Hucmscs

Commercially available hUCMSCs (Cyagen Biosciences, China) were cultured in serum-free mesenchymal stem cell medium (StemRD, USA) and incubated in 5% CO₂ at 37 °C. When cell confluence reached 80%–90%, they were digested and seeded in 48-well plates (2 × 10⁴/well). After cell adherence, the control group was cultured in DMEM (Gibco, USA) proliferation medium (PM) containing 10% FBS (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA), while the experimental group was cultured in the osteogenic induction medium (OM) including PM and osteoinductive supplements: 10 mM β-glycerophosphate (Sigma, USA), 0.2 mM ascorbic acid (Sigma, USA) and 100 nM dexamethasone (Sigma, USA). The following assays were conducted to validate whether hUCMSCs bore osteogenic differentiation properties.

hUC‐MSCs were purchased from Cyagen Biotechnology Inc. The culture medium used to expand cells was DMEM/F12 (Gibco, USA) containing 10% FBS (Gibco), and culturing was performed at 21% O₂, 5% CO₂, and 37°C. Cells at passage 5 were used for the experiment. When hUC‐MSCs reached 80% confluency, they were incubated with 0.25% trypsin–EDTA solution for 2 min. After adding a 2 mL culture medium to stop the trypsin digestion, the cells were centrifuged at 1000 rpm for 3 min. Subsequently, they were cultured in exosome‐depleted medium at 1 × 10⁵/mL, and 24 h later, the medium was collected. They were isolated from the medium using multiple ultracentrifugation steps, and the exosome pellet was suspended in 1000 μL lysis buffer or sterile PBS, depending on subsequent experiments. The concentration and size distribution of the isolated exosomes were confirmed via the nanoparticle tracking analysis using the NanoSight NS300 (Malvern Panalytical). The morphology was detected using transmission electron microscopy (TEM). Western blotting was performed to detect the levels of exosome markers, such as Flotillin‐1, CD63, and TSG101.

The hUCMSCs were purchased from Haixing Biosciences (Suzhou, Jiangsu, China). The hUCMSCs were cultured in accordance with a previously described method[45 (link)]. Afterward, the adipogenic, osteogenic, chondrogenic capacity of these cells was performed. Passages 3–7 of hUCMSCs were cultured in osteogenic, adipogenic or chondrogenic differentiation medium (Cyagen, China, HUXUC-9004) as described by manufacturer. Oil red O staining, Alizarin red staining and Alcian blue staining were used to evaluate adipogenesis, osteogenesis, and chondrogenesis. Detection of hUCMSCs surface markers by a FACSVerse instrument (BD Bioscience, San Jose, CA, USA) is performed as follows. The hUCMSCs were stained with human anti-CD14, anti-CD19, anti-CD34, anti-CD45, anti-CD73, anti-CD90, anti-CD105, or anti-HLA-DR. Identical concentrations of PE-conjugated mouse IgG isotype antibodies were used as negative controls (all from BD Biosciences). Data were analyzed using the software FlowJo V10 (FlowJo).

hUC-MSCs used in this experiment were purchased from Cyagen Biotechnology (Guangzhou, China). The cells were cultured with Dulbecco’s modified Eagle medium (DMEM)/F12 containing 5% fetal bovine serum (FBS) and 1% penicillin–streptomycin (P/S), placed in a cell incubator at a constant temperature of 37 °C, and continuously maintained at a concentration of 5% CO₂. All hUC-MSCs used in the experiments were P2–P4 generations. The culture medium for hUC-MSC chondrogenic differentiation was purchased from Cyagen Biotechnology.