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Alexandria Hofmann, Praveen Balne, Ratnakar Tripathi, Sabeeh Kamil, Suneel Gupta, Prashant R. Sinha, Nishant Rajiv Sinha, Rajiv R Mohan; Effects of matrix-stiffness on human corneal stromal fibroblast differentiation. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2926.
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Matrix environment affects cell fate and function during wound repair. Fibroblasts are known to sense and respond to changes in matrix-stiffness. Currently, no in vitro model exists to study the role of matrix-stiffness in corneal wound healing to the best of our knowledge. This study aimed to identify in vitro matrix-stiffness conditions promoting corneal stromal fibroblasts differentiation to myofibroblasts using hydrogels of varying elasticity.
Primary human corneal stromal fibroblasts (hCSFs) were generated from healthy human donor corneas obtained from Eye Banks. The hCSFs generated from corneal explants were seeded in collagen-1 pre-coated hydrogel cell culture plates with soft (4kPa) and stiff (50kPa) matrix and incubated at 37oC for 72h in a 5% CO2 incubator. RNA and total protein were extracted from cells and expression of alpha smooth muscle actin (αSMA), collagen type III, and decorin were analyzed using qRT-PCR and enzyme linked immunosorbent assay (ELISA).
Primary human corneal stromal fibroblasts grown on the soft matrix in vitro showed typical flat, elongated, spindle-shaped fibroblastic morphology up to the longest tested time (72h). Conversely, same cells grown on stiff matrix demonstrated typical corneal myofibroblastic phenotype, contractile large flat cells with bundles of microfilament/stress fibers and high αSMA, up to the longest tested time (72h). A significantly increased mRNA levels of the two tested pro-fibrotic factors, αSMA and collagen type III, and decreased levels of one tested anti-fibrotic factor, decorin, were observed in cells grown on stiff matrix compared to cells grown on soft matrix (p<0.05). Similarly, ELISAs demonstrated significantly increased αSMA and collagen-III proteins, and decreased decorin protein expression in cells grown on stiff matrix compared to the cells grown on soft matrix (p<0.05).
Tested hydrogels could be used to study the role of matrix-stiffness and mechano-transduction pathways involved in corneal stromal wound healing.
This is a 2020 ARVO Annual Meeting abstract.
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