Elsevier

Biomaterials

Volume 28, Issue 5, February 2007, Pages 851-860
Biomaterials

Fibronectin terminated multilayer films: Protein adsorption and cell attachment studies

https://doi.org/10.1016/j.biomaterials.2006.09.037Get rights and content

Abstract

Electrostatically driven layer-by-layer (LbL) assembly is a simple and robust method for producing structurally tailored thin film biomaterials, of thickness ca. 10 nm, containing biofunctional ligands. We investigate the LbL formation of multilayer films composed of polymers of biological origin (poly(l-lysine) (PLL) and dextran sulfate (DS)), the adsorption of fibronectin (Fn)—a matrix protein known to promote cell adhesion—onto these films, and the subsequent spreading behavior of human umbilical vein endothelial cells (HUVEC). We employ optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microgravimetry with dissipation (QCMD) to characterize multilayer assembly in situ, and find adsorbed Fn mass on PLL-terminated films to exceed that on DS terminated films by 40%, correlating with the positive charge and lower degree of hydration of PLL terminated films. The extent and initial rate of Fn adsorption to both PLL and DS-terminated films exceed those onto the bare substrate, indicating the important role of electrostatic complexation between negatively charged protein and positively charged PLL at or near the film surface. We use phase-contrast optical microscopy to investigate the time-dependent morphological changes of HUVEC as a function of layer number, charge of terminal layer, and the presence of Fn. We observe HUVEC to attach, spread, and lose circularity on all surfaces. Positively charged PLL-terminated films exhibit a greater extent of cell spreading than do (negatively charged) DS-terminated films, and spreading is enhanced while circularity loss is suppressed by the presence of adsorbed Fn. The number of layers plays a significant role only for DS-terminated films with Fn, where spreading on a bilayer greatly exceeds that on a multilayer, and PLL-terminated films without Fn, where initial spreading is significantly higher on a monolayer. We observe initial cell spreading to be followed by retraction (i.e. decreased cell area and circularity with time) for films without Fn, and for DS-terminated films with Fn. Overall, the Fn-coated PLL monolayer and the Fn-coated PLL-terminated multilayer are the best performing films in promoting cell spreading. We conclude the presence of Fn to be an important factor (more so than film charge or layer number) in controlling the interaction between multilayer films and living cells, and thus to represent a promising strategy toward in vivo applications such as tissue engineering.

Introduction

Living cells receive important signals through biological recognition events at their surface. Biomaterials capable of mimicking these signals are potentially valuable as tissue-engineering substrates, biosensing surfaces, and drug delivery vehicles. Materials must be capable of signal transmission and also meet stringent structural, mechanical, and degradation requirements—a scenario best realized through a coating approach where bulk material and surface properties are effectively decoupled. Multilayer nanofilms, formed by the layer-by-layer (LbL) method [1], [2], [3], [4], [5], [6], [7], are promising in this regard. LbL assembly occurs through the alternate adsorption of positively and negatively charged polyelectrolytes, and the resultant multilayer films can be rendered bioactive through the adsorption of protein or other biological molecules capable of transmitting signals to contacting cells [8], [9], [10], [11], [12], [13]. Since only physical interactions are involved, LbL assembly represents a simple, controllable, and broadly applicable route to bioactive thin film materials.

Fibronectin (Fn) is an extracellular matrix protein known to promote cell attachment and spreading [14]. The mechanism is thought to involve attachment of α5β1 transmembrane integrin receptors to Fn's cell binding site, located on the 10th type-III repeat module and containing the specific amino acid sequence RGD, as well as to synergy sites located on the 8th and 9th type-III repeats. Thus, materials coated with Fn are promising for a variety of cell-contacting applications. Indeed, a number of studies attest to the enhanced cell attachment and spreading of surfaces coated with Fn, compared to identical materials in the absence of Fn [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36].

Despite the promise of multilayer films in cell-contacting applications, and the proven utility of Fn as a cell adhesion motif, only a few investigations of Fn-coated multilayer films have appeared [37], [38], [39], [40], [41]. Ai et al. showed cerebellar neurons to attach, grow, and differentiate on silicone coated with multilayer films consisting of three bilayers of poly(styrene sulfonate) (PSS)/poly(ethylene imine) (PEI) and four bilayers of Fn/poly(d-lysine) (PDL) [37]. Interestingly, similar cell behavior occurred on silicone coated with a monolayer of PDL without any Fn. Ngankam et al. analyzed Fn adsorption to poly(allylamine hydrochloride) (PAH)/PSS films and found the extent of adsorption to be significant: films of mass 1.1 and 0.5 μg/cm2 were observed on PAH and PSS terminated films, respectively, with considerable surface aggregation present in the latter system [38]. Kreke et al. investigated osteoblast adhesion strength, via a flow-induced detachment assay, onto Fn-coated PAH/heparin sulfate films formed at various values of solution pH [39]. Although Fn adsorption was modest (always less than 0.01 μg/cm2), they observed the force required for cell detachment to scale with quantity of adsorbed Fn. Li et al. found a Fn coating to enhance smooth muscle cell attachment and growth on a PAH/PSS film, and the degree of enhancement to increase with the number of layers [40]. In contrast, Olenych et al. found only a minor influence of adsorbed Fn on the spreading and motility of smooth muscle cells cultured on multilayer films ranging from cell adherent to cell resistant [41].

We investigate here the formation and cell attachment properties of Fn-terminated multilayer films composed of the linear polyelectrolytes poly(l-lysine) (PLL) and dextran sulfate (DS). Films composed of biological polymers, such as polyamino acids and polysaccharides, are ideal for in vivo applications (e.g. tissue engineering) owing to their biological origin and biodegradability. We employ optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microgravimetry with dissipation (QCMD) to measure the kinetics of LbL assembly and Fn adsorption, and phase contrast optical microscopy to analyze the attachment and spreading of human umbilical endothelial cells (HUVEC) in contact with multilayer films. Our goal is to understand the time-dependent morphological response of a model cell system—in contact with a multilayer film composed of polyelectrolytes of biological origin—in terms of number of polyelectrolyte layers, the charge of the terminal layer, and the presence of Fn.

Section snippets

OWLS

OWLS is a highly sensitive method (precision ∼1 ng/cm2) for measuring macromolecular adsorption kinetics at the solid–liquid interface [42], [43], [44], [45]. Detection is based on excitation of guided modes via a polarized laser light beam directed upon a grating coupler at the surface of an optical waveguide. The mass and thickness of an adsorbed layer can be related to changes in the guided modes through an optical model, such as one assuming an optically uniform adsorbed layer [46]. Solvent

Multilayer film formation

Fig. 2A shows OWLS and QCMD measurements of film mass versus time for the LbL growth of PLL–DS films, as described in Section 2. OWLS provides a measure of the polymer contribution to the film mass (i.e. the “dry mass”); the increase in dry mass is essentially irreversible during both PLL and DS adsorption steps, and plateau levels scale roughly exponentially with layer number. (Schaaf, Voegel, and co-workers have recently linked exponential film scaling to intra-film mobility of at least one

Discussion

We investigate the LbL film formation of a polyamino acid/polysaccharide multilayer film, the adsorption of the matrix protein Fn to films composed of various numbers of layers, and the time-dependent morphological changes of a model cell system (HUVEC) as functions of layer number, charge of terminal layer, and the presence of Fn. The PLL/DS multilayer film grows exponentially with layer number, probably due to the intra-film mobility of PLL, and contains a significant quantity of water, most

Conclusion

We investigate the LbL formation of a multilayer film composed of polyelectrolytes of biological origin; the adsorption of the matrix protein Fn to these films; and the morphological response of a model cell system in terms of number of polyelectrolyte layers, the charge of the terminal polyelectrolyte layer, and the presence of Fn. We find Fn to adsorb more strongly to (positively charged and less hydrated) polycation-terminated films, and for cells to generally spread—to a greater extent and

Acknowledgements

We gratefully acknowledge the National Institutes of Health for financial support through R01-EB00258.

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