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Tan, et al.
harvesting devices, to prosthetic bionic skins . remains edible, which is the requirement for 3D
[3]
The adoption of 3D printing technology offers food printing. In addition, enzymatic crosslinking
multiple advantages over traditional techniques does not produce free radicals and uncrosslinked
to fabricate scaffolds, including uniformity and monomers derived from photocrosslinking
reproducibility in manufacturing, reduction of reactions that might adversely affect the cellular
user error, and precise control over scaffold pore conditions. Both photocrosslinking and enzymatic
size, connectivity, and geometry . Among the crosslinking are permanent and confer mechanical
[6]
materials used in extrusion-based printing, natural strength to the crosslinked gelatin chains required
biopolymers such as collagen, gelatin, and chitosan for 3D printing. TG is often added to gelatin
are promising candidates for bioprinting and food to facilitate the formation of the peptide bond
printing due to their excellent biocompatibility between the γ-carbonyl group of glutamine
and abundance of cell recognition sites [7,8] . In residue and the ε-amino group of a lysine residue
addition, natural biopolymers such as gelatin within the gelatin . The formation of peptide
[9]
are inexpensive to fabricate, and they can be bonds stabilizes the structure of the printed
synthesized with relative ease . Essential physical scaffold and improves its structural integrity.
[9]
properties (such as stiffness and water content) This enzymatic crosslinking is favored among
can be altered to obtain similar elastic modulus other available methods of crosslinking (such as
of native tissues such as skeletal muscle, which chemical crosslinking) due to the low occurrence
was previously reported to be within the range of side reactions (due to substrate specificity) and
of 10 – 50 kPa . Finally, gelatin is a generally no cellular toxicity of the enzymes. The use of
[10]
edible material and can eventually be used for enzymes also eliminates the need for specialized
food printing as a ready-to-eat product without equipment and other photo-sensitive additives that
the need for post-processing. These properties may be toxic in nature .
[9]
make the gelatin as a desirable candidate for the DIW 3D printing has been widely demonstrated
application in 3D printing. in 3D printing for polymers, foods, hydrogels [13, 14] .
3D printing of gelatin would require adequate Polymers such as polyvinyl alcohol were 3D printed
control over the physical properties of the gelatin to serve as coating layers for controlling the
inks. The melting point of gelatin is 30 – 37°C, drug release of active ingredients . For the
[15]
depending on their bloom strength, pH, and application in food printing, rheology-modified
concentration ; gelatin is unable to hold/retain food inks have been printed using a DIW 3D
[11]
its structure and would melt at physiological printer to model complex 3D shapes without
temperature. Permanent peptide bonds need to temperature control . Potato starch-containing
[5]
be formed between amino acids to ensure the anthocyanin and lemon juice was 3D printed as
integrity of the structure in a liquid medium and a two-part gel system exhibited time-dependent
at physiological temperature. Among the most color change in response to the diffusion of
employed techniques to achieve permanent hydrogen ions from the lemon juice layer into the
crosslinking are photocrosslinking and enzymatic anthocyanin layer . Such 3D-printable hydrogels
[16]
crosslinking. Photocrosslinking between included photo-curable polyethylene glycol
methacryloyl groups in Gelatin-Methacryloyl acrylate, poly(2,6-dimethyl-1,4-phenylene oxide)
happens relatively fast (in the order of seconds), acrylate, gelatin-methacrylate, and hyaluronan-
which rapidly confers structural stability to the methacrylate [17-19] . In addition, gelation can be
printed scaffold . Enzymatic crosslinking (i.e., triggered by printing a hydrogel-forming polymer
[12]
using transglutaminase [TG]), on the other hand, solution into a bath of the reactive substance. It was
takes place relatively slower (in the order of reported that alginate printed into calcium solution
minutes) than photocrosslinking. Although slower, using this method has yielded complex cell-laden
TG has been widely used as a meat glue to mediate 3D structures maintaining cell viability . DIW
[20]
the crosslinking of gelatin; the crosslinked product of these hydrogel-forming polymers offers a

International Journal of Bioprinting (2020)–Volume 6, Issue 4 119