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Jennifer L Wehmeyer, Anthony J. Johnson, David Zamora; Development of a lyophilized amniotic membrane allograft for the treatment of ophthalmic injury. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3480.
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© ARVO (1962-2015); The Authors (2016-present)
Amniotic membrane (AM) has been shown to enhance corneal wound healing and, as such, has significant clinical utility as a biological dressing for a number of ophthalmic applications. Most commercially available AM products, however, have strict refrigeration requirements thus limiting the availability of the tissue. The goal of this study was to develop a sterile, “off-the-shelf,” AM tissue scaffold utilizing a novel SCCO2 sterilization process in conjunction with lyophilization, for use in corneal wound management.
Placentas were provided by Dr. Leslie Myatt of the University of Texas Health Science Center at San Antonio, San Antonio, TX. The AM was isolated from the placenta, rinsed in saline, and placed epithelial side up on nitrocellulose paper prior to sterilizing with supercritical CO2 (Nova 2200; NovaSterilis, Lansing, NY). Frozen AM (F-AM) was stored at -80 °C while lyophilized AM (L-AM) underwent a 24h lyophilization process before being stored at room temperature. Water content and oxygen permeability (Dk) of re-hydrated native AM, F-AM and L-AM were measured following ISO standard 9913-1. In addition, the permeability of native AM, F-AM and L-AM to antibiotics (Vigamox) was assessed using a Franz diffusion cell apparatus. The concentration of Vigamox in solution was determined by spectrophotometry. The biocompatibility of F-AM and L-AM was evaluated in vitro using human corneal epithelial cells (HCECs). Cell viability and proliferation was monitored over 7 days using the MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-dipenyltetrazolium bromide) assay.
The water content of native AM, F-AM and L-AM was 92%, 92% and 88%, respectively. Using this information, the Dk was calculated and found to be greater than 99 x 10-11 ml O2 cm/sec cm2 mmHg, for all AM groups tested. F-AM and L-AM demonstrated increased permeability to Vigamox as compared to native AM. HCECs readily attached to all AM groups evaluated, however, L-AM exhibited increased cell proliferation as compared to that of F-AM.
Our results show that L-AM maintains desirable properties of an ocular bandage and demonstrated improved biocompatibility as compared to F-AM. Our method of AM sterilization combined with lyophilization renders a tissue graft which can be used as a bandage or a scaffold for ocular therapeutic applications without the need for costly and limiting storage requirements.
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