The effect of decellularization protocols on characterizations of thermoresponsive and light-curable corneal extracellular matrix hydrogels.

To compare the effects of two decellularization protocols on the characteristics of fabricated COrnea Matrix (COMatrix) hydrogels. Porcine corneas were decellularized with Detergent (De) or Freeze-Thaw (FT)-based protocols. DNA remnant, tissue composition and α-Gal epitope content were measured. The effect of α-galactosidase on α-Gal epitope residue was assessed. Thermoresponsive and light-curable (LC) hydrogels were fabricated from decellularized corneas and characterized with turbidimetric, light-transmission and rheological experiments. The cytocompatibility and cell-mediated contraction of the fabricated COMatrices were assessed. Both protocols reduced the DNA content to < 0.1 µg/mg (native, > 0.5 µg/mg), and preserved the collagens and glycosaminoglycans. The α-Gal epitope remnant decreased by > 50% following both decellularization methods. We observed more than 90% attenuation in α-Gal epitope after treatment with α-galactosidase. The thermogelation half-time of thermoresponsive COMatrices derived from De-Based protocol (De-COMatrix) was 18 min, similar to that of FT-COMatrix (21 min). The rheological characterizations revealed significantly higher shear moduli of thermoresponsive FT-COMatrix (300.8 ± 22.5 Pa) versus De-COMatrix 178.7 ± 31.3 Pa, p < 0.01); while, this significant difference in shear moduli was preserved after fabrication of FT-LC-COMatrix and De-LC-COMatrix (18.3 ± 1.7 vs 2.8 ± 2.6 kPa, respectively, p < 0.0001). All thermoresponsive and light-curable hydrogels have similar light-transmission to human corneas. Lastly, the obtained products from both decellularization methods showed excellent in vitro cytocompatibility. We found that FT-LC-COMatrix was the only fabricated hydrogel with no significant cell-mediated contraction while seeded with corneal mesenchymal stem cells (p < 0.0001). The significant effect of decellularization protocols on biomechanical properties of hydrogels derived from porcine corneal ECM should be considered for further applications.