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James W. Foster, Roanne R. Jones, Vitaliy Khutoryanskiy, Bruce Caterson, Che J. Connon, Stem Cell & Nanomaterials Laboratory; Using Collagen Density To Restore The Quiescent Phenotype Of Keratocytes. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1076.
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Keratocytes make up the bulk of the cellular component of the corneal stroma. They usually reside between the collagen fibrils and are critical for maintaining the clarity and function of the cornea. We hypothesise that by culturing keratocytes in a 3D environment that mimics the normal corneal stroma a more quiescent phenotype will be maintained.
Both Compressed and Uncompressed Collagen gels were seeded with low passage human central keratocytes and to create a 3D artificial corneal stroma1,2. These constructs were monitored over time for changes in hydration and contraction. Cell Phenotype was quantified by qPCR and protein analysis, alamar blue were used to examine proliferation. Rheology was used to monitor changes in stiffness and TEM was used to analyse fibril organisation.
Keratocytes in compressed collagen gels expressed more of the quiescence markers Lumican and Keratocan at levels that are comparable with that seen in native tissue, we have demonstrated that this is due to factors inherent to the compressed collagen gel as although the uncompressed gels did contract to a similar degree of Hydration and Structure they were unable to retain cells in an undifferentiated state.
We have demonstrated that encapsulating keratocytes in compressed collagen gels induces a de-differentiation from an aggressive myo-fibroblastic cell type to a more quiescent one. The improved mechanical properties make our constructs more suitable for tissue engineering applications and provide an improved model of the stroma that recreates the native environment of the stroma better than existing models such as contracted collagen gels.
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