Purchase this article with an account.
C.S. Kamma, S. Hayes, C. Boote, J. Albon, K.M. Meek, M.E. Boulton; An in vitro Study of Corneal Wound Healing: Cellular and Collagen Ultrastructural Changes . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2726.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To investigate the cellular and ultrastructural changes in corneal stroma in trephine wounded bovine corneas.
Nine bovine corneas were centrally wounded using a 5mm trephine, which created a wound penetrating halfway through the stroma. Nine unwounded corneas acted as controls. The corneas were excised and organ cultured for up to 14 days. For X–ray diffraction experiments the corneas were removed from the organ culture at 0, 7 and 14 days and snap–frozen in liquid N2. Small and wide angle X–ray scattering was performed at stations 2.1 and 14.1, respectively, at the UK Synchrotron Radiation Source. X–ray scattering patterns were analysed to quantify the collagen fibril diameters, centre–to–centre spacing and orientation. For histology and immunohistochemistry corneas were removed from the organ culture at 0, 1, 3 and 7 days, fixed in 10% formalin and embedded in paraffin wax. Sections were stained with Haematoxylin and Eosin to assess overall morphology. Immunolocalisation of vimentin and α–smooth muscle actin was used to differentiate between keratocytes and myofibroblasts respectively and cytokeratin to monitor epithelial cell differentiation wound repair.
14 days after injury there was a significant increase of the fibril diameter and the interfibrillar spacing (p<0.05–one–way ANOVA). These variables remained unchanged for the control (i.e. non–injured) corneas during the organ culture time–span (p>0.2). A greater amount of collagen was accumulated at the wound area. This area was shown to have an increase in the proportion of the aligned collagen. Collagen fibrils were orientated in a circular pattern around the wound area. Epithelial migration was observed 24 hours after injury. There was an obvious difference in the keratocytes’ shape adjacent to the wound 24 hours post–wounding. Myofibroblasts were observed at the 7 days time–point.
The in vitro organ culture model used effectively preserves control of corneal hydration provided by the corneal endothelium and following wounding shows changes in the proportion of aligned collagen fibrils and their orientation to the wound. These changes correlated with re–epithelialisation and changes in keratocyte morphology. Ultrastructural changes became obvious 14 days post–wounding suggesting that wound healing is accompanied by a significant swelling of the stromal matrix.(*Authors contribute equally in research)
This PDF is available to Subscribers Only