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

摘要

To overcome the limitations of the low stability of tissue collagens and possible toxicity of chemical crosslinkers, 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.