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

Abstract

To overcome the drawbacks associated with limited cross-linking efficiency of carbodiimide modified amniotic membrane, this study investigated the use of l -lysine as an additional amino acid bridge to enhance the stability of a nanofibrous tissue matrix for a limbal epithelial cell culture platform. Results of ninhydrin assays and zeta potential measurements showed that the amount of positively charged amino acid residues incorporated into the tissue collagen chains is highly correlated with the l -lysine-pretreated concentration. The cross-linked structure and hydrophilicity of amniotic membrane scaffolding materials affected by the lysine molecular bridging effects were determined. With an increase in the l -lysine-pretreated concentration from 1 to 30 mM, the cross-linking density was significantly increased and water content was markedly decreased. The variations in resistance to thermal denaturation and enzymatic degradation were in accordance with the number of cross-links per unit mass of amniotic membrane, indicating l -lysine-modulated stabilization of collagen molecules. It was also noteworthy that the carbodiimide cross-linked tissue samples prepared using a relatively high l -lysine-pretreated concentration (ie, 30 mM) appeared to have decreased light transmittance and biocompatibility, probably due to the influence of a large nanofiber size and a high charge density. The rise in stemness gene and protein expression levels was dependent on improved cross-link formation, suggesting the crucial role of amino acid bridges in constructing suitable scaffolds to preserve limbal progenitor cells. It is concluded that mild to moderate pretreatment conditions (ie, 3–10 mM l -lysine) can provide a useful strategy to assist in the development of carbodiimide cross-linked amniotic membrane as a stable stem cell niche for corneal epithelial tissue engineering.

Limbal Stem Cell Deficiency: Current Treatment Options and Emerging Therapies

“…Chemical crosslinking of HAM may enhance mechanical and thermal stability, optical transparency, and resistance to collagenase digestion [ 122 – 126 ]. The crosslinking agents that have been investigated are Glutaraldehyde, (L-Lysine-modulated) Carbodiimide, and Al 2 (SO 4 ) 3 [ 122 – 126 ].…”

“…Chemical crosslinking of HAM may enhance mechanical and thermal stability, optical transparency, and resistance to collagenase digestion [ 122 – 126 ]. The crosslinking agents that have been investigated are Glutaraldehyde, (L-Lysine-modulated) Carbodiimide, and Al 2 (SO 4 ) 3 [ 122 – 126 ]. In vitro experiments showed that Glutaraldehyde conferred a higher degree of cytotoxicity than Carbodiimide [ 123 ], whereas the addition of L-lysine to the Carbodiimide crosslinking enhanced mechanical and thermal strength, the ability to support LESCs, and resistance to enzymatic digestion, though higher concentrations could compromise transparency and biocompatibility [ 126 ].…”

Universal Corneal Epithelial-Like Cells Derived from Human Embryonic Stem Cells for Cellularization of a Corneal Scaffold

“…Efforts also have been explored to seek engineered biomaterials, for example, modified human amniotic membrane, collagen, fibrin, poly(epsilon-caprolactone), silk fibroin-chitosan, and chitosan-gelatin as corneal cell carriers. 20 – 26 However, they often do not meet the clinic standards for transparency, mechanical strength, biocompatibility, and biosafety. In contrast, decellularized cornea (DC) have emerged as a relatively safe and sustainable scaffold for cell delivery, since it not only maintains the corneal architecture, strength, and optical properties, but also retains native matrix ultrastructure.…”

Cryopreservation in Ophthalmology

“…Shortly thereafter, Mueller injected a preservation solution containing DMSO into the anterior chamber of an eyeball, placing the eyeball in a preservation solution containing glycerol. The cornea was removed before surgery for full-thickness transplantation [100,101]. In 1965, Capella [102] used DMSO as an antifreeze to improve a cryopreservation method, ensuring corneal graft activity.…”