Eccentric lamellar keratolimbal grafts harvested with a manually guided microkeratome

Background: To perform lamellar keratolimbal allograft transplantation in a one- step procedure with a single graft, we investigated the feasibility of harvesting eccentric lamellar keratolimbal grafts from conventionally processed corneoscleral buttons using a manually guided microkeratome in conjunction with an artificial anterior chamber system. Methods: We used the Moria LSK- One microkeratome and the automated lamellar therapeutic keratoplasty ( ALTK) system ( Antony, France). Ten human donor eyes were used to obtain single- piece lamellar keratolimbal grafts. Specimens were processed for light and electron microscopy. Results: Eccentric keratolimbal grafts could be obtained from all human donor buttons. Grafts include a crescent- shaped limbal and a large corneal portion. No visible damage to the limbal region was discernible. Conclusion: Our data show that the LSK- One microkeratome in conjunction with the ALTK system allows harvesting eccentric keratolimbal grafts from donor corneoscleral buttons. Copyright (c) 2007 S. Karger AG, Basel

Changes in Corneal Basal Epithelial Phenotypes in an Altered Basement Membrane

To examine the corneal epithelial phenotype in an altered basement membrane.Corneas from 9 patients with symptoms of continuous unstable corneal curvature (CUCC) were harvested by penetrating keratoplasty and subjected to histology examination and immunohistochemical staining with transactivating and N-terminally truncated pP63 transcript (ΔNp63), cytokeratin 3 (Krt3), ATP-binding cassette sub-family G member 2 (ABCG2), connexin 43 (CX43), p38 mitogen-activated protein kinases (p38MAPK), activating protein 2 (TFAP2), and extracellular signal-regulated kinase (Erk1/2) monoclonal antibodies. Positive immunostaining with ABCG2, p38MAPK, and TFAP2 monoclonal antibodies was observed in the basal epithelial cells of CUCC patients, and CX43 and ΔNp63 were detected in the full-thickness epithelial cells of CUCC patients.Our results indicate that alteration of the corneal basement membrane induces a de-differentiation-like phenotype in corneal basal epithelial cells

Gamma-irradiated human amniotic membrane decellularised with sodium dodecyl sulfate is a more efficient substrate for the ex vivo expansion of limbal stem cells

yesThe gold standard substrate for the ex vivo expansion of human limbal stem cells (LSCs) remains the human amniotic membrane (HAM) but this is not a defined substrate and is subject to biological variabil-ity and the potential to transmit disease. To better define HAM and mitigate the risk of disease transmis-sion, we sought to determine if decellularisation and/or c-irradiation have an adverse effect on culture growth and LSC phenotype. Ex vivo limbal explant cultures were set up on fresh HAM, HAM decellularised with 0.5 M NaOH, and 0.5% (w/v) sodium dodecyl sulfate (SDS) with or without c-irradiation. Explant growth rate was measured and LSC phenotype was characterised by histology, immunostaining and qRT-PCR (ABCG2, DNp63, Ki67, CK12, and CK13). Ƴ-irradiation marginally stiffened HAM, as measured by Brillouin spectromicroscopy. HAM stiffness and c-irradiation did not significantly affect the LSC phe-notype, however LSCs expanded significantly faster on Ƴ-irradiated SDS decellularised HAM (p < 0.05) which was also corroborated by the highest expression of Ki67 and putative LSC marker, ABCG2. Colony forming efficiency assays showed a greater yield and proportion of holoclones in cells cultured on Ƴ-irradiated SDS decellularised HAM. Together our data indicate that SDS decellularised HAM may be a more efficacious substrate for the expansion of LSCs and the use of a c-irradiated HAM allows the user to start the manufacturing process with a sterile substrate, potentially making it safer