Optical mechanical refinement of human amniotic membrane by dehydration and cross-linking
ABSTRACT The aim of this study was to develop a method for refining the optical and mechanical properties of human amniotic membrane (AM) to provide ophthalmic transparent implants for use during severe donor cornea shortages. AM was allowed to gradually dehydrate at 4-8 °C with and without chemical cross-linking. To improve the transparency of AM, a simple dehydration process using a refrigerator at 4-8 °C overnight was examined. For further improvements, dehydrated AM was then cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxy-succimide before rehydration. Light transmittance and tensile strength of individual specimens were evaluated. Light transmittance of AM improved from 50.9-77.7% at 550 nm by this simple low temperature dehydration process. Its high light transmittance was partially maintained at 70.1%, even after rehydration with normal saline. Interestingly, chemically cross-linked AM showed a significantly greater light transmittance of 81.5% under wet conditions. In addition, tensile strength was significantly increased after cross-linking from 2.32 N/mm(2) (native tissue) to 11.78 N/mm(2) (cross-linked specimens). Light transmittance and tensile strength were successfully improved by these approaches, including low temperature dehydration with and without chemical cross-linking. The use of refined AM could be feasible for use in current and future ophthalmic treatments. Copyright © 2012 John Wiley & Sons, Ltd.
Full-textDOI: · Available from: Yuji Tanaka, May 06, 2015
- SourceAvailable from: Alex J Shortt[Show abstract] [Hide abstract]
ABSTRACT: Limbal epithelial stem cell (LESC) deficiency can cause blindness. Transplantation of cultured human limbal epithelial cells (hLE) on human amniotic membrane (HAM) can restore vision but clinical graft manufacture can be unreliable. We have developed a reliable and robust tissue equivalent (TE) alternative to HAM, Real Architecture for 3D Tissue (RAFT). Here, we aimed to optimise the optical and mechanical properties of RAFT TE for treatment of LESC deficiency in clinical application. The RAFT TE protocol is tunable; varying collagen concentration and volume produces differing RAFT TEs. These were compared with HAM samples taken from locations proximal and distal to the placental disc. Outcomes assessed were transparency, thickness, light transmission, tensile strength, ease of handling, degradation rates and suitability as substrate for hLE culture. Proximal HAM samples were thicker and stronger with poorer optical properties than distal HAM samples. RAFT TEs produced using higher amounts of collagen were thicker and stronger with poorer optical properties than those produced using lower amounts of collagen. The 'optimal' RAFT TE was thin, transparent but still handleable and was produced using 0.6ml of 3mg/ml collagen. Degradation rates of the 'optimal' RAFT TE and HAM were similar. hLE achieved confluency on 'optimal' RAFT TEs at comparable rates to HAM and cells expressed high levels of putative stem cell marker p63α. These findings support the use of RAFT TE for hLE transplantation towards treatment of LESC deficiency. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.Acta biomaterialia 06/2015; 91. DOI:10.1016/j.actbio.2015.06.007 · 5.68 Impact Factor