Dexamethasone (dex)

Dehydrocurvularin and galiellalactone were obtained by fermentation of the producer strains Penicillium sp. IBWF3-93 and the ascomycete IBWFA111-95, respectively. Isolation of the compounds from the culture fluid by extraction with ethyl acetate and preparative HPLC was performed as described previously (Weidler et al., 2000 (link); Rudolph et al., 2012 (link)). The purity of the compounds was >98% as determined by HPLC equipped with diode-array detection and mass spectrometry analysis. Dexamethasone was obtained from Applichem (Germany).
In initial studies, the concentrations of each compound were chosen according to earlier studies. 10.3 μM (2 μg/ml) galiellalactone, 10.3 μM (3 μg/ml) dehydrocurvularin, 2.5 μM (1 μg/ml) Dexamethasone which were used on ENS cells were tested in the perfusion model with little to no effect (data not shown). Therefore, higher compound concentrations were used for intestinal perfusion. In the perfusion model 20.7 μM (4 μg/ml) dehydrocurvularin, 20.6 μM (6 μg/ml) galiellalactone and 5.1 μM (2 μg/ml) Dexamethasone were used. Due to the complexity of organ resection and perfusion, only one concentration of every compound was evaluated in this model.

Cells (30,000/foam) were seeded onto the sterilized unmodified, fibronectin modified, and laminin modified PBS foams. Cell seeded foams were incubated in a CO₂ incubator at 37°C, in 5% CO₂ and 90% humidity throughout 20 days. At day 3, osteogenic medium (Dulbecco's Modified Eagle Medium (DMEM: 4.5 g/liter glucose)) (Gibco Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco-Invitrogen, USA), 100 units/mL Penicillin-Streptomycin-Amphotericin (PSA) (Lonza, Switzerland), 50 μM ascorbic acid (AppliChem, Germany), 100 nM dexamethasone (AppliChem, Germany), and 10 mM β-glycerophosphate (AppliChem, Germany) was added into the wells and it was changed twice a week. All tests were carried out in triplicate throughout the whole study.

7.5 × 10³ cells /cm² CTR-hGMSCs and CBD-hGMSCs, were seeded at 2nd passage in a 6-well cell culture plate (Falcon) and incubated overnight at 37°C, in a humidified 5% CO₂ atmosphere. The culture medium was replaced with osteogenic induction medium represented of MSCGM-CD medium supplemented with 100 nM dexamethasone (Applichem GmbH, Darmstadt, Germany), 10 nM β-glycerol-phosphate (Applichem) and 0.05 mM 2-phosphate-ascorbic acid (Sigma-Aldrich). The medium was replaced every 3 days. At days 21 Alizarin Red S staining (Sigma-Aldrich Co.) was performed to detect calcium formation (Trubiani et al., 2015 (link)).