Amphotericin b

¹H and ¹³C NMR spectra were recorded, respectively, at 300 and 75 MHz in a Bruker Avance III spectrometer using CDCl₃ and DMSO-d₆ (Aldrich) as solvent and internal standard. Silica gel (Merck, 230–400 mesh) and Sephadex LH-20 (Aldrich) were used for column chromatographic separation, while Silica gel 60 PF₂₅₄ (Merck) was used for analytical TLC (0.25 mm). Amphotericin B (Cristalia, Brazil) and Glucantime (Sanofi-Aventis, Brazil) were solubilized in sodium chloride 0.9% (w/v).

For chondrogenic differentiation, 500,000 MSCs were centrifuged for 5 min at 800× g into a pellet and then incubated in chondrogenic induction medium (94% DMEM high glucose, 40 µg/mL transferrin, 40 µg/mL sodium selenite, 1 µM dexamethasone, 0.17 mM ascorbic acid 2-phosphate, 1 mM sodium pyruvate, 0.35 mM proline, 1.25 mg/mL bovine serum albumin (all Sigma-Aldrich, Steinheim, Germany), 100 µg/mL penicillin/streptomycin, 2.2 µg/mL amphotericin B, 0.1375 IE/mL insulin glargine (Sanofi-Aventis, Frankfurt am Main, Germany), and 10 ng/mL transforming growth factor β1 (Abcam, Berlin, Germany) for 42 days. Afterwards the pellets were fixed for 2 h in 4% paraformaldehyde (Merck, Darmstadt, Germany) and then dehydrated for 2 h in 70%, 96% and 100% 2-propanol, followed by a 30 min incubation in 100% acetone (all Carl Roth, Karlsruhe, Germany). The pellets were then transferred into paraffin and processed into sections for histological evaluation by Safranin-O/Fast Green (Waldeck, Muenster, Germany) staining. A qualitative analysis for orange stained proteoglycans and glycosaminoglycans as a marker for the development of cartilage tissue was microscopically conducted. One sample was analyzed for each donor in the individual setting and one sample in total for the pooled setting. Results are shown representatively (Figure 2).