Study On The Effect Of Surface Modification On The Biocompatibility Of316L Stainless Steel Via Grafting Methoxy Poly(ethylene Glycol)

Background:Coronary heart disease (CHD) is a major cause of human death, andcoronary stent implantation had become the preferred method for thetreatment of the patients with severe CHD. In2013the number of globalpercutaneous coronary intervention (PCI) is4.3million including morethan400,000in China. However, endovascular stents still havedeficiencies. The first generation of stent is bare metal stent (BMS) with ahigh rate of in-stent restenosis because of vascular endothelial cellproliferation. The introduction of carrier (polymer) and drug system ofthe second-generation drug-eluting stents (DES) inhibits the neointimahyperplasia with antiproliferative agents in the polymeric coating;however, it requires long-term dual antiplatelet therapy and has the risk of(very) late stent thrombosis associated with incomplete stentendothelialization, long-term endothelial dysfunction, chronic arteryinflammation, allergic reaction etc. The third-generation drug-elutingstents are mainly bioabsorbable stents, which are concerned about acutestent recoil and radial strength. Therefore, the development of coronarystent with good radial strength and biocompatibility is an urgent task.AISI316stainless steel (SS) has been extensively used as stentmaterials owing due to its excellent corrosion resistance and superiormechanical properties in medical field. The surface modification byhydrophilic monomers, such as PEG molecule, can enhance thehemocompatibility of biomedical devices and may be the answer to the allergic reaction, tera-togenic, and carcinogenic dangers caused by thereleased nickel ions. Among the hydrophilic molecules, polyethyleneglycol (PEG) molecules, linear or branching polyether polymercompounds, are composed of ethylene glycol monomer polymerizationwith hydroxyl end. PEG is the most widely used for the surfacemodification, because of its unique properties such as hydrophilicity, highexclusion volume in water, nontoxicity, and nonimmunogenecity. Whenthe PEG molecules are combined with other molecules or materials, theunique property will be transferred to the new materials. Some studiesalso prove that PEG is the most effective molecules in protecting thesurface against the protein adsorption and bacterial adherence because ofthe hydrophilic molecular and the large excluded volume.Surface modification methods vary from physical adsorption tocovalent attachment to the surface. The covalent attachment method is amore promising method with a better stability, which is an essentialcharacter for blood-contact devices. A number of strategies have beenintroduced for coating stainless steel, including surface silanization withepoxy precursors followed by grafting of PEG chains, PEG spin-coatingwith RF-plasma mediated crosslinking and polyethyleneimine thin filmsmodified with aldehyde-terminated PEG chains. Covalent attachmentwith PEG de-rivatives is the most advantageous method for the simpleprocedure and stable attachment. Therefore, the surface modification ofstainless steel by mPEG through covalent attachment is indispensable inenhancing biocompatibility.The purpose of research is testing the effect of mPEG layer modifiedon the surface of stainless steel on the biological compatibility and lay theexperimental and theoretical basis for the new type of drug-eluting stents.Materials and methods：(i) Synthesis of silanized mPEG: Silanized mPEG was prepared according to the literature procedure. mPEG (0.01mol) were dried underreduced pressure at110C for2h and then dissolved in200mL of THFin a500mL three-neck round-bottom flask under dry nitrogenatmosphere. IPTS (0.025moL) and dibutyltin dilaurate (0.001moL) weresequentially added to the mPEG solution. The resulting mixture wasstirred continuously for48h under dry nitrogen. Silanized mPEG wasprecipitated from THF with hexane twice after the reaction and driedovernight in vacuo, and characterized by FTIR and1H-NMR.(ii) Modification of SS by mPEG: AISI316SS sheets (10×10×2mm3) were cleaned by sequential sonication in acetone, ethanol andMilliQ water after polished, and then, immersed in15%HCl overnight.The SS were immersed in piranha solution for20min. The steel sheetswere cleaned thoroughly by rinsing with MilliQ water, followed byblowing with nitrogen, and then immediately immersed in the silanizedmPEG solution (50mg/mL) for3h. The silanized mPEG solution wereprepared by dissolving them in a ethanol-water solution (95:5V/V%),and the pH of the solution was adjusted4.5with acetic acid.The solutionwas stirred for24h. The grafted samples were sequentially rinsed withdistilled water and ethanol before curing at110C for1h. Each samplewas washed thoroughly with ethanol–water solution in an ultrasonic bathfor5min to physically remove the adsorbed silanized mPEG and driedunder nitrogen. The silanized mPEG grafted on the SS surface wascharacterized by contact angle, X-ray photoelectron spectroscopy (XPS)and atomic force microscopy (AFM).(iii) The biocompatibility of the mPEG modified SS was evaluatedwith fibrinogen adsorption, platelet activation and adhesion, humanumbilical vein endothelial cell (HUVEC) adhesion and cytotoxicity test. Results:(i) Silanized mPEG was synthesized directly by coupling IPTS tomPEG with dibutyltin dilaurate as a catalyst. The silanized mPEG weregrafted onto the SS surface through Si-O bonds.(ii) The results obtained from X-ray pho-toelectron spectroscopy(XPS) confirm that the mPEG modified steel contained more C/Si andless Fe/Cr on the surface. the analysis of the O1sand C1speak exhibitedincreased hydrocarbon and decreased metal oxides. The hydrophilicity ofthe SS was improved obviously confirmed by contact anglegoniometry.Atomic force microscopy (AFM) exhibited a morphologicalchange and decrease in the contact angle.(iii) In vitro biocompatibility test by fibrinogen absorption, plateletactivation and adhesion showed fibrinogen absorption, plateletactivation,and adhesion were clearly suppressed on the surface modifiedsteel. Human umbilical vein endothelial cell (HUVEC) could adhere andproliferate on the surface of the mPEG-modified stainless steel and themodification of SS sheet with mPEG increases nontoxicity.Conclusion：(i)316L SS surfaces could be successfully modified with thesilanized mPEG through Si-O bonds.(ii) The surface morphology and hydrophilicity changed obviouslyafter the mPEG modification, which improved biocompatibility.(iii) The mPEG layer could suppress fibrinogen adsorption, plateletactivation and adhesion and support HUVEC adhering and proliferatingon the surface, showing an excellent biocompatibility.