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A Review on Bioinks and their Application in Plant Bioprinting
spreading [52-54] . Meanwhile, hyaluronic acid (HA) is the added to the printing wells. Finally, the printing was
primary component of the ECM owing to its excellent initiated .
[51]
biocompatibility and ease of modification, which This adaptable multiscale biofabrication approach can
allows the control of the biochemical and biophysical be used to create 3D anisotropic fibrous microenvironments
characteristics of the microfibers [55-57] . This composite to engineer therapeutic connective tissues.
system using NorHA microfibers provides a distinctive Future research must consider the effect of cellular
approach as it utilizes fibrous components that are easily alignment on cell matrix deposition and function, which
manipulated [57,58] . is outside the scope of this review .
[51]
To produce NorHA microfibers, HA was first
transformed to tetrabutylammonium (TBA) salt (HA- 4. Biomaterials commonly used in 3D
TBA) through a 2 h proton exchange reaction with bioprinting
a Dowex 50W proton exchange resin [59] . The resin
was removed through filtration, and the filtrate was The development of bioink is one of the most difficult
[61]
neutralized with tetrabutylammonium hydroxide issues in the 3D bioprinting process . In general terms,
(TBA-OH) to a pH of ∼7.02 – 7.05 before being the ink should indeed satisfy the physical, mechanical, and
frozen and lyophilized. HA-TBA was changed with biological necessities of the printing process. To initiate,
norbornene groups by esterifying it with 5-norbornene- the ink must be biocompatible while also allowing cell
2-carboxylic acid (3 equivalent), 3-(dimethylamino) proliferation and adhesion. Physically, the ink must be
pyridine (1.5 equivalent), and ditertbutyl decarbonate viscous enough to dispense from the print head. Finally,
(0.4 equivalent) for 20 h at 45°C in the presence of the most important mechanical requirement is to provide
nitrogen. The reaction was then quenched with water and sufficient strength and stiffness to ensure that the ink
remains structurally intact after printing.
dialyzed against water for 7 days at room temperature According to the biological necessities, bioinks can
using 0.25 g NaCl l of deionized water (DI) before be categorized as natural bioinks, synthetic bioinks, and
−1
being lyophilized. Electrospun fiber mats were then hybrid bioinks, which retain components of both natural
created [57,58] . Fiber mats were cut into 1 mm pieces and and synthetic bioinks (Figure 4).
3
soaked in PBS for 30 min to produce microfibers. Mats Natural biomaterials mimic the ECM structure or
were then sheared following hydration by continuously composition, biocompatibility, biodegradability, and self-
passing the solution through a needle. Then, 18 g (×40), [3]
21 g (×40), and 23 g (×40) needles were used to shear assembling ability, making them ideal synthetic biomaterials .
the mats. The microfiber solution was first filtered using Synthetic biomaterials have their own advantages, such as
a 40 μm cell filter (BD, 352340), then through a 5 μm photocrosslinking ability, stable pH, mechanical stability,
pluriStrainer after fragmentation. The residual solution and stable temperature responses; however, their poor
®
was collected, centrifuged at 18,000 RCF, and kept at cellular adhesion and biocompatibility, toxic byproducts,
4°C for up to 2 months in the dark. and mechanical property loss during degradation limit
[62]
GelMA was sterilized using a germicidal lamp under their applications . Overall, the combination of both
a laminar flow hood for 30 min before being dissolved in natural biomaterials and synthetic biomaterials, that is,
hybrid materials, is necessary to produce bioinks capable of
sterile solutions of photoinitiator (0.05 wt percent Lithium mimicking both animal and plant tissues .
[63]
phenyl-2,4,6-trimethylbenzoylphosphinate [LAP]) and
PBS for a final concentration of 5 wt%, unless otherwise 4.1. Natural biomaterials
stated, for bioink and suspension bath formulations .
[51]
GelMA was dissolved by heating solutions to 37°C for Natural bioinks are polymers derived as biomaterial from
40 min. Unless otherwise noted, fibers were added at a naturally occurring resources. Several natural biomaterials
concentration of 43 × 10 mL and cells were introduced are commonly used as bioinks in 3D printing, including
7
−1
at a concentration of 5 × 10 cell mL . The formulation agarose, alginate, cellulose, chitosan, collagen, dECM,
6
−1
was placed in the printer using a 1 mL syringe. For and silk.
agarose suspension baths, 0.5 wt% agarose was mixed
with deionized water and autoclaved for 1 h on the liquid 4.1.1. Agarose
cycle at 120°C . The solution was immediately placed Agarose is a natural polysaccharide obtained from
[60]
on a stir plate and stirred at 700 rpm until it reached a red seaweed that comprises repeating β-D-galactose
temperature 25°C. This stock solution was then kept at and 3,6-anhydro-α-L-galactose disaccharide units [64,65]
4°C for up to 3 months before use. Unless otherwise (Figure 5). As a member of the carbohydrate family,
noted, the solution was diluted to 0.25 wt% with sterile agarose has a thermo-reversible gelling mechanism and
PBS before bioprinting. The solution was then centrifuged high biocompatibility, thus agarose is frequently used in
at 500 × g for 5 min following dilution and before being tissue engineering applications [40,66,67] .

178 International Journal of Bioprinting (2022)–Volume 8, Issue 4