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X. Q. Guo, A. E. K. Hutcheon, D. Karamichos, J. D. Zieske; Further Development of an in vitro Model of Human Corneal Fibrosis. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4533.
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© ARVO (1962-2015); The Authors (2016-present)
We have demonstrated that a Vitamin C derivative (VitC) can stimulate human corneal fibroblasts to secrete and assemble matrix in a manner mimicking corneal development. We postulate that addition of transforming growth factor-β (TGF-β) would generate a model of corneal scarring.
Human corneal fibroblasts were grown in four media conditions for 4 or 8 wks: (Group 1 or G1) Vit C only; (G2) Vit C for 4 wks, then VitC + TGF-β1 for 4 wks; (G3) VitC + TGF-β1 for 1 wk, then VitC only for 3 or 7 wks; and (G4) VitC + TGF-β1 for the entire time. Cultures were analyzed with TEM and indirect-immunofluorescence. Cellular fibronectin (cFN), -smooth muscle actin (SMA) and collagen type III (type III) were localized.
The thickness of the constructs increased with the addition of TGF-β1. Compared to G1, G2 increased 1.7-fold, G3 1.8/2.0-fold and G4 2.1/3.1-fold at 4 and 8 wks. In all TGF-β treated cultures, the cells became very long and flat, numerous cells with filaments were seen, the collagen levels increased, and long collagen fibrils could be seen. (4 wks) cFN is present in G1 at very low levels, whereas, in G4, cFN is present at high levels and G3 appears to be intermediate. Type III is present at very low levels in G1 and G3 in the lower half of the construct. However, in G4, Type III is found in the upper 1/5 of the construct. SMA positive cells are present throughout G3 and G4, while G1 only has a few positive cells at the bottom of the construct. (8 wks) Type III is found in the middle of the construct in G1, in the top half at high levels in G2 and G4, and in the middle of the construct in G3. SMA is present at low amounts in G1, throughout the construct at low levels in G3, and in numerous cells in G2 and G4. cFN in G1 and G3 is found at low levels in the top and bottom of the construct; whereas, G2 and 4 have cFN in high amounts at the top of the construct.
The presence of TGF-β in the cultures for even one week resulted in elevated levels of matrix, cFN and myofibroblasts. Interesting, TGF-β stimulated the generation of aligned matrix, and fewer lamellae then control cultures.
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