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

Abstract

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-(Nmaleimidomethyl) 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 maleimideactivated 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/cm 2 ). Coupling of the sulfo-SMCC crosslinker (∼0.03 nmol/cm 2 ) and peptides (∼0.004 nmol/cm 2 ) 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/cm 2 ) 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 concentrationdependent manner. The grafting scheme presented has generality in coupling thiol-specific molecules to silicon or titanium surfaces.

In vitro characterization of peptide-modified p(AAm-co-EG/AAc) IPN-coated titanium implants

“…23 Furthermore, these data agree with the reported threshold (0.01 < G attach/spread ¼ 0.62 pmol/cm 2 ) required for maximal osteoblast attachment and spreading on amino-functionalized SiO 2 /Si surfaces modified with the bsp-RGD(15) peptide. 24 Although the threshold for maximal alkaline phosphatase activity was the same as for attachment and proliferation, normalization of the activity to cell number suggests that this is not causative, but coincidental. Although no difference was found in ultimate activity between the 0.1 and 10 pmol/cm 2 bsp-RGD(15) modified surfaces, the time to reach that maximum shifted (14 vs. 21 days).…”

Attachment of hyaluronan to metallic surfaces

“…The most common ones use silane coupling agents that react with the oxide layer formed on metal surfaces. [21][22][23][24][25][26][27] In most cases, the silane coupling agent consists of a trimethoxy or triethoxy silane where the fourth bond of the silane molecule is an organo-functional group that can be further reacted to attach a variety of organic compounds or polymers. The methoxy or ethoxy groups hydrolyze, leaving the silanol to react with the metal oxides.…”

“…These and other adhesive proteins contain short peptide sequences such as RGD, REDV, YIGSR, PHSRN, KNEED, and PRRARV, [37][38][39][40][41][42][43][44][45][46][47][48][49] which are bound by the integrins to promote cell adhesion, spreading, and proliferation. Although significant work has been done to attach proteins and peptides to metal oxide particles 21 and silicon oxide on Si wafers, 26 relatively little research has been reported on attachment of polypeptides to macroscopic metallic surfaces. 23,24,26,27,50 -53 With respect to short peptides, Xiao et al 27 attached an RGDcontaining peptide to titanium using aminopropyl silane and maleimide chemistry.…”

“…23,24,26,27,50 -53 With respect to short peptides, Xiao et al 27 attached an RGDcontaining peptide to titanium using aminopropyl silane and maleimide chemistry. Rezania et al 26 attached the RGD sequence to quartz using similar chemistry, and showed that the RGD retained its activity in promoting the attachment and spread of rat calvaria osteoblast-like cells. 26 Surfaces with cell-adhesive peptides could be used in applications such as bone fixation devices where bone ingrowth is desirable or for selective adhesion of endothelial cells to antithrombogenic vascular stents.…”

Adhesion of MC3T3‐E1 cells to RGD peptides of different flanking residues: Detachment strength and correlation with long‐term cellular function

“…A maleimide heterobifunctional crosslinker that allows for a selective conjugation with -NH 2 and -SH groups of peptides [8,9] was applied to 3-aminopropyltriethoxysilane (APTES) SAMs. A large, residual non-specific interaction was identified on these maleimide-functional substrates that could be minimized by a simple modification of the attachment chemistry.…”