Enhancement of the growth of human endothelial cells by surface roughness at nanometer scale

This study investigated whether a nanometer scale of surface roughness could improve the adhesion and growth of human endothelial cells on a biomaterial surface. Different molecular weights or chain lengths of polyethylene glycol (PEG) were mixed and then grafted to a polyurethane (PU) surface, a model smooth surface, to form a nanometer (nm) scale of roughness for PU-PEG surfaces (PU-PEG_mix) while PEG with a molecular weight of 2000 was also grafted to PU to form PU-PEG₂₀₀₀ for comparison. In addition, the concept was tested on cell-adhesive peptide Gly-Arg-Gly-Asp (GRGD) that was photochemically grafted to PU-PEG_mix and PU-PEG ₂₀₀₀ surfaces (e.g., PU-PEG_mix-GRGD and PU-PEG ₂₀₀₀-GRGD surfaces, respectively). To prepare GRGD-grafted PU-PEG_mix and PU-PEG₂₀₀₀ surface, 0.025M of GRGD-SANPAH (N-Succinimidyl-6-[4′-azido-2′-nitrophenylamino]-hexanoate) solutions was grafted to PU-PEG_mix and PU-PEG₂₀₀₀ by surface adsorption of the peptide and subsequent ultraviolet (UV) irradiation for photoreaction. The grafting efficiencies for GRGD to PU-PEG_mix and PU-PEG₂₀₀₀ surfaces were about 67% for both surfaces, semi-quantitatively analyzed by an HPLC. The surface roughness, presented with a roughness parameter, R_a, and the topography of the tested surfaces were both measured and imaged by an atomic force microscope (AFM). Among the R_a values of the films, PU was the smoothest (e.g., R _a=1.53±0.20nm, n=3) while PU-PEG_mix was the roughest (e.g., R_a=39.79±10.48nm, n=4). Moreover, R _a values for PU-PEG_mix and PU-PEG_mix-GRGD surfaces were about 20nm larger than those for PU-PEG₂₀₀₀ and PU-PEG₂₀₀₀-GRGD, respectively, which were consistent with the topographies of the films. Human umbilical vein endothelial cells (HUVECs) were adhered and grown on the tested surfaces after 36h of incubation. Among the films, HUVEC's adhesion on the surface of PU-PEG_mix-GRGD was the densest while that on the surface of PU-PEG₂₀₀₀ was the sparsest. Also, the adhesion and growth of HUVECs for the roughness surfaces were statistically significantly better than that of smooth surface for both GRGD grafted and un-grafted surfaces, respectively. The viability for the growth of HUVECs on the tested surfaces analyzed by MTT assay also confirmed the efficacy of the increased surface roughness. In conclusion, increased surface roughness of biomaterial surfaces even at 10-10²nm scale could enhance the adhesion and growth of HUVECs on roughness surfaces that could be useful for applications of tissue engineering.