Corneal Regeneration by Deep Anterior Lamellar Keratoplasty (DALK) Using Decellularized Corneal Matrix

Abstract

The purpose of this study is to demonstrate the feasibility of DALK using a decellularized corneal matrix obtained by HHP methodology. Porcine corneas were hydrostatically pressurized at 980 MPa at 10°C for 10 minutes to destroy the cells, followed by washing with EGM-2 medium to remove the cell debris. The HHP-treated corneas were stained with H-E to assess the efficacy of decellularization. The decellularized corneal matrix of 300 μm thickness and 6.0 mm diameter was transplanted onto a 6.0 mm diameter keratectomy wound. The time course of regeneration on the decellularized corneal matrix was evaluated by haze grading score, fluorescein staining, and immunohistochemistry. H-E staining revealed that no cell nuclei were observed in the decellularized corneal matrix. The decellularized corneal matrices were opaque immediately after transplantation, but became completely transparent after 4 months. Fluorescein staining revealed that initial migration of epithelial cells over the grafts was slow, taking 3 months to completely cover the implant. Histological sections revealed that the implanted decellularized corneal matrix was completely integrated with the receptive rabbit cornea, and keratocytes infiltrated into the decellularized corneal matrix 6 months after transplantation. No inflammatory cells such as macrophages, or neovascularization, were observed during the implantation period. The decellularized corneal matrix improved corneal transparency, and remodelled the graft after being transplanted, demonstrating that the matrix obtained by HHP was a useful graft for corneal tissue regeneration.

Fig 1. Surgical procedure of DALK using decellularized corneal matrix in a rabbit eye.

(A) Normal cornea. (B) The recipient cornea was trephinated at approximately one and one fourth in depth using the 6.0 mm diameter Hessbarg-Barron vacuum trephine. (C) Hydrodelamination was performed through the perforated corneal stromal area. (D) Following the removal of stromal tissue, Descemet’s membrane was completely exposed in the transplantation area. (E) A decellularized corneal graft sutured to the recipient bed with interrupted sutures.

Fig 2. Histological images of native porcine cornea (A, B) and decellularized corneal matrix (C, D) stained with haematoxylin and eosin (H-E) and anti-α-Gal antibody, respectively.

Scale bar: 200 μm.

Fig 3. Macroscopic images and images of the rabbit eye stained with fluorescein immediately (A, F), at 2 weeks (B, G), at 1 month (C, H), at 2 months (D, I), and at 6 months (E, J) following the transplantation of decellularized corneal matrix.

Arrows indicate the boundaries between decellularized corneal matrix and recipient cornea. Scale bar: 2 mm.

Fig 4. Changes in haze grading of decellularized corneal matrix after DALK over a 6-month follow-up period (A).

Time course of corneal epithelial wound healing of the decellularized corneal matrix after deep anterior lamellar keratoplasty. (B). The epithelial defect areas calculated from fluorescein-stained images.

Fig 5. Representative histological sections of decellularized corneal matrix at 6 months after surgery.

Sections were stained with H-E (A), antibody to macrophages/monocytes marker (CD68) (B), and antibody to vascular smooth muscle cells marker (α-SMA) (C). Scale bar: 500 μm (A-C). Higher magnification shows the edge (D, E, F) and centre (G, H, I) of the transplantation region. Histological images of decellularized corneal matrix stained with keratin and PCNA. (J-M). Scale bar: 100μm.