Ca am

Cell-laden spongy-like hydrogels were incubated with calcein-AM (Ca-AM, 1 μg mL⁻¹, Invitrogen, USA), propidium iodide (PI, 2 μg mL⁻¹, Invitrogen, USA) and Hoechst (20 mM, Thermo Fisher Scientific, USA) for 1 h at 37 °C. Cell viability was observed with a confocal laser scanning microscope (SP8, Leica Microsystems CMS GmbH). In the analysis, cells stained with calcein-AM were categorized as live cells, while cells stained with PI were classified as dead cells. To determine the counts of live and dead cells for each condition, we conducted quantification across nine randomly selected images using the Cell Counter plugin within ImageJ 1.54b (National Institutes of Health, Bethesda, MD, USA). The percentage of live cells was calculated according to the following formula:

After three and seven days, cell-laden spongy-like hydrogels were incubated with calcein-AM (Ca-AM, 1 μg/mL, Invitrogen, Carcavelos, Portugal) and propidium iodide (PI, 2 μg/mL, Invitrogen, Carcavelos, Portugal) for 1 h at 37 °C in a humidified tissue culture incubator with 5% CO₂ atmosphere. Then, constructs were fixed and counterstained with DAPI (0.02 mg/mL) and the percentage of live/dead cells was assessed with a Leica TCS SP8 confocal microscope. The number of live and dead cells for each condition was quantified in five random images (three independent experiments) using the Cell Counter plugin of FIJI for ImageJ. The percentage of live cells was calculated as followed (Equation (3)):
For visualization of the cytoskeleton F-actin fibers and nuclei, cells were fixed and stained with phalloidin-TRITC (0.1 mg/mL, Sigma-Aldrich, Loures, Portugal) and DAPI (0.02 mg/mL), respectively. Samples were observed with a Leica TCS SP8 confocal microscope.