Abstract
Purpose
To develop a new decellularization technique of porcine cornea using freezing-thawing-centrifugation (FTC) and to examine the characteristics of acellular porcine cornea (APC) for xenograft material.
Methods
Two-hundred micrometer thickness porcine corneas were decellularized with DNase/RNase, followed by 3 freezing-thawing-centrifugations (FTC, group 1), lyophilized FTC-APC (group 2), and chemical enzyme treated APC (CE-APC, group 3). Histologic evaluation to examine cells and collagen matrix, comparison of transparency, and cultivation to determine the viability of stromal cells was performed in fresh porcine cornea and 3 experimental groups.
Results
Decellularization occurred successfully in all experimental groups. Decellularization was confirmed by H&E staining and cultivation. Transparency of group 1 was similar to the normal porcine cornea but transparency of group 2 and group 3 was decreased. Collagen fibers of CE-APC (group 3) were not as well arrayed as FTC-APC (group 2).
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![]() | Figure 1.Optical transparency of the porcine cornea. Freezing-thawing-centrifugation-decellurized acellular porcine cornea (FTC-APC) was transparent (B), similar with fresh porcine cornea (A). But Transparency of chemical enzyme-decellularized acellular porcine cornea (CE-APC) decreased about one-third compared to fresh porcine cornea (D). Lyophilized FTC-APC was visually opaque (C). |
![]() | Figure 2.H&E staining showed that no cells were present in Freezing-thawing-centrifugation-decellurized acellular porcine cornea (FTC-APC) (B), lyophilized FTC-APC (C), and chemical enzyme-decellularized acellular porcine cornea (CE-APC) (D), while many keratocytes were observed in fresh porcine cornea (A). The thickness of CE-APC was thinner than fresh porcine cornea and FTC-APC (A, B, C, D: 200×). |
![]() | Figure 3.M-T staining showed that collagen fibers were not well arrayed after decellularizing procedure (B: Freezing-thawing-centrifugation-decellurized acellular porcine cornea (FTC-APC), C: lyophilized FTC-APC, D: chemical enzyme-decellular-ized acellular porcine cornea (CE-APC)) than fresh cornea (A). Collagen fibers of CE-APC were most irregular (A, B, C, D: 200×). |
![]() | Figure 4.Keratocytes grew from fresh porcine cornea rapidly (A: Day 1, B: Day 5, C: Day 9). But keratocyte was not grown from Freezing-thawing-centrifugation-decellurized acellular porcine cornea (FTC-APC) (D), lyophilized FTC-APC (E), and chemical en-zyme-decellularized acellular porcine cornea (F) until culture day 9. |