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Ricardo Martins Gouveia, Elena González-Andrades, Juan Cardona, Ana Ionescu, Miguel Alaminos, Miguel González-Andrades, Che John Connon; Using template anisotropy to optimize the structure and function of corneal stromal substitutes. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1999. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The impact of anisotropy on tissue performance constitutes an important parameter in biofabrication. This is particularly critical for corneal stromal tissue with its highly-organized collagen fibrils. Our aim was to create stromal substitutes with native-like composition and structure through a bottom-up strategy. For this, we used smart templates with distinct anisotropies to control cell alignment and subsequent deposition of corneal stromal extracellular matrix (ECM). The templates were also designed to allow the physiological self-release of tissues thereby designated as corneal stromal Self-Lifting Analogous Tissue Equivalents (SLATEs).
Human keratocytes were cultured on templates with different anisotropies made from peptide amphiphiles containing a protease-responsive sequence followed by the integrin-binding amino acid motif RGDS. SLATEs fabricated by keratocytes during 3 months in serum-free conditions were allowed to self-detach by removing retinoic acid from the medium for increased protease expression. The scaffold-free SLATEs were analyzed for their structure, composition, mechanics, and biological function.
SLATEs generated on anisotropic templates showed a dense, native-like ECM with aligned collagen (A-SLATEs). In contrast, tissues on isotropic templates comprised cells and matrix oriented at random (R-SLATEs). Both A- and R-SLATES were highly transparent and maintained high cell viability after self-release, which allowed create thicker constructs through stacking (S-SLATEs). However, A-SLATEs were significantly (p<0.01) denser, thicker, and stiffer than R-SLATEs, making them easier to handle and more resistant to degradation, both in vitro and after implantation in rabbit corneas. Importantly, SLATEs also supported attachment and growth of corneal limbal epithelial cells, but elicited distinctive effects on their phenotype. Specifically, stiffer A-SLATEs promoted epithelial cell differentiation and stratification, whereas the softer R-SLATEs maintained their limbal phenotype.
We show that the intrinsic properties of artificial tissues can be controlled solely through surface templating, and that these properties can in turn define tissue function, such as the ability to support the growth and differentiation of other cell types, or to resist enzymatic degradation. Such finding has evident impact in devising more sophisticated corneal substitutes.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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