# Heparin-Eluting Tissue-Engineered Bioabsorbable Vascular Grafts
## 3 Heparin Conjugation Methods
### Physical Conjugation
#### 3.1.2 Mixing Heparin with Sulfated Biopolymers  
Conjugating heparin through gas plasma results in lower heparin bioactivity, drug instability in high-pressured blood flow, and tissue toxicity [32]. The advantage of this conjugation technique is that it can be adopted quickly and is inexpensive compared to other methods. However, it has the disadvantage that heparin does not adhere to the graft polymer strongly and/or uniformly, so the initial burst when exposed to blood is unavoidable. [PERSON] et al., investigated dual-layered grafts made of an outer electrospun PCL layer and inner PLCL sponge layer with heparin [28]. Heparin was dissolved and separated by thermally induced phase separation (TIPS) to make a wet porous PLCL inner sponge (Figure 1). In order to evaluate the amount of heparin conjugated to the graft and how it affects platelet deposition, they activated sheep platelets in vitro and compared the extent to which they deposited on the graft in the heparin group and control group. A comparison was made using direct observation of the lumen by SEM and LDH assay methods for quantitative evaluation [33]. The results showed that platelet absorption was significantly suppressed in the heparin group. Heparin did not inhibit platelet aggregation; therefore, we considered that heparin may have changed the charge on the luminal surface. They quantitatively examined the release of heparin using the toluidine blue method [34; 35]. A large initial burst of heparin release occurred within 24 h. However, continuous, albeit low, heparin release was observed for the next 14 days. They implan  
Figure 1: Manufacturing process of heparin-eluting graft: (**A**) Heparin-eluting TEVGs were created with an outer poly-\(\varepsilon\)-caprolactone (PCL) electrospun nanofiber layer with a 15 \(\upmu\)m average pore Scheme 50. poly(l-lactide-co-\(\varepsilon\)-caprolactone) copolymer (PLCL). SEM showed the inner heparin eluting layer; (**B**) Surgical implantation of TEVG to sheep carotid artery, 3 out of 5 Control grafts were occluded within 1 week. On the other hand, heparin-eluting grafts were obvious throughout the observation period; (**C**) Histological analysis of 15 \(\upmu\)m TEVG and occluded control TEVG, red bar = 200 \(\upmu\)m, grey bar = 500 \(\upmu\)m. HE, heparin-eluting; TEVG, tissue-engineered vascular graft. Adopted with permission from [PERSON] et al. in sheep carotid arteries and maintained patency for up to one year. The positives of this method are that it is simple and cost-effective. [PERSON] et al., fabricated a tissue-engineered small intestine, using a subcritical CO2 exposure system to slowly release heparin-binding EGF-like growth factor (HBF-GF) embedded in a PGA/PLLA scaffold. Heparin-binding EGF-like factor (HBF-GF) embedded in PGA/PLLA scaffolds was successfully released slowly in an active form [36] and was able to stimulate crypt growth in vitro and intestinal mucosa formation in vivo. A notable aspect of this technique is that exposure to subcritical CO2 does not deform the scaffold structure and does not impair its mechanical properties [37; 38]. [PERSON] et al., has also attempted to embed already-submerged heparin particles deep into the scaffold by using subcritical CO2 exposure in the inner PLCL layer of our dual-layered grafts. Furthermore, it was found that reducing the heparin burst in the early stages in vitro increased the heparin release profile (Figure 2). This has the potential to further improve graft patency by embedding to a deeper level with the addition of a subcritical CO2 exposure technique. Future implantation using scaffolds larger than 5 cm are needed. [PERSON] et al. have shown that polyhydroxybutyrate/valerate(PHBV)/polycaprolactone (PCL) grafts modified with growth factors such as VEGF, bFGF, and SDF-1\(\alpha\) (PHBV/ PCL-GFmix) performed well when implanted in the rat abdominal aortas [29]. Subsequently, they used a sheep model, which is considered to be more suitable for the in vivo testing of cardiovascular implants [39].