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Ricardo Martins Gouveia, Valeria Castelletto, Ian W Hamley, Che John Connon, Stem Cells and Nanomaterials Group; Fabrication of a Bio-Prosthetic Cornea from Human Keratocytes. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5140.
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We aim to create a bio-prosthetic human cornea using a bottom-up strategy. For this, we developed a smart enzyme-responsive biomaterial template to direct adhesion, proliferation, alignment, and stratification of human corneal stromal keratocytes. Cells thus instructed are expected to produce native-like stromal tissue, with extracellular matrix (ECM) composed of aligned collagen fibrils and appropriate proteoglycan moieties deposited in lamellae.1 But most importantly, the fabricated corneal tissue will be able to enzymatically degrade the template and control its own release.
The enzyme-responsive template was made from self-assembling peptide amphiphile (PA) molecules composed of a hexadecyl lipid chain attached to a peptide headgroup containing a metalloprotease (MMP)-responsive sequence followed by the integrin-binding motif Arg-Gly-Asp-Ser. Keratocytes were grown on these templates in serum-free media for up to 90 days, and the resulting tissue was analysed for stromal-specific ECM markers at transcriptional and protein levels. Retinoic acid (RA) at 1×10-5 M was added to culture media during growth to inhibit MMP expression from keratocytes,2 and then removed to elicit PA template degradation and tissue self-release.
We successfully fabricated human corneal tissue in vitro solely by culturing human donor-isolated keratocytes on a smart template (Fig. 1). This template promoted integrin-mediated adhesion, and keratocyte proliferation and stratification. The addition of RA to serum-free media further enhanced these effects, and significantly increased the production of keratocyte markers such as collagen type-I, keratocan, lumican, and ALDH1. Furthermore, RA reduced MMP activity from culture supernatant to less than 5% of control, thus ensuring the integrity of the PA template throughout the entire period in culture and allowing the formation of structurally coherent, ECM-enriched, robust corneal tissue. By resuming MMP expression after RA removal, we were able to control template degradation and promote tissue self-release, minimizing damage from manipulation.
This study demonstrates a feasible bottom-up strategy to create native-like bio-prosthetic corneal tissue entirely fabricated by human keratocytes in vitro. This has evident applications in regenerative medicine. 1. Gouveia et al., J Mater Chem B, 2013,1: 6157 2. Gouveia and Connon, Invest Ophthalmol Vis Sci, 2013, 54: 7483
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