To fully characterize the bioengineered cornea models (HHC and HOC) and compare them with control native human corneas, we used histological, histochemical, and immunohistochemical analyses. For light and electron microscopy, HHC, HOC, and control human corneas were fixed in either 4% paraformaldehyde or 2.5% glutaraldehyde in 0.1 M phosphate buffer. For light microscopy, samples were embedded in paraffin and 4-μm sections were obtained. To analyze the artificial cornea stromas, we used immunohistochemistry for vimentin to detect keratocytes and histochemistry with Alcian blue, PAS, picrosirius red, Gomori's reticulin, and Verhoeff methods to identify ECM proteoglycans, glycosaminoglycans, mature collagen fibers, and reticular and elastic fibers, respectively. To characterize the bioengineered corneal stroma and epithelium, we used immunofluorescence with primary antibodies for type-I collagen and cornea cytokeratins (CK3/12), intercellular junction desmosomes (plakoglobin [PKG]), tight junctions (zonula occludens 1 [ZO1]), and gap junctions (connexin 43 [CX43]), respectively. To analyze crystallin expression in both the epithelial and stromal layers of the artificial cornea models, we used immunofluorescence for alpha A (Cry-αA), alpha B (Cry-αB), beta (Cry-β), zeta (Cry-ζ), beta-gamma 3 (Cry-βγ3), and lambda 1 (Cry-λ1). For this purpose, paraffin-embedded tissue sections were deparaffinized and rehydrated in decreasing concentrations of alcohol, and incubated with the primary antibodies. Then the samples were washed in PBS and a secondary anti-mouse or anti-rabbit antibody labeled with a fluorescent pigment (FITC or Cy3) was applied for 60 minutes, followed by rinsing with PBS. Finally, the nuclei were counterstained with 4′,6-diamidino-2-phenylindole and samples were covered with glass coverslips and analyzed in a Nikon Eclipse 90i fluorescent microscope (Nikon, Tokyo, Japan) and a Leica DMI6000 confocal microscope (Leica, Solms, Germany).
For scanning electron microscopy, samples were dehydrated, prepared for critical point, dried and mounted on aluminum stubs, sputter-coated with gold according to preestablished protocols,
15 and examined in a Quanta 200 scanning electron microscope (FEI, Eindhoven, The Netherlands).