HMF proliferation in mTG Hydrogels

HMFs were encapsulated in 7.5% gelatin gels cross-linked with 20, 30, or 60 μg/ml of mTG as previously described. Cell proliferation was assessed in triplicate on days 1, 3, 5, and 7 during the incubation period and was evaluated using the WST-1 assay (Roche; Mannheim, Germany) according to the manufacturer’s instructions. Briefly, the WST reagent was added directly to cell culture media in a 1:10 dilution. HMF encapsulated gels were allowed to incubate in WST reagent for 3 h in a humidified atmosphere at 37 °C and 5% CO₂. Results were evaluated at a wavelength of 450 nm using a Synergy HT spectrophotometer. These assays were performed twice on triplicate samples for each of the gel conditions and time points. To account for any influence on the absorbance reading from the gel itself, the WST-1 assay was performed on control gels, lacking HMFs, cross-linked with 20, 30, and 60 μg/ml of mTG. The absorbance values from control gels were obtained, averaged, and then subtracted from the absorbance values of the gels with encapsulated HMFs.