Materials taking
advantage of the characteristics of biological
tissues are strongly sought after in medical science and bioscience.
On the natural corneal tissue surface, the highly soft and lubricated
surface is maintained by composite structures composed of hydrophilic
biomolecules and substrates. To mimic this structure, the surface
of a silicone hydrogel contact lens was modified with a biomimetic
phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine)
(PMPC), and the nanoscaled morphology and mechanical properties of
the surface were confirmed with advanced surface characterization
and imaging techniques under an aqueous medium. Concavities and convexities
on the nanometer order were recognized on the surface. The surface
was completely covered with a PMPC layer and remained intact even
after 30 days of clinical use in a human ocular environment. The mechanical
properties of the natural corneal tissue and the PMPC-modified surface
were similar in the living environment, that is, low modulus and frictional
properties comparable to natural tissues. These results show the validity
of material preparation by biomimetic methods. The methodologies developed
in this study may contribute to future development of human-friendly
medical devices.
Previous studies have shown that the corneal epithelium will close a limbus to limbus scrape wound in four to five days, but requires 10 days to become firmly attached to the stroma. In order to determine if the restoration of the corneal epithelial barrier follows a similar sequence, we have used freeze-fracture to study tight junction reformation in a rabbit epithelial wound model. Dutch-belted rabbit corneal epithelium was removed with a limbus to limbus scrape wound and sampled from 0 to 30 days post-wounding. A minimum of 3 animals from each time point were processed for electron microscopy and freeze-fracture. Freeze-fracture showed that the cells at the wound margin had a reduction in the number of intramembrane particles on their apical surface. In areas adjacent to the wound edge, fragments of tight junctions were first observed on two days post-wounding specimens. The junctions became progressively more complex in the area behind the wound edge until wound closure at four days when the junctions were also present in the central region of the cornea. The maturation of the junctions continued and at five days after surgery they resembled control junctions. This sequence suggests that the the establishment of morphologically mature tight junctions may be necessary before the corneal epithelium can firmly reattach to the stroma.
The increasing importance of in vitro models has presented new challenges in SEM processing techniques. The present study has evaluated the quality of preservation of cultured human lens epithelial cells processed by critical point, Peldri II, and tert-butyl alcohol drying. Specimens processed by critical point drying produced specimens with severe cracking of cell processes and microcracks across cell membrane surfaces. Peldri II and tert-butyl alcohol drying eliminated breakage of the filopodia and lamellipodia as well as eliminating the microcracks across the apical membrane surface. The morphology of lens epithelial cells grown on Cytodex 3 beads appeared rounded with convoluted membrane surfaces. These morphological features were present for cells processed by all three methods. Cytodex 3 beads were subsequently shown to shrink 52% in diameter during dehydration, which results in an 89% reduction in volume for the bead. Cells grown on Biosilon beads, which do not shrink, had a morphology similar to the cells grown on a flat substrate. These results indicate that Peldri II and tert-butyl alcohol drying offer an attractive alternative to critical point drying when preparing cultured cells for SEM. Interpretation of cultured cell morphology must consider shrinkage of the substrate material as a possible contributor to artifact.
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