Page 43 - IJB-3-2

Miaomiao Zhou, et. al.

lance between printability and biological properties mined the gelling behaviour and viscosity of 10%
of hydrogels towards 3D bioprinting. Increasing the GelMA solution treated with MTGase, as well as the
polymer concentration results in a highly viscous mechanical properties of hydrogels formed by enzymatic
hydrogel precursor and a quick gelation into a cross- crosslinking and photo crosslinking.
linked hydrogel, which provides good printability and 2. Materials and Method
high shape fidelity [10,11] , but a dense polymer network
can inhibit the formation of new ECM and matrix
remodelling as well as cell migration [12,13] . Therefore, 2.1 Materials
the development of a hydrogel system with appropriate Methacrylic anhydride (MAAnh), Irgacure 2959
balance of printability and cell support will promote (I2959), deuterium oxide (D O) and gelatin (gel
2
hydrogel application in 3D bioprinting. strength ~ 175 g Bloom, Type A, from porcine skin)
Large numbers of natural- or synthetic-derived h y- were purchased from Sigma-Aldrich (St. Louis, MO,
drogels have been studied for 3D bioprinting such as USA). MAAnh is a reactant for synthesizing gelatin
alginate [14] , collagen [15] , gelatin [16] and poly(ethylene methacryloyl. I2959 is the most reported photo initiator
glycol) diacrylate . Among those materials, gelatin is for GelMA due to its water solubility and relatively low
[17]
an attractive material with biological cues containing cytotoxicity compared with other photo initiators [31] .
cell-adhesion motifs (arginine-glycine-aspartic The microbial transglutaminase (MTGase) was ob -
acid (RGD) sequences) and target sites for matrix tained from Ajinomoto (Tokyo, Japan). The microbial
metallo proteinase (MMP) in cell remodelling and transglutaminase powder with sodium caseinate and
[18]
degradation . The thermally sensitive ability of gelatin maltodextrin additives has an enzymatic activity of 100
can support the printing process [19–22] . Moreover, gelatin U/g.
can be modified with methacrylamide and a minority of
methacrylate groups, resulting in a photo-crosslinkable 2.2 Synthesis of GelMA
material–gelatin methacryloyl (GelMA) [23] . GelMA The synthesis of GelMA was carried out under
retains biofunctionality from gelatin [18] and its photo- the optimized condition according to the previous
crosslinkable property enables quick formation of a work [32,33] . Briefly, GelMA was prepared by reaction
covalently crosslinked hydrogel, which maintains the of type A gelatin with methacrylic anhydride as in the
printed construct permanently, thus becoming stable following: 7.95 g of Na CO and 14.65 g of NaHCO
3
2
3
[24]
under physiological temperature . were dissolved in 1 L distilled water to produce 0.25 M
GelMA has been demonstrated as a suitable printing carbonate-bicarbonate (CB) buffer solution. Following
material for 3D bioprinting. Printing GelMA requires that, 50 g of gelatin was dissolved into 500 mL of the
relatively high polymer concentrations due to low vis- as-prepared buffer. The pH value of the gelatin solution
cosity at 37 °C [24] ; however, previous work has shown was gradually adjusted to 9 by adding 5 N NaOH
that the high polymer concentration could compromise solution in a dropwise manner. MAAnh was added to
cell viability [24–26] . Nichol et al. studied cell viability the solution to achieve an MAAnh:gelatin ratio of 0.05
of NIH 3T3 fibroblasts encapsulated in 5%–15% mL/g. The reaction proceeded at 50 °C for 3 h. 1 N
GelMA, and high cell viability (>80%) was generally HCl was added and the reaction was stopped when the
observed in below 10% GelMA [27] . Additionally, to pH value of the solution was adjusted to 7.4. The crude
improve the printability of GelMA, precise control product was filtered and dialyzed using Minimate TFF
of the nozzle temperature and the cooling down the system (Pall Corporation, New York, NY, USA) with a
platform have been conducted to successfully print 10K MWCO cassette to remove any unreacted MAAnh
GelMA, but then the hardware becomes important [28] . and methacrylic acid by-product. Finally, GelMA was
Thus, the development of a smart system with improved lyophilized to obtain a dried product and stored at −20
rheological properties is imperative for using GelMA in °C for future use.
3D bioprinting. 1
In this work, an enzymatic crosslinking process 2.3 H NMR Characterization
2+
triggered by a Ca -independent microbial transglu- The methacryloylation of gelatin was measured by
taminase (MTGase), a nontoxic crosslinker with high using H NMR spectroscopy. The GelMA solution had a
1
specific activity [29] , was introduced to catalyse the concentration of 50 mg/mL in D O and H NMR spectra
1
2
iso peptide formation between the γ-carboxamides were repetitively collected for three times. Purely ab-
of glutamine residues and ε-primary amino of lysine sorptive signals were corrected by phase correction.
residues in chains of GelMA [30] . We hypothesize that The areas of the peaks were integrated after baseline
this enzymatic crosslinking method could improve correction. The degree of methacryloylation (DM) was
the rheological properties and printability. We exa- defined by Equation 1 where the percentage of ε-amino

International Journal of Bioprinting (2017)–Volume 3, Issue 2 131