Interrelationship between cross-linking structure, molecular stability, and cytocompatibility of amniotic membranes cross-linked with glutaraldehyde of varying concentrations

The aim of this study was to investigate the interrelationship between cross-linking structure, molecular stability, and cytocompatibility of glutaraldehyde (GTA)-treated amniotic membrane (AM). With increasing cross-linker concentration from 0.001 to 0.1 mmol GTA per mg AM, the cross-linking index and number of cross-links per unit mass significantly increased. Inversely, the average molecular weight of polymer chains between two consecutive junctions was significantly decreased with an increase in cross-linking density. The shrinkage temperature and weight remaining of the chemically modified collagenous tissues were respectively used for the assessment of their resistance to thermal denaturation and enzymatic degradation. Our results demonstrated that the collagen molecular stability was enhanced considerably in the presence of higher amounts of non-zero-length cross-linkers. The AM matrices treated with low cross-linker concentrations (i.e., <0.03 mmol GTA per mg AM) displayed good compatibility with human corneal epithelial cells. In contrast, the samples with the greatest extent of cross-linking may cause marked alteration in cell morphology and a decrease in cell viability. Although the biological tissues cross-linked with GTA still possessed anti-inflammatory activities, the interleukin-6 cytokine levels in lipopolysaccharide-stimulated cells were increased with increasing cross-linking density. The up-regulated stemness gene expression was positively correlated with the degree of GTA cross-linking, suggesting the chemical cross-linking structure-mediated preservation of limbal epithelial progenitor cells. It is concluded that during cross-linking treatment, the GTA concentration is critical to tailor the properties of chemically modified AM for a limbal stem cell niche.