Experimental Research On The Novel Small-caliber Vascular Prosthesis Basing On Controlled Release Of Heparin From Mesoporous Channel

Vascular reconstructive surgery, implanting artificial vessels instead of lesion vessels, is the efficient treatment of cardiovascular disease and peripheral vessel disease; however, the challenge comes from constructing long-term patent small-caliber vascular prosthesis (φ<6mm) that can prevent thrombus formation and intimal hyperplasia, which are the two inevitable causes that yield the restenosis of the vascular reconstruction, and ultimately contribute to long-term failure in vascular prosthesis.In order to meet the clinical needs, developing novel long lasting antithrombotic small-caliber vascular prosthesis to improve the long-term patency is essentially required. Heparin is a routinely used powerful anticoagulant and has been verified to be capable to prevent neointimal proliferation, enhance the biocompatibility; therefore heparinization has been proved to be an obvious and efficient way to improve the performance of artificial vessels. Heparinization can be achieved by ionic or covalent binding but with limited success. A heparin release system is more beneficial for heparinization of artificial surface, and the key step is to achieve the slow and long lasting release of heparin to the surface of grafts without the lost of the activity and the change of spatial conformation.Mesoporous materials own large BET surface area and pore volume, tunable pore size in the range of2-50nm in accord with the molecular size of heparin, the width of which is1～2nm and the length is several nanometer. It can be through space limitation effect originated from mesoporous channels not chemical binding that heparin is adsorbed and released, thus avoiding the demolishment of the activity of heparin.Herein, we develop a new strategy to prepare mesoporous vascular prosthesis and then utilized in heparinization to improve the long-term antithrombogenicity. The study is composed of four parts.Part I. The fabrication and heparinization of heparin-released mesoporous-based vascular prosthesis(HMBVP)Objective To fabricate the mesoporous-based vascular prosthesis and load heparin into mesoporous channel, and to study their properties to offer an experimental foundation for the further differentiation and actual application.Methods Mesoporous vascular prosthesis were prepared by injecting mesoporous solution was injected into ePTFE grafts. heparin-released mesoporous-based vascular prosthesis was realized by soaking into heparin solution. Scanning electron microscopy (SEM) images of ePTFE grafts and MVP-n series were characterized by using HITACHI S4800microscopes. The elemental mapping and EDX analysis were obtained on the above instrument. Nitrogen adsorption isotherms were measured. The amounts of heparin released from HMBVP-n samples were determined by toluidine blue method. The activity of immobilized heparin was determined by chromogenic assay.Results The heparin-released mesoporous-based vascular prosthesis weas synthesized successfully, the SEM images of ePTFE grafts and MVP-n materials show that ePTFE grafts have3D net-linked frameworks composed of wide nodes and thin fibers. Observed both from the outer surface and cross-section of MVP-n materials, mesoprous silica is uniformly coated on the surface as well as the internal fibers throughout the whole ePTFE grafts. X-ray mapping images further clearly confirm the homogeneously dispersion of the particles, meaning the similar dispersion and content of mesoporous silica particles on MVP-n materials. MVP-n materials have large BET surface area and average pore size located in mesoporous range. The three MVP-n materials have same content of mesoporous silica particles but with different textural properties. Due to the introduction of meosopore, large amount of heparin can be adsorbed on MVP-n materials and released continuously for a long time. After a month release, there is still more than75%of heparin remained on each HMVP-n sample, much slower and sustained than previous release system. The chromogenic assay confirmed that heparin-loaded keep lasting activity.Conclusion The heparin-released mesoporous-based vascular prosthesis weas synthesized successfully, realizing long-time release of heparin and sustaining activity of heparin.Part Ⅱ. Biocompatibility research of heparin-released mesoporous-based vascular prosthesisObjective To observe hemocompatibility and Biocompatibility of heparin-released mesoporous-based vascular prosthesis.Methods Static water contact angles on the graft surface were detected for testing material hydrophilicity. Platelet-adhesion assay could be used to detect the activation of platelets and fibrin clots. APTT and PT were determined using a automatic blood coagulation analyzer to evalute long-term anticoagulant activity of bonded heparin of material. The coagulation time of recalcified blood plasma in contact with the sample sheets was determined to study activation of the intrinsic coagulation pathway. Protein adsorption onto the surface of the biomaterial was determined to evaluate hemocompatibility. Percent hemolysis, LDH Cytotoxicity Assay and stent materials in vivo cultivation of experiments were administered to evalution histocompatibility of vascular prosthesis. Results The contact angle of original ePTFE film was decreased from112.3°to16°after heparinization with mesoporous. The hydrophilicity of loaded-heparin mesoporous-ePTFE grafts obviously was improved. The results for platelet adhesion demonstrated that heparin-loaded mesoporous immobilized onto the ePTFE grafts did not increase adherence and aggregation of platelets compared with virgin material. The coagulation time of recalcified blood plasma in contact with the sample sheets and APTT,TT was delayed, indicating a long-term anticoagulan performance of material. The adsorption of fibrinogen on the surface of HMVP-n membranes is effectively reduced compared with unmodified ePTFE graft, but HMVP-n grafts attracted higher amount of albumin than the unmodified ePTFE graft(P<0.05). LDH released from cultured cells was detected for evaluating cytotoxicity of biomaterials. there was not significant difference of OD values between modified material and unmodified material. it is notable that mesoporous-ePTFE grafts contain heparin facilitate the adhesion and growth of tissue cells without cytotoxicity, improving cytocompatibility of the surface of ePTFE graft. The materials in vivo cultivation of experiments show that All tissues had no necrosis without systemic toxicity reaction.Conclusion The heparin-released mesoporous-based vascular prosthesis had good biocompatibility and hemocompatibility.Part III. Endothelialization of heparin-released mesoporous-based vascular prosthesisObjective In vitro the isolation, cultivation, identification and mark of rabbit endothelial progenitor cells (EPCs) were administrated to offer an experimental foundation for the further study to realize endothelialization of heparin-released mesoporous-based vascular prosthesisMethods EPCs were isolated from rabbit heart blood and purified by density centrifuge and adhered to the cultural plastic in vitro. Then, the cells were proliferate by culture. The proliferation and growth characteristics were observed with phase contrast microscope. EPCs quality was evaluated with immunocytochemistry, fluorescent microscope and scanning electron microscope(SEM). The passage3cells were tested which expressed positive for fik-1,vWF and CD34by immunohistochemistry. The flow cytometer was used for characterization of cells phenotype with D14, CD133, CD45, CD31,CD34surface antigen. Adhesion and growth of EPCs on HMBVP-n materials were tested by MTT assay, SEM and adhere rate. EC Seeding and a flow system for shear stability testing was administrated to evalute adhere capability with shear force.Results EPCs had a typical morphological fibroblast and powerful proliferation potential. The primary cells can form clusters and take up acetylated low-density lipoprotein and bind to UEA-I, and they uniformly expressed positive for fik-1,vWF and CD34, indicating that they are indeed endothelial cells. EC-like cells expressed positive for CD14, CD45, while EOCs expressed positive for CD31, CD34. EPCs can produce NO and possess capability of capillary tube formation. Proliferation potential of EPCs seeded in on mesoporous-ePTFE Graft was evaluated by the MTT assay. OD values of EPCs seeded on HMVP-n films was not obviously different from the positive control. However, the OD values of EOCs seeded on the HMVP-1was higher than that on the HMVP-2and HMVP-3. EPCs on the different mesoporous-ePTFE membranes produced similar amounts of NO, while there was few NO production of EPCs on original ePTFE film. All positive fluorescent staining of cells expressed vWF and exhibited EPCs phenotype and characteristic. The confluent EPCs monolayer with CM-Dil fluorescent staining and the SEM images of EPCs proliferation on HMVP-n films was show, implying mesopororous and loaded heparin maintain shear stability and improved cytocompatibility of the surface of ePTFE graft.Conclusion EPCs could be isolated and cultivated through density centrifuge successfully. The phenotypical properties and characteristics in EPCs was tested. EPCs can be adhere well to HMBVP-n materials, keeping excellent morphology and function, heparin-released mesoporous-based vascular prosthesis is promising fo endothelialization of ePTFE. Part IV. Experimental research on abdominal aorta transplantation with EPCs-seeded heparin-released mesoporous-based vascular prosthesis in rabbitsObjective To develop an experimental model of abdominal aorta-Iliac artery bypass procedure by transplantation with heparin-released mesoporous-based vascular prosthesis in rabbits and to detect the change of histomorphology, thrombosis formation and intima hyperplasia of grafts.Methods Rabbits were operated by abdominal aorta-Iliac artery bypass procedure with about40mm with EPCs-seeded heparin-released mesoporous-based vascular prosthesis according to sterile and no-touch doctrines. Histochemical staining and scan microscopy was manipulated to detect the aortas intima. The patency rate of transplanted grafts were detected. Rabbits were killed3,15,30days after operation to observe histopathologic analysis. Thrombosis formation and intima hyperplasia of grafts was abserved by SEM and Masson staining.Results The patency rates of EPCs-seeded heparin-released mesoporous-based vascular prosthesis were significantly higher than that of simple EPCs-seeded ePTFE scaffolds group (66.7%vs0%, P<0.05). Histopathological observation,30days after transplatation, shows that no obvious neontimal and no thrombosis formation could be seen with heparin-released mesoporous-based vascular prosthesis as control group. The endothelial layer had been covered completely by EPCs in test group. The intima hyperplasia for HMBVP-n was obvious lower than that for simple EPCs-seeded ePTFE scaffolds group.Conclusion The patency rates of EPCs-seeded heparin-released mesoporous-based vascular prosthesis group were significantly higher than that of simple EPCs-seeded ePTFE grafts group. These results suggest EPCs-seeded heparin-released mesoporous-based vascular prosthesis could be excellent scaffolds for small calibre vascular prosthesis.