Cultivation of the cartilage layer

1. Resuspend passage 5 adipose-derived stromal/stem cells (ASCs) in chondrogenic medium [high-glucose Dulbecco’s modified Eagle medium (DMEM; Gibco) supplemented with TGF-β3 (10 ng/ml; PeproTech), dexamethasone (100 nM; Sigma-Aldrich), ascorbic acid 2-phosphate (50 µg/ml; Sigma-Aldrich), sodium pyruvate (100 µg/ml; Corning), proline (40 µg/ml; Sigma-Aldrich), 1% insulin, transferrin, sodium selenite (Corning) mix, and 1% penicillin/streptomycin (P/S; Gibco)].
2. Dilute cell suspension to 2.5 × 10⁵ cells/ml to form condensed mesenchymal bodies (CMBs).
3. Aliquot cell suspension into deep round-bottom 96-well plates (Nunc 96-Well Polypropylene DeepWell Storage Plates; Thermo) at 1 ml per well and centrifuge at 250g for 5 min.
4. Allow the CMBs to self-assemble over 3 days in the incubator at 37°C, 5% (v/v) CO₂ with daily media changes.
5. Determine the desired surface area and thickness of the cartilage region and calculate how many CMBs (measuring 1 mm in diameter) will be required, e.g.
   a. The cartilage region covering the articulating surface of the condyle was sized to 1 mm in thickness.
   b. The scaffold condylar surface areas determined by the SOLIDWORKS software were 4 to 5 cm² for the anatomical scaffolds and 0.70 cm² for the small scaffolds.
   c. This corresponds to 800 CMBs per anatomical scaffold and 120 CMBs per small scaffold.
6. Cut a polycarbonate track etch (PCTE) membrane (1.0 µm pore size; Sterlitech) to match the condylarsurface.
7. Place the membrane in the void of the condylar PDMS block, place the CMBs onto the PCTE membrane, and press the decellularized scaffold onto the CMB layer.
8. Allow 1 day for the CMB layer to attach to the scaffold under static culture conditions, with the bioreactor oriented with the condylar end facedown.
9. Perfuse chondrogenic media for 5 weeks over the condylar surface at a flow rate of 1 ml/min, separated from direct contact with the CMB layer by the PCTE membrane to reduce shear stress.
10. Change culture medium twice per week.
11. Introduce osteogenic cells to the graft 2 weeks after the initial seeding of the CMB layer, by which time a compact cartilage layer has been formed.

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