Thincert well inserts

Oscillatory shear rheometry was performed on a Discovery Hybrid 2 (DHR-2) rheometer (TA Instruments, USA) using a 20 mm parallel plate geometry with a gap size of 500 µm. Samples were prepared by pipetting 300 μL of hydrogel into ThinCert well inserts (1 μm pore size Greiner Bio-One Ltd, Gloucestershire, UK). The inserts were then placed into 12-well plates and incubated at 37 °C overnight in either 1 mL of ddH₂O or bovine nucleus pulposus cell culture media. After media exposure, samples were removed from the inserts by peeling-off the bottom membrane of the insert and transferred onto the rheometer plate. The upper rheometer head was then lowered to the desired gap size and samples were left to equilibrate for 3 min at 37 °C. Frequency sweep experiments were performed at 0.2% strain, within the linear viscoelastic region in the frequency range: 0.01–10 Hz. For shear-thinning recovery experiments, samples were tested in time-sweep mode by alternating cycles of recovery (5 min at 0.2% strain and 1 Hz frequency) with cycles of high-shear (5 min at 100% strain and 1 Hz frequency). For all experiments a solvent trap was used to minimise sample evaporation. All measurements were repeated at least three times to ensure reproducibility.

Hydrogel morphology and cell-hydrogel interaction were evaluated by Scanning Electron Microscopy (SEM). Briefly, 100 µL of the pre-gel solutions with and without Hap were pipetted into ThinCert well inserts (0.4 µm pore size Greiner Bio-One Ltd, UK). The inserts were then placed into 24-well plates and incubated at 37 °C with a total volume of 1.3 mL DMEM to fully crosslink the hydrogels. The following day Raw 264.7 cells were seeded onto the hydrogels as described above. After 7 days, cells were washed in PBS and fixed in 2.5 % (w/v) glutaraldehyde (Sigma-Aldrich, UK) and 4% (w/v) paraformaldehyde (Sigma-Aldrich, UK) in 0.1 M HEPES buffer (Sigma-Aldrich, UK). After rinsing the samples in PBS, for cell observation all samples were dehydrated in a graded ethanol (EtOH) series (25, 50, 75, 95, and 100 % v/v EtOH/water). Samples were maintained at 100 % EtOH and dried in a K850 Critical Point Drier (CPD, Quorum Technologies, UK). After the CPD step, samples were transferred into metallic pins and coated with gold palladium alloy using an SC7620 Mini Sputter Coater (Quorum). Samples were then imaged on a Quanta 250 FEG SEM (Thermo Fisher Scientific) at 20 kV.

The morphologies of Fmoc-FF/S hydrogels with and without incorporated collagens were analysed by Scanning Electron Microscopy (SEM, Thermo Fisher Scientific, Loughborough, UK). Briefly, hydrogels were prepared by pipetting ~300 μL of the pre-gel solutions into Thin-Cert well inserts (0.4 μm pore size Greiner Bio-One Ltd., Stonehouse, UK). The inserts were then placed into 24-well plates and incubated at 37 °C with a total volume of 1.3 mL PBS containing the protein of interest to fully crosslink the hydrogels. After 24 h, hydrogels were fixed in 2.5% (w/v) glutaraldehyde (Sigma-Aldrich, Welwyn Garden City, UK) and 4% (w/v) paraformaldehyde (Sigma-Aldrich, Welwyn Garden City, UK) in 0.1 M HEPES buffer (Sigma-Aldrich, Welwyn Garden City, UK). After rinsing the samples in PBS, all samples were dehydrated in a graded ethanol (EtOH) series (25, 50, 75, 95, and 100% v/v EtOH/water). Samples were maintained at 100% EtOH and dried in a K850 Critical Point Drier (CPD, Quorum Technologies, Lewes, UK). After the CPD step, samples were transferred into metallic pins and coated with gold palladium alloy using an SC7620 Mini Sputter Coater (Quorum, Lewes, UK). Samples were then imaged on a Quanta 250 FEG SEM (Thermo Fisher Scientific, Loughborough, UK) at 20 kV.