# Heparin-Eluting Tissue-Engineered Bioabsorbable Vascular Grafts
## 3 Heparin Conjugation Methods
### Chemical Conjugation
#### 3.2.1 Covalent Interaction  
Heparin which is covalently bonded to surfaces has been proven to last longer than electrostatically bonded heparin. Covalent bonding of heparin is the best option to solve the problem of burst release. Additionally, this approach promotes resistance to mechanical forces at the blood and luminal surface interface [45]. However, the carboxyl groups present on heparin molecules contribute to the drug's mobilization, which limits binding to proteins and may reduce its anticoagulant properties. The lack of heparin bioactivity may be due to ineffective immobilization methods. Heparin conjugated with covalent or ionic bonds is not easily removed by water rinsing, but it has also been suggested that heparin may be depleted over time in the presence of blood flow, exposing a surface without anticoagulant [47]. Chemical conjugation of heparin can be achieved using covalent cross-linkers between the carboxyl groups in the heparin molecule, and amino covalent heparin immobilization to scaffolds is often intended to mediate the addition of growth factors via electrostatic interactions between heparin and the added protein (growth factor such as VEGF) (Figure 4). With the addition of growth factors, heparin not only exhibits a sustained release profile (details in the next section), but also the highest activated partial thromboplastin time (APTT) to date, with a reported synergistic effect of sustained growth factor release rate and no burst release for up to three weeks. As mentioned in the previous section, the negative charge of heparin may inhibit the fibrinogen adsorption [24]. [PERSON] et al., hypothesized that increasing the hydrophilicity of heparinized electrospun PCL scaffolds and negative charge of heparin may inhibit the fibrinogen adsorption [27]. In vivo studies in a canine model have shown that their heparin-containing scaffold remains patent after one month, and a nearly confluent layer of ECs was observed in the graft lumen. The high cytocompatibility of the scaffolds may be attributed to heparin molecules, possibly promoting cell adhesion and proliferation by entrapping VEGF in the blood. As also mentioned in physical conjugation, it is possible to exploit the flexibility and structural integrity of PLLA and PLCL polymers to fabricate heparinized nanofibers [34]. Recently, the [PERSON] group prepared various P(LLA-CL) blends using the TIPS method, and then conjugated them with heparin using carbodiimide chemistry/EDC/NHS, which utilizes the esterification reaction between the terminal hydroxyl groups of PLLA and PLCL and the carboxyl groups of heparin molecules (Figure 3) [48]. In another study, VEGF was covalently bonded to hydrophilic heparin molecules. The VEGF-heparin molecules were then coaxially electrospun with P(LLA-CL) as the shell, and then combined to a collagen-elastin core material [31]. Heparin had an initial burst release causing a cumulative release of 48% on day one, although it continued to be released for 22 days; they concluded that the burst release of VEGF and structures in collagen may be the reason for the rapid growth of seeded endothelial cells. There is also an innovative method that uses photopolymerization reactions, which are part of covalent bonding. Heparin-functionalized bioabsorbable microspheres and nanoparticles have been widely reported for the delivery of several growth factors such as basic fibroblast factor (bFGF) and vascular endothelial growth factor (VEGF) [49; 50; 51]; in these nanostructures, the development of nanosponge technology is remarkable. Nanosponges refer to a class of nanomaterials with nanoporous structures and excellent absorption/composite properties. Various \"nanosponges\" based on organic/polymeric and inorganic materials been reported [52; 53; 54; 55; 56]. These have high volume expansions in addition to being porous [57]. [PERSON] et al., searched for a chemically stable vehicle for the sustained release of various growth factors.