## 2 Material and Methods
### Natural Polymeric Biomaterials  
Conceptually, hybrid scaffolds are bio-based materials that enhance biocompatibility and cellular infiltration by incorporating components of a native extracellular matrix (collagen, gelatin, elastin, fibrin). In addition, naturally occurring polymers such as chitosan and silk can improve the overall structural integrity of a graft. Many biological-based scaffold materials have been investigated in small animal models [28; 29].  
Scaffolds fabricated from naturally occurring polymers offer the potential of improved biocompatibility through enhanced cellular interactions and reduced likelihood of a foreign body reaction. Nevertheless, the biodegradation profiles and mechanical properties of these polymers can be less than optimal. Therefore, natural biomaterials are often combined with biodegradable synthetic polymers or are chemically modified. Of particular note to tissue engineering are elastin and chitosan.  
Elastic is a primary constituent of the extracellular matrix and confers compliance to vessels, thereby ensuring smooth blood flow by the storing and releasing of elastic energy. Elastin itself is highly insoluble and a difficult material to handle when creating scaffolds [30]. Hence, there are few scaffolds composed of elastin. Instead of creating an entire scaffold made out of elastin, it is used primarily to supplement a scaffold composed of another material. [PERSON] et al. have reported successful results in a rabbit model with a two-layer scaffold consisting of an inner layer of recombinant human tropoelastin and an outer layer of PCL [31]. These grafts show similar mechanical strength to a human intrathoracic artery and display low thrombogenicity. The crosslinking of tropoelastin is paramount to increasing its mechanical strength, and these grafts appear promising, however further research is needed to evaluate their scaffold characteristics.  
Chitosan (CS) is a natural polymer obtained from shellfish, and in addition to the ease with which it can be chemically modified, it shows good strength, a porous structure, and antibacterial characteristics. CS has been successfully used in bone, cartilage, and skin tissue engineering constructs [32].  
[PERSON] et al. conducted a study on an electrospun PCL-chitosan nanofiber TEVG in a sheep carotid artery model which showed patency of 67% (\(n=6\)) [33]. When integrated within a scaffold, rapidly degrading chitosan is thought to promote better cell invasion and neovascular remodeling due to their large pore sizes.  
Another technique used to incorporate the strength of naturally occurring extracellular matrix (ECM) into a scaffold is tissue decellularization. Decellularization has been evaluated in a multitude of studies and involves removing most of the cellular and antigenic components of a tissue. The decellularized tissue would then theoretically leave an intact ECM with preserved mechanical properties. A heterologous TEVG constructed with small intestinal submuc has been successfully transplanted into a sheep model with good results [34]. In this study, the group seeded with endothelial cells (ECs) showed good patency, but the unseeded control group occluded within 15 days. These studies indicate that decellularized vascular scaffolds will result in thrombosis unless the scaffold is endothelialized or receives additional modifications.  
In addition, ECM elements can be exposed to physical and chemical stress during a decellularization process, which can adversely affect the biomechanical properties of an ECM. The disadvantages of decellularized materials include the inability to alter the content and structure of an ECM, variability between donor sources, and risk of viral transmission from animal tissues.  
The most sophisticated decellularized approach has been successfully implemented by [PERSON] et al. Their human decellularized TEVG is prepared in vitro using allogeneic human vascularSMCs [9]. This TEVG has undergone a multicenter clinical trial and will be further described in detail in Section 4.