In vivo confocal microscopic observation of lamellar corneal transplantation in the rabbit using xenogenic acellular corneal scaffolds as a substitute

Abstract

Background: The limiting factor to corneal transplantation is the availability of donors. Research has suggested that xenogenic acellular corneal scaffolds (XACS) may be a possible alternative to transplantation. This study aimed to investigate the viability of performing lamellar corneal transplantation (LCT) in rabbits using canine XACS.

Methods: Fresh dog corneas were decellularized by serial digestion, and LCT was performed on rabbit eyes using xenogeneic decellularized corneal matrix. Cellular and morphological changes were observed by slit-lamp, light, and scanning electron microscopy at 7, 30 and 90 days postoperatively. Immunocytochemical staining for specific markers such as keratin 3, vimentin and MUC5AC, was used to identify cells in the graft.

Results: Decellularized xenogenic corneal matrix remained transparent for about 1-month after LCT. The recipient cells were able to survive and proliferate into the grafts. Three months after transplantation, grafts had merged with host tissue, and graft epithelialization and vascularization had occurred. Corneal nerve fibers were able to grow into the graft in rabbits transplanted with XACS.

Conclusions: Xenogenic acellular corneal scaffolds can maintain the transparency of corneal grafts about 1-month and permit growth of cells and nerve fibers, and is, therefore, a potential substitute or carrier for a replacement cornea.

Figure 1

(a) Xenogenic acellular corneal scaffolds (XACS) was ivory white after the decellularized procedure; (b) Plastic cup as the control; (c) XACS was turned to be transparent after the dehydration, the under character could be seen clearly; (d) XACS became swellen after 1 h rehydration; (e-f) H and E staining of XACS (original magnification, ×10), no blue-stain cell nucleus or cell debris was found in the matrix for H and E staining by LM. The collagens were lined up in a loose formation but similar to the normal cornea matrix fiber; (e) Surface is the original corneal epithelium layer. (f) Surface is the original corneal endothelium layner; (g-h) Masson staining of XACS (original magnification, ×20).

Figure 2

The results of scanning electron microscopic examination showed that the collagen fiber diameter similar to normal corneal fiber and inter-connected to network, formed collagen bundle regular and parallel to the corneal surface.

Figure 3

Investigate the biocompatibility of xenogenic acellular corneal scaffolds (XACS) for rabbit cornea. The XACS was inserted into a rabbit corneal stromal pocket and observed at 1 week, 1 month and 3 months of postsurgical period. The cornea showed a mild haze at 1 week, optically clear slit-lamp images at 1 month and 3 months. The fresh dog cornea stroma is presented in the left column as a control, which is similar to XACS group at 1-week. Haze became severe in fixed dose combination group at 1 month and neovascularization appeared at 3 months.

Figure 4

Clinical observation: investigate xenogenic acellular corneal scaffolds (XACS) as a lamellar cornea substitute in rabbit. Corneal grafts following the lamellar corneal transplantation using XACS were observed by slit-lamp microscopy with or without fluorescein staining at 1 week (a-c), 1 month (e-g) and 3 months (i-k) after operation, respectively. Morphology of the rabbit cornea in different time points after the transplantation (Hematoxylin and eosin staining, ×20) (d, h, l): (d) A thin layer of corneal epithelium cells were noted. Few spindle-like cells in matrix were seen; (h) 1 month after operation. Cobble stone-like cells were located at the surface of the cornea; (l) 3 months after the operation, new vessels were noted in the corneal matrix in graft.

Figure 5

Immunochemistry study of xenogenic acellular corneal scaffolds group at different postoperative period: Keratin 3 positive staining cells were noted in the graft surface and vimentin positive cells were observed in the graft stroma. All epithelium cells were observed MUC5AC negative (original magnification, ×10).

Figure 6

In vivo Heidelberg Retina Tomograph-II confocal microscopy in normal rabbit cornea (a-d): (a) Subepithelium nerve fiber; (b) Langhan's cell with high reflection; (c) Cornea nerve fiber in the corneal stroma; (d) The epithelium layer and anterior stroma layer. One week postoperatively in xenogenic acellular corneal scaffolds (XACS) group (e-h): (e) No cells were detected in the anterior stroma, which was in a loose network formation; (f) Few long spindle like cells were in the posterior of the stroma of the graft; (g) There was no space between the interface of the XACS graft and the recipient rabbit cornea. Some high reflection dots were detected in the graft; (h) The endothelium layer was similar to the normal rabbit cornea. One month postoperatively in XACS group (i-l): (i) Corneal epithelial cells on the graft; (j) Activated langhan cells in the subepithelial layer without any corneal nerve detected; (k) High reflex cells with small nuclei in the anterior part of the graft, especially around the suture area; (l) Regenerated corneal nerve fiber grew into the graft in anterior stroma. Cells were less than the normal cornea. Three months postoperatively in XACS group (m-p): (m) Spindle-like cells were detected in the posterior part of the graft; (n) Cells in the anterior part of the graft in a parallel formation; (o) Tortuous corneal nerve in the graft matrix; (p) Tortuous corneal nerve.