Surface modification of Ti45Nb alloy by immobilization of RGD peptide via self assembled monolayer.

A new low modulus beta Ti-Nb alloy with low elastic modulus and excellent corrosion resistance is currently under consideration as a surgical implant material. The usefulness of such materials can be dramatically enhanced if their surface structure and surface chemistry can be controlled. This control is achieved by attaching a self assembled monolayer (SAM) based on 11-chloroacetyl-1-undecylphosphonic acid, CAUDPA, to the surface and immobilization of a peptide to the monolayer. The SAM is characterized by Fourier Transform Infrared Spectroscopy (FTIR) and X-ray Photoelectron Spectroscopy (XPS) at two different takeoff angles. The CAUDPA molecules were covalently bonded on the substrate in a configuration in which the phosphonic group turns toward the Ti45Nb while the acetyl chloride end group tail turns to the topmost surface. In such configuration sequential in situ reaction is possible by exchange between the chloride and a biological molecule. Such biological molecule is the arginine-glycine-aspartic acid-cysteine, RGDC, small amino acid sequence present in many molecules of the extracellular matrix. Preliminary cell culture in-vitro result shows an improvement of the response of osteoblast cells to Ti45Nb after the peptide immobilization.

Cell type-specific modulation of fibronectin adhesion functions on chemically-derivatized self-assembled monolayers.

Cell type-specific responses (microfilament stress fibers for fibroblasts or neurites for neuroblastoma cells) were evaluated in culture on inert and chemically-derivatized silane substrata adsorbed with fibronectin (Fn). Substrata of self-assembled monolayers contain a 14-17 carbon aliphatic chain terminating with different chemical endgroups -- [CH3], [C=C], [Br], [CN], [Diol], [COOH], [NH2], [SH], [SCOCH3], or [SO3H]. Fn adsorbed effectively to all derivatized surfaces. 3T3 fibroblasts or neuroblastoma cells attached equivalently to all surfaces preadsorbed with Fn, indicating availability of receptor binding sites on Fns. However, transmembrane signaling from Fn(adsorbed): receptor(cell) surface complexes yielded a range of abilities for generating F-actin stress fibers in fibroblasts or neurites in neuroblastoma cells. Efficiency for stress fiber formation was very different from that of neurite extension. The same chemical endgroups on glass, titanium, or germanium yielded the same patterns of cellular physiological responses, indicating that inert substrata do not act at a distance and that only chemical endgroups regulate Fn signaling functions. When adhesion-inert albumin is co-adsorbed with Fn, efficiency of neurite extension is improved on some surfaces or diminished on others. These results indicate that the conformation of Fn(adsorbed) changes in specific ways on derivatized substrata. Change in Fn conformation was confirmed by FTIR/ATR spectroscopy experiments of Fn(adsorbed). Overall, these studies indicate changes in Fn conformations on chemically-derivatized self-assembled monolayers leading to up- or down-regulation of cell type-specific physiological responses from receptors via their signaling pathways. They also offer predictability for regulating responses of specific cell types when these cells interact with biomaterial implants in vivo.

Problems and approaches in covalent attachment of peptides and proteins to inorganic surfaces for biosensor applications.

Some of the fundamental problems in covalent attachment of peptides and proteins to putative biosensor surfaces are reviewed and specific approaches to these problems discussed. In addition, selected aspects of our recent work utilizing self-assembled monolayer (SAM) systems designed to react selectively with the thiol side chain of Cys in proteins are presented. Uniform attachment of a 21-amino acid peptide antigen through a single Cys residue with retention of biological function (antibody binding) has been attained. Further work with this system may lead to solutions for some of the problems which currently prevent the development of reliable biosensors for industrial and medical use.

Cell-type-specific adhesion mechanisms mediated by fibronectin adsorbed to chemically derivatized substrata.

Plasma fibronectin (pFN) adhesion mechanisms on inert substrata were evaluated for murine fibroblasts (3T3) and human neuroblastoma (Platt) cells using glass coverslips chemically derivatized with a self-assembled monolayer of aliphatic chains terminated with a specific endgroup to interact with adsorbed pFN: [CH3], [SH], [SCOCH3], [NH2], [SO3H], or underivatized glass [SiOH]. All surfaces bound similar amounts of pFN and facilitated attachment of both cell types within narrow ranges. However, spreading/differentiation responses of cells differed considerably among the surfaces. While 3T3 cells spread and developed microfilament stress fibers comparably on all surfaces, inclusion of an RGDS-containing synthetic peptide in the medium revealed variation in resistance to stress fiber formation mediated by an RGDS-recognizing integrin: [NH2] greater than [CH3] much greater than [SiOH],[SH],[SCOCH3]. Different patterns of neurite formation were observed for neuroblastoma cells: [SiOH], [SO3H] greater than [SCOCH3],[SH] much greater than [CH3] greater than [NH2]. Similarity in cell responses to both [CH3] and [NH2] surfaces argues against a pattern dependent upon the hydrophobicity of substrata. When pFN was diluted to a subsaturable concentration with albumin for adsorption, neuroblastoma responses changed significantly from those observed on pFN-saturated surfaces, for both spreading and neurite generation: [NH2],[SO3H] much greater than [SH], [SCOCH3] greater than [SiOH],[CH3]. Responses to the pFN: albumin mixture were markedly improved from responses after sequential adsorptions, demonstrating "optimization" of pFN conformation (not merely binding) by coadsorption of albumin molecules. In most cases, the [NH2] surface yielded responses distinctively different from the other surfaces. Overall, these data suggest many variations in the conformation of pFN molecules adsorbed to specific inert surfaces, as well as variations in the responses of cell surface receptors to conformationally specific pFNs. They also reveal cell-type-specific changes in differentiated cell responses on derivatized substrata, mediated by different classes of cell surface receptors for the two cell types, and provide optimism for regulating FN-dependent adhesion mechanisms in either positive or negative contexts on biomaterial surfaces derivatized with one or more of these chemical end-groups.

Modulation of cell adhesion by modification of titanium surfaces with covalently attached self-assembled monolayers.

The surface of titanium has been modified by covalent attachment of an organic monolayer anchored by a siloxane network. This coating completely covers the metal and allows controlled modification of surface properties by the exposed chemical endgroups of the monolayer forming surfactant. The attachment of such a film allows different bulk materials (e.g., glass and titanium) to have identical surface properties and this can be used in regulating cell adhesion responses. This control over surface functionality can modulate the functions of fibronectin in regulating attachment and neurite formation by neuronal cells. The effect on bacterial adherence that is achieved by using such monolayers to vary surface hydrophilicity is also assessed.

Modulation of fibronectin adhesive functions for fibroblasts and neural cells by chemically derivatized substrata.

Adhesion responses of fibroblasts (Balb/c 3T3 cells) and human neuron-derived (Platt neuroblastoma) cells have been examined with plasma fibronectin (pFN) adsorbed to glass surfaces derivatized with an alkyl chain and six chemical end groups interfacing with the bound pFN to test regulation of pFN function. Using new derivatization protocols, the following surfaces have been tested in order of increasing polarity: [CH3], [C = C], [Br], [CN], [Diol], [COOH], and underivatized glass [( SiOH]). For all substrata, pFN bound equivalently using either a supersaturating amount of pFN or a subsaturating amount in competition with bovine albumin. Attachment of both cell types was also equivalent on all substrata. However, spreading/differentiation responses varied considerably. F-actin reorganization was tested in 3T3 cells with rhodamine-phalloidin staining. While stress fibers formed effectively on pFN-coated [SiOH] and [Br] substrata, only small linear bundles of F-actin and a few thin stress fibers were observed on the [COOH], [Diol], and [CN] substrata; the hydrophobic substrata [( CH3] and [C = C]) gave an intermediate response. When a synthetic peptide containing the Arg-Gly-Asp-Ser sequence required for integrin binding to FNs was included in the medium as an inhibitor, additional differences were noted: Stress fiber formation was completely inhibited on [SiOH] but not on [Br] and stress fiber formation was very sensitive to inhibition on the hydrophobic substrata while the F-actin patterns on the [CN] and [COOH] substrata were unaffected. Evaluation of neurite outgrowth by neuroblastoma cells on these substrata revealed both qualitative and quantitative differences as follows: [Diol] = [COOH] greater than [SiOH] much greater than [CN] = [Br] greater than [CH3] = [C = C]. While there was poor cytoplasmic spreading and virtually no neurites formed on the hydrophobic surfaces when pFN alone was adsorbed, neurite formation could be "rescued" if a mixture of pFN with an excess of bovine albumin was adsorbed, demonstrating complex conformational interactions between substratum-bound pFN and adhesion-inert neighboring molecules. In summary, these studies demonstrate that different chemical end groups on the substratum modulate pFN functions for cell adhesion, principally by affecting the conformation of these molecules rather than the amounts bound. Furthermore, these studies confirm multiple-receptor interactions with the FN molecules in cell type-specific adhesion patterns.(ABSTRACT TRUNCATED AT 400 WORDS)