Purchase this article with an account.
C.J. Connon, C. Boote, N. Koizumi, S. Kinoshita, K.M. Meek; Collagen Fibril Structure and Organization Within Amniotic Membranes Used in Ocular Surface Regeneration . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2719.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Human amniotic membrane (HAM) is increasingly used in ophthalmic surgery both as a corneal bandage and a carrier for corneal epithelial transplants. Presently there is little consideration of the variation in membrane structure both within the tissue, between donors or of the significance these differences might have on subsequent epithelial cultivation. Previously, a decrease in extracellular collagen content has been associated with poor epithelial attachment, stratification and morphology. Therefore we applied our previous experience in quantifying collagen arrangement within the corneal stroma to the extracelluar matrix (ECM) of the amniotic membrane to establish the suitability of our methods to quantify structural differences within HAM.
HAM was collected following elective caesarean section, washed in phosphate buffered saline (PBS, pH 7.2), cut into 5 cm squares and stored at –80ºC. HAM was fixed for electron microscopy in 2.5% glutaraldehyde in PBS for 1 hour at room temperature followed by 1 hour in 1% osmium tetroxide and embedded in Araldite resin. Ultrathin sections were counter–stained with 2% uranyl acetate and 2% lead citrate before examination in a Jeol 1010 transmission electron microscope. Small–angle synchrotron x–ray scattering was carried at the Synchrotron Radiation Source at Daresbury, U.K. on defrosted HAM. The patterns were analysed to produce scattering intensity plots from which we calculated the interfibrillar spacing, fibril diameters and degree of fibril order of the fibrillar collagen within the HAM.
Transmission electron micrographs confirm the structural similarity between corneal stroma and HAM ECM. Both are composed of regularly aligned collagen fibrils (to a lesser extent within HAM). Fast Fourier Transforms of representative areas of both corneal stroma and HAM (taken from electron micrographs of fibrils in cross section) showed similar spatial periodicities suggesting a significant degree of order in HAM's fibrillar arrangement. X–ray diffraction patterns from the HAM gave quantitative measurements of average fibril diameter (43nm) and interfibrillar Bragg spacing (46nm). These measurements are of the same order as those taken from the cornea at a similar hydration.
We believe that synchrotron x–ray diffraction can be successfully applied to HAM to accurately quantify inter– and intra–sample structural differences. We propose that these structural differences, once quantified, may influence the amniotic membrane’s ability to nurture epithelial growth.
This PDF is available to Subscribers Only