Bioactivation of Metal Oxide Surfaces. 1. Surface Characterization and Cell Response

Silicon and titanium oxide surfaces (SiO2/Si and TiO2/Ti) were covalently modified with bioactive molecules (e.g., peptides) in a simple three-step procedure. Bioactive surfaces were synthesized by first immobilizing N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS) to either polished quartz disks, polished silicon wafers, or sputter-deposited titanium films. Subsequently, a maleimide-activated surface amenable to tethering molecules with a free thiol (e.g., cysteine) was created by coupling sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) to the terminal amine on EDS. In particular, Cys-Gly-Gly-Asn-Gly-Glu-Pro-Arg-Gly-Asp-Thr-Tyr-Arg-Ala-Tyr (-RGD-) and Cys-Gly-Gly-Phe-His-Arg-Arg-Ile-Lys-Ala (-FHRRIKA-) peptides with terminal cysteine residues were immobilized on maleimide-activated oxides. X-ray photoelectron spectroscopy (XPS) and spectroscopic ellipsometry were used to assess the chemistry, thickness, and surface density of the grafted layers. EDS deposited from anhydrous methanol produced reaction site-limited monolayers (∼0.28 nmol/cm2). Coupling of the sulfo-SMCC cross-linker (∼0.03 nmol/cm2) and peptides (∼0.004 nmol/cm2) resulted in an order of magnitude drop in surface density for each stage of the reaction scheme. Peptide-modified surfaces with densities varying over 2 orders (0.01−4 pmol/cm2) of magnitude were synthesized to study the effect of the peptides on mammalian cell function. The adhesion and spreading of cells derived from mammalian bone, in contact with the peptide-modified surfaces, was dependent on the specific peptide sequence grafted in a concentration-dependent manner. The grafting scheme presented has generality in coupling thiol-specific molecules to silicon or titanium surfaces.