Preparation Of Small-diameter Heparinized Arterial Grafts With The Potential Of In Situ Vascular Regeneration

Cardiovascular disease (CVD), which threatens the health of mankind heavily,isone of the leading reasons for mortality and morbidity in the world. In recent years,with the improvement of people，s living level, the change of diet structure, and theaging of the population, the morbidity of CVD is a rising trend year by year. Vasculartransplantation using autografts is an effective strategy and has been accepted as the"gold standard". But this strategy has often been limited by availability or shortage ofdonors. Therefore, there is an urgent demand for artificial grafts as substitutes. Thefunction of artificial vascular grafts in clinical application is adequate for using inlarge-diameter graft (＞6mm), but poor when used in procedures requiringsmall-diameter vascular grafts (＜6mm), such as coronary artery and peripHeralartery. Beacause the small-diameter vascular have many disadvantages, such asthrombus formation and following restenosis. In addition to the above architecturalfeature, grafts for vascular tissue engineering should be biocompatible,nonimmunogenic, and should possess appropriate biomaechanical properties. In thisstudy, decellularized vascular graft is prepared from umbilical artery via theenzyme-detergent decellularization. Heparinized vascular graft is prepared fromdecellularized vascular graft via cross-linking agent EDC and NHS.Methods:1. The preparation of decellularized vascular graft: Umbilical arteryswere treated by enzyme-detergent decellularization. Tissue samples were thenobserved by HE staining and Hoechst staining to confirm the removal of cells andECM.2. The preparation of heparinized vascular graft: Decellularized vascular graftand heparin were crosslinked by EDC and NHS, which was confirmed by toluidineblue staining and ATR-FTIR.3. MorpHology and structure of umbilical arterys,decellularized vascular graft and heparinized vascular graft were observed by HEstaining, Masson trichrome staining and VG staining; the lumen microstructure ofthese grafts were observed by scanning electron microscope(SEM); the retention ECM of these grafts were analyzed by determination of collagen and proteoglycan;the bomechanics of the dealed grafts were evaluated by breaking force and tensilecurve.4. The blood compatibility of heparinized vascular graft was observed byplatelet adhesiveness test and AV-shunt.Results:1. From the HE staining and Hoechst staining, we can seedecellularized vascular graft was completely cell free, and the structure of ECM wasretained completely.2. From toluidine blue staining and ATR-FTIR, we can seeheparin was immonilized on the graft succesfully, using EDC and NHS as cross-linker.3. From the HE staining, Masson trichromestaining and VG staining, the ECMstructure,collagen and elastic fibers of heparinized vascular graft were well preserved;From SEM, the lumen of grafts became smooth from porous structure, viaheparinization processing; Heparinized vascular graft were well kept the maincomposition of natural vascular ECM by determination of collagen and proteoglycan;The mechanical properties of heparinized vascular graft were well retained comparedto umbilical artery and decellularized vascular graft.4. Heparinized vascular graftshowed better blood compatibility than decellularized vascular graft via plateletadhesiveness test and AV-shunt.Conclusions:1. A small–diameter decellularized vascular graft were preparedsuccessfully in vitro via enzyme-detergent decellularization.2. A small–diameterheparinized vascular graft were prepared successfully in vitro via crosslinking heparin.3. The heparinized vascular graft remain original vascular structures, the mainECM（including collagens, proteoglycan and elastin），and mechanical properties.4.The effect of the heparinized vascular graft on the anti-thrombosis property wasconfirmed by in vitro and ex vivo test.