EVALUATION OF CROSS-LINKING TIME FOR POROUS GELATIN HYDROGELS ON CELL SHEET DELIVERY PERFORMANCE

To overcome the drawbacks posed by surgical manipulation of bioengineered corneal endothelial cell (CEC) sheets, a simple stirring process combined with freeze-drying method was recently developed for the production of cross-linked porous gelatin hydrogels that can provide the support structure and improve the aqueous humor circulation. In this study, we further evaluated the influence of cross-linking time (0-48 h) on the delivery performance of carbodiimide modified gelatin carriers. It was found that smaller pore size, lower porosity, and larger superficial area were associated with increasing extent of cross-linking of the carrier discs. Although the hydrogels treated for short reaction time (i.e., below 6 h) had low resistance to initial nutrient permeation, these materials exhibited rapid swelling, implying a potential anterior segment tissue squeezing effect for use as intraocular implants. In addition, the delivery carriers with limited extent of cross-linking degraded too fast to be effective for retention of cell sheet grafts at the site of injury. By contrast, the gelatin samples with cross-linking degrees greater than 50% showed slower degradation rates and smaller porous structure, thereby possibly causing a significant inhibition of CEC proliferation. Cell sheet transfer studies demonstrated that the carrier discs with a high cross-linking degree (59.4 +/- 1.3%) were more difficult to achieve stable cell attachment than their counterparts with a low cross-linking degree (48.3 +/- 1.5%). Our findings suggest that among the cross-linked porous samples studied, 12 h is the best cross-linking reaction time for preparation of cell sheet carriers with suitable delivery performance.