Page 253 - IJB-9-5

International Journal of Bioprinting

when MA exceeded 150 μL. The main reason for this and amide III band (1220–1330 cm ). In Figure 2A, it
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phenomenon was that both ASF and MA are hydrophobic. showed that both gel photocuring process and ethanol
The excessive addition of MA would lead to an increase in soaking process could significantly alter the bands of
molecular weight and coagulation. To find the appropriate amides I, II, and III.
amount of MA, ASF-MA was manufactured by adding To analyze the secondary structural fraction of
10, 25, 50, and 100 µL MA separately to the ASF solution. materials, we used the Peakfit v4.12 software for multi-
[31]
The properties of ASF-MA hydrogels were related to the peak separations which contained four types of common
amount of MA. The nucleophilic addition reactions of MA proteins in secondary structure. They were β-sheet (1615–
to the primary amine of lysine in ASF described above 1640 cm , 1680–1690 cm ), β-turn (1660–1680 cm ),
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were confirmed by H-NMR (Figure 1B). We found that random coil (1640–1650 cm ), and α-helix (1650–
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the characteristic resonances within the methacrylamide 1660 cm ). In the amide I region (1600–1700 cm ), it
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vinyl group (δ = 5.5–6.5 ppm) and the methyl group (δ = was mainly the stretching vibration of the carbonyl group
1.8 ppm) of MA appeared by adding MA. In addition, (C=O), which was sensitive to the change of the secondary
the lysine methylene signal (δ = 2.9 ppm) at the MA structure. The amide I band was quantified by using the
gradually decreased as the MA volume increased, which second derivative of a Gaussian function fit (Figure 2B).
substantiated the modification of lysine residues in the The relative content of each secondary structure was
ASF by MA. The results of the TNBS assay are shown in calculated according to the peak area of each fitting curve
Figure 1C. According to the calculated results, 1 g of ASF (Figure 2C). The content of MA had a specific influence on
contained about 0.28 mmol of free amino groups. The the secondary structure level of ASF. As the MA content
degree of substitution was 13.85%, 28.81%, 35.80%, and increased, the β-sheet decreased from 25 ± 0.79% to 19.5 ±
45.98% with 10, 25, 50, and 100 μL added MA, respectively. 0.78%. Meanwhile, the α-helix and random coil increased
We proved that the methacryloyl content of the ASF slightly from 59.07 ± 0.72% to 66.8 ± 0.96%, while the
molecular chain increased with the addition of MA. β-turn angle did not change significantly. The β-sheet
We used SEM to observe the internal structure of percentage increased significantly to 26.6 ± 0.63%, after
the hydrogel (Figure 1D) for ASF-MA H O hydrogels ASF-MA10% hydrogel formed by photopolymerization
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immersed in water that was formulated by the liquid was lyophilized. The β-sheet percentage increased
nitrogen fast freezing method. As the concentration significantly to 26.6 ± 0.63%, after ASF-MA10% hydrogel
increased, the cross-section of the hydrogel became denser. formed by photopolymerization was lyophilized. After
The pore diameter of 10% hydrogel was about 15.15 ± 2.10 soaking the hydrogel in 75% ethanol for 4 h, the β-fold
μm. Compared with 10% hydrogel, 20% hydrogel showed content dramatically rose to 65.5 ± 0.60%, which was
smaller nanopores. The pore diameter of 20% hydrogel about 1.5 times more than the β-fold content of the
was about 6.18 ± 3.30 μm, and the difference of the 20% unprocessed hydrogel. At the same time, the total amounts
hydrogel was relatively significant. We observed that part of α-helix and random coil dropped drastically to 26.39
of them were at the nanometer level, while the rest of them ± 0.60%. The proportions of both α-helix and random
were large. The 30% of ASF-MA hydrogel had pore size coil showed decreasing trends, especially the quantity of
measured in nanometers. ASF-MA EtOH hydrogels were the random coil which showed a reduction from 43.3 ±
prepared by the critical point drying method. The pore size 1.20% to 13.4 ± 0.81%. In summary, the modification of
was significantly different from the liquid nitrogen freeze- MA could affect the secondary structure of ASF. Still, the
dried samples, and most of them were dense nanopores. photopolymerization and ethanol immersion had a greater
The cross-section of the material became denser with effect on it and resulted in a significant increase in the
increasing concentration of ASF-MA. The method of β-sheet and crystalline state fraction.
soaking in alcohol also made the hydrogel structure denser. Then, the compressive properties of hydrogels with
different concentrations and degrees of substitution
3.2. Physical properties of ASF-MA were measured. As shown in Figure 2D, the compressive
We further performed a detailed FTIR data analysis to properties of the ASF-MA PBS hydrogels enhanced with
characterize the chemical modification of ASF by MA and the degree of methacryloylation and concentration. The
found that the secondary structure of proteins was affected compressive strength of 30% ASF-MA 10% PBS could
by various factors. Moreover, the secondary structure of reach 269 kPa (35% deformation). However, 10% ASF-
the protein was related to the mechanical properties of MA 10% PBS and 30% ASF-MA 2.5% PBS under the same
the hydrogel. There were three main characteristic areas of deformation were only 5.6 and 15.1 kPa, respectively. The
protein in the infrared spectrum, which were the amide I compressive strength of 20% ASF-MA 10% PBS hydrogel
band (1600–1700 cm ), amide II band (1480–1575 cm ), (about 75.7 kPa) was superior to that of other hydrogels
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Volume 9 Issue 5 (2023) 245 https://doi.org/10.18063/ijb.760