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Jennifer McDaniel, Anthony Johnson, David O. Zamora; AMNIOTIC MEMBRANE ALLOGRAFTS MAINTAIN KEY BIOLOGICAL PROPERTIES POST SCCO2 AND LYOPHILIZATION PROCESSING. Invest. Ophthalmol. Vis. Sci. 2017;58(8):133.
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© ARVO (1962-2015); The Authors (2016-present)
Amniotic membrane (AM) has been widely used in the clinic to treat corneal wounds due to its ability to reduce inflammation and scarring and enhance regeneration in injured tissues. While AM has many wound healing benefits, processing and storage hurdles remain with current commercially available products. Therefore, the goal of this study was to develop a sterile, shelf-stable AM tissue graft utilizing a novel supercritical carbon dioxide (SCCO2) sterilization process in conjunction with lyophilization for use in corneal wound management.
Placentas were provided by the Cooperative Human Tissue Network. AM was isolated, rinsed in saline, placed epithelial side up on nitrocellulose paper and either stored at -80°C (N-AM), SCCO2 sterilized and placed in -80°C (S-AM) or SCCO2 sterilized followed by lyophilization (L-AM) and stored at variable temperatures for 1 wk, 1, 3 and 6 months. Total protein from N-, S-, and L-AM stored samples was isolated at different time points, quantified and analyzed for growth factor expression using a human growth factor (GF) antibody array. Western blot analysis of AM samples was performed to determine the presence of PTX3 over time. Histological staining (H&E, Masson’s trichrome) was done on paraffin embedded N-, S-, and L-AM samples to assess if changes in structural morphology occurred.
The profile of human GFs detected in N-, S-, and L-AM protein samples demonstrated similar patterns across all conditions analyzed. Furthermore, six months post-processing PTX3, an important mediator of inflammation and regeneration, was still detected in processed AM samples. Histological analysis of N-, S-, and L-AM revealed an organized, single-epithelial layer with an underlying, thicker basement membrane, albeit increasingly more condensed in the S-AM and L-AM samples.
SCCO2 is an emerging tissue sterilization technology that provides a novel method for AM graft preparation and sterilization while preserving the inherent biochemical and structural properties of the tissue. When combined with a lyophilization step, L-AM maintains key biological molecules such as the presence of PTX3 and other GFs found in N-AM without the need for costly and logistically challenging storage requirements. The generation of a sterile, shelf-stable AM graft for use as an ocular graft would be of immense value to both the civilian and military populations.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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