All polymer components were UV-sterilized for 3 hours before usage for in vitro experiments. PdBT and P(NIPAAm-co-GMA) were dissolved at triple the intended final concentrations in PBS at pH 7.4 and kept on ice until the point of hydrogel fabrication. At the point of fabrication, the two polymer solutions and an MSC suspension in media were mixed at 1:1:1 volume ratio and then pipetted at 30 μl per gel construct into autoclaved cylindrical Teflon molds of 8-mm diameter and 1-mm height, producing constructs with a final cell density of 15 × 106 cells/ml (36, 37). The hydrogel-containing molds were then placed in a petri dish, moved to a 37°C, 5% CO2 incubator, and given 75 min to form and cross-link. After this, hydrogels were removed from the molds and placed in 1 ml of each of the aforementioned cell culture media, with replacement of media every 2 to 3 days. At time points of interest, hydrogels were soaked in PBS for 15 min, weighed, and processed for either a DNA PicoGreen assay or LIVE/DEAD imaging.

For the DNA PicoGreen assay, hydrogels were placed in 300 μl of filtered ultrapure H2O and then homogenized using a QIAGEN (Hilden, Germany) TissueLyser II at 26 s−1 for 5 min. Double-stranded DNA of live cells was then quantified in each hydrogel sample using an Invitrogen (Eugene, OR) PicoGreen assay, according to kit instructions. For LIVE/DEAD imaging of MSCs within each hydrogel, ~0.5-mm cross-sectional slices were acquired from each PBS-soaked hydrogel using a handheld razor blade and stained with 250 μl of a 2 μM calcein AM and 4 μM ethidium homodimer-1 solution. After 30 min of incubation at room temperature in the dark, representative images of the slices were taken using excitation/emission filters of 494/515 nm (green, live) and 528/617 nm (red, dead) under a 10× objective on an A1-Rsi confocal microscope from Nikon Instruments (Tokyo, Japan).

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