Carbodiimide cross-linked amniotic membranes for cultivation of limbal epithelial cells.
In ophthalmic tissue engineering, amniotic membrane (AM) is one of the most prevalent natural matrices used for limbal epithelial cell (LEC) cultivation and transplantation. However, the application of AM as a scaffold is limited by its low biomechanical strength and rapid biodegradation. The present study reports the development of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) cross-linked AM as an LEC carrier. The collagenous tissue materials were modified with varying cross-linker concentrations (0-0.25 mmol EDC/mg AM) and were characterized by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), ninhydrin assays, electron microscopy, light transmission measurements, mechanical and in vitro degradation tests, as well as diffusion permeability and cell culture studies. Our results showed that chemical cross-linking approaches saturation at concentrations of 0.05 mmol EDC/mg AM. The formation of cross-links (i.e., amide bonds) in the samples treated with 0.05 mmol EDC/mg AM may cause significant aggregation of tropocollagen molecules and collagen microfibrils without affecting cell morphology of biological tissues. With the optimum concentration of 0.05 mmol EDC/mg AM, chemical cross-linker could significantly enhance the mechanical and thermal stability, optical transparency, and resistance to collagenase digestion. Continuous permeation of albumin through the cross-linked AM would be helpful to cell growth over the matrix surface. In addition, the EDC cross-linked samples were able to support LEC proliferation and preserve epithelial progenitor cells in vitro and in vivo. It is concluded that the AM cross-linked with 0.05 mmol EDC/mg AM may be a potential biomaterial for regenerative medicine.
Ma
David Hui-Kang
DH
Limbal Stem Cell Laboratory, Department of Ophthalmology, Chang Gung Memorial Hospital, Taoyuan 33305, Taiwan, ROC.
Lai
Jui-Yang
JY
Cheng
Hsiao-Yun
HY
Tsai
Chen-Chi
CC
Yeh
Lung-Kun
LK
D000650:Amnion / Q000737:chemistry*; D000818:Animals; D002234:Carbodiimides / Q000737:chemistry*; D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000737:chemistry; D004847:Epithelial Cells / Q000166:cytology*; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology; D011817:Rabbits; D023822:Tissue Engineering / Q000379:methods*
D002234:Carbodiimides; D003432:Cross-Linking Reagents