Ascorbic acid

Osteogenic differentiation was induced with 10⁻⁸M dexamethasone (Sigma D1756), 50 μg/ml ascorbic acid (Sigma A4406) and 10⁻²M β-glycerophosphate (Fluka 50020) for 7 and 14 days in alpha MEM with 10% FBS. Adipogenic differentiation was induced with alpha MEM (with 10% FBS) consisted of 0.05 mM indomethacin (Sigma I7378), 10 μg/ml insulin (Sigma I0516), 10⁻⁸M dexamethasone, 50 μg/ml ascorbic acid and 0.45 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma I5879) for 7 or 14 days. For chondrogenic differentiation, 2.5 ×10⁵ cells/ml was transferred into 15-ml tube (Orange Scientific 5540300) and spin down. After 24 h, the cell pellet will form the ball-like micromass and transfer the medium into DMEM (Hyclone SH30081) with 10⁻⁵M dexamethasone, 1% NEAA (Sigma M7145), 0.1% ITS + (Sigma I2521), 50 μg/ml ascorbic acid, 40 μg/ml l-proline (Sigma P5607), 100 μg/ml sodium pyruvate (Sigma P5280) and 10 ng/ml TGF-β1 (Sigma T7039) contained. The micromass was cultured by shaking every other day to prevent adherence to the tube and culture for 7 or 21 days. Osteogenic, adipogenic differentiation and chondrogenic micromass sections were detected by staining with Alizarin red S (Sigma A5533), Oil red O (Sigma O9755) and Alcian blue (Sigma B8438) / nuclear fast red (Sigma N3020), respectively.

We screened multiple UlaA homologs from different prokaryotic species in order to obtain an inward-facing conformation structure. Those genes were cloned into pET21b (Novagen) with a C-terminal His x 8 tag. The transformed C43 (DE3) (Lucigen) cells were grown in Luria broth at 37 ℃ and induced with 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG) after the OD₆₀₀ reached 1.2. Cells were disrupted with a French Press with two passes at 15,000 p.s.i., in buffer A containing 25 mM Tris-Cl, pH 8.0, and 150 mM NaCl. After a low-speed centrifugation, the resulting supernatant was centrifuged at high speed to sediment a membrane fraction, which then was incubated in buffer A with 1% (w/v) n-dodecyl β-d-maltopyranoside (DDM, Anatrace) and 2 mM ascorbic acid (Sigma-Aldrich) for 1 h at 4 °C. The lysate was centrifuged again, and the supernatant was loaded onto a Ni²⁺-NTA affinity column (Qiagen). After three washes, the protein was eluted with 25 mM Tris-Cl, pH 8.0, 150 mM NaCl, 250 mM imidazole, 2 mM ascorbic acid and 0.56% (w/v) n-nonyl- β-D-maltopyranoside (NM, Anatrace), and it was then concentrated by Amicon Ultra (Millipore) for subsequent gel filtration in buffer A with detergent and 2 mM ascorbic acid. The UlaA domain from Pasteurella multocida showed a high yield and good behavior in gel filtration. The peak fractions were collected for crystallization.

Cell-laden hydrogels were either cultured in pro-inflammatory
media, which consisted of the cell expansion medium supplemented with
50 μg/mL ascorbic acid (Millipore Sigma) and 20 ng/mL recombinant
human IL-1β (Peprotech) to simulate the osteoarthritic environment,^{49 (link)} or in noninflammatory media, which consisted
of the cell expansion medium supplemented with 50 μg/mL ascorbic
acid and 40 ng/mL dexamethasone (Millipore Sigma).^{50 (link)} Hydrogels were cultured in 48-well plates with media exchanged
and collected every 2 days. fbACs were encapsulated at a final cell
concentration of 2 million cells/mL.