ADA was synthesized from sodium alginate (MW 100,000–200,000 g/mol, Sigma-Aldrich, USA) following the process reported by Zehnder et al. . According to previous work of our group, this process results in a degree of oxidation of ADA of approx. 30% . To prepare ADA–GEL, equal volumes of filtered 5% (w/v) ADA and 5% (w/v) GEL Type A (300 Bloom, Sigma, USA) solutions were stirred together for 10 min. The mixture was then casted, crosslinked by covering with 0.1 M CaCl2 (VWR, Belgium) or 0.1 M BaCl2 (Merck KGaA, Germany) for 15 min, and washed three times with Hank’s balanced salt solution (HBSS, Sigma-Aldrich, USA).
Mechanical properties of ADA–GEL were determined by using disc-shaped samples (n = 3, 16 mm of diameter and thickness of approximately 1 mm) subjected to a frequency sweep in compressive deformation mode to determine storage (E′) and loss (E″) moduli at room temperature by DMTA. A suitable pre-load (40 g) and strain amplitude (0.1%) were determined by previous amplitude sweeps.
Degradation of ADA–GEL was evaluated by incubating ADA–GEL disks in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Germany) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin (both Sigma-Aldrich, Germany) (the same DMEM was used for cell growth) under cell culture conditions (37 °C, 95% relative humidity, 5% CO2). Mass change was calculated at defined time points. In parallel, the chemical composition of samples which were incubated for 7, 14, 21, 28 days was investigated by using attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR) (IRAffinity-1S, Shimadzu, Japan). The medium was changed three times a week following the same procedure used for cell culture studies.
Cell encapsulation in ADA–GEL ring structures
Sacrificial gel containing 9% (w/v) MC (Sigma, USA) and 5% (w/v) GEL was used. This particular composition of the sacrificial gel was obtained from the printing tests of different gel formulations consisting of various concentrations of MC and GEL (data not shown here). Sacrificial gel was transferred into an autoclaved cartridge with a conical nozzle (G22, Nordson EFD, Germany). The cartridge was then placed in a 3D printer (BioScaffolder GeSiM 2.1, GeSiM, Germany). The sacrificial gel was printed as two concentric rings with diameters of 4.4 and 10 mm. The formed well between them was then filled with 70 μl ADA–GEL containing osteosarcoma cells MG-63 (Sigma-Aldrich, Germany) at the concentration of 1 × 106 cells/ml. The samples were crosslinked by covering with CaCl2 or BaCl2 and washed three times with HBSS. The ring-shaped constructs were afterwards incubated in DMEM for 21 days under cell culture conditions.
Cell activity monitoring
Cell viability was monitored by using the WST-8 assay kit (Sigma-Aldrich, Germany) which was applied according to the manufacturer’s protocol. SYTOX™ green nucleic acid stain and rhodamine phalloidin (both Invitrogen™, Molecular Probes® by Life Technologies™, USA) were used for staining cell nuclei and actin filaments, respectively, for fluorescence microscopy (Axio Observer Scope D1, Carl Zeiss AG, Germany).
Statistical analyses were performed by one-way analysis of variance (ANOVA) with Bonferroni means comparison.