3.2 The effect of crosslinking time on GelMA properties
The number of double bonds in GelMA increased with increasing crosslinking time (Chen et al., 2012) (Figure 2A).Treated with different crosslinking time, GelMA showed different physical states. While the GelMA with only 5 s crosslinking time was changed from liquid to preliminary solid, which cannot maintain its shape for a longer time. However, samples with 10, 20 and 40 s crosslinking time maintained good shape (Figure 2B) lasting for long time.
Fourier transform infrared spectroscopy (FT-IR) was used to identify the correlation peaks in GelMA with different crosslinking times. A peak at 1525 cm-1 corresponded to C-N stretching and N-H bending; 1640 cm-1 to O-H bonding; 2922 cm-1 to C-H stretching; and 3300 cm-1 to O-H stretching. GelMA with different crosslinking times showed different peaks at these positions (Dursun Usal, Yucel, & Hasirci, 2019). We evaluated the FTIR of gelatin, uncrosslinked GelMA, and GelMA with different crosslinking times. The peaks of the two amino groups at 1540 and 1640cm-1 in uncrosslinked GelMA were significantly increased compare with gelatin, which indicated that the amino groups in gelatin were successfully replaced by methacrylic acid. After UV irradiation for different times, the two peaks gradually decreased. In addition, the -OH stretching vibrations at 3300 cm-1 also decreased, which was related to the decrease of total -OH caused by UV curing (Figure 2C).
GelMA with different crosslinking time was shaken in DI water at 37 ℃ for two days. The uncrosslinked GelMA was removed, and the remaining GelMA in the structure expanded which led to the pore size increasing (Figure 2D). The average pore sizes were 292.2 ± 25.00 (5 s), 308.9±23.53 (10 s), 175.8±15.66 (20 s), 85.06±8.977 (40 s), 82.07±9.089 (100 s) (Figure 2E). Gelatin, glycerin, and hyaluronic acid contained in crosslinked GelMA bioink will not affect the pore sizes of the constructs.
In order to characterize the physical properties of GelMA bioink with different crosslinking times, oscillatory rheological measurements were used. The storage modulus (G’) and viscosity coefficient of GelMA bioink increased with the crosslinking time, and these kept steady when the crosslinking time reached 40 s (Figure 2F-G). In a certain range, the storage modulus remained stable with changing of frequencies and strains, which indicated the structure of crosslinked GelMA was stable under the shear force (Figure 2H-I). In addition, the storage modulus of GelMA bioink at each crosslinking time was much greater than the loss modulus which indicated that crosslinked GelMA was in a solid state (Figure 2J).
GelMA bioink with different crosslinking times showed different swelling ratios in DPBS and DI water. GelMA’s swelling ratio was 23 times in DPBS with 5 and 10 s crosslinking times. With increasing of crosslinking time, the swelling ratio decreased. The swelling ratio was 17.5 times with 20s crosslinking time, and it was 15 times with 40s at which point it stabilized (Figure 2K). The swelling ratio was more remarkable in DI water. The swelling ratio was 400 times in 5 and 10 s, 200 times with 20 s and closed to 100 times with 40 s (Figure 2L). This trend indicated that an increased number of double bonds result in a more stable framework.
The residual mass indicated the amount of residual GelMA in structure. With the increased of crosslinking time, residual mass increased gradually. The average residual masses obtained were 75.32±2.12% (5 s), 86.16±0.95% (10 s), 94.34±1.16% (20 s), 97.79±0.44% (40 s), 97.65±1.221% (60 s), 80s is 98.05±0.05% (80 s), and 97.18 ± 0.45% (100 s) (Figure 2M).