Micropattern-Immobilization Of Heparin And Their Effects On Cell Behavior In Vitro

The anatase titanium oxide films (Ti-O) were synthesized on silicon substrates by Unbalance Magnetron Sputtering. The Ti-O was modified by 3-aminopropylphosphonic acid (APPA), and tested by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), water contact angle, scanning electron microscopy (SEM) and Quartz crystal microbalance (QCM). In addition, heparin was modified by 4-azidoaniline to obtain photoreactive heparin, which confirmed by ultraviolet absorbance spectra and FTIR. The heparin was immobilized on APPA modified Ti-O (APPA/Ti-O) using a UV lamp with a wavelength of 365 nm. The amount of heparin immobilized on the surface was determined using the toluidine blue assay. And activated partial thromboplastin time (APTT) was tested in order to optimize the concentration of photoactive heparin solution. The patterns of heparin with four different sizes of stripes were fabricated by photo-immobilization of photoreactive heparin on APPA/Ti-O in presence of photomask. The surface morphology of the patterned samples dyed by toluidine blue was examined using optical microscopy and surface profiler. The influences of patterns on the platelets and endothelial cells behaviour were investigated by in vitro tests.After treatment with APPA, two weak peaks of methylene of APPA could be found in FTIR spectrum of Ti-O. It was observed from XPS spectra that surface content of N and P increased. The surface became more hydrophilic, and the particle size of surface diminished. The quality increased after APPA assembly which was tested by QCM. The above results revealed that APPA had assembled on Ti-O surface.After heparin was modified, a maximum absorption assigned to azidophenyl group was observed in the ultraviolet spectrum, and stretching vibrations of C-N3 and Ar-H were found in the FTIR spectrum. The results above confirmed that the azide group had been introduced into heparin molecule.The photoactive heparin with a concentration of 40 mg/ml was used to make heparin patterns, and the non-patterned heparin sample (HEP40) was used as control. Upon UV irradiation, the heparin was immobilized on APPA/Ti-O surface which confirmed by FTIR results. The results of toluidine blue assay showed that the amount of heparin immobilized on HEP40 was 3.8±0.7μg/cm2. The APTT of HEP40 was prolonged compared to plasma. Optical images of patterned heparin showed the patterns were clear and could reproduce the mask pattern precisely. The height of pattern was about 30 nm confirmed by surface profiler.The results of platelets and endothelial cells adhesion showed that APPA modified Ti-O surface could enhance both endothelial cell growth and platelets adhesion. Nonpatterned heparin surface could reduce adhesion and activation of platelets but inhibit the proliferation of endothelial cell as well. Heparin pattern was fabricated in proportion with above two surfaces (APPA and heparin). The patterned heparin samples could reduce the platelet adhesion which represented the similar effect as HEP40. At the same time, the patterned heparin promoted cell adhesion, spreading and proliferation. Due to the compositive effects of two surfaces, the size of pattern could make different effects on the growth of endothelial cells. The micro stripes with a similar size as cell could induce the cells to elongate and arrange along the stripe direction. But the patterns with smaller size were benefit for proliferation of endothelial cell. The experiment of cell adhesive force indicated that the pattern could induce endothelial cells arranged as the direction of flow and improve the adhesive force between the cells and substrate. It may suggest a new modification method for blood-contacting device, providing the functions of both inhibiting thrombus formation and accelerating endothelialization simultaneously.