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Alvena Kureshi, Christine Putri, James Phillips, James L Funderburgh, Julie T Daniels; RAFT tissue equivalent model of keratoconus: A novel 3D system useful for interrogating disease phenotype and functional differences of corneal stromal cells.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2359.
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© ARVO (1962-2015); The Authors (2016-present)
Keratoconus (KC) is a corneal disorder characterized by progressive structural thinning, resulting in a conical-shaped cornea causing loss of vision. Cell-extracellular matrix interactions are thought to control normal tissue organization and transparency and it is expected that these interactions may be perturbed in KC. The aim of this study was to develop a 3D RAFT tissue equivalent (TE) model of KC to compare the phenotypic and functional behavior of stromal cells.
Stromal cells were characterised by phenotypic assessment using a variety of stromal cell markers and a contraction assay to compare their contractile capacity. RAFT TE were seeded with normal and KC stromal cells (n=3) and cultured for 3 weeks. Functional characterization of RAFT TE included investigations of material properties using an Electroforce Bose tensile testing device; MMP activity in stromal cells using gelatin zymography and ELISA; collagen production using a Sircol collagen assay and assessment of collagen fibril organization using scanning electron microscopy.
A 3D in vitro model of KC was achieved using RAFT TE. Normal and KC stromal cells both exhibited stromal cell markers in culture. KC stromal cells exhibited a significantly greater contractile capacity at 72 hours of culture with 75 ± 3% compared to normal cells at 29 ± 7% and acellular gels at 3 ± 1% (n=3) (p<0.0001). Statistically significant differences in RAFT TE material properties were observed. Mean break strength of KC and normal RAFT TE were 0.17 ± 0.02 MPa and 0.25 ± 0.03 MPa respectively (n=3) (p<0.05). A reduced stiffness of 0.01 ± 0.002 MPa in KC RAFT TE compared to 0.02 ± 0.002 MPa in normal RAFT TE was observed. Increased activity of MMP-2 in normal RAFT TE was observed but no differences in pro-MMP-1 or collagen production. Collagen fibril ultrastructure in KC RAFT TE appeared markedly thinner and loosely packed compared to normal RAFT TE.
To our knowledge, this is the first 3D tissue equivalent model of KC. RAFT TE serves as an ideal biomimetic model to investigate normal and abnormal tissue organization in the corneal stroma. Significant differences in contractile capacity of KC cells and strength and stiffness of KC RAFT TE suggests the remodeling capacity of stromal cells is perturbed in KC, leading to ultrastructural changes of the corneal stroma.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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