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A dual crosslinking strategy to tailor rheological properties of gelatin methacryloyl
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groups of gelatin modified with methacryloyl groups shear rate from 0.1 to 1000 s at 37 °C.
was calculated. Frequency sweep was carried out to test the visco-
elastic properties of the hydrogels by using a parallel
(1) plate geometry with 10-mm diameter. The mechanical
spectra were recorded with 2% strain over a frequency
2.4 Preparation of Enzymatic Crosslinked range from 0.1 to 10 Hz at 37 °C.
GelMA (MTGase-GelMA) Solutions 3. Results and Discussion

GelMA solution was prepared by dissolving the
lyophilized GelMA at a concentration of 10% (w/ 3.1 Methacryloylation of Gelatin
v) in phosphate-buffered saline (PBS) solution. Dif -
ferent amounts of MTGase were separately added The chemical structures of unmodified gelatin and
to the as-prepared GelMA solutions so that the final GelMA are shown in Figure 1a–b. Compared with
concentrations were 1, 3 and 5 U/mL. The incubation the spectrum of unmodified gelatin, GelMA sample
temperature was 37 °C. formed new functional groups, marked as green “a”
and blue “c” in Figure 1b, which can be confirmed by
2.5 Preparation of Hydrogels with Different the H NMR spectra (Figure 1c). The peaks at around
1
Crosslinking chemical shifts (δ) of 5.3 and 5.6 ppm were assigned
to the acrylic protons (2H) of the grafted methacryloyl
The enzymatic crosslinked hydrogels were prepared group, and another peak at δ = 1.9 ppm was attributed
by pouring 200 μL 10% (w/v) GelMA with 3 U/mL to the methyl group (3H) of the grafted methacryloyl
MTGase into a cylindrical mould, and sealed and group. Meanwhile, there was a decrease of intensity
incubated at 37 °C for 12 h. The photo-crosslinked at 2.9 ≤ δ ≤ 3.1 ppm, which was assigned to the lysine
hydrogels were prepared by pouring 200 μL of 10% (w/ methylene (2H) and marked as pink “b”. As lysine is the
v) GelMA solution containing 0.1% (w/v) Irgacure 2959 reaction site, this trend could be used to quantify DM,
into a cylindrical mould at 37 °C and exposed to UV which yielded to be 53.5% ± 0.9%. The remaining lysine
2
light (λ = 365 nm with an intensity of 1.5 mW/cm ) for groups could be utilized for enzymatic crosslinking as it
5 min. The dual crosslinked hydrogels were prepared is an acyl acceptor.
by a mixture of the above steps; after incubating the
MTGase-GelMA solution at 37 °C for 12 h, the samples 3.2 Rheological Characterization
were then UV-cured for 5 min. All the samples were
taken out of the mould for further experiments, of which 3.2.1. Gelling Period of GelMA Incubated with MTGase
the dimensions were 8 mm in diameter and 3 mm in The mechanism of enzymatic crosslinking is that
height. MTGase catalyses the inter- and intra-molecular bonds
2.6 Rheological Properties formation between the γ-carboxamides of glutamine
residues and ε-primary amino of lysine residues in the
The rheological properties of the enzymatically cross- chains of GelMA (Figure 2). The growth of a connected
linked hydrogels were tested by a rheometer (MCR 501, structure will eventually lead to formation of a chemical
Anton Paar Germany GmbH, Ostfildern, Germany) with gel.
a 25-mm cone-plate geometry and with an angle of 2°. To determine that the reaction took place, a time
To study the formation of gel network, the time sweep sweep test was performed at the incubation temperature
test was performed where storage modulus (G’) and loss (37 °C). The effect of the MTGase concentration on
modulus (G”) were monitored as a function of time at a the gel formation of 10% GelMA was examined. There
fixed frequency of 1 Hz and strain of 3%. To avoid the were two moduli generated from these experiments:
evaporation of water, the MTGase-GelMA solutions G’ representing the deformation energy stored, and
were sealed in the tube and incubated at 37 °C, then G” being the energy dissipated during shear. They are
sequentially loaded onto the rheometer at an interval of 1 sensitive to molecular structure evolution, especially the
hour and tested for 1 min. During testing, measurements formation of network.
were taken every second, and the average of the 60 data Figure 3 shows that, at the beginning, the GelMA
points represented the average modulus of the sample at solution with MTGase behaved liquid-like, where G”
different incubation time periods. The time-dependent is higher than G’. When gelation takes place, there
viscosity during the enzymatic crosslinking proceeding is a crossover of G’ and G” (whereby the value of G’
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was tested at 37 °C under the shear rate of 100 s , in a is higher), which could be seen for the 10% GelMA
similar way of data collection described earlier. The flow incubated with 5 U/mL MTGase. The effect of the con-
behaviour of solutions was examined within the range of centration of MTGase on the gelling period is sum-

132 International Journal of Bioprinting (2017)–Volume 3, Issue 2