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Microsphere-Based Bioink for Large Tissue with Angiogenesis
diameters to further form on-demand nutrient channels respectively. The testing results are shown in Figure 2.
and angiogenesis distribution. The viscosity of the preheated electrospraying ink in
30°C testing temperature continuously increased within
3.2. Rheological properties of gelatin the 1200 s testing process and failed to get the stable
electrospraying ink state. However, in the 37°C testing temperature, the

The reversible crosslinking process of TSMs was viscosity maintained stable and was at the lower level, as
achieved by low temperature. Since TSMs would undergo shown in Figure 2G. Based on this, the flow sweep was
many processes such as centrifugation for separating carried out and the result showed that the electrospraying
from silicon oil, mixing with preheated GelMA precursor ink owned obvious shear-thinning property as shown in
solution and extruding bioprinting of centimeter-scale Figure 2H and would own lower viscosity when flowed
3D structure, for TSMs to finally play their role in from the nozzle tip, which was suitable for microdroplet
constructing nutrient channels and angiogenesis, it is electrospraying.
necessary to maintain its original gelation state until In terms of the low-temperature crosslinking process
GelMA precursor solution is fully photocrosslinked. of TSMs, the received gelatin microdroplet in the silicon
Throughout these processes, there are two main potential oil would be crosslinked at 4°C. Therefore, the thermo-
factors probably leading to the instability of the gelation crosslinking duration needs to be determined to get
state of TSMs. In terms of the loaded cells, before the microdroplets totally crosslinked from 37°C to 4°C. Here,
electrospraying of TSMs, HUVECs should be initially low amplification oscillation time sweep was carried out.
mixed in gelatin precursor solution to form 3D vascular The results as shown in Figure 2I demonstrated that, at
network in the final centimeter-scale tissue. During the the beginning 60 s, the electrospraying ink maintained
thermo-crosslinking process, gelatin molecules have to fluid state (G’<G’’). At 60 s, gelatin electrospraying ink
bypass these cells and form hydrogel network, which was at semi-gelation state. During 60 s~400 s, G’’ further
causes the network inside TSMs to be interrupted to some increased and G’ decreased, demonstrating the gelation
extent. In addition, the movement and metabolic heat of state was further enhanced. After 400 s, G’ and G’’ tended
HUVECs can also damage the in the process of preparing to be stable and the microdroplets was totally crosslinked
TSM-B, TSMs will be mixed with preheated GelMA to be TSMs. Therefore, the thermo-crosslinking treatment
precursor solution to guarantee the uniformity of the duration should be above 400 s to get solid TSMs for
bioink. Besides, during the extruding process, to ensure further extruding bioprinting.
the printability and the formation of stable and uniform In terms of the extruding bioprinting process, the
filament, GelMA precursor solution in TSM-B should be TSMs should be mixed with GelMA precursor solution.
at semi-gelation state. In the published study of extrusion According to the discussion above, this process would be
printing based on GelMA based bioink, the printing carried out at the range of 20°C~24°C. Thus, the sol-gel
temperature was often set to 20 – 24°C, which would state of the crosslinked microspheres in this temperature
cause TSMs to gradually undergo a solation transferring range should be tested. Here, low amplification oscillation
due to high temperature, losing its original 3D shape [32-36] . time testing was carried out and the testing temperature
Therefore, it is necessary to increase the concentration of was set as 24°C, as shown in Figure 2J. It could be
the gelatin precursor solution appropriately to increase the found that even though G’ decreases and G’’ increased
quantity and strength of the hydrogel network in TSMs, gradually, G’’ was greater than G’, indicating that the
so that they would maintain a stable gelation state in electrospraying ink maintain gelation state, proving that
subsequent “secondary printing.” Here, the concentration the prepared TSMs could be further used as an auxiliary
of gelatin precursor solution was set as 15% w/v. component in TSM-B to participate in subsequent
In terms of the electrospraying process, the flow step extruding printing.
measurement of gelatin electrospraying ink was carried 3.3. Rheological properties of TSM-B with
out to explore the stabilization time of the viscosity different recipes
in the electrospraying environment. In our previous
research on the low-concentration GelMA microsphere To form stable and uniform filament during the extruding
electrospraying, the environment temperature was set bioprinting, the temperature control method of TSM-B
as 30°C to get enough fluidity and shorter viscosity needs to be properly arranged. In the preparation process
stabilization duration . However, because the of TSM-B, to ensure that the original shapes of the added
[31]
concentration of the applied gelatin electrospraying ink TSMs, it is necessary to initially reduce the temperature
was much higher, its viscosity stabilization time may be of the preheated GelMA precursor solution to weaken
greatly extended in this temperature. Here, the preheated the solation transferring level. Meanwhile, in the cooling
electrospraying ink (37°C) was added to rheometer process, TSMs should be added when the GelMA
and the testing temperature was set to 30°C and 37°C, precursor solution is still with a low viscosity so that

International Journal of Bioprinting (2022)–Volume 8, Issue 4 21