The Evaluation Of Hydroxybutyl Chitosan As Cardiovascular Stent Coating

Coronary artery disease is always caused by blockages of coronary arterieswhich was responsible for approximately20%of all deaths in2005in the UnitedStates. The percutaneous transluminal coronary angioplasty (PTCA) was introducedfor the management of coronary artery disease which used together with a stent tomaintain the shape of vascular. The stent was developed from bare-metal stents (BMS)to drug-eluting stents (DES) which could release functional drugs to the artery, but thesafety of DES is also doubtful, it may lead to the in-stent restenosis (ISR) or stentthrombosis. Therefore, it is urgent to develop a new stent to avoid these side effects.In this paper, we prepared a chitosan derivative, hydroxybutyl chitosan (HBC),by etherifying modification method, the product is water soluble and have thetemperature-sensitive property of gelling. Here, we measured its physic-chemicalproperty, and evaluated the cytocompatibility of the solution, the possibility to be usedfor3-dimentional cell culture and used as the coating of cardiovascular stent.In this study, HBC was synthesized in the alkaline conditions by the reaction ofchitosan and1,2-epoxy butane, three HBC materials were prepared at differentreaction conditions which have different molecular weight (MWs),290kDa,530kDaand960kDa, respectively. The rheological measurement was used to determine thegel temperature (Tgel) of HBC. HBC of290kDa could not transform into gel even thetemperature was up to50°C, while the530kDa could transform into gel at40°C and960kDa at26°C, the gelling happened without any crosslinking agent and the phasetransformation was reversible, with the increase of MWs, the gelling was more easilyand the Tgelwas lower. SEM was used to observe the internal morphology of thehydrogel and a porous network structure was observed, with a smooth and dense porewalls and the size of the pore was about50-200μm, with the increase of MWs, thepore was more regular and tight.MTT assay and NO determination were used to evaluate the cytocompatibility of HBC water solution and the results shown that HBC solution could promote theproliferation of Human Umbilical Vein Endothelial cells (HUVECs) and the secretionof NO. In detail, for different MWs,290kDa,530kDa and960kDa, with theincrease of the MWs, the promotion effect to the proliferation was enhanced and theamount of NO released was increased; for different concentration of HBC solution,25,50,250,500and1000μg/ml, with the increase of the concentration, the promotioneffect to the proliferation was enhanced and the amount of NO released was increasedtoo; for different acting time,24h,48h and72h, with the increase of the acting time,the effect was the same like the MWs and the concentration. In a word, HBC solutionhas good cytocompatibility to HUVECs.HBC hydrogel was used for cell culture to evaluate the feasibility as a3-dimentional matrix. HUVECs were inoculated on the surface of hydrogel, Ca-AMwas used to stain cells and fluorescence pictures were taken to show the morphologyof cells. Cells on the surface of the hydrogel appeared normal round or polygonalshape but with some extent of aggregation, cells could be passaged and growth well.The gelling of HBC was temperature-sensitive and reversible which could be used forthe3-dimentional culture of cells. At low temperature, cells was added into HBCsolution (3%wt), and then increase the temperature to37°C, HBC solutiontransformed into gel and cells were loaded inside the hydrogel which could becultured in a3-dimentional condition. Cells were stained and the fluorescenceobservation shown that cells could grow well in the hydrogel with normalmorphology and proliferation activity. The reversible phase transformation could beuse to passage the cell which avoid the use of trypsin.The platform of cardiovascular stent, Co-Cr alloy, was treated with strongalkaline to make the surface unsmooth which could benefit the adhesion of HBCcoating. HBC (0.3%wt) was coated on the surface of treated metal for5times to get acompact coating. The coating was intact after immersed in Hanks’ for21days withshake. The hemolysis rate of coated surface was less than bare metal and thecoagulation time was prolonged, these two assay illuminated that the coated stenthave good hemocomatibility than bare metal. HUVECs were inoculated onto the surface of bare metal and HBC coated stent, after several days culture, fluorescenceobservation was performed, cells on the coated surface shown a low beginningadhesion but a quick proliferation rate than bare metal, which lead to the same cellnumber after5days culture, but cells on the coated surface appear some extent ofaggregation which need more deeper study. Consequently, HBC have the potential tobe used as cardiovascular stent.