Preliminary studies of constructing a tissue-engineered lamellar corneal graft by culturing mesenchymal stem cells onto decellularized corneal matrix

Abstract

Aim: To construct a competent corneal lamellar substitute in order to alleviate the shortage of human corneal donor.

Methods: Rabbit mesenchymal stem cells (MSCs) were isolated from bone marrow and identified by flow cytometric, osteogenic and adipogenic induction. Xenogenic decellularized corneal matrix (XDCM) was generated from dog corneas. MSCs were seeded and cultured on XDCM to construct the tissue-engineered cornea. Post-transplantation biocompatibility of engineered corneal graft were tested by animal experiment. Rabbits were divided into two groups then underwent lamellar keratoplasty (LK) with different corneal grafts: 1) XDCM group (n=5): XDCM; 2) XDCM-MSCs groups (n=4): tissue-engineered cornea made up with XDCM and MSCs. The ocular surface recovery procedure was observed while corneal transparency, neovascularization and epithelium defection were measured and compared. In vivo on focal exam was performed 3mo postoperatively.

Results: Rabbit MSCs were isolated and identified. Flow cytometry demonstrated isolated cells were CD90 positive and CD34, CD45 negative. Osteogenic and adipogenic induction verified their multipotent abilities. MSC-XDCM grafts were constructed and observed. In vivo transplantation showed the neovascularization in XDCM-MSC group was much less than that in XDCM group postoperatively. Post-transplant 3-month confocal test showed less nerve regeneration and bigger cell-absent area in XDCM-MSC group.

Conclusion: This study present a novel corneal tissue-engineered graft that could reduce post-operatively neovascularization and remain transparency, meanwhile shows that co-transplantation of MSCs may help increase corneal transplantation successful rate and enlarge the source range of corneal substitute to overcome cornea donor shortage.

Keywords: acellular corneal matrix; mesenchymal stem cells; neovascularization; tissue-engineered cornea; xenogenic decellularized corneal matrix.

Figure 1. Isolation and identification of rabbit MSCs

A: MSC morphology 7d after seeding in petri dish observed by inverted microscope (×10); B: Flow cytometry result showed positive express of CD90; C: Flow cytometry detected no expression of CD34 and CD45; D: Cell morphology after 21-day osteogenic induction under inverted microscope (×20); E: Von Kossa staining image (×40) showed calcium nodules (black); F: Lipid droplets appeared in the cytoplasm 4d after adipogenic induction (×20); G: Oil-red O staining showed large numbers of lipid droplets (red) in cytoplasma at 14th day of induction. Scale bar: 25 µm.

Figure 2. XDCM-MSCs morphology under inverted microscope and SEM

A-B: Morphology of MSCs at 1^st and 7^th day after seeding onto XDCM observed by inverted microscope (×10). Scale bar: 100 µm. C-F: Morphology of MSCs 1, 3, 7, and 14d after seeding onto XDCM observed by SEM (×500). C1-F1: Morphology details with greater magnification respectively.

Figure 3. Post-operative observation by slit lamp

A-H: Images with diffuse light source; A1-H1: Images with slit light source; A2-H2: Images with fluorescein staining. Fluorescent green area represented corneal epithelial defect.

Figure 4. Corneal surface evaluation between XDCM and XDCM-MSC groups

A: Corneal neovascularization scores; B: Corneal opacity scores; C: Percentage of corneal epithelial defect (%). ^aP<0.05; ^bP<0.01.

Figure 5. In vivo confocal microscopy images anterior, inside and posterior of grafts

A-D: XDCM-MSC group. A: Epithelium; B: Superficial part inside transplanted grafts; C: Deep part inside transplanted grafts; D: Rabbit corneal stroma; E-H: XDCM group. E: Epithelium; F: Superficial part inside transplanted grafts; G: Deep part inside transplanted grafts; H: Rabbit corneal stroma. Short arrows: Nerves inside grafts. Long arrows: Neovessels inside grafts.