Stabilization of collagen nanofibers with L-lysine improves the ability of carbodiimide cross-linked amniotic membranes to preserve limbal epithelial progenitor cells.
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.
Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan, Republic of China ; Biomedical Engineering Research Center, Chang Gung University, Taoyuan, Taiwan, Republic of China ; Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan, Republic of China.
Jui-Yang
JY
Lai
Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan, Republic of China.
Pei-Ran
PR
Wang
Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan, Republic of China.
Li-Jyuan
LJ
Luo
Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan, Republic of China.
Si-Tan
ST
Chen
D000650:Amnion / Q000737:chemistry*; D000818:Animals; D002234:Carbodiimides / Q000737:chemistry; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry* / Q000494:pharmacology; D003432:Cross-Linking Reagents; D004847:Epithelial Cells / Q000166:cytology*; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology; D008239:Lysine / Q000737:chemistry* / Q000494:pharmacology; D057139:Nanofibers / Q000737:chemistry*; D011817:Rabbits; D013234:Stem Cells / Q000166:cytology*; D023822:Tissue Engineering; D054457:Tissue Scaffolds / Q000737:chemistry*
D002234:Carbodiimides; D003432:Cross-Linking Reagents; D003094:Collagen; D008239:Lysine
amniotic membrane; collagen nanofiber stabilization; corneal epithelial tissue engineering; l-lysine-pretreated concentration; limbal progenitor cell preservation