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Mariana Garcia, Kevin E. Healy, Christine F. Wildsoet; In Vitro Cytocompatibility Of Mammalian Scleral Fibroblasts In A Biomimetic Extracellular Matrix. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6309.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a battery of in vitro assays to characterize the adhesion and proliferation patterns of mammalian scleral fibroblasts in a synthetic biomimetic extracellular matrix, with an intended therapeutic application for myopia control. The biomaterial comprises a thermoresponsive enzymatically-degradable semi-interpenetrating polymer network (edsIPN) that can be degraded by native matrix metalloproteinases and also presents binding sites to the integrins α3β1, α5β1, αvβ1, and αvβ5, all of which have been found in mammalian sclera (Metlapally et al, Mol. Vis., 2006).
Scleral fibroblasts were isolated from 2-month old guinea pigs (Cavia porcellus). EdsIPN were synthesized in the individual wells of 48-well plates, in sufficient volume as to uniformly cover the bottom of the wells. Adhesion assay: Cells were seeded onto either tissue culture polystyrene (TCPS) or edsIPN, and the number of suspended cells in sampled medium counted with a hemocytometer over 0 to 5h post-seeding, as an indirect measure of cell adherence. Proliferation assay: Cell proliferation in the edsIPN was monitored over a period of 12 days using the Cell Titer 96 Aqueous non-radioactive proliferation assay (Promega). Cell shape was also monitored over time using phase microscopy.
Adhesion assay: At t=0 h, significantly more cells were attached to the edsIPN than to the control polystyrene surface. In all subsequent time points, no significant differences were observed. Proliferation assay: Guinea pig scleral fibroblasts took significantly longer to enter a logarithmic growth phase when seeded onto the edsIPN compared to TCPS, possibly as a result of the lower modulus of the substrate.
Completed experiments demonstrated the utility of the above assays in characterizing the interactions between edsIPN and scleral fibroblasts. These assays will be used to assess the biocompatibility of different edsIPNs with scleral fibroblasts , to optimize their physical properties and chemical composition for the intended therapeutic application.
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