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J.W. Ruberti, S.A. Melotti, G.J. Braithwaite; Nanoscale Engineering of Type I Collagen Fibrils to Mimic the Multiple Layers of Aligned Lamellae in Cornea . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4218.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To generate a strong, biomimetic, transparent stromal construct for use in corneal replacement or repair. Methods: It was hypothesized that the physics of spin coating flows might guide the self-assembly of type I collagen monomers to generate a thin layer of aligned fibrils. It was further postulated that multiple layers of aligned fibrils could be stacked with control over the alignment direction for adjacent lamellae. To test this hypothesis, type I collagen monomers (Vitrogen®) were polymerized on the surface of a rotating substrate. The substrate was housed in an environmental chamber which maintained the humidity at above 95%, and the temperature between 36°C and 41°C. At these conditions collagen is known to self-assemble. In these experiments, the rate of injection of monomer, and the rotation rate of the substrate, were controlled to maintain a thin film of solution on the rotating substrate (~10 microns). The resulting shear rate at the solution/substrate interface was 700/sec which we calculate to be sufficient to substantially favor alignment of the growing collagen fibrils. Additionally, the thin film confinement induced by the flow regime on the spin coater surface also promotes strong orientation by limiting the space available for rotation of the growing fibrils. Results: SEM images of the polymerized films exhibit areas of total alignment of type I collagen fibrils with a reasonably uniform diameter (~150 nanometers). SEM images also demonstrate that thin, uniform thickness lamellae of aligned fibrils can be produced with this methodology. Further SEM results show that additional layers with different fibril alignment directions can be stacked sequentially, thus demonstrating the feasibility of approximating corneal lamellae. However, there were regions on the substrate where fibril alignment was not observed. It is postulated that non-aligned areas were not in the flow field for the complete duration of the experiment (i.e. instability of the wetting film). Conclusions: This study has demonstrated the feasibility of generating multiple layers of aligned collagen fibrils with each layer having a different principle alignment direction. The potential for such a construct to be used in the repair or replacement of diseased corneas is apparent. However, the existence of patches of random fibril alignment in some areas and lack of control over the fibril diameter of the construct requires further refinement of the technique.
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