Methylene blue solution

Wells were coated with COL1. After incubation with BSA solution (1%) for 1 h, 20,000 cells were seeded to a total volume of 100 µL medium per well and supplemented with DPBS, 45 min at 37 °C and 5% CO₂. Afterwards, wells were gently washed to remove nonadherent cells. Adherent cells were fixated by formaldehyde (3%, Merck, Darmstadt, Germany) and stained with methylene blue solution (1%, Merck). After washing with DPBS, cells were lysed using 0.1 M HCl and methylene blue was quantified at 620 nm using a plate reader (Thermomultiscan EX, Thermo Fisher Scientific Inc).

HaCaT cells were first cultured in H-Ca medium and maintained in an incubator under 5% CO₂ at 37 °C. The short-term cell culture was used for the HaCaT cell migration assay, with incubation for 48 h in L-Ca medium. After 48 h culture in L-Ca medium, involucrin expression was minimal, similar to cells cultured in undifferentiated conditions. A cell migration assay was performed using a transwell chamber plate (6.5 mm D.I., 8.0 μm pore size, polycarbonate membrane; Transwell Permeable Supports 3422, Corning Inc., New York, NY, USA) as previously described [12 (link)]. Cells subjected to short-term cell culture as described above were seeded at a density of 2 × 10⁴ cells/well in the upper chamber supplemented with 200 μL H-Ca medium, while 750 μL H-Ca medium with or without experimental reagents (Sema3A, kCer, or histamine) was added to the lower chamber compartment. After 48 h incubation at 37 °C, the unmigrated cells in the upper chamber were gently removed with a cotton swab, and the cells that had migrated to the lower compartment of the membrane were fixed with cold absolute methanol for 10 min and stained with GIEMSA’S AZUR EOSIN Methylene Blue solution (109,204, Merck, Darmstadt, Germany) as described previously [17 (link)].

Release behaviour of SN and DN hydrogels was studied using methylene blue (MB) solution (Sigma-Aldrich, St. Louis, MO, USA). The pieces of hydrogels (≈2 cm²) were dried at room temperature for three days and then in a vacuum oven until it reached the constant weight. Dried hydrogels were immersed in MB solution (0.15 g/L) at room temperature and left for two days to attain equilibrium swelling condition. The concentration of MB molecules inside hydrogels c₀ was determined from the mass of dried and swollen hydrogels. The hydrogels were then removed from MB solution to phials filled with 4 mL of water placed in water bath thermostated at 45 °C. Time-dependence of MB concentration was detected with UV-Vis spectrophotometer (Hitachi U-2900, Hitachi, Tokyo, Japan) at 664 nm using MB signal of maximum intensity at 664 nm. At regular time intervals, MB solution released from hydrogel was pipetted out and the concentration of MB c(t) was measured. The fraction of released MB f(t) was calculated as
$f\left(t\right)=\frac{c(t)}{c_0}$
Obtained time-dependencies of the fraction of released MB f(t) was fitted with expression
$f\left(t\right)=f_{\mathrm{MB}}-b\exp \left(-\frac{t}{\tau }\right)$
where f_MB is the equilibrium fractions of released MB, τ is the release delay time and b is a coefficient.