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Rachel Gomes, Siran Wang, Chiara E Ghezzi, Whitney Stoppel, James L Funderburgh, Kenneth Kenyon, David L Kaplan; In vitro study: Corneal stromal stem cells cultured on cornea ECM blended silk films. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5311.
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© ARVO (1962-2015); The Authors (2016-present)
In this in vitro study, the goal is to investigate the influence of human corneal ECM protein incorporated into silk protein films on human corneal stromal stem cell (hCSSCs) functions. We will investigate the role of cECM components on keratocyte cellular behavior. We will focus on the isolation of endogenous cECM from human corneas, assessing the formation of cornea stroma like tissue in terms of composition and structural organization over time, as well as cellular phenotype.
Human cornea (donor cornea not suitable for transplantation) were decellularized in SDS, and then pepsin digested before lyophilization to form an ECM powder. Glycosaminoglycans and protein content in ECM powder were assessed. The cECM proteins were stabilized in the silk materials via water annealing, which affects crystallinity/mechanical properties. The collagens and proteoglycan content were quantified via mass spectroscopy and ELISA. Corneal stromal stem cells were seeded onto the modified silk films and cultured up to 6 weeks in vitro. The tissues formed were compared to plain silk and RGD-functionalized silk films, both without cECM, related to cell growth and new ECM secretion and organization. Histology, fluorescence microscopy, immunohistochemistry, qPCR and proteomic analysis were used quantify the behaviour of the hCSSCs cultured on the cECM blended silk films and controls. Detailed proteomics analysis will be conducted to investigate ECM formation by the cells on these silk-cECM substrates (collagen I, collagen V, collagen VI, lumican, keratocan, decorin).
A pepsin-based protein extraction protocol to achieve high yield of cECM components from human corneal tissues was successfully implemented. A content of 1.4% sulfated glycosaminoglycans per dry weight of corneal proteins was found and is critical for corneal tissue behavior (cGAGs comprise 2 to 5 wt% in native human cornea. hCSSCs are expected to grow and proliferate on all the substrates; although improved outcomes are anticipated in the presence of the cECM in the films.
This study has defined a novel experimental system for examining the effect of extracellular environment in the production of stromal matrix. We expect that future studies using this system to provide important details as to the role of the extracellular matrix in guiding corneal cell differentiation.
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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