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Extrusion of two-vasculature scaffold for angiogenesis
A B

Figure 11. The cell viability of the two-vasculature-embedded scaffold according to the culture conditions (A) without the additional GFs
and (B) with the additional GFs.

highest viability at day 0 and early days among all the highest number at day 7 among all the time points in
three culture conditions. The additional GFs did not the same culture condition. In the case of the length, it
significantly influence the cell viability in all three presented a gradual increasing tendency.
culture conditions. The flowing condition showed the most active
angiogenesis. It was assumed that the GF diffusion
3.5. Angiogenic sprouting from the near hollow channel stimulated HUVECs
Figure 12 exhibited the angiogenic sprouting of the more than the GF diffusion from the outside media.
embedded HUVECs in the generated two-vasculature- The glass capillary of the obstructing condition looked
embedded scaffold up to day 10 with F-actin restricting the GF diffusion into the embedded HUVECs.
(Figure 12A-C) and CD31 (Figure 12D-F) markers. It was demonstrated that below 65% cell viability in the
The F-actin and CD31 are well-known biomarkers to obstructing condition could affect no angiogenesis at
check cytoskeleton and angiogenesis, respectively. day 10.
The cells in the obstructing condition angiogenically
sprouted from day 3 to day 7 (Figure 12A and D). 4. Discussion
Those sprouting looked no significant directionality. On The formulated scaffolds consisted of two separated
day 10, all the sprouts disappeared, and no new sprouts channels and the outer shell. One channel was filled with
were recognized. The cells in the soaking condition the mixture of HUVECs and type I collagen. The other
presented the angiogenic sprouting from day 3 to day channel was the hollow channel. The outer shell material
10 (Figure 12B and E). Considerate directionality was
not observed as well. The cells in the flowing condition was the composite of gelatin and alginate. The laboratory
angiogenically sprouted during all the time points from made connecting device linked the generated scaffold
day 1 to day 10 (Figure 12C and F). In particular, to a syringe pump for 10 days without any leakage.
significant directionality of all the new sprouts to the The flowed media diffused from the hollow channel
hollow channel was observed, which diffused the high GF to the embedded HUVECs. Due to the diffused media,
media into the HUVEC vessel. Furthermore, connections aggregation of the individual HUVECs developed into
among adjacent sprouts were found on day 7 and day 10. the vascular structure. The angiogenic sprouting was
Sprouting number and length were quantitatively induced from the HUVEC vessels to the hollow channel
analyzed, as shown in Figure 12D and E. The flowing by the gradient of the additional GFs.
media inside presented an increasing tendency in both the Scaffold material could be divided into natural,
number and the length. Except for the sprouting number synthetic, hybrid, and decellularized extracellular
at day 7, the cells in the flowing condition showed the matrix. Each group has its own advantages and
highest number, the longest length among all the three disadvantages. Gelatin, a natural polymer, has been used
conditions. On day 7, the sprouting number of the in tissue engineering due to its high biocompatibility
obstructing condition was slightly higher than that of the and biodegradability. However, the poor mechanical
flowing condition. On day 7, the obstructing condition properties of gelatin restrict its application. Fortunately,
exhibited the highest number and the longest length alginate is generally used to improve its mechanical
among all the time points in the same culture condition. characteristics. Taking into consideration the previous
The sprouting number of the soaking condition showed investigations [27-29] , the composite of gelatin-alginate

62 International Journal of Bioprinting (2022)–Volume 8, Issue 3