Alginate acid sodium salt

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), D, L-dithiothreitol (DTT), and polyvinyl alcohol (PVA, Mw: 72,000 Da) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Doxorubicin (DOX), a cationic anticancer drug, was purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). Alginate lyase was purchased from Hubei Xinmingtai Chemical Co., Ltd. (Wuhan, China). Alginate acid sodium salt (from brown algae, Mw: 12–58 kD, cell culture tested), dioctylsulfosuccinate (AOT), cystamine dihydrochloride (Cys), and gold(III) chloridehydrate (HAuCl₄·3H₂O) were purchased from Sigma–Aldrich (USA).

ATDC5 cells were cultured in alginate spheres according to Culbert et al. (2014 (link)). Briefly, cell suspensions at 6.7×10⁶ cells/ml in 1.2% alginate acid sodium salt (Sigma-Aldrich, Buchs, SG, Switzerland) solution were extruded through 16-gauge needles as ∼30 µl drops in 30 ml of 102 mM CaCl₂ (Sigma-Aldrich, Buchs, SG, Switzerland) in order to allow sphere formation. After drop solidification, cells/alginate spheres were washed with PBS and cultured in chondrogenic medium, replenishing every 3 days. A number of alginate spheres for each condition were formalin-fixed and processed for histological stainings and immunohistochemical assays. In parallel, spheres were also incubated with 55 mM sodium citrate (Sigma, Buchs, SG, Switzerland) to recover cells for total RNA extraction and expression analysis of markers specific for chondrogenesis such as Runx2, Sox9, Col II, Col X, aggrecan and also ACVR1 by RT-qPCR with TaqMan Assays probes (Life Technologies, Thermo Fisher Scientifics, Waltham, MA, USA) (see Table S3 for specification).

Freeze-drying technique was used to prepare porous matrices. Polymers were added to purified water to obtained separately solutions of pullulan 15% w/w (abcr, Karlsruhe, Germany) and alginate acid sodium salt 4% w/w (Sigma, Steinheim, Germany) respectively. Compounds were homogenized through mechanical stirring (L366, Labinco BV, Breda, The Netherlands) with stirrer speed fixed to avoid air bubbles. Forward, glycerol 95% (Sigma, Steinheim, Germany), methylcellulose (4000 cp, Sigma, Steinheim, Germany), and methylene blue (Alfa Aesar, Kandel, Germany) were added to gain the concentrations in dry mass according to Table 1. Then mixture was aeration and homogenized by mix and whipping at 2340 rpm (Gako e/s, Eprus, Bielsko-Biała, Poland) for 10 min to get a foam, 40.0 g of mixture was transferred to a Petri dish (teflon-coated) and was frozen at −26 °C for 48 h and then freeze-dried at room temperature with ultimate vacuums of 9.0 × 10⁻² to 1.3 × 10⁻¹ mBar (Lyovac GT2, Steris, Köln, Germany) for 20 h. Matrices after drying are depicted on Figure 1a,b. were preserved from moisture and light at ambient temperature for another characterization.