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Jemin Jeremy Chae, Yoo Jung Shin, Gyeong Woo Cheon, Okhee Jeon, Jennifer Elisseeff; Reconstructed decellularized tissue for corneal regeneration. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5309. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Although the decellularized cornea has potential to be used a corneal substitute, decellularization procedures often reduce the function of the cornea by decreasing its transparency and disrupting the concave shape. We have developed methods of reconstructing decellularized cornea that improves corneal transparency and maintains its natural shape.
Fresh porcine corneas were harvested and treated with by chemical (sodium dodecyl sulfate and Triton-X) and enzymatic (fetal bovine serum) methods. The reconstruction procedures, vitrification and riboflavin crosslinking, were carried out within a pair of curved molds that have similar curvature to the rabbit cornea.The reconstructed corneas (n=5) and native porcine corneas (n=5) were evaluated using the light transmittance test with the same thickness, Optical Coherence Tomography (OCT), pathological examination with H&E staining and transmission microscopy (TEM). The curvature of the molds and the reconstructed corneas were calculated with 2D OCT images. To quantify density and the diameter of collagen fibrils in the microstructure, ten TEM images were randomly chosen in each cornea (n=50), and areas were defined using a 1µmX1µm square. The collagen fibrils were evaluated using ImageJ software. Data is presented as mean±standard deviation (SD).The two-tailed Student’s t-test or Mann-Whitney test were used for statistical analysis depending on the size of the sample set.
After the reconstruction procedures, light transmittance in the visible wave length was significantly increased. The curvature of the reconstructed cornea (7.613±0.136mm) is identical with that of the mold (7.615±0.138mm). In the assessment of macro- and micro-morphology, the reconstructed cornea showed similar structure with that of the native cornea. The reconstruction procedures significantly increased the density (107.6±23.0 to 307.0±18.0/µm2, p=0.001) of collagen fibrils and significantly decreased diameter of collagen (40.7±2.5 to 31.2±3.4nm, p=0.001) of the decellularized cornea, which may correspond to the improvement in transparency.
We successfully reconstructed the decellularized porcine cornea with vitrification and riboflavin crosslinking using a pair of concave molds. This method produces a decellularized cornea with improved transparency and curvature for applications in corneal regeneration which may serve as substitutes for conventional human allograft transplantation.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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