Relative importance of surface wettability and charged functional groups on NIH 3T3 fibroblast attachment, spreading, and cytoskeletal organization

Abstract

Understanding the relationships between material surface properties, adsorbed proteins, and cellular responses is essential to designing optimal material surfaces for implantation and tissue engineering. In this study, we have prepared model surfaces with different functional groups to provide a range of surface wettability and charge. The cellular responses of attachment, spreading, and cytoskeletal organization have been studied following preadsorption of these surfaces with dilute serum, specific serum proteins, and individual components of the extracellular matrix. When preadsorbed with dilute serum, cell attachment, spreading, and cytoskeletal organization were significantly greater on hydrophilic surfaces relative to hydrophobic surfaces. Among the hydrophilic surfaces, differences in charge and wettability influenced cell attachment but not cell area, shape, or cytoskeletal organization. Moderately hydrophilic surfaces (20-40 degree water contact angle) promoted the highest levels of cell attachment. Preadsorption of the model surfaces with bovine serum albumin (BSA) resulted in a pattern of cell attachment very similar to that observed following preadsorption with dilute serum, suggesting an important role for BSA in regulating cell attachment to biomaterials exposed to complex biological media.

Figure 1

NIH 3T3 fibroblast attachment to model surfaces preadsorbed with 5% serum. Model surfaces are listed along the x axis in order of increasing hydrophobicity as determined by static water contact angle. N = 6 surfaces; * = p < 0.05 relative to oxidized thiol.

Figure 2

NIH 3T3 fibroblast area and aspect ratio on model surfaces preadsorbed with 5% serum. N = 3 surfaces with 21 cells analyzed per surface.

Figure 3

Cytoskeletal organization of NIH 3T3 fibroblasts representative of the predominant morphologies on oxidized thiol, quaternary amine, and amine surfaces (a,c) and thiol and methyl surfaces (b,d). Cells were stained with rhodamine-phalloidin for actin (a,b) and by indirect immunofluorescence for vinculin (c,d). Original magnification 400×.

Figure 4

NIH 3T3 fibroblast attachment to oxidized thiol/thiol surfaces preadsorbed with bovine serum albumin (BSA, 2.1 mg/mL), high density lipoprotein (HDL, 65 μg/mL), immunoglobulin G (IgG, 0.5 mg/mL), type IV collagen (33 μg/mL), fibronectin (FN, 20 μg/mL), and lamina (LN, 20 μg/mL). N = 3 surfaces. * = P < 0.05.

Figure 5

NIH 3T3 fibroblast attachment to model surfaces preadsorbed with 2.1 mg/mL of BSA. N = 3 surfaces. * = P < 0.05 relative to oxidized thiol.

Figure 6

NIH 3T3 fibroblast attachment to model surfaces without preadsorbed protein. N = 3 surfaces. * = P < 0.05 relative to oxidized thiol.