Transforming growth factor β3

Dynamic compressive loading was performed in a Mechano-Active Transduction and Evaluation (MATE) bioreactor (Apex Biomedical LLC, Wilsonville, OR, United States) (Figure 1). Dynamic cyclic compression was applied to cell-loaded gels with controlled strain and displacement within a tissue culture incubator (37°C, 5% CO₂) (Iseki et al., 2019 (link)). To mimic a cadence of daily walking, the MATE system was operated at 1 Hz with different strains (5 and 25%) for 1 h per day over 14 consecutive days. All samples were maintained in 3 ml chondrogenic medium [CM: high-glucose DMEM supplemented with 1% Antibiotics-Antimycotics, 40 μg/mL L-proline (Sigma, St. Louis, MO, United States), 10 μg/ml ITS+ (Thermo Fisher Scientific), 50 μg/ml ascorbate 2-phosphate, 10 ng/ml transforming growth factor (TGF)-β3 (Peprotech, Rocky Hill, NJ, United States)]. Medium for all groups was changed every 3 days. The non-loaded samples in static culture were used as the control. Chondrogenesis was analyzed after 14 days.

Approximately 2 × 10⁴ cells were seeded in 12-well plates in LG-DMEM culture medium. After incubation for 24 h, the cells were treated with GA-D and replaced with osteoblast-conditioned medium (HG-DMEM (Gibco, New York, USA) with 10% FBS, 100 nmol/L dexamethasone (Sigma, SL, USA), 50 mg/L vitamin C (Solarbio, Beijing, China), and 10 mmol/L β-glycerophosphate (Solarbio, Beijing, China)) and chondrocyte-conditioned medium (HG-DMEM with 10 ng/L transforming growth factor- (TGF-) β3 (Peprotech, NJ, USA), 1 × 10⁻⁷ mol/L dexamethasone, and 50 mg/L vitamin C) for 7 days, as described in previous reports [26 (link), 28 (link)]. Osteogenic differentiation was preliminarily evaluated by alkaline phosphatase staining, and chondrogenesis was assessed by toluidine blue staining.

Pellets were created by centrifuging 2.5 × 10⁵ cells/pellet in 1 mL of media at 300 g for 3 min in 1.5-mL microcentrifuge tubes. Pellets were formed and cultured in their standard MM and in prochondrogenic medium (PChM) consisting of the cells' MM with reduced FBS (1% [v/v]), further supplemented with 10 ng/mL transforming growth factor (TGF)-β3 (PeproTech), 0.1 μM dexamethasone, 40 μg/mL l-proline (Sigma), 50 μM ascorbic acid phosphate (Sigma), 1% (v/v) insulin, transferrin, selenium (Sigma), and 1% (v/v) sodium pyruvate (Sigma). Pellets were cultured for 20 days with twice weekly media changes. Pellets were fixed and used at days 1 and 20 for microcomputed tomography (μCT) analysis and histology, and either frozen at −80°C, or processed immediately at days 0 and 20 for sulfated glycosaminoglycans (sGAGs), DNA, and gene expression quantification. Spent culture media were also processed for sGAG quantification.

The MPCs underwent multi-lineage differentiation analysis to determine their osteo/chondro/adipo-genic capacity^{30 (link),34 (link)}.
Osteogenesis: For each replicate, 5 × 10⁵ cells were seeded into each well in a 24-well plate and then placed into DMEM/F-12 media that contained Dexamethasone (final concentration (FC): 100 nM) (Sigma), L-Ascorbic Acid (FC: 50 μg/mL) (Sigma), β-Glycerolphosphate (FC: 10 mM) (Sigma).
Adipogenesis: For each replicate, 5 × 10⁵ cells were seeded into each well in a 24-well plate and then placed into DMEM/F-12 media that contained Dexamethasone (FC: 1 μM) (Sigma), Insulin (FC: 10 μM) (Sigma), Indomethacin (FC: 200 μM) (Sigma), and Isobutylmethylxanthine (FC: 500 μM) (Sigma).
Chondrogenesis: For each replicate, 5 × 10⁵ cells were pelleted through centrifugation and placed into DMEM/F-12 media that contained Dexamethasone (FC: 10 nM) (Sigma), L-Ascorbic Acid (FC: 50 μg/mL) (Sigma), MEM Non-Essential Amino Acids (FC: 1%) (MEM-NEAA Gibco), Transforming growth factor (TGF)-β3 (FC: 10 ng/mL) (Peprotech), Bone morphogenetic protein (BMP)-2 (FC: 500 ng/mL) (Peprotech), Insulin transferrin selenium (FC: 1%) (Lonza- BioWhittaker), and sodium pyruvate (FC: 1%) (ThermoFisher). Media was adjusted to neutral pH (7.0–7.6).
After 21 days, differentiation was assayed using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and histological staining.

MSCs (1 × 10⁶) were placed in polypropylene tubes and centrifuged at a speed of 300 × g for 6 min. The pellets were cultured in 1 ml complete chondrogenic medium consisting of Dulbecco’s modified Eagle’s medium (Life Technologies, Gaithersburg, MD, USA) supplemented with 10⁻⁷ M dexamethasone (Sigma), 1% insulin-transferrin-sodium selenium (Sigma), 50 μM ascorbate-2-phosphate (WAKO), 50 μg/ml proline (WAKO), 20 ng/ml transforming growth factor-β3 (Life Technologies), and 1% antibiotics (WAKO)39 (link). The polypropylene tubes were maintained at 37 °C in a 5% CO₂ incubator, and the medium was changed every 3 days. The pellets were used for immunostaining or qPCR at each time. Data were normalized to the average mRNA level at day 0 (set at 1) and are presented as means ± SEM.

The samples were cultured for 7 days in basal media. After this period, the differentiation process was started, when relevant. Samples for differentiation studies were cultured in chondrogenic media consisting of high glucose (4.5 mg/mL) DMEM (Dubelcco`s Modified Eagle Medium) with 100 μg/mL of sodium pyruvate (Gibco), 0.2 mM l-ascorbic acid-2-phosphate (Sigma-Aldrich), 1% 100x ITS (insulin-transferrin-selenium) liquid media supplement (Thermo Fisher Scientific), 40 μg/mL proline (Sigma-Aldrich), 100 U/mL penicillin/streptomycin, and 100 nM dexamethasone (Sigma-Aldrich). To the complete media and right before addition to the cultures, 0.01ug/mL of transforming growth factor-β3 was supplemented (Peprotech). Media for both maintenance and differentiation conditions were changed every second day.