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B. Klenkler, A. Ragheb, M. A. Brook, H. Sheardown; Thiol Modification Inhibits Corneal Epithelial Cell Growth on Polydimethylsiloxane Surfaces. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1885.
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© ARVO (1962-2015); The Authors (2016-present)
The control of epithelial cell growth over polymeric biomaterials is critical for the successful design of ocular prostheses such as artificial corneas. In this work, the effect of surface modification of polydimethylsiloxane (PDMS) with thiol groups, and subsequent tethering of pegylated epidermal growth factor (EGF), on corneal epithelial cell coverage is examined.
PDMS was functionalized with surface thiol groups by reaction with 3-mercaptopropyltrimethoxysilane (MPS) in methanol/potassium hydroxide. The surface thiol density was measured using Ellman’s Reagent. EGF was reacted with NHS-PEG-maleimide in solution, and subsequently bound to the thiolated PDMS at varying EGF concentrations. Human corneal epithelial cells were seeded on the surfaces and cultured in serum-free medium. Cell growth was monitored and quantified by trypsinization and counting with a Coulter counter. Thiolated PDMS surfaces were defunctionalized by reaction of the free thiol groups with 3-maleimidopropionic acid in acetonitrile.
Upon optimization of MPS concentration and reaction time, a maximum surface thiol density on the PDMS of 76 nmol/cm2 was attained. Subsequent binding of pegylated EGF, at concentrations of 1 and 10 µg/mL, to the thiolated surfaces resulted in 24 and 65 ng/cm2 of surface-bound EGF, respectively; approximately 78% of the EGF was covalently bound. At five days of culture, quantities of corneal epithelial cells on thiolated PDMS surfaces were approximately 10% of those on unmodified PDMS, and cells were round and non-viable. Similarly low levels of cell growth were found on thiolated surfaces modified with both concentrations of pegylated EGF. In comparison, cell growth was enhanced by the presence of adsorbed EGF on non-thiolated PDMS surfaces. Reaction of the thiolated PDMS with 3-maleimidopropionic acid resulted in the complete elimination of free surface thiol groups. Cell growth on these defunctionalized surfaces was increased to 60% of that observed on non-thiolated PDMS; cells showed a normal, spread morphology.
The presence of free surface thiol groups inhibits the growth of corneal epithelial cells and the stimulatory effect of bound EGF on PDMS substrates. Thiol modification may thus be used for components of ocular prostheses where the prevention of epithelial cell attachment is desired. EGF can be tethered to PDMS via surface thiol groups; the subsequent defunctionalization of remaining free thiols using 3-maleimidopropinic acid may allow for retention of the proliferative effect of the EGF.
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