**1. Introduction**

In the field of tissue engineering, one methodology that is often used is to integrate signal factors, cells and biomaterial constructs to replace or improve functional tissues [1,2]. In this common practice, biomaterial constructs act as the basic structure to support and guide cell growth under the influence of some biologically active molecules [3]. An ideal biomimetic structure must have properties (e.g., biocompatibility, elasticity, extensibility) similar to those of the extracellular matrix (ECM) of the native tissue [4]. The selection of the structure and the materials of the biomimetic construct is crucial for e ffectively mimicking the ECM of human body.

Coronary bifurcation is vulnerable to atherosclerosis as a result of the distinct local blood flow patterns [5] caused by the bifurcated structure of the coronary artery [6]. The blood flow through the

artery exerts shear stress onto this particular area, which makes this area susceptible to lesions [7], and which subsequently leads to the coronary artery bifurcation lesion [8], one common lesion of the coronary artery. The treatment of coronary artery bifurcation lesions is a major challenge for interventional cardiology [9]. There exist several interventional techniques for the treatment of this disease, such as the kissing balloon dilatation, crush technique, amongs<sup>t</sup> others [10]. However, the operation process of these techniques remains complex, and the complication rates are usually high. The results of the interventional techniques are usually not satisfactory [11]. For example, the bifurcation area is often subjected to occlusion after operation, and the restenosis rate is very high. Instead of employing traditional interventional techniques, the development of a substitution of the coronary arteries for transplantation is a new method that holds grea<sup>t</sup> promise [12].

As far as the treatment of vascular diseases such as the abovementioned coronary bifurcation lesions is concerned, autografts are an ideal candidate in transplantation operations [13]. They possess a benign biocompatibility to the host patients, and the rejection rates are usually low. However, the number of autografts is often rare because of the lack of suitable donors and harvest sites [14]. Synthetic grafts composed of polytetrafluoroethylene and Dacron perform well in replacing large-diameter blood vessels (inner diameter > 6 mm), but they are not suitable for small-diameter conditions [15]. In the channel of small-diameter vessels, the blood flow rate is low and the shear stress is high, which often causes thrombosis and neointimal hyperplasia. In contrast, sca ffolds made of natural materials such as gelatin and chitosan often perform better [16].

Some of the recent tissue engineered vascular graft (TEVG) fabricating methods tend to mimic the native blood vessels, which feature a multilayered structure, multi-branches and a spatial configuration [17–19]. Like the vessels in vivo, the multilayered structure is helpful for aligning di fferent types of cells, such as endothelial cells, fibroblasts, and smooth muscle cells, in di fferent layers and improving the interaction between them [20], which is a tremendous progress in better mimicking the structure of a native blood vessel. Additionally, in human tissue, the number of vessel branches increases while their inner diameters decrease [21]. This morphology could ensure the mass transportation within the whole vascular network. Therefore, when designing the relatively large-diameter portion of a vessel network, the branching factor must be taken into consideration. Finally, for the purpose of realizing an even distribution of the vessel network in a three-dimensional space, the configuration of the large-diameter channel should be designed as a three-dimensional structure rather than a planar one. However, due to the limitations of the fabrication techniques and materials, it still currently remains a challenge to produce bioengineered vascular constructs that meet all of the abovementioned characteristics.

Among materials commonly used for biological tissue fabrication, gelatin is a natural polymer similar to collagen; it possesses benign biocompatibility and biodegradability [22,23]. Due to these properties, gelatin could be an ideal candidate for cell delivery and could serve as the substitute for ECM. Some recent studies have used microbial transglutaminase (mTG) to crosslink gelatin to increase the biocompatibility and mechanical strength of engineered constructs [24–26]. Enzymatically crosslinked gelatin gels were proven to be biocompatible with human cells. For this reason, a gelatin/mTG biopolymer was usually chosen as the ECM-like material for fabricating biomimetic constructs. Besides, Pluronic F127, which is composed of polyoxyethylene (PEO) and polyoxypropylene (PPO), undergoes thermally reversible gelation above a lower critical solution temperature (LCST) [27,28]. Therefore, it is suitable as the fugitive ink when constructing a hollow channel.

In the present study, a novel additive and subtractive hybrid manufacturing technique combining 3D printed molds, casting hydrogel and sacrificial material was proposed. Compared to current fabrication techniques, the proposed method fabricated multilayered TEVG with a curved structure and multi-branches for the first time, to our best knowledge. The material of the construct was enzymatically crosslinked gelatin. The vascular geometry design was based on the model of the left coronary artery (LCA), hence the fabricated vessel-like construct possessed an interconnected channel

whose diameter varied in the range of 1.8 to 3 mm. The proposed technique is a promising low-cost approach for tissue engineering.

### **2. Materials and Methods**
