1 Introduction
Cardiovascular diseases (CVDs) are considered the major cause of death world widely (Zoghbi et al., 2014). The main treatment methods include medical therapy, endovascular intervention, and surgical transplantation (Buchanan et al., 2014). Autologous blood vascular transplantation remains to be the gold standard for surgical transplantation (Rodriguez, Pavlovsky, & Del Pozo, 2016). Although surgical technique continues to be improved, the success rate of autologous blood vascular transplantation is only around 50% (de Vries, Simons, Jukema, Braun, & Quax, 2016). The reason for failure is the embolization of vessels due to the difference of diameter and mechanical properties between grafts and cardiovascular (Y. Zhang et al., 2017). Tissue engineered vascular grafts (TEVGs) have been explored as potential candidate for the treatment of CVDs. TEVGs, larger than 6mm has been widely used in cardiovascular surgery. However, due to the limitation of manufacturing technology, the construction of small-diameter blood vessel (SDBVs) grafts is still a challenge (Yamanaka, Yamaoka, Mahara, Morimoto, & Suzuki, 2018).
Most of the vessels are composed of three concentric layers: intima, media and adventitia (Elliott & Gerecht, 2016; Nemeno-Guanzon et al., 2012). Intima is the innermost layer of vessels, which is consisted of a monolayer of endothelial cells. It directly contacts the blood and is a barrier between vessels and blood flow to prevent blood infiltration and thrombosis. The middle layer is composed of collagen I, collagen II, proteoglycan, elastin, and smooth muscle cells. Smooth muscle and collagen are arranged in a spiral pattern which is conducive to maintaining the elasticity and structural support. The adventitia is composed of fibroblasts and a loose collagen structure, which can keep the vascular structure intact and prevent tearing (Lesman, Rosenfeld, Landau, & Levenberg, 2016; Tomasina, Bodet, Mota, Moroni, & Camarero-Espinosa, 2019). There are many methods for making TEVGs, including electrospinning, phase separation, dissolution casting and so on (Jing et al., 2015; J. S. Miller et al., 2012; Pattanaik et al., 2019; Wang et al., 2018). These processes simulate blood vessels in mechanical properties, but they are still insufficient in biological properties.
3D bioprinting, a newly developed technology that integrates digital modeling, electromechanical control, information control, biomaterials, and chemistry, can accurately locate the cells and biomaterials into the complex multi-scale structure and simulate the complex tissue (Kolesky, Homan, Skylar-Scott, & Lewis, 2016; Murphy & Atala, 2014). In the past decade, 3D bioprinting has been widely used in the field of vascular regenerative medicine (Hong et al., 2020; Kolesky et al., 2014). Rotary bioprinting is a convenient method. Fibrin-based vascular structures are fabricated through a new self-designed rotary 3D bioprinter. During two months of the cultures, mechanical strength and collagen deposition are observed and the burst pressure of the structure reaches 52% of human saphenous vein (Freeman et al., 2019). Yet, the structure constructed in this way is relatively single, which cannot meet the complexity of natural vessels. 2 cm long, 4 mm diameter lumen heterogeneous bilayer bionic blood vessels are bioprinted by vertical stacking in one step. The dense inner layer containing HUVECs and the loose outer layer containing SMCs are formed by using the two separate concentrations of GelMA in different layers (Xu et al., 2020). The above research provides a theoretical possibility for us to create small-diameter vessels. However, there is also a big deficiency in this study. The construct is easy to collapse during the printing process due to the gravity of hydrogels, and the length and inner diameter of vessels are limited. Therefore, a new approach needs to be found to provide support and overcome these shortcomings.
Sacrificial hydrogels such as Agarose, gelatin and pluronic F127 are usually used to create complex tubular structure. Pluronic F127 containing HUVECs and Ca2+ is used as the coaxial inner layer and catechol-functionalized, gelatin methacrylate (GelMA/C) containing SMCs is used as the coaxial outer layer. The GelMA/C undergoes rapid oxidative crosslinking in situ when touching the Ca2+ during the printing process and a vascular structure with high tissue affinity, perfusability and permeability is formed (Cui et al., 2019). Agarose is applied as the temporary support of the scaffold and various vascular cell types are successfully self-assembled by a rapid prototyping bioprinting method to form a vessel construct with controllable diameter (Norotte, Marga, Niklason, & Forgacs, 2009). Although sacrificial hydrogels can be used as a support in the bioprinting process, each sacrifice hydrogel has its own disadvantages. Agarose has a melting point of 60-70°C which will lead the cell destruction in the process of removal (Jordan S. Miller et al., 2012). Gelatin has poor mechanical property and cannot fulfill the support of complex structure. Due to good quality in printing and wonderful support property, pluronic F127 is used in common. However, the concentration of pluronic F127 used in the bioprinting needs to reach 30-40% which will lead the water separate out from the low concentration bioink. Thus, the accuracy of the structures and the cell viabilities will be affected (Wu, DeConinck, & Lewis, 2011).
Based on this, to overcome the defects of sacrificial hydrogel and improve the accuracy of the processing. We herein present a new approach to fabricating bilayer small-diameter vascular vessel without sacrifice bioink by using two-step crosslinking process. To achieve this, ¼ lumen wall of bioprinted GelMA-based flat structure were bioprinted and undergo a short time UV light curing to obtain a certain strength. Then the flat structure can be turned over to the uncrosslinked GelMA of concave structure. Treated with long time UV curing, the two independent structures can be combined. Through the experiments, we studied the properties of GelMA bioink with different crosslinking time first and investigate the connectivity of the two-step crosslinking structure. Furthermore, we fabricated complex small-diameter vascular vessels with HUVECs and SMCs by the two-step crosslinking method. To our knowledge, this work represents a new methodology for constructing complicated bionic vascular vessel.