Purchase this article with an account.
Yu Jung Shin, Jemin Jeremy Chae, Jennifer Elisseeff; Optimization of Decellularized Bovine Cornea for Corneal Reconstruction. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1394.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
In previous studies, we have shown that the reconstructed decellularized porcine cornea is capable of corneal tissue regeneration. However, the size limitation of porcine cornea prevents a complete coverage of human cornea. Additionally, porcine tissues are prohibited in certain cultures. In order to address these issues, we have optimized a decellularization process specific to the bovine cornea. The purpose of this study is to optimize a non-immunogenic tissue based material which can be reconstructed into highly transparent corneal substitute.
Native bovine corneal tissues were dissected from fresh bovine eyes and washed in anti-bacterial and antifungal solutions. Afterwards, decellularization procedure was performed with 1% sodium dodecyl sulfate treatment (24hrs) followed by 1% Triton-X treatment (24hrs, 48hrs, 72hrs). Each set of cornea (N=5) was immersed in same amount of solutions and incubated under the same stirring speed (150rpm) to ensure identical diffusion and shear stress. Efficiency of decellularization method was assessed by Hochester dye (1ug/ml in TNE buffer) DNA quantification with fluorometer. Qualitative analysis of decellularization was conducted using H&E staining. Statistical analysis was performed by two tailed student’s t test and the data was presented with mean ± standard deviation.
DNA content of decellularized cornea was significantly reduced at all 48hr, 72hr, and 96hr time points (p=0.000) compared to that of native cornea (2.969±0.086 µg/mg). The lowest mean DNA content was observed after 72 hours of SDS-Triton-x treatment (0.296±0.026 µg/mg) compared to mean DNA contents after 48 hours (0.391±0.027 µg/mg) and 96 hours (0.394±0.106 µg/mg) of treatment. Student’s t test showed a significant decrease in DNA content between 48hr and 72hr time points (p=0.001). However, no significant difference was measured between 72hr and 96hr time points (p=0.142). The efficiency of 72hr SDS-Triton-x treatment was further supported by H&E staining. The 72hr sample did not show any hematoxylin staining, suggesting effective removal of cells.
Efficiency of decellularization is crucial in engineering a non-immunogenic material. Our results show that we have successfully optimized the decellularization condition for bovine corneal tissues. Through further vitrification and molding process, we aim to optimize a reconstructed decellularized bovine cornea with enhanced functions.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
This PDF is available to Subscribers Only