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Purpose Amniotic Membrane (AM) has gained increasing popularity as a useful carrier for ex-vivo expanded cells for tissue engineering, particularly in ocular surface reconstruction. However, current methods employed for denuding AM are highly variable and the consequent effects on the structural and molecular composition of the AM basement membrane (BM) are ambiguous. We compare the effects of the main denuding procedures, and propose a highly effective standardised alternative. Methods AMs preserved for transplantation were denuded using published EDTA and Dispase-based methodologies and our novel thermolysin-based procedure. Scanning and Transmission electron microscopy and immunohistochemistry, for BM components (Collgens IV, and VII, laminin 5, and inegrins α6 and -β4) were used to assess effectiveness of denuding epithelium, whilst maintaining the integrity of the BM. Results EDTA and Dispase-based denuding techniques resulted in the disaggregation and even destruction of the BM structure and molecular composition. Employing thermolysin effectively denuded epithelium whilst maintaining BM structural and molecular integrity. Conclusion Current procedures for preparing AM are variable and often ineffective, resulting in non standard membranes. Our novel thermolysin-based technique effectively denudes the AM whilst preserving an essentially intact and consistent BM. Therefore, we propose this novel thermolysin procedure may potentially improve overall generation of tissue engineered constructs using AM.

Purpose When transplanted as a graft AM is incorporated into the cornea. Our aim was to ascertain by immunohistology the fate of the incorporated membrane over time. Methods Corneal buttons from 8 eyes treated by AMT for bullous keratopahty and subsequently had penetrating keratoplasty were examined by electron microscopy and by immunohistology with markers for keratocytes (CD34), fibroblasts (vimentin) and myofibroblasts (SMactin). Time from AMT to PK was between 2 to 32 months Results Amnion tissue was covered with stratified corneal epithelium with well-defined hemidesmosomes. At places a fluid cleft was seen between amnion and underlying Bowman’s zone. Transformed keratocytes/fibroblasts could be seen migrating from the anterior stroma, through breaks in the Bowman’s zone, into connective tissue of the amniotic membrane. Immunohistology showed that the cells populating amnion stroma were CD34 negative but positive for vimentin and smooth muscle actin. In 2 samples where corneal transplants were performed more than 1 year following AMT, some cells in the amniotic stroma showed CD34+ staining Conclusion The amniotic basement membrane facilitates epithelial cell migration and adhesion. Corneal stromal keratocytes can migrate through breaks in Bownans zone, into the amniotic tissue thus integrating it with the host. Despite the presence of large fluid clefts between amniotic membrane and Bowman’s zone, the overlying epithelium remained compact. Repopulation of the amniotic stroma by corneal keratocytes allows for rebuilding of corneal stroma with an indication that over time they may revert to the resting keratocyte immunophenotype.