J Korean Med Sci.  2006 Jun;21(3):544-549. 10.3346/jkms.2006.21.3.544.

Ultrastructural Analysis of in vivo Expanded Corneal Epithelium on Amniotic Membrane

• Ha HS
• Song KY
• Kim JC

• KMID: 1778442
• DOI: http://doi.org/10.3346/jkms.2006.21.3.544

Abstract

The purpose of this study is to characterize and compare the ultrastructural changes occurring during the in vivo cultivation of corneal epithelium on amniotic membrane (AM) at several different time points. Corneal burn patients (n=7) with a corneal epithelial defect and severe limbal damage were selected. Initially, AM transplantation with limbal autograft was performed at the acute stage of corneal burn to reconstruct the damaged ocular surface. One to six (mean interval; 3.3+/-1.2) months later, the central part of AM containing an in vivo expanded corneal epithelium was excised and retransplanted in adjacent lesions. The excised epithelium with AM was examined by electron microscopy and immunohistochemical study. By electron microscopy, one and two months after expansion, cultivated epithelium on AM showed an undifferentiated epithelium and an incomplete basement membrane (BM). But, after three months, the cultivated epithelium began to differentiate into a multilayered epithelium with a continuous BM with increased hemidesmosomes. These findings were further confirmed by immunohistochemical study, that cytokeratin K3 was expressed in the cultivated corneal epithelium and newly formed BM was partially positive of collagen IV at three months. At least 3 months may be needed for the proliferation and differentiation of in vivo cultivated corneal epithelium on AM.

Keyword

Amnion; Biological Dressings; Amniotic Membrane; Epithelium, Corneal; Tissue Expansions

MeSH Terms

Stem Cells/cytology
Stem Cell Transplantation/*methods
Middle Aged
Microscopy, Electron
Male
Keratin-3/biosynthesis
Immunohistochemistry
Humans
Epithelium, Corneal/cytology/*metabolism/*pathology/*transplantation
Corneal Diseases/*therapy
Burns/*surgery/therapy
Biological Dressings
Amnion/*ultrastructure
Adult

Figure

• 

  Fig. 1 First month after in vivo cultivation on amniotic membrane (AM). (A) The expanded epithelium on AM consists of 4, 5 cell layers of corneal epithelium and looks normal in appearance, though there are partial detachments (asterisk) between basal cells and AM (H&E, ×200). (B) Cultured corneal epithelium stained poorly for cytokeratin K3 (×200) and growing BM does not stain for collagen IV (inset: ×200).

• 

  Fig. 2 Third month after in vivo cultivation on AM. (A) The cultured epithelium exhibits well differentiated five to six stratified cell layers and is attached firmly to the AM (H&E, ×200). (B) Whole suprabasal corneal epithelium is stained strongly with cytokeratin K3 (×200) and newly formed BM is stained partially with collagen IV (arrowhead in inset: ×200).

• 

  Fig. 3 EM findings of epithelial basal cells (asterisk) and hemidesmosomes (arrow) and basement membrane development. (A, B) First month (C, D) Second month (E, F) Third month. The basal cells of in vivo cultivated corneal epithelium are differentiated from rudimental round cell with large N/C ratio at the 1st month to normal cuboidal cells after 3 months of cultivation. The electron-dense structure of BM began to appear at 2 months and became distinct continuous layers with well-formed hemidesmosomes. The density of hemidesmosomes increased with time, i.e., 1.3, 2.2, and 3.4 per micrometer on average after 1, 2, 3 months of in vivo cultivation, respectively.

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