The effect of surface microtopography of poly(dimethylsiloxane) on protein adsorption, platelet and cell adhesion

https://doi.org/10.1016/j.colsurfb.2009.02.018Get rights and content

Abstract

Chemical homogeneous poly(dimethylsiloxane) (PDMS) surface with dot-like protrusion pattern was used to investigate the individual effect of surface microtopography on protein adsorption and subsequent biological responses. Fibrinogen (Fg) and fibronectin (Fn) were chosen as model proteins due to their effect on platelet and cell adhesion, respectively. Fg labeled with 125I and fluorescein isothiocyanate (FITC) was used to study its adsorption on flat and patterned surfaces. Patterned surface has a 46% increase in the adsorption of Fg when compared with flat surface. However, the surface area of the patterned surface was only 8% larger than that of the flat surface. Therefore, the increase in the surface area was not the only factor responsible for the increase in protein adsorption. Clear fluorescent pattern was visualized on patterned surface, indicating that adsorbed Fg regularly distributed and adsorbed most on the flanks and valleys of the protrusions. Such distribution and local enrichment of Fg presumably caused the specific location of platelets adhered from platelet-rich plasma (PRP) and flowing whole blood (FWB) on patterned surface. Furthermore, the different combination of surface topography and pre-adsorbed Fn could influence the adhesion of L929 cells. The flat surface with pre-adsorbed Fn was the optimum substrate while the virgin patterned surface was the poor substrate in terms of L929 cells spread.

Introduction

Protein adsorption, the first response after biomaterials contacting the biological environment, plays a crucial role in mediating the subsequent reactions such as platelet adhesion and in determining the final biocompatibility of biomaterials [1]. Besides, the designs of biosensor [2], medical diagnosis device and drug delivery vehicle should also take protein adsorption into account. Therefore, it is vital to investigate and understand the interaction between protein and material's surface.

During the past several decades, many efforts have been focused on controlling protein adsorption to biomaterials by chemical modification [3], [4], [5], [6], [7], whereas fewer reports have been involved with the simplex effect of surface topography on protein adsorption. In fact, protein adsorption depends not only on the surface chemistry but also on the surface topography. Due to the complexity of the surface properties, it is difficult to separate the effect of surface topography from that of surface chemistry. Very recently, coarse surfaces with distinct roughness [8], silica particles with various diameters [9], [10] and titanium surfaces with particular patterns [11] were used as model substrates to investigate the impact of surface topography on protein adsorption. These studies indicated that chemical homogeneous surfaces with nanotopographical features including roughness, curvature and geometrical figures, could affect protein adsorption behavior [12].

However, large numbers of polymeric biomaterials have micro-scale surface topographies or patterns. These surface features are either unconsciously introduced during material processing or intentionally fabricated for biomedical application such as scaffolds used in tissue engineering. Plenty of researches are focused on the relationship between surface microtopography and cell behaviors including adhesion [13], growth [14], differentiation [15], etc. Nevertheless, protein adsorption, the first response after surface microtopography contacting the biological environment, is being overlooked probably due to the standpoint that micro-scale topography is indistinctive to nano-scale individual protein [16]. When considering the vast number of proteins involved in adsorption, the complicated adsorption process, and the fourth dimension-time [17], surface microtopography might influence cell behaviors by virtue of its initial effects on previous adsorbed protein. Thus, it may be an effective way to clarify this intricate relation by tailoring specific micropattern bearing homogeneous chemical composition on biomaterials surfaces.

Based on the above hypothesis, we proposed a simple and straightforward method to investigate the individual effect of surface mircotopography on protein adsorption and subsequent biological response. Microtopography on poly(dimethylsiloxane) (PDMS), a widely used biomaterial, was fabricated as the method used in soft lithography technique [18]. Instead of being used as a stamp to transfer spheres [19], [20], [21], chemical and biological molecules [22], PDMS with specific microtopography but homogeneous chemical composition was directly utilized as substrate. Fibrinogen (Fg) and fibronectin (Fn) were used as model proteins due to their pivotal effects on platelet and cell behaviors. Fg adsorption and subsequent platelet adhesion from platelet-rich plasma (PRP) and flowing whole blood (FWB) to patterned PDMS surface were studied. In addition, the effect of patterned PDMS surface on adsorbed Fn was indirectly evaluated in terms of the morphology of adherent L929 cells.

Section snippets

Materials and reagents

PDMS from the Sylgard 184 silicone elastomer kit (Dow Corning, Midland, MI) was used to prepare substrates. Fibrinogen (Plasminogen-Depleted, Human Plasma) was obtained from CalBioChem (CAS No. 9001-32-5). Fibronectin (From human plasma) was purchased from Sigma (Product No. F2006). Na125I was bought from China Isotope Corporation. Fluorescein isothiocyanate (FITC), penicillin, streptomycin, trypsin and trypanblau were purchased from Amresco. Sephadex G-50 was acquired from Sigma–Aldrich.

Surface characterization

PDMS is an excellent material for biomedical applications [25] and widely used as stamp to form chemical or biological heterogeneous patterns on many substrates [22]. Herein, patterned PDMS surface with dot-like protrusions was prepared as shown in Fig. 1 and its dimensional parameters were summarized in Table 1. Spontaneously, the chemical composition of resulting surface was identical. Obviously, when the patterned surface directly contacts protein solution, the individual effect of surface

Conclusions

In conclusion, chemical homogenous micropatterned PDMS surface was prepared by a universal and simple method. The amount and distribution of adsorbed Fg were influenced by such microtopography, suggesting that microtopography could also affect protein adsorption and should not be neglected. The location of platelets adhered from PRP and FWB on patterned surface followed the similar pattern of adsorbed Fg. In addition, the surface topography and pre-adsorbed Fn influenced the morphology of

Acknowledgments

The authors thank John Brash's group in McMaster University for help with flowing whole blood experiment. This work was financially supported by the National Natural Science Foundation (20534040, 90606013, 20634030), the Ministry of Education (107080), and the Ministry of Science and Technology of China (2008CB617510).

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