells had been fastidiously harvested and picked up in centrifuge tubes after being washed with PBS. To attenuate non-specific antigen binding, the cells had been then incubated with 3% BSA for 1 h. After centrifugation, the cells had been resuspended in PBS and incubated at 37 °C in a light-protected surroundings together with the next fluorescently-labeled antibodies: FITC-conjugated anti-F4/80 antibody and PE-conjugated anti-CD86 antibody (BD Biosciences, USA) for 30 min [58]. Subsequently, a radical washing step was carried out to take away any unbound antibodies, and the samples had been subjected to stream cytometry for evaluation.
miRNA sequencing
Whole RNA was extracted utilizing the mirVana miRNA Isolation Equipment (Ambion). The RNA amount was assessed with Nanodrop 2000 (Thermo Fisher Scientific Inc., USA), whereas its integrity was evaluated utilizing the Agilent 2100 Bioanalyzer (Agilent Expertise, USA). To assemble small RNA libraries, the NEBNext Small RNA Library Prep Set for Illumina package (NEB, USA) was utilized. After confirming the high-quality libraries with the Agilent 2100 Bioanalyzer, sequencing was carried out on the Illumina Novaseq 6000 platform. For the evaluation of differentially expressed miRNAs, the factors used for filtering had been a q-value < 0.05 and fold change (FC) > 2 or FC < 0.5. The DEG algorithm from the R package deal was employed to calculate q-values. Goal gene prediction was carried out utilizing the miranda software program, with the parameters set as follows: S ≥ 150, ΔG ≤ -30 kcal/mol, and strict demand for five’ seed pairing. Lastly, differential expression miRNAs’ goal genes had been subjected to GO enrichment and KEGG pathway enrichment analyses utilizing R packages. All small RNA sequencing and knowledge analyses had been carried out by Eurofins Genomics (Shanghai, China).
Animals
A complete of 48 male Sprague-Dawley rats, aged 10 weeks, had been generously supplied by the Chinese language Academy of Sciences Animal Heart. Moral approval for all animal experiments was obtained from the Wenzhou Medical College Animal Ethics Committee, with the approval quantity wydw2023-0355. The experimental teams had been fastidiously designed as follows: the sham surgical procedure group (present process solely joint incision), the OA group (induced by anterior cruciate ligament transection and medial meniscus resection), the OA + MSCs-Exo group, and the OA + F-MSCs-Exo group, every comprising 6 rats [59]. Briefly, ranging from the fourth week after the institution of the OA mannequin, the OA + MSCs-Exo group and OA + F-MSCs-Exo group obtained intra-articular injections of MSCs-Exo or F-MSCs-Exo (10 μl/week), respectively [60]. The primary goal of this examine was to research and evaluate the therapeutic results of MSCs-Exo and F-MSCs-Exo within the rat mannequin of osteoarthritis.
To elucidate the position of miR-146b-5p in F-MSCs-Exo, the rats had been additional divided into 4 teams: the sham surgical procedure group, the OA group, the OA + F-MSCs-Exo group, and the OA + F-MSCs-Exo + Antagomir-146b-5p group, with 6 rats in every group. In abstract, ranging from the fourth week after the institution of the OA mannequin, the rats within the OA + F-MSCs-Exo group obtained weekly intra-articular injections of F-MSCs-Exo (10 μl/week). Moreover, from the fourth week, the rats within the OA + F-MSCs-Exo group and the OA + F-MSCs-Exo + Antagomir-146b-5p group had been handled with intra-articular injections of a damaging management (nc) (5 nmol) or Antagomir-146b-5p (5 nmol) immediately into the joint cavity. The experiment was carried out for a complete of eight weeks post-surgery. On the finish of the eight weeks, the rats had been humanely euthanized beneath anesthesia, and knee joint samples had been collected to evaluate the illness development.
Microcomputed tomography scans
Following the humane euthanization of the rats, knee joint specimens had been fastidiously collected and preserved by in a single day fixation in 4% paraformaldehyde. Subsequently, the mounted knee joint specimens underwent meticulous micro-computed tomography (micro-CT) utilizing the superior SkyScan-1276 micro-CT system (Bruker micro-CT, Kontich, Belgium). The micro-CT scans had been carried out in three planes for every knee joint, encompassing the sagittal, transverse, and coronal planes. The acquired photographs had been then employed to reconstruct the intricate 3D illustration of the knee joint. Leveraging the 3D reconstructed photographs, complete three-dimensional structural parameters had been employed to meticulously consider the subchondral bone residing inside the tibial plateau. This complete analysis facilitated an in depth and insightful evaluation of each the cartilage and the underlying bone structure inside the knee joint.
Histological evaluation
After an 8-week period, the knee joints of the rats had been fastidiously harvested and stuck in 4% paraformaldehyde for twenty-four h. Following fixation, a 4-week decalcification course of was carried out utilizing a ten% EDTA answer (Solarbio, China). Subsequently, the tissues underwent meticulous processing, together with dehydration, paraffin embedding, and sectioning. The obtained sections had been then subjected to histological staining utilizing Hematoxylin and Eosin (H-E) staining (Beyotime, China) in addition to Safranin O-Quick Inexperienced (S-O) staining (Beyotime, China) for morphological evaluation [61]. Excessive-quality photographs of the stained sections had been captured utilizing an optical microscope. To comprehensively assess the situation of the cartilage, the internationally acknowledged Osteoarthritis Analysis Society Worldwide (OARSI) scoring system was employed [62]. This established scoring system enabled an in depth analysis of structural adjustments within the cartilage and supplied invaluable insights into the extent of osteoarthritis pathology.
Immunohistochemical evaluation
The knee joints of the rats had been mounted in 4% paraformaldehyde, adopted by a collection of processing steps, together with decalcification, paraffin embedding, and sectioning. After dewaxing and dehydration, antigen retrieval was carried out utilizing 3% hydrogen peroxide. Subsequently, the sections had been blocked with 10% goat serum (Solarbio, China) for 30 min at 4 °C after which incubated in a single day at 4 °C with the first antibodies (diluted at 1:200) for MMP-13, P62, and INOS. On the next day, the sections had been incubated with HRP-conjugated secondary antibodies at 37 °C for 1 h [63]. Lastly, visualization was carried out to look at the immunohistochemical evaluation of the tissues.
Statistical evaluation
All knowledge had been expressed as imply ± customary deviation (SD) and analyzed utilizing SPSS 20.0 software program (Chicago, IL, USA). To determine intergroup variations, impartial t-tests had been carried out, whereas one-way evaluation of variance (ANOVA) was used for a number of group comparisons. Every experiment was carried out with a minimal of three organic replicates to make sure robustness and reliability. The importance ranges had been indicated as follows: *P < 0.05, **P < 0.01, and ***P < 0.001, representing the respective ranges of statistical significance.
