Interactions of corneal cells with transforming growth factor beta 2-modified poly dimethyl siloxane surfaces.

The downgrowth of corneal epithelial cells at the interface of an artificial cornea and the host eye tissue poses a significant problem to be overcome in developing a successful implant. As a means of inhibiting the proliferation of corneal epithelial cells on the stromal surface of the implant, we examined the immobilization of transforming growth factor beta-2 (TGF-beta2) via a bifunctional poly ethylene glycol (PEG) spacer to poly dimethyl siloxane (PDMS) surfaces. Growth factor immobilization was confirmed by modification with (125)I-labeled TGF-beta 2. The modified surfaces were also characterized by advancing water contact angles, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Although the amount of growth factor covalently bound to the surface was difficult to quantify apparently due to strong interactions between the growth factor and the PEG layer and high levels of adsorption, differences in the modified surfaces, suggestive of the presence of a significant amount of TGF-beta 2, were found. In vitro interactions of the modified surfaces with human corneal epithelial and stromal cells were examined. Growth factor surface concentrations as well as culture in the absence and presence of serum and other adhesive proteins were examined. Corneal stromal and epithelial cells cultured on the TGF-beta 2-modified surfaces consistently gave results opposite to those expected. Likely, the most notable and surprising result was the almost complete lack of adhesion of the stromal cells, with coverage averaging between 3 and 5%. In comparison, corneal epithelial cell growth appeared to be promoted by the presence of the immobilized growth factor, with cell coverage averaging 50-60% at 7 days of culture. A TGF-beta 2 concentration effect was noted with both cell types in the absence of serum, with increases in the coverage at higher TGF-beta 2 concentrations. The observed cell growth appeared to be the result of interactions between the cells and active growth factor, because the addition of anti-TGF-beta 2 to the culture medium reduced cell coverage to levels similar to those noted on control surfaces. Therefore, although TGF-beta 2-modified surfaces may not be suitable as corneal epithelial cell inhibiting surfaces, interactions of surface immobilized growth factor and corneal cells are complex and should be further examined.

Interactions of corneal epithelial cells and surfaces modified with cell adhesion peptide combinations.

In order to facilitate the adhesion of corneal epithelial cells to a poly dimethyl siloxane (PDMS) substrate ultimately for the development of a synthetic keratoprosthesis, PDMS surfaces were modified by covalent attachment of combinations of cell adhesion and synergistic peptides derived from laminin and fibronectin. Peptides studied included YIGSR and its synergistic peptide PDSGR from laminin and the fibronectin derived RGDS and PHSRN. Surfaces were modified with combinations of peptides determined by an experimental design. Peptide surface densities, measured using 125-I labeled tyrosine containing analogs, were on the order of pmol/cm2. Surface density varied as a linear function of peptide concentration in the reaction solution, and was different for the different peptides examined. The lowest surface density at all solution fractions was obtained with GYRGDS, while the highest density was consistently obtained with GYPDSGR. These results provide evidence that the surfaces were modified with multiple peptides. Water contact angles and XPS results provided additional evidence for differences in the chemical composition of the various surfaces. Significant differences in the adhesion of human corneal epithelial cells to the modified surfaces were noted. Statistical analysis of the experimental adhesion results suggested that solution concentration YIGSR, RGDS, and PHSRN as well as the interaction effect of YIGSR and PDSGR had a significant effect on cell interactions. Modification with multiple peptides resulted in greater adhesion than modification with single peptides only. Surface modification with a control peptide PPSRN in place of PHSRN resulted in a decrease in cell adhesion in virtually all cases. These results suggest that surface modification with appropriate combinations of cell adhesion peptides and synergistic peptides may result in improved cell surface interactions.

Adhesion of corneal epithelial cells to cell adhesion peptide modified pHEMA surfaces.

Epithelialization of a corneal implant is a desirable property. In this study we compared surface modification of poly (2-hydroxyethyl methacrylate) (pHEMA) with the cell adhesion peptides RGDS and YIGSR. Various parameters in the tresyl chloride activation and modification reactions were considered in order to maximize surface coverage with the peptide including tresyl chloride reaction solvent. tresyl chloride reaction time, tresyl chloride concentration, peptide concentration, and peptide reaction pH. Surface chemistry and corneal epithelial cell adhesion to the modified surfaces were examined. X-ray photoelectron spectroscopy data suggested that while peptide modification had occurred, surface coverage with the peptide was incomplete. Acetone was found to result in a higher fraction of nitrogen and surface bound carboxyl groups compared to dioxane and ether. Furthermore, corneal epithelial cell adhesion to the surfaces for which acetone was used for the activation reaction was significantly greater. Statistical analysis of the various samples suggests that lower peptide concentrations and higher tresyl chloride reaction times result in better cell adhesion. Furthermore, modification with YIGSR resulted in higher surface concentrations and better cell adhesion than modification with RGDS. Little or no cell adhesion was noted on the unmodified pHEMA controls. Protein adsorption results suggest that the differences in cell adhesion cannot be attributed to differences in serum protein adsorption from the culture medium. We conclude that YIGSR modified surfaces have significant potential for further development in corneal applications.