Glutaraldehyde cross-linking of amniotic membranes affects their nanofibrous structures and limbal epithelial cell culture characteristics

Abstract

Given that the cells can sense nanometer dimensions, the chemical cross-linking-mediated alteration in fibrillar structure of collagenous tissue scaffolds is critical to determining their cell culture performances. This article explores, for the first time, the effect of nanofibrous structure of glutaraldehyde (GTA) cross-linked amniotic membrane (AM) on limbal epithelial cell (LEC) cultivation. Results of ninhydrin assays demonstrated that the amount of new cross-links formed between the collagen chains is significantly increased with increasing the cross-linking time from 1 to 24 hours. By transmission electron microscopy, the AM treated with GTA for a longer duration exhibited a greater extent of molecular aggregation, thereby leading to a considerable increase in nanofiber diameter and resistance against collagenase degradation. In vitro biocompatibility studies showed that the samples cross-linked with GTA for 24 hours are not well-tolerated by the human corneal epithelial cell cultures. When the treatment duration is less than 6 hours, the biological tissues cross-linked with GTA for a longer time may cause slight reductions in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, and anti-inflammatory activities. Nevertheless, significant collagen molecular aggregation also enhances the stemness gene expression, indicating a high ability of these AM matrices to preserve the progenitors of LECs in vitro. It is concluded that GTA cross-linking of collagenous tissue materials may affect their nanofibrous structures and corneal epithelial stem cell culture characteristics. The AM treated with GTA for 6 hours holds promise for use as a niche for the expansion and transplantation of limbal epithelial progenitor cells.

Stabilization of collagen nanofibers with l-lysine improves the ability of carbodiimide cross-linked amniotic membranes to preserve limbal epithelial progenitor cells

“…33 In our laboratory, collagen nanofiber-based AM materials have been chemically cross-linked by exogenous carbodiimide. 8,13,14 However, a technique involving use of EDC/NHS to catalyze the formation of zero-length covalent cross-links between protein molecules allows only limited cross-linking. Herein, we investigated further use of lysine as a cross-linking bridge to enhance the EDC/NHS cross-linking efficiency of AM collagen while maintaining its biocompatibility.…”

“…8 Cells with a density of 5×10 4 cells/well were seeded into 24-well tissue culture polystyrene plates and maintained in regular growth medium served as control groups. After incubation with various sterilized AM samples at 37°C for 5 days, total RNA was isolated from the cells using TRIzol reagent as described elsewhere.…”

“…7 More recently, we also studied the effect of glutaraldehyde cross-linking on the nanostructure of AM materials and found that the cross-linking chemistry-mediated alteration in fibrillar collagen is critical to determining the performance of biological tissue scaffolds for maintenance of stemness in LECs. 8 The enhanced collagen molecular stability of glutaraldehyde-treated AM is positively correlated with the amount of cross-linkers in the reaction system. 9 Nevertheless, a method involving chemical modification of AM with glutaraldehyde probably raises risk of safety, especially when the extent of cross-linking exceeds a critical value.…”

“…9 Nevertheless, a method involving chemical modification of AM with glutaraldehyde probably raises risk of safety, especially when the extent of cross-linking exceeds a critical value. 8,9 In comparison with a non-zero-length cross-linker such as glutaraldehyde, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) is a zero-length cross-linker that activates carboxylic acid groups and then forms peptide linkages with the amino groups of proteinaceous matrices. 10,11 Since the modification with carbodiimide does not introduce foreign structures into the biomaterial network, this crosslinking technique is considered more biocompatible.…”

Carbodiimide cross-linking of amniotic membranes in the presence of amino acid bridges

“…ACCEPTED MANUSCRIPT 4 nanoscale modification strategy based on carbodiimide chemistry can fabricate gelatin/chondroitin sulfate scaffolds for controlled three-dimensional corneal stromal cell culture [8]. In addition, during EDC cross-linking of gelatin molecules, the solvent composition is found to greatly affect the performance of delivery carriers for retinal sheet transplantation [9].…”