Baltec

HCEnCs were seeded on FNC-coated glass coverslips and maintained in M5-Endo for at least 48 hours. Following a four-day storage, coverslips were removed and fixed with neutrally buffered 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) at 4 °C for 4 hours. Alternatively, samples were placed at 37 °C for 48 hours recovery, and subsequently fixed. Samples were processed as previously described^{17 (link)}. Briefly, samples were post-fixed in 1% osmium tetroxide at room temperature for 1 hour. The samples were then dehydrated, critical point dried (BALTEC, Balzer, Liechtenstein) and mounted onto a metal stub using carbon adhesive tabs. Samples were sputter-coated for 160 seconds with a 22 nm layer of gold-phalladium alloy (BALTEC) and examined under a scanning electron microscope (Quanta 650FEG; FEI, Hillsboro, OR, USA).

The endothelial denuded area along the laser trephination was evaluated with scanning electron microscopy (SEM). Corneas were fixed in neutral buffered 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield, USA) at 4 °C for 24 h. After rinse with 1 × PBS, they were cut into halve and post-fixed in aqueous solution of 1% osmium tetroxide at room temperature for 30 min. The samples were then dehydrated under an increasing alcohol gradient: 25% ethanol for 5 min, 50% ethanol for 5 min, 75% ethanol for 5 min, 95% ethanol for 5 min, 3 × 100% ethanol for 10 min each, followed by critical point drying (BALTEC, Balzer, Liechtenstein). Dehydrated samples were then mounted onto a metal stub using carbon adhesive tabs. Samples were sputter-coated with a 25-nm layer of gold–palladium alloy (BALTEC), and examined under a scanning electron microscope (Quanta 650FEG; FEI, Hillsboro, OR). The denuded endothelial area, alongside the laser path for each cornea sample, was measured using ImageJ (National Institutes of Health, Bethesda, MD). The denuded area was defined as regions absent of endothelial cells from the laser cut.

Using a Philips-XL-30 instrument (FEI, Hillsboro, OR, USA), the surface morphology of the 3DP scaffolds and 3DP with ESP collagen film scaffolds was analyzed by SEM. The samples were fixed in 2% glutaraldehyde for 2 h at 4 °C, washed twice in phosphate buffer solution (PBS), and post-fixed in 1% osmic acid for 2 h at 4 °C. After two PBS washes, the samples were dehydrated in a graded series of ethanol, dried to a critical point (HCP-2; Hitachi, Tokyo, Japan), mounted, sputter-coated with gold (BAL-TEC; FEI), and analyzed by SEM.