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

Abstract

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)

Proteins at Interfaces

“…Thus, for example, Horbett has shown that the amount of competing protein required to inhibit the adsorption of fibrinogen to solid surfaces from binary mixtures depends on the absolute concentration of the species involved, suggesting that the apparent competitive effectiveness is occupancy dependent (79). Such complexities notwithstanding, the extreme surface activity of a few proteins seems indisputable.…”

“…Such complexities notwithstanding, the extreme surface activity of a few proteins seems indisputable. Among these should be mentioned hemoglobin (79) and high molecular weight kininogen (20). Convincing, structurally based explanations of the high surface activity of these proteins have not so far been proposed, although the positively charged histidine-rich domain of HMWK (27) may account for its adsorption to negatively charged surfaces.…”

“…Modulation of the biologic activity of adsorbed fibronectin is indicated by several studies showing that fibronectin adsorbed to various surfaces is not equivalent in its interactions with cells, despite the presence of similar amounts of adsorbed fibronectin (77)(78)(79)(80)(81)(82). For example, fibronectin adsorbed to tissue culture grade polystyrene supports BHK cell attachment and spreading, whereas fibronectin adsorbed to ordinary polystyrene does not support spreading unless some albumin is added to the fibronectin solution (77).…”

Characterization of the interface between adsorbed fibronectin and human embryonic stem cells

“…As soon as a material is placed in a biological environment, there is a thermodynamic driving force that influences the adsorption of proteins onto the surface, and it is this layer that interacts with the cell via the cell membrane. Thus, the characteristics of the adsorbed protein layer in terms of the chemical, topographical or mechanical cues presented to the cells can have a controlling influence on the cell response [1][2][3].…”

The competitive adsorption of human proteins onto natural-based biomaterials

“…It is therefore likely that the results obtained for HSA and HFN will be representative of a large number of serum proteins. Moreover, authors have reported the ability of albumin to 'rescue' fibronectin molecules influencing its molecular conformation on hydrophobic surfaces (Grinnell & Feld 1981), which was also reported to facilitate cell adhesion-dependent processes (Lewandowska et al 1989).…”