[Methods for sealing of corneal perforations]. / Metode de sigilare a perforatiilor corneene.

A variety of corneal pathology can lead to corneal ulcers and perforations. A deep corneal ulcer may need surgical treatment to allow good volume restoration and reepithelisation. Corneal perforation must be sealed and when the perforation is large, the task of repairing the defect can be underwhelming. The elegant solution is the corneal transplant, but this is not always readily available, especially in undeveloped countries. We present here two cases with different solutions to seal the perforated cornea: the first one has a large peripheral defect and it is successfully sealed with scleral patch and the second one is central with small perforation and is successfully sealed with multilayered amniotic membrane. Both cases are followed for over 12 months and demonstrate good corneal restoration (both on clinical examination and corneal topography). Sclera and amniotic membrane can be used to seal corneal defects when corneal transplant is not readily available.

Cultivation and characterization of limbal epithelial stem cells in rabbits.

PURPOSE: In the last decades, strong evidence emerged regarding the presence of stem cells located at the corneal limbus. Our objective was to find a way to isolate and cultivate rabbit corneal stem cells in vitro, into an epithelial tissue. MATERIALS AND METHODS: Two in vitro systems were developed to culture rabbit corneal stem cells: (1) limbal biopsies used as explants and cultivated on fresh denuded amniotic membrane and (2) a monolayer culture obtained by enzymatic treatment of the corneal biopsies. Genetic characterization (PCR) was performed. Specific triggers were used to induce differentiation of corneal stem cells. RESULTS: At four weeks, 16 explant samples out of 18 cultures showed good expansion, ranging from 1 cm to 2 cm. Genetic characterization showed similar expression of genetic stem markers for corneal stem cells and placental stem cells, previously characterized (stem cell factor, Oct3/4, Vimentin, Nestin and Neurofilament). Corneal stem cells showed high Rhodamine efflux and were effective progenitors for neuronal, myocardial, osteogenic and endothelial lineage. CONCLUSIONS: In one month, it was possible to grow enough epithelial tissue with preserved proliferative state to allow transplantation on the cornea.

In vitro expansion and characterization of corneal stem cells isolated from an eye with malignant melanoma.

PURPOSE: The objective of this study was the identification, characterization and in vitro replication of the human corneal stem cells, taking into consideration the difficulties in obtaining sufficient corneal material from living donors. The study explored a variety of stem cell markers, usually found in embryonic or adult mesenchymal stem cells. Culture medium and replication substrates had to be identified, with no data available on this subject in our country (there are no other reports on corneal stem cells in Romania, to our knowledge). MATERIALS AND METHODS: Corneal epithelial limbus was harvested from an enucleated eye, containing also a choroid malignant melanoma. Stem cells from the limbus were isolated and cultivated in vitro. Expression of specific stem cell markers was evaluated with immunocytochemistry. RESULTS: Corneal stem cell expansion in primary culture was slow, achieving 70-80% confluence after 28 days. Stem cells were easily isolated in standard medium, showed fibroblastoid morphology and were positive for certain stem cell specific markers in immunocytochemical staining: Oct3÷4, SOX2, Nanog, SSEA4, CD44, CD90, CD133, and CD34. They also expressed pan-cytokeratin. Donor age (72 years) and the presence of a malignant tumor close to limbal stem niche could have had an impact on the proliferation rate and the characteristics of the corneal stem cells. CONCLUSIONS: Isolated limbal cells were adult type stem cells with an epithelial orientation. The characterization of these cells with immunocytochemistry allowed us to observe surface markers that other stem cells also express.