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.