Effect of riboflavin concentration on the development of photo-cross-linked amniotic membranes for cultivation of limbal epithelial cells

To overcome the limitations of the low stability of tissue collagens and possible toxicity of chemical cross-linkers, the amniotic membrane (AM) was photo-cross-linked by ultraviolet (UV) irradiation in the presence of riboflavin. This study aims to investigate the effect of photoinitiator concentration on the preparation of photo-cross-linked AM materials for cultivation of limbal epithelial cells (LECs). The number of cross-links per unit mass of collagen matrix was significantly increased with increasing riboflavin concentration from 0.1 to 10 mg ml^-1. In addition, the equilibrium water content, ultrastructure, nanotopography, and enzymatic degradability of AM samples were found to be associated with the cross-linked structure of UV-irradiated biological tissues. Human corneal epithelial cellular responses to photo-cross-linked AM were assessed by measuring the mitochondrial dehydrogenase activity and interleukin-6 expression levels. Irrespective of the riboflavin concentration, the test samples were fully biocompatible and retained anti-inflammatory activities, probably due to the absence of exogenous cross-linker molecules in the proteinaceous matrices following cross-linking reaction. The results of the quantitative real-time reverse transcription polymerase chain reaction and western blot analyses showed that the LECs cultured on the AM substrates with different cross-linking densities had varying levels of enhanced stemness. The expressions of ABCG2 at mRNA and protein levels were significantly up-regulated with increasing surface roughness of physically cross-linked biological tissue materials. For the first time, here we report that the riboflavin concentration may play an important role in the modulation of properties of photo-cross-linked AM as a new LEC carrier.