Simple oral mucosal epithelial transplantation in a rabbit model

Abstract

This study investigated a rabbit model of autologous simple oral mucosal epithelium transplantation (SOMET) for limbal stem cell deficiency (LSCD). LSCD was created in the SOMET group and the Control group. In the SOMET group, oral mucosa harvested from the buccal region was treated with dispase, cut into small pieces, and placed on the exposed corneal stroma without using graft sutures, amniotic membrane, and/or glue. A soft contact lens was positioned and tarsorrhaphy was performed in both groups. Postoperative corneal neovascularization and fluorescein staining scores were evaluated by slit lamp microscopy in both groups. At 2 weeks postoperatively, eyes were excised and subjected to immunohistochemical staining for CK3, CK13, CK15, and p63. In the SOMET group, transplantation of oral mucosa led to complete recovery of LSCD, as indicated by low neovascularization scores, low fluorescein staining scores, and detection of stratified K3/K13-positive cells on the stroma at 2 weeks after surgery. In contrast, corneal epithelial defects persisted in the Control group at 2 weeks. SOMET achieved re-epithelialization of the corneal surface in this rabbit LSCD model. It is a simple technique that does not require culture and could be a promising option for ocular surface reconstruction in bilateral LSCD.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Hematoxylin-eosin staining of harvested rabbit oral mucosal tissue. The surface layer was removed from rabbit oral mucosal tissue with 2 pairs of forceps, dividing the tissue into 2 layers. (A) The upper five to six layers of cells in the mucosal epithelium are shown. (B) The lower layer of oral mucosa contained basal cells with a high N/C ratio (▲) in the basal layer and near the submucosal connective tissue. (C) After treatment with 1.2 IU of Dispase II, submucosal connective tissue was separated from the mucosa. The stratified oral mucosal epithelium contained an irregular basal layer. (D) Submucosal tissue and resident mononuclear cells after treatment with 1.2 IU of Dispase II. There are no basal cells in the submucosal connective tissue.

Figure 2

Culture of oral mucosal epithelial cells after dispase treatment. Colony formation was noted around the clumps of cultured cells. Gradual proliferation around the clumps of cells formed round areas of epithelium by day 5 of culture. Scale bar = 1000 µm.

Figure 3

Postoperative slit lamp findings in the SOMET and Control groups. In the SOMET group, epithelium is expanding over the corneal surface around the grafts at 1 week after surgery and epithelialization is complete by 2 weeks. No fluorescein staining of the cornea is observed at 2 weeks (bottom left). In the Control group, neovascularization is present at the periphery of the cornea. Epithelialization of the corneal surface shows no progression and most areas are still fluorescein-positive at 1 and 2 weeks after surgery (bottom right).

Figure 4

Postoperative clinical scores. Clinical scores were compared between the SOMET group (n = 3) and the Control group (n = 4). At 1 week after surgery, the fluorescein staining score (*p = 0.01) and the total scores (**p = 0.02) were significantly lower in the SOMET group compared with the Control group. At 2 weeks after surgery, the corneal neovascularization score (**p = 0.02), fluorescein staining score (**p = 0,02), and total scores (***p = 0.03) were significantly lower in the SOMET group than in the Control group.

Figure 5

Immunohistochemical staining of oral mucosal epithelium. Submucosal connective tissue was removed by dispase treatment and the superficial tissues were used for immunohistochemistry. Apart from 3 to 5 basal layers, the oral mucosal epithelial cells are diffusely stained by K3 (top left) and K13 (top right), consistent with the phenotype of oral mucosal epithelium. Some basal cells show cytoplasmic positivity for K15 (bottom left), while there is nuclear P63 positivity in the lowest basal cell layer (bottom right).

Figure 6

Immunohistochemistry of cultured oral mucosal epithelium. Oral mucosal epithelium was cultured with B-27 and EGF to form epithelial sheets that were subjected to immunohistochemistry. On hematoxylin eosin staining, the cultured oral mucosal epithelial sheets had a smooth five-layer structure. Immunohistochemical staining showed cytoplasmic K3 positivity in all of the cell layers and K13 positivity is all layers except the basal layer. Nuclear positivity for p63 was seen in the basal layer. On the other hand, K15 was completely negative.

Figure 7

Immunohistochemistry of rabbit eyes. Frozen normal and postoperative eye specimens were used for immunohistochemical examination. The left column shows images of a normal cornea (A–E). (A) Hematoxylin-eosin (HE) staining reveals five to six layers of corneal epithelial cells in the central region. K3 is positive in the cytoplasm of cells in the surface layer of the epithelium (B), while K13 (C), p63 (D), and K15 (E) are negative in all layers. The central column shows images of a normal limbus and conjunctiva (F–K). HE staining of the normal limbus and conjunctiva is displayed in (F,G), respectively. K3 is negative in the normal conjunctiva (H), while K13 shows cytoplasmic positivity in the upper layer of the normal limbus (I). Nuclear p63 positivity is seen in the basal layer of the limbus (J), along with cytoplasmic K15 positivity in basal layer cells (K). The right column shows the central cornea of a SOMET group eye (L–Q). HE staining of the central cornea reveals 3–5 layers of flat epithelial cells (L). HE staining also shows that the transplanted oral mucosal graft consists of stratified squamous epithelium (M). In the deeper layer at the graft site, basal cells with a high N/C ratio exist close to the basal layer (M). In the expanding epithelium, the surface layer of cells demonstrates cytoplasmic K3 positivity (N) and K13 is also positive in the cytoplasm of superficial epithelial cells (O). Basal layer cells at the graft site show nuclear staining for p63 (P) and cytoplasmic staining for K15 (Q). In the Control group, HE staining revealed no epithelial proliferation and multinucleated spheres were attached to the exposed corneal stroma (R). Neither K3 positivity (S) nor K13 positivity (T) was detected in the Control group.

Figure 8

Anterior segment of an eye in the SOMET group immediately after placement of the grafts. The grafts of oral mucosal epithelium were washed with saline and arranged on the denuded corneal stroma (not around the limbus or bare sclera) with the epithelial surface upward. Grafts were placed without any glue or sutures.