Tgf β3

CD4⁺ T cells were purified from spleen and lymph nodes (LNs) with anti-CD4 microbeads (Miltenyi Biotech) and then further sorted as naive CD4⁺CD44^loCD62L^hi T cells using a FACSAria III sorter (BD Biosciences). Sorted cells were activated with plate-bound anti-CD3 (4 μg/mL) and soluble anti-CD28 (2 μg/mL, both BD Biosciences) in the presence of polarizing cytokines. For T_H17 differentiation, the following reagents were used: 2.5 ng/mL recombinant human TGFβ1 (eBioscience) and 20 ng/mL recombinant mouse IL-6 (R&D Systems) for nonpathogenic T_H17 cells. Pathogenic T_H17 cells were differentiated with 25 ng/mL of IL-1β (R&D Systems), 20 ng/mL of IL-6, and 20 ng/mL of IL-23 (R&D Systems). Alternatively, as indicated in the text, TGFβ1-induced pathogenic T_H17 cells were differentiated in the presence of 2.5 ng/mL TGFβ1, 20 ng/mL IL-6, and 20 ng/mL of IL-23. TGFβ3-induced cells were generated with 2.5 ng/mL TGFβ3 (R&D Systems) and 20 ng/mL IL-6. Cells were cultured for 3 days and collected for RNA, intracellular cytokine staining, and flow cytometry. FICZ (Enzo Life Sciences; 100 nM) and CH223191 (Sigma-Aldrich; 30 μM) were added at the start of the cultures where indicated.

Gelatin from porcine skin, paraffin oil, SPAN80, EDC, and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (USA). Glutaraldehyde and acetone were purchased from Daejung Chemicals and Metals (South Korea). TGF-β3 and matrilin-3 were purchased from R&D System (USA). TGF-β3 antibody was purchased from Proteintech (USA) and FITC-labeled matrilin-3 antibody was purchased from Biorbyt (USA). An Alexa Fluor 594 antibody labeling kit was obtained from Life Technologies (USA). StemFit 3D® was purchased from Microfit (South Korea). All culture media were purchased from Hyclone (GE Healthcare, UT, USA). A cell counting kit-8 (CCK-8) assay was purchased from Dojindo (Japan). Trizol reagent was purchased from Qiagen (Hilden, Germany), while the reverse transcription kit and SYBR® Green were purchased from Takara (Japan).

TMSCs were subjected to two types of treatment: TGF-β3 and mechanical strain. TMSCs were cultured at 1 × 10⁴ cells/cm² in six-well UniFlex plates coated with COL1. TMSCs were incubated for 1, 3, 7, and 14 days in DMEM/low-glucose containing 10% FBS and 50 μg/mL L-ascorbic acid 2-phosphate (Sigma-Aldrich, MO, USA), with 10 ng/mL TGF-β3 or mechanical strain (R&D Systems, Minneapolis, MN, USA). During harvesting, the medium was changed, and TGF-β3 was supplemented every 2–3 days. The mechanical strain was applied to TMSCs once for 1 h per day using a Flexcell FX-5000 Tension system (Flexcell®, Burlington, NC, USA). The cells were subdivided into three groups: Static, 2, and 5%. The mechanical strain was expressed as a sine wave with a frequency of 0.5. Each experiment was repeated twice per patient.

To assess the multilineage differentiation capacity, the obtained bone marrow stem cells (BMSC) underwent osteogenic, adipogenic, and chondrogenic induction by different culture media.
For osteogenic differentiation, cells were cultured with osteogenic medium with α-MEM supplemented with 10% FBS, 10⁻⁷ M dexamethasone (Sigma-Aldrich), 10 mM β-glycerol phosphate (Sigma-Aldrich), and 50 mM ascorbate-2-phosphate (Sigma-Aldrich). After 3 weeks of differentiation, the mineralization was stained by Alizarin Red S staining. For adipogenic differentiation, cells were cultured with α-MEM supplemented with 10% FBS, 10⁻⁶ M dexamethasone, 0.5 μM isobutylmethylxanthine (IBMX, Sigma-Aldrich), and 10 ng/mL of insulin (Sigma-Aldrich) for 2 weeks. Lipid accumulation was identified by Oil Red O staining. For chondrogenic differentiation, cells (1 × 10⁶) were seeded in polypropylene tubes with DMEM supplemented with 10⁻⁷ M dexamethasone, 1% insulin-transferrin-selenium (ITS, Sigma-Aldrich), 50 μM ascorbate-2-phosphate, 1 mM sodium pyruvate (Sigma-Aldrich), 50 μg/mL of proline (Sigma-Aldrich), and 20 ng/mL of TGF-β3 (R&D Systems, Minneapolis, MN, USA). After 3 weeks in culture, the pellets were fixed in 10% buffered formalin for 48 h and embedded in paraffin. Then, 4 μm thick sections were processed for toluidine blue staining (Sigma-Aldrich).