Histopathology and Ultrastructure of Human Corneas After Amniotic Membrane Transplantation

In recent years, amniotic membrane (AM) transplantation (AMT) has been used increasingly to treat various types of ocular surface pathologies.¹ Diverse techniques of AMT have been proposed for corneal diseases. One or more layers of AM can be used as a patch, graft, or sandwich (combination of ≥1 graft and a patch)^2,3 (Figure 1). Despite a growing body of literature on clinical applications of AM, little is known about the pathology of human corneas after AMT.^4,5 We offer histopathological, immunohistochemical, and ultrastructural evidence of the corneal epithelial (CE) growth patterns in relation to the AM.

We describe 3 patients with different ocular surface pathologies and histories; each of these patients required a different technique of AMT. In all cases we transplanted the AM with the epithelial side up, the AMs were fixed with interrupted 10-0 nylon sutures, and a therapeutic contact lens was applied for 1 month.

Excised corneal buttons obtained by means of penetrating keratoplasty (PKP) were fixed in paraformaldehyde and processed for light microscopy (embedded in paraffin and stained with periodic acid–Schiff, Masson trichrome, and acidic mucopolysaccharide stains). Monoclonal mouse antihuman cytokeratin (DAKO, Hamburg, Germany) immunohistochemistry distinguished the epithelial layers. For transmission electron microscopy, specimens were postfixed in 2% buffered osmium tetroxide, dehydrated in graded alcohol concentrations, and embedded in epoxy resin (Epon 812; Fluka Chemie AG [a division of Sigma-Aldrich Corp], Buchs, Switzerland). Ultrathin sections were stained with a combination of uranyl acetate and lead citrate and examined under a transmission electron microscope (Leo EM906E; Carl Zeiss, Oberkochen, Germany).

In the right eye of a 38-year-old man, a single graft sandwich AMT (3-mm graft within the CE defect plus a 12-mm patch) was applied because of a persistent corneal ulcer. Penetrating keratoplasty had been performed previously for vascularized scars due to limbal insufficiency in sporadic aniridia. Penetrating keratoplasty à chaud was necessary 60 days after AMT because of focal penetrating corneal melt along one of the graft sutures. On biomicroscopic evaluation, the superficial AM patch was absent but the AM graft remained (Figure 2A).

On light microscopy, a subepithelial layer of condensed AM stroma with translucent cuboid AM epithelial cells was seen in the excised corneal button. A multilayered CE was on top of the AM epithelium (Figure 2B).

Transmission electron microscopy showed condensed AM stroma covered by a monolayer of cuboid electron-transparent AM epithelial cells and topped by 5 to 7 layers of electron-dense CE cells (Figure 2C and D). The AM epithelial cells disclosed a discontinuous basement membrane with hemidesmosomes. Thin anchoring fibrils were occasionally observed to connect short basement membrane segments with the stromal collagen fibers (Figure 2E). Along the interface between the AM and CE, interdigitating microvillous cell processes and numerous well-differentiated desmosomes were observed between the 2 epithelial layers (Figure 2F); occasional gap junctions were also found.

In the right eye of a 70-year-old man, a double-graft AMT (1 corneal graft and 1 corneal/conjunctival graft) was applied to treat an 80% deep midperipheral ulcus serpens corneae due to Pseudomonas aeruginosa, following resorption of the hypopyon due to medical treatment. Penetrating keratoplasty à chaud was required 19 days after AMT because of corneal perforation. Biomicroscopic evaluation revealed that both of the AM grafts were present but retracted (Figure 3A).

On light microscopy, 2 layers of AM stroma were identified (Figure 3B). A layer of CE was noted between the grafts, and another plaque of CE was observed below the inner AM graft (Figure 3C). Corneal epithelial cells were present on a monolayer of translucent cuboid AM epithelial cells on top of the outer AM graft (Figure 3B).

Transmission electron microscopy disclosed a condensed AM stroma, which was covered by a monolayer of translucent cuboid AM epithelium that had a multilayered squamous electron-dense CE above it (Figure 3D). Accordingly, interdigitating plasma processes and abundant, well-differentiated desmosomes could be found between the epithelial cell layers (Figure 3E).

In the right eye of a 55-year-old woman, a single-patch (12-mm) AMT was indicated following endothelial corneal transplant rejection with persistent epithelial defects after PKP for vascularized herpetic scars. On biomicroscopic evaluation 78 days after AMT, most of the AM patch remained but was severely retracted (Figure 4A). Elective repeat PKP was performed to improve visual acuity.

Light microscopy showed that the Bowman layer was covered by 3 distinct layers of CE (Figure 4B and C). The first layer consisted of 1 or 2 layers of original or ingrowing CE cells. The second layer was adjacent to an optically empty space and consisted of a squamous epithelial multilayer attached to the AM stroma. The third layer was on top of the AM epithelium and consisted of a multilayer of squamous CE.

Transmission electron microscopy disclosed that the AM epithelium, which lacked a continuous basement membrane, appeared partly degenerative and disconnected from the underlying stromal connective tissue (Figure 4D and E). The interface between the AM and CE was characterized by intertwining cellular processes, numerous desmosomes, and occasional gap junctions (Figure 4F). The CE layer covering the posterior surface of the AM stroma showed an irregular basal cell surface that was loosely attached to the AM stromal collagen. Along the basal cell surface, small plaques or short segments of a basement membrane–like material, hemidesmosomes, and small bundles of microfibrils resembling anchoring fibrils were observed (Figure 4G and H). Corresponding structures and a discontinuous basement membrane also were observed along the basal aspects of the multilayered CE covering the Bowman layer, which showed large interruptions (Figure 4I).

The AM graft is intended to serve as a new basement membrane for CE cells; the patch is intended to promote CE growth beneath a biological shield supported by growth factors within the AM. The present study confirms that AM may be integrated in toto into the corneal surface and preserved there for months. This study focuses on the intercellular junctions between residual AM and the CE. The unexpected presence of short segments of cell-to-cell communication in addition to newly produced basement membrane and small bundles of filaments resembling abundant, well-differentiated desmosomes between CE and AM epithelial cells in all 3 cases indicates firm adherence between the 2 epithelial layers. In addition to the interdigitating microvillouslike cell processes, these adhesion structures serve as an anchor for the overgrowing CE. The second unexpected finding was the ability of the CE to grow on the stromal side of the AM. No basal membrane was demonstrated in this growth pattern when assessed with transmission electron microscopy.

These cases elucidate that the condition of the ocular surface and the technique of AMT used may determine the integration pattern of each transplant. Despite the well-established clinical benefits of AMT for persistent corneal ulcers, the exact mechanisms of achieving permanent epithelial closure in such eyes still are not completely understood and need further investigation.

Correspondence: Dr Seitz, Department of Ophthalmology, University Hospital of Saarland, Kirrberger Strasse, D-66421 Homburg/Saar, Germany (berthold.seitz@uniklinikum-saarland.de).

Financial Disclosure: None reported.

Funding/Support: This study was supported in part by Eötvös Scholarship 63/2004 from the Hungarian Ministry of Education, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the Neurocenter, University of Erlangen.