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International Journal of Bioprinting Bioprinting cell-laden protein-based hydrogel

structures into the tertiary 3D configuration. Notably, the microenvironment may affect the protein structures.
proteins have functional amino and carboxyl groups that For example, remodeling of the initial hydrogel matrix
can be utilized to convert these materials into hydrogels by can happen as a function of incorporating the cells and
enzymatic, chemical, and physical crosslinking methods. secretion of native ECM [100] . Besides, hydrogel degradation
The unfolding of protein and its accumulation into a gel and swelling can impact the bioprinted construct’s
matrix is the most frequent process in protein gelation, integrity [101] . Thus, careful consideration should be given to
leading to alterations in conformation from the third to the several important biophysical cues, such as composition,
second structure and the subsequent rise in random coil biodegradation, porosity-related parameters, and
content. Specifically, the structural changes in proteins for crosslinking process. Furthermore, chemical structure,
the PBHs’ formation are owing to the secondary structure the presence of GFs and signaling molecules, as well as
content’s alterations, and proteins’ carboxyl and amino cell signaling, must be taken into account in the case of
groups can form a hydrophilic environment, resulting biochemical cues. All in all, the PBHs should meet several
[21]
in an inherent, great swelling property of PBHs . One biophysical and biochemical requirements to be applied in
crucial factor in the bioprinting of PBHs is the hydrogel’s bioprinting (Figure 2).
mechanical characteristics specified via its network 3.1. Biophysical factors
structure and crosslinking density. Generally, β-sheet
structures are known to provide hydrogels with more 3.1.1. Composition
robust mechanical features than α-helix structures due to One of the most significant biophysical cues is the
their higher degree of crosslinking and stability; therefore, composition of PBHs, which must be taken into account
a greater content of β-sheet structures in PBHs can make when employing them as bioinks for cartilage and bone
them more suitable for bioprinting. As an instance, a group TE. In this regard, several aspects must be considered;
of researchers performed the gelatin-silk fibroin bioink firstly, PBHs must be innately non-immunogenic, non-
[99]
gelation employing enzymatic and physical crosslinking; cytotoxic, and minimally pro-inflammatory to ensure
to elucidate, the physical crosslinking (sonication) the health of cells [102] . Gelatin and collagen hydrogels are
could accelerate the silk fibroin macromolecules’ self- the most commonly employed PBHs for cartilage and
assembly in β-sheet crystals, which in turn improved the bone regeneration due to their excellent biocompatibility
bioink rheology. and desirable capability to support cell growth and
differentiation in these tissues [103] . In the case of PBHs,
Physical and chemical factors are essential for the gelatin molecules are able to form partial triple-helical
bioprinting process, especially the bioprinting of cell-laden conformations by hydrogen bonds below a critical
PBHs. In this case, cells are incorporated into PBHs, and temperature, resulting in a sol-gel transition; however,

Figure 2. An overview of biophysical and biochemical considerations for the use of PBHs as bioinks.

Volume 9 Issue 6 (2023) 471 https://doi.org/10.36922/ijb.1089