Experimental Study Of Endothelial Prohealing Drug Eluting Stent

Part I Studies on Biocompatibility between Chitosan Coat and CD133+ Endothelial Progenitor CellsObjective To investigate the effect of chitosan/heperin layer-by-layer self assembly coat film to the adhesion,proliferation and differentiation of CD133+ endothelial progenitor cells(EPCs), and to use molecular biologial technique to investigate its feasibility as coating material of drug eluting stent.Methods (1) Use gravity gradient centrifugation to isolate mononuclear cells from human umbilical cord blood, and use magnetic activated cell sorting to sort CD133+ cells. (2) To fabricate chitosan/heperin layer-by-layer self assembly coat film,1% gelatin coat and bare glass cell culture dish as control group, then culture the cells. (3) Immunofluorescen assessment of the cultured cells.(4) To extract RNA from the cells, and to use RT-PCR method to test mRNA expression levers of eNOS,VE-cadhern,KDR,PECAM-1,Sirtuin-1,Thrombomodulin in different culture mediums.Results (1) Gravity gradient centrifugation and magnetic activated cell sorting methods can obtain pure CD133+ EPCs, and can well different to endothelial cells induced by VEGF; (2) Compared with bare glass, Chitosan coating film can significantly promote the adhesion,proliferation and differentiation of EPCs.Conclusion Chitosan coating film can promote the adhesion, proliferation and differentiation of EPCs, with good biocompatibility, and can be a promising biomaterial.Part II The Preparation and Related Properties Study of Chitosan /Heparin Layer-by-layer Self Assembly (LBL) and LBL-Drug StentsObjective To prepare chitosan / heparin layer-by-layer self assembly stent (LBL), LBL-Drug stent, and to test its physical and chemical properties as mechanical properties, toughness, hydrophilic, drug release characteristics, and cell adhesion abilities.Methods (1) To prepare LBL stent by soaking method, and prepare Rapamycin eluting stent by spraying method, and prepare LBL-Drug stent by asymmetric coating method; (2) To observe the performance of stents by observing the stents after pression and expansion pressure via electron microscope, performing stent tough experiment, determining the stents contact angle, performing drug release test of LBL-Drug stents in vitro, and endothelial cell adhesion in vitro experiments.Results (1) Successfully prepare chitosan / heparin layer-by-layer self-assembly (LBL) stent, LBL-Drug stent, and test its physical properties and of drug release characteristics. The properties of our prepared stents meet the requirements of stent production process. The coating of the stent is smooth and and well-distrubuted without cracking after pression and expansion. No significant difference in the expansion between the dry and wet conditions. The contact angle analysis shows that the hydrophilicity of stents enhenced after self-assembly technique treatment, with good biocompatibility. (2) The sirolimus releasing characteristics of LBL-Drug eluting stent is explosive release curve characteristics, and conform releasing characteristics of the current on sale rapamycin drug-eluting stent. (3) In vitro cell adhesion test shows thar the cell adhesion capacity of the stent is enhanced by LBL processing treatment, and stent coating material has good biocompatibility.Conclusion (1) The coronary stent technology meets the basic requirements of scaffold preparing technology; (2) Both the hydrophilicity and biocompatibility of stent enchanced by the LBL processing handling; (3) By the LBL processing handling, the stent gain the ability of promoting cell adhesion, and can promote early endothelial repairment. Part III Animal experimental study on efficacy and safety of promoting endothelial repair drug-eluting stentObjective To observe the efficacy and safety of promoting endothelial repair drug-eluting stent in the minipigs model.Methods (1) The BMS,LBL,Drug and LBL-Drug stents were implanted in pig left anterior descending, circumflex and right coronary artery, formed by over-expansion model of coronary injury. stent endothelial coverage, intimal hyperplasia, implant site inflammation were observed after 1 week,1 month,3 months and 6 months of operaion; (2) To evaluate endometrial hyperplasia 6 month by intravascular ultrasound after 6 month of operation; (3) At the time of 1,3 and 6 month follow-up coronary angiography after coronary injection of acetylcholine, endothelial function was evaluated by observe endothelium-dependent vasoconstriction.Results 1. Each group has a different rate of stent endothelial coverage. LBL and LBL-Drug group have the fastest coverage speed, and endothelial begin to cever after 1 week which is faster than BMS group. And Drug group has the lowest endothelial cover rate. At 1 month, all group have complete endothelial cover, of which, LBL group and LBL-Drug group have relative smooth and regular endothelial compared with BMS group and Drug group. And endothelial hyperplasia leads to some extent of stenosis in BMS group. 2. By pathological observation and computer-aided measurement, Drug-LBL group and the Drug group have wider lumen area at 1 month, 3 months and 6 months, when compared with BMS group and LBL group,the difference is statistic significance(P<0.05), and no difference between BMS group and LBL group; compared with BMS group and LBL group, LBL-Drug group and Drug group have lower neointima area and neointima thickness at 1,3 and 6 months, and the difference is significance (P<0.05), while the BMS group and LBL group are no significant difference (P> 0.05). Stenosis rate at 1 month,3 months and 6 months of LBL-Drug group and Drug group are lower than the other two groups, the difference is statistically significant (P<0.05), BMS group was the highest of 4 groups.3. Judging from the infiltration of inflammatory cells,1 month Drug stents has the most severe inflammatory infiltration, with a highest inflammation score. Inflammation score of each group at 1 month are:BMS 0.71±0.69, LBL 0.5±0.5, Drug 1.71±0.69, LBL-Drug 0.71±0.69. Drug group has the highest inflammation score, the difference is statistically significant (P <0.05), other groups have no significant difference (P> 0.05), At 3 months and 6 months, the infiltration of inflammatory cells reduced when compared with 1 month, the inflammation score in each group is no significant difference (P<0.05); 4. At 6 month, IVUS examination revealed BMS group of significant in-stent neointimal hyperplasia and stenosis rate of 60%, LBL Group only mild in-stent neointimal hyperplasia, no significant intimal hyperplasia in Drug stent group and LBL-Drug stent group; 5. Acetylcholine endothelium-dependent vasoconstriction observation test suggests that, each stent group is affected most notable by acetylcholine at 3 months. Therefore, the response begins to recover at 6 month. The endothelial function effect in Drug group is of the largest and longestm of 4 groups, while the LBL coating stents with endothelial function is affected relatively small, and at 3 months, begin to recover, and recovered at 6 months. The endothelial function effect in LBL stent is little and the recovery is earlier compared with other groups.Conclusion (1) The implantation of prepared coronary stent of the four groups of in the minipig model is feasible. evaluate the safety and efficacy of the stents by 1-6 months of follow-up. (2) At the time of 1 month,3 months and 6 months, LBL-Drug group and Drug group have a wider lumen area and lower stenosis rate compared with BMS group and LBL group, and their neointimal area and neointimal thickness are less than that of the BMS group and LBL group. At 1 month after implantation, Drug group has the greatest inflammatory response. Compared with BMS,LBL and Drug stent, LBL-Drug Stent has a relatively complete and regular intimal coverage with no in-stent restenosis, and inflammatory reaction around the stent is slight,which can be thought to a satisfactory results. (3) Using asymmetric coating technology (LBL-Drug stents), inner layer of stent can promote endothelial cells repairment, and outer layer of stent apply of biodegradable scaffold and anti-proliferation of materials, which can inhibit smooth muscle cells proliferation and reduce the inflammatory response, with good biocompatibility. These animal datas will provide the development of prohealing endothelial stent with core parameters, and will make a theoretic and experimental basis for further research and application.