Flow-manipulated, crosslinked collagen gels for use as corneal equivalents

Our aim was to construct a mechanically stable and optically transparent collagen gel from an acidified atelocollagen solution which is suitable for use as a corneal stromal equivalent. Light transmission and mechanical testing were conducted on variously crosslinked constructs at different pH levels. Ultrastructural analysis was performed to assess directionality of the molecular arrangement produced by flow manipulation, as well as the amount of collagen fibrillogenesis which resulted from different pH and/or crosslinking conditions. Clinical and histological integration of the gels with living tissue was examined following implantation into rabbit corneal intra-stromal pockets. Transmission electron microscopy revealed the importance of the fine control of pH levels during gel formation and indicated that the stage at which collagen fibrillogenesis is halted within the constructs was critically dependent on the pH of the collagen solution. Transparency testing disclosed that high levels of collagen fibrillogenesis, as well as high levels of crosslinker concentration, detrimentally affected the transparency of the construct. As a result, a dual titration was required to achieve good light transmission through the gels. It was also evident that the amount of crosslinking required to gelate the collagen solution was reduced as the level of fibrillogenesis progressed. Thus, it was necessary to establish a balance between the solution pH and crosslinker concentration. Implantation of the collagen constructs into partial depth intra-stromal pockets in rabbits was followed up for 6 months, and demonstrated favourable biocompatibility. This showed that gels which had lower levels of both fibrillogenesis and crosslinking were degraded more readily by the host tissue. The collagen gels described here are mass-production friendly, and have promise as potential functional stromal equivalents for use in stromal grafting, or in constructing full thickness artificial corneas.