The variation in transparency of amniotic membrane used in ocular surface regeneration

Background/aims: Scant consideration has been given to the variation in structure of the human amniotic membrane (AM) at source or to the significance such differences might have on its clinical transparency. Therefore, we applied our experience of quantifying corneal transparency to AM. Methods: Following elective caesarean, AM from areas of the fetal sac distal and proximal (ie, adjacent) to the placenta was compared with freeze-dried AM. The transmission of light through the AM samples was quantified spectrophotometrically; also, tissue thickness was measured by light microscopy and refractive index by refractometry. Results: Freeze-dried and freeze-thawed AM samples distal and proximal to the placenta differed significantly in thickness, percentage transmission of visible light and refractive index. The thinnest tissue (freeze-dried AM) had the highest transmission spectra. The thickest tissue (freeze-thawed AM proximal to the placenta) had the highest refractive index. Using the direct summation of fields method to predict transparency from an equivalent thickness of corneal tissue, AM was found to be up to 85% as transparent as human cornea. Conclusion: When preparing AM for ocular surface reconstruction within the visual field, consideration should be given to its original location from within the fetal sac and its method of preservation, as either can influence corneal transparency

Comparative cytotoxicity of Acanthamoeba castellanii-derived conditioned medium on human corneal epithelial and stromal cells

Soluble factors in the secretome of Acanthamoeba castellanii play crucial roles in the pathogenesis of Acanthamoeba keratitis (AK). Investigating the pathological effects of A. castellanii-derived conditioned medium (ACCM) on ocular cells can provide insights into the damage inflicted during AK. This study examined ACCM-induced cytotoxicity in primary human corneal stromal cells (CSCs) and a human SV40 immortalized corneal epithelial cell line (ihCECs) at varying ACCM concentrations (25 %, 50 %, 75 %, and 100 %). MTT, AlamarBlue, Sulforhodamine B, lactate dehydrogenase, and Caspase-3/7 activation assays were used to assess the impact of ACCM on the cell viability, proliferation and apoptosis. Additionally, fluorescent staining was used to reveal actin cytoskeleton changes. ACCM exposure significantly decreased cell viability, increased apoptosis, and disrupted the actin cytoskeleton, particularly at higher concentrations and longer exposures. Proteases were found to mediate these cytopathogenic effects, highlighting the need for characterization of A. castellanii proteases as key virulence factors in AK pathogenesis

The biomechanics of amnion rupture: an X-ray diffraction study

Pre-term birth is the leading cause of perinatal and neonatal mortality, 40% of which are attributed to the pre-term premature rupture of amnion. Rupture of amnion is thought to be associated with a corresponding decrease in the extracellular collagen content and/or increase in collagenase activity. However, there is very little information concerning the detailed organisation of fibrillar collagen in amnion and how this might influence rupture. Here we identify a loss of lattice like arrangement in collagen organisation from areas near to the rupture site, and present a 9% increase in fibril spacing and a 50% decrease in fibrillar organisation using quantitative measurements gained by transmission electron microscopy and the novel application of synchrotron X-ray diffraction. These data provide an accurate insight into the biomechanical process of amnion rupture and highlight X-ray diffraction as a new and powerful tool in our understanding of this process