Abstract
Abstract: :
Purpose:The successful integration of keratoprostheses (KPros) within the cornea depends on the skirt host keratocyte adhesion to anchor the implant and prevent epithelial downgrowth. Currently available artifical KPros use either PMMA, p(HEMA) or PTFE as a skirt but are prone to complications e.g. implant extrusion and retroprosthetic membranes. The aim of this study was to assess the suitability of different materials as possible keratoprosthetic skirts using a recently avaliable modified human keratocyte cell line. Methods:Human keratocytes were seeded on to discs made of Hydroxyapatite (HA), p(HEMA), HAPEX, PTFE and glass. The cells were incubated for 4 hours, 24 hours and 1 week at 37ºC with triplicate discs for each time point. Assays for viablility / cytotoxicity cell adhesion were performed using solutions of calcein–AM and ethidium homodimer–1. Cells were then counted using 5 fields of view on each disc. Immunofluorescence was also performed using primary antibody to Beta–1 Integrin, Vinculin and and Phalloidin. Cells were also prepared to undergo scanning electron microscope examination. Results:The viability/cytotoxicity assays indicated increased cell viability on HA>glass>PTFE with increasing duration of incubation. pHEMA and HAPEX had the lowest cell counts. However, cytotoxicity assays were higher in PTFE>HAPEX>pHEMA. Immunofluorescence showed qualitatively greater expression of ß1 integrin and vinculin on HA > GL > PTFE, after 24 hours of culture on the different materials. Stress fibres were equally prominent on HA and glass but less prominent on PTFE. Scanning electron microscopy, at 24 hours, on HA the cells were spread thinly that the surface texture of the HA was clearly visible through flattened lamellipodia. Lamellipodia of cells on glass were not as thin as those on HA but were, well spread and showed more filopodia than cells cultured on HA. Cells cultured on PTFE produced numerous long cell extensions and were less spread than those on HA or glass. Conclusions:HA was the most viable material for keratocyte adhesion. Other materials e.g. PTFE, pHEMA either had low cell counts or high numbers of cell cytotoxicity. The quality of cellular adhesions as assessed by focal contacts demonstrated by immunofluorescence confirmed high levels of expression of ß1 integrin and vinculin on HA. The enhanced cellular morphology of human keratocytes on HA was shown by scanning electron microscopy.
Keywords: keratoprostheses • cornea: basic science • cell adhesions/cell junctions