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.
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ABSTRACT: Differential scanning calorimetry (DSC) was used to study the thermal stability of native and synthetically cross-linked rat-tail tendon at different levels of hydration, and the results compared with native rat-tail tendon. Three cross-linking agents of different length between functional groups were used: malondialdehyde (MDA), glutaraldehyde and hexamethylene diisocyanate (HMDC). Each yielded the same linear relation between the reciprocal of the denaturation temperature in Kelvin, T(max), and the water volume fraction, epsilon (1/T(max)=0.000731epsilon+0.002451) up to a critical hydration level, the volume fraction of water in the fully hydrated fibre. Thereafter, water was in excess, T(max) was constant and the fibre remained unchanged, no matter how much excess water was added. This T(max) value and the corresponding intrafibrillar volume fraction of water were as follows: 84.1 degrees C and 0.48 for glutaraldehyde treated fibres, 74.1 degrees C and 0.59 for HMDC treated fibres, 69.3 degrees C and 0.64 for MDA treated fibres, and 65.1 degrees C and 0.69 for untreated native fibres. Borohydride reduction of the native enzymic aldimines did not increase the denaturation temperature of the fibres. As all samples yielded the same temperature at the same hydration, the temperature could not be affected by the nature of the cross-link other than through its effect on hydration. Cross-linking therefore caused dehydration of the fibres by drawing the collagen molecules closer together and it was the reduced hydration that caused the increased temperature stability. The cross-linking studied here only reduced the quantity of water between the molecules and did not affect the water in intimate contact with, or bound to, the molecule itself. The enthalpy of denaturation was therefore unaffected by cross-linking. Thus, the "polymer-in-a-box" mechanism of stabilization, previously proposed to explain the effect of dehydration on the thermal properties of native tendon, explained the new data also. In this mechanism, the configurational entropy of the unfolding molecule is reduced by its confinement in the fibre lattice, which shrinks on cross-linking.Journal of Molecular Biology 03/2005; 346(2):551-6. · 3.96 Impact Factor
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ABSTRACT: The lamellar architecture found in many natural fibrous tissues has a significant bearing on their specific functions. However, current engineered tissues have simultaneously no realistic structures and no adequate functions. This study demonstrates a two-step process for obtaining structurally mimicking laminates in natural fibrous tissues with good optical and mechanical characters from purified-clinically- safe collagen molecules. Stacked lamella structures can be created by repeating flow casting, with the controlling parallel/orthogonal directionalities of each thin single-layer (2e5 mm in thickness). The transparency of laminates is successfully improved by a unique multi-cyclic vitrification with chemical cross-linking. The directionalities of optical and mechanical functions in laminates are strongly related with the preferential collagen alignments in the laminates. The tensile strength of laminates is extremely higher than any other engineered materials as well as native cornea, which exhibit an orthogonal laminated collagen structure and a good optical transmission.Biomaterials 02/2011; 32:3358-3366. · 8.31 Impact Factor
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ABSTRACT: Our aim was to construct a mechanically stable and optically transparent collagen gel from an acidified atelocollagen solution which is suitable for use as a corneal stromal equivalent. Light transmission and mechanical testing were conducted on variously crosslinked constructs at different pH levels. Ultrastructural analysis was performed to assess directionality of the molecular arrangement produced by flow manipulation, as well as the amount of collagen fibrillogenesis which resulted from different pH and/or crosslinking conditions. Clinical and histological integration of the gels with living tissue was examined following implantation into rabbit corneal intra-stromal pockets. Transmission electron microscopy revealed the importance of the fine control of pH levels during gel formation and indicated that the stage at which collagen fibrillogenesis is halted within the constructs was critically dependent on the pH of the collagen solution. Transparency testing disclosed that high levels of collagen fibrillogenesis, as well as high levels of crosslinker concentration, detrimentally affected the transparency of the construct. As a result, a dual titration was required to achieve good light transmission through the gels. It was also evident that the amount of crosslinking required to gelate the collagen solution was reduced as the level of fibrillogenesis progressed. Thus, it was necessary to establish a balance between the solution pH and crosslinker concentration. Implantation of the collagen constructs into partial depth intra-stromal pockets in rabbits was followed up for 6 months, and demonstrated favourable biocompatibility. This showed that gels which had lower levels of both fibrillogenesis and crosslinking were degraded more readily by the host tissue. The collagen gels described here are mass-production friendly, and have promise as potential functional stromal equivalents for use in stromal grafting, or in constructing full thickness artificial corneas.Biomaterials 12/2010; 31(34):8996-9005. · 8.31 Impact Factor