Abstract
Purpose: :
Improved corneal epithelial cell growth over keratoprostheses is required to prevent downgrowth and device extrusion. In this work, epidermal growth factor (EGF), a mitogen for epithelial cells, was immobilized to polydimethylsiloxane (PDMS) substrates, and the cellular response was analyzed.
Methods: :
Three methods were used to bind EGF to PDMS via polyethylene glycol (PEG) tethers. 1) EGF was first reacted with NHS2PEG and then bound to allyamine–modified PDMS. 2) PDMS was modified with allyl–PEG–NHS and then EGF was attached to the surface–bound PEG. 3) EGF was first reacted with NHS–PEG–maleimide and then bound to thiol–modified PDMS. The amount of bound EGF was varied by altering the EGF solution concentration, and the EGF–PEG yield optimized using SDS–PAGE. 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. Expression of matrix proteins and α6–integrins was assessed by immunostaining and confocal microscopy.
Results: :
In Method 1, 46 to 91 ng/cm2 of EGF was bound, however of this, 70% was adsorbed even under optimal EGF–PEG reaction conditions. Cells grew to confluence on these surfaces, and cell counts increased from 1.2x104 to 7.8x104 by Day 5. On unmodified and allylamine–only control surfaces, cell coverage was incomplete, and Day 5 counts were 1.9x104 and 2.5x104 respectively. With a tenfold lower initial seeding density, cell colonies were larger on the EGF–modified surfaces than on controls, indicating that EGF caused higher cell proliferation as well as initial adhesion. Fibronectin and laminin expression was greater on EGF–modified surfaces than controls at each time point, and α6–integrins were highly localized at the cell–material interface indicating enhanced adhesion of cells to the modified PDMS. Method 2 resulted in 5 to 190 ng/cm2 of bound EGF, with less than 10% adsorption. However cell confluence was not achieved, and the highest growth was at 45 ng/cm2. The high underlying PEG density on these surfaces is inhibitory to cell growth, as indicated by zero coverage on PEG–only controls.
In Method 3, 54 nmol/cm2 of thiols were attached to the PDMS. Subsequent EGF binding via heterobifunctional PEG may allow improved control over covalent EGF attachment and cell growth than the other two methods.
Conclusions: :
Tethering of EGF to PDMS can significantly improve the growth of corneal epithelial cells, with effects dependent on the underlying substrate modification.
Keywords: keratoprostheses • cornea: epithelium • growth factors/growth factor receptors