4 Discussion
Scientists have been rapidly developing tissue engineered artificial
blood vessels in the past decade, and used multiple ways to construct
them(Morin, Smith, Davis, & Tranquillo, 2013). 3D bioprinting has
become a research hotspot because of precision spatial control and
uniform cell distribution (Hong et al., 2020). 3D bioprinting is a rapid
prototyping and additive manufacturing technology, which considers
various design aspects such as imaging, modeling, printer selection,
bioink selection, culture conditions and 3D structure (Murphy & Atala,
2014; Y. S. Zhang, Oklu, Dokmeci, & Khademhosseini, 2018). However,
there are still difficulties in the fabrication of complicated
small-diameter blood vessels. The accuracy of 3D printing technology is
limited in some scenarios for medical application, and current bioinks
have varier limitations in the most targeted applications. Printable
materials with good biocompatibility tended to have poor mechanical
properties. In bioprinting matrix loss and structural collapse often
lead to failure of the printed construct (Ozbolat & Hospodiuk, 2016).
Normally, sacrificial hydrogels offer a temporary support in the
fabrication of small-diameter vessels. But all sacrificial hydrogels
have disadvantages (Bertassoni et al., 2014; Tocchio et al., 2015).
Therefore, we developed a two-step crosslinking method to fabricate
tunable structures. This method avoids the use of sacrificial hydrogels
and produces a vessel structure with using single photocurable hydrogel
– GelMA.
Hydrogel, as a material for loading cells and supporting structures, is
particularly important in 3D bioprinting (Hoelzl et al., 2016). An ideal
hydrogel needs to have biological and mechanical properties similar to
its intended tissue and should be adaptable to the printing process. In
addition, hydrogel structures need to facilitate cell proliferation and
differentiation after bioprinting. Naturally derived hydrogels
(including agarose, alginate, collagen, fibrin, gelatin and hyaluronic
acid) provide an effective growth environment for cells (Cui, Nowicki,
Fisher, & Zhang, 2017). However, these hydrogels lack sufficient
mechanical strength to create the complicated 3D structure of a tubular
structure. GelMA as a biocompatible polymer is produced by modified
gelatin with methacrylic anhydride (MA), and is able to be crosslinked
with UV light to obtain higher physical strength (Liu & Chan-Park,
2010). Our preliminary experiments showed that uncrosslinked GelMA
hydrogel can be stacked up to 20 printed layers. In this study, 5% m/v
GelMA was used to fabricate the tubular structure. The pore size at this
concentration could hold SMCs in the spot of three dimensional space,
and was beneficial to the proliferation of the cells (Jeong et al.,
2007). We compared the properties of GelMA solution with different
curing time. Although GelMA with 5 s crosslinking could get semisolid
immediately, it could not keep its shape for a longer time. 10 s
crosslinking time for the GelMA was used at the first crosslinking step
due to maintain its spatial form, open the least double bonds, and then
lay a proper foundation for the second crosslinking step. Moreover, the
water absorption capacity of GelMA with 5 s and 10 s crosslinking time
could reach 400 times of its own mass in DI water, and its volume also
expands accordingly. Therefore, the low degree of crosslinked GelMA was
also a good swelling material to be applied in the investigation of
tissue engineering.
Pre-crosslinking can enhance the physical property of photocurable
hydrogel. In the process of vertical bioprinting, the printed bottom
GelMA bioink was partly crosslinked by continuous UV light to avoid cell
leakage and structure collapse (Xu et al., 2020). A user-defined,
complexity cell-laden channel was fabricated by a sequential printing
approach. The photocurable hydrogels were briefly exposed layer-by-layer
to increase support performance. The sacrificial hydrogels were printed
into the desired layer and fully crosslinked. With the removal of
sacrificial hydrogels, precise and complex channels could be constructed
(Ji, Almeida, & Guvendiren, 2019). In our study, GelMA was made
semisolid after first-step crosslinking process. The semisolid GelMA
with required physical strength could be bonded tightly with
uncrosslinked GelMA. The combination was received second-step
crosslinking process which produced a longer UV exposure time. A bionic
vascular vessel with small diameter was built successfully with using
the two-step crosslinking method, which maintains consecutive tubular
structure.
Furthermore, this method might be not only suitable for vessels, but
also for other complicated structures such as hepatic sinusoid, nephron
and pulmonary alveoli. The hydrogel can be partly cured, and the
two-step curing can integrate two independent parts into a whole. This
method may be suitable to photocurable hydrogel with better mechanical
properties and make structures that are difficult to be formed in one
step by forming them in two or more steps. In addition, different kinds
of photocuring materials containing double bonds may also be used with
this two-step crosslinking method. In this way, we could make the
connection between different tissues in bioprinting, such as the
superior vena cava bioprinted by one photocuring material connected with
the heart which is bioprinted by another photocuring material.
Human blood vascular vessel have three distinct layers: intima
(endothelial cells), media (smooth muscle cells), and externa
(fibroblasts) (Tomasina et al., 2019). We created a small diameter
vascular structure (Ø < 6mm) with the spatial distribution of
two different cells, which include bioprinted SMCs within bioink and
subsequent perfusion of HUVECs into the lumen space. SMCs could
aggregate, spread out, and proliferate to form outer layer of the
construct, in that rich collagen area was found. Perfused HUVECs formed
the connected inner layer that covers entire intralumenal surface with
dense junctions. The SMCs were lined longitudinally along the structure
which may be caused by axial traction during printing. During the
culture of bioprinted vessel structure, the previous inner square shape
turned to smooth gradually, that has been reported in previously
publication (Esch, Post, Shuler, & Stokol, 2011).
Through this two-step crosslinking method, we have successfully
bioprinted multiple complicated tubular structures and bionics vessels
with inner monolayer endothelium surrounded by outer layer SMCs. This is
a new fabrication method for tissue engineering of small diameter
vessel. In addition, the alternatives of two-step crosslinking method
might be adopted to create more complex structures.