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González, et al.

Figure 6. The most frequently reported cells used in bioinks.

Non-animal cells have also been used in bioink second place, followed by gelatin and collagen in third
formulations. Bacterial bioprinting is an emerging field and fourth place, respectively. These polymers have a
that is gaining momentum [137] and will enable exciting common origin, namely collagen from animal tissues
applications in the coming years. Recent papers illustrate including mammals [155,156] , fish [157] , and poultry [158] . As
the use of bioprinting techniques for the fabrication of a group, they are the most prevalent type of hydrogels
bacterial biofilms with different functionalities [138-141] or used in bioprinting applications. GelMA and gelatin
the re-creation of complex bacterial communities [66,102,142] . are simpler versions of collagen, and both are friendlier
Microalgae [143,144] have also had representation within materials to process and handle than collagen when
the bioprinting literature. Recent experimental evidence formulating bioinks. They are also less costly; for
shows that the symbiotic coexistence of microalgae and instance, a gram of collagen from Sigma-Aldrich costs
mammalian cells in thick bioprinted tissue constructs is US$ 2360, while a gram of GelMA and gelatin costs US$
a feasible alternative for enabling a sustainable supply of 206 and US$ 0.342, respectively. GelMA is a chemically
oxygen within the constructs. modified gelatin that cross-links upon exposure to
ultraviolet or visible light in the presence of a suitable
4.2. Hydrogel formulations used in bioinks photoinitiator, such as Irgacure [153,159] , lithium phenyl-
Bioinks may consist solely of cells [94,145,146] . However, 2,4,6-trimethylbenzoylphosphinate (LAP) [160,161] , or eosin
13 of the analyzed documents used cell aggregates or Y [162,163] , the end result is a solid hydrogel held together
cell spheroids as bioinks. Most bioinks are cell-laden by covalent bonds [154] . In fact, Irgacure, LAP, and eosin
hydrogels and the hydrogel matrix has a starring role in Y appear in the top 10 most used cross-linking methods.
the functionality of the bioink which contributes to the Gelatin [164] , in contrast, forms physical hydrogels
success of the bioprinting technology. Hydrogels must in response to low temperatures [165] . In this case, weak
provide the right environment for living cells while still intermolecular forces between the gelatin chains, rather
exhibiting the physicochemical properties (i.e., rheology, than covalent cross-links, hold the hydrogel together.
stability, molecular integrity) needed to facilitate their Indeed, temperature occupies the third place in Figure 7B
processability or manipulation [147] . We identified 156 as a “cross-linking method” (in this case, no covalent
different hydrogels and 48 cross-linking methods within or ionic bonds are involved, only weak molecular
the analyzed literature. Figure 7 presents the top 10 most interactions).
frequently reported hydrogels and cross-linking methods A technological advantage of using GelMA instead
used for bioink formulations. of gelatin is that, while gelatin melts under incubation
The most popular combination used in bioink conditions (37°C), a photo-cross-linked GelMA construct
formulations is alginate (a carbohydrate extracted from remains stable [166] .
brown algae) and its preferred cross-linking agent However, neither gelatin nor GelMA conserves
(calcium chloride; CaCl ) [148] . Alginate [149-151] is an anionic the tertiary and quaternary structure of collagen, which
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carbohydrate-based polymer that cross-links efficiently may represent a disadvantage for many applications.
(easily and rapidly) in environments rich in divalent However, they both preserve the arginine-glycine-
cations such as aqueous solutions of CaCl or calcium aspartate (RGD) domains (arginine, glycine, and
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sulfate (CaSO ). [152] The ease and speed of this type of aspartate) required for cell anchoring, which is a
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cross-linking make alginate a convenient working matrix. central attribute when designing cell scaffolds for tissue
Gelatin methacryloyl (GelMA) [120,153,154] comes in engineering [166] , as promoting cell anchoring to the bioink
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