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.