# Heparin-Eluting Tissue-Engineered Bioabsorbable Vascular Grafts
## 4 Discussion  
This review describes two methods to improve small-diameter arterial graft patency. One, the heparin conjugation method, improves the patency of grafts made from bioabsorbable materials. The second is a method of extending the suspended release period of heparin to maintain the graft patency rate for a longer time. Adequate and sustained heparin release is considered a means of increasing graft patency, while the long-term endothelialization process is completed. Heparin has been shown to prevent thrombosis associated with thrombin suppression, inhibition of vascular smooth muscle cell (VSMC) proliferation, and prevention of graft occlusion [61; 62], but that is not all. Heparin can also stabilize physiologically unstable growth factors in the heparin-binding domain [63; 64]. Heparin has the potential to overcome the challenges associated with growth factor instability in solution, self-aggregation, low bioavailability, short half-life in vivo, and in vivo delivery [65; 66; 67]. This has led to many advances in the patency of the smallest diameter tissue-engineered arterial grafts. In addition, to test the efficiency of TEVG grafting in these studies, small animal models are not suitable for preclinical testing due to the differences in metabolism and arterial pressure from humans. It is necessary to test the efficacy and safety of these materials in large animal models such as sheep or pigs prior to clinical trials. In fact, in this review, we have focused on experiments performed on large animal models. Although we have no choice but to use animals, we must always consider the three Rs of animal use (reduction, refinement, and replacement) in order to protect the animals. For instance, adequate mechanical strength should be verified before implantation in the animals. Before implanting the grafts to the animals, some in vitro tests need to be performed. For example, the tensile strength test alone is not sufficient to ensure that the graft will not cause an aneurysm in an arterial environment. Thus, beating tests simulating arterialpressure [68], and evaluation of heparin release using the toluidine blue method must be performed [27; 28]. In vitro evaluations of biocompatibility, such as platelet deposition on the luminal surface of the graft and the LDH assay [26], are necessary. For the biodegradability, although different reagents are used for different polymers, we should verify the degradation rates of different biodegradable grafts using serial accelerated degradation studies [69]. Due to the nature of tissue engineering grafts, some studies expect neo-tissue formation using materials with fast degradation rates [70; 71]. In the arterial environment, these grafts tend to dilate relatively quickly. Hence, materials with a longer degradation period are preferable at this point. Animal studies should be performed with grafts that meet the above in vitro criteria. Many studies have shown that endothelialization is important to maintain graft patency. However, these studies shown transanal endothelial growth at most of 1 to 2 cm [44]. This might be the reason why TEVG has been relatively successful in experimental models such as sheep carotid or porcine femoral arteries, where 2-5 cm prostheses have been evaluated (currently, the recommended length of TEVG is limited to 10 times the diameter (4-6 cm for ovine carotid grafts), but clinically, coronary and lower limb bypass graft prostheses are, on average, 15 cm in length [72]. Hematogenous or percutaneous endothelialization is a time-consuming process, and large-animal experimental designs need to use grafts \(>\) 5 cm in length to validate clinical importance. After grafting, if patency can be maintained without graft dilatation, long-term observation for about one year is desirable. In regenerative medicine research, the assessment of tissue regeneration and inflammation typically requires animal sacrifice and graft harvesting for histological evaluation.