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

β-sheet structure, and aqueous silk fibroin solution contains the laser light; these phenomena inflict less damage to
hydrophobic domains that self-assemble into 3D hydrogels. the protein structure and cell membrane, which improves
As an amphiphilic material, it is capable of entrapping cell survival [222] .
water and forming bioinks suitable for preventing cell Rhee et al. [106] focused on high-density cell-laden
dehydration in bioprinting. Thus, these kinds of PBHs PBHs. In this regard, primary fibrochondrocytes from
are ideal bioinks for the bioprinting process since they are bovine joints (10 × 10 cells/mL) were mixed with the
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biocompatible, tunable, biodegradable, and capable of self- collagen hydrogel, and the bioprinted constructs were
assembly [209,210] . It is also important to note that spidroins then tested for cell viability and mechanical properties.
1 and 2 are two major ampullates (draglines) in silk with a Accordingly, cells within the constructs were generally
highly repetitious amino acids’ core sequence, conjugated 90% viable immediately after printing. In addition, neither
to the non-repetitive N-and C-terminal domains on either the cell numbers nor their viability varied with time over
side, resulting in a bulging protein structure. By virtue of 10 days. A compressive modulus of 30 kPa was achieved
this property, hydrophilic domains are enclosed within the at the highest printing concentration of collagen hydrogel
micelles, whereas hydrophobic terminal domains build (17.5 mg/mL). Therefore, their constructs demonstrated
the edges, ensuring that proteins are stable. This feature is excellent mechanical stability and could support and
essential for creating precise cell-laden PBHs that can be maintain cell growth. In another pioneering investigation,
bioprinted employing this technology [211,212] . silk-glycidyl methacrylate (Silk-GMA) loaded with
Regarding the impact of bioink concentration on cell human chondrocytes was fabricated by Hong et al. [223]
viability, high concentrations can cause more pressure on for producing engineered cartilage with functional and
the printing nozzle, followed by the generation of high efficient features. They bioprinted hydrogels (5 × 5 × 2 mm )
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level of shear stresses that are damaging to cells. As a containing human chondrocytes (1 × 10 cells/mL) and
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result, the survival of cells should be evaluated at various NIH 3T3 fibroblast cells combined with the Silk-GMA
levels of bioink concentration to enhance the cellular bioink. Likewise, 30% Silk-GMA solution comprising
performance [213] . In order to induce solidification by sol- human chondrocytes (10 × 10 cells/mL) was bioprinted
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to-gel transition, factors like pH changes, temperature, in the shape of a trachea ring (external diameter: 7 mm,
and crosslinking approaches may rupture cell membranes, internal diameter: 5 mm, and height: 2 mm) for in vitro
cause apoptosis and necrosis, or denature biological cartilage TE. Within a novel research, Ren et al. used
[91]
components (e.g., GFs and proteins) that are mixed with an extrusion-based bioprinter to fabricate collagen type
the bioink for developing biomimetic tissues [214-216] . II hydrogel constructs embedding chondrocytes from
New Zealand White rabbits’ knee joints. Three groups were
High cell viability during bioprinting and maintenance
of cellular survival for extended periods are of high created based on the density of total cells incorporated
into the collagen type II pre-gel (20 × 10 , 10 × 10 , and
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priority [217] . As the printing process progresses, numerous 5 × 10 cells/mL). The constructs were crosslinked for 30
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external factors influence the viability of cells, such as min at 37°C in a humidified incubator and were cultured at
printing modules and materials concentrations. Besides, 0, 1, 2, and 3 weeks. Interestingly, 98 ± 1% of chondrocytes
the bioprinting process exerts mechanical forces on cells, were alive. Viability tests were conducted on the first day
causing deformation and breaching their membranes. following synthesis in order to evaluate the damage caused
Despite of cells’ ability to resist specific force levels, their by bioprinting to cells; 93 ± 3% of living cells were present
integrity can be lost if subjected to excessive stresses [218] . in varying groups with no significant difference between
Therefore, recognizing the cell damage mechanisms them (Figure 5A). Based on the results, bioprinted hydrogel
during the bioprinting process to maintain cell viability, constructs with biomimetic cell density gradients can be
one of the basic requirements of bioprinting, is critical. utilized to fabricate engineered cartilage tissues.
Cell viability can also be affected by thermal and shear
stresses that create cell-laden PBH drops. Scientists have The experiment, explained earlier, on engineering P3
reported that cells under local temperatures of up to 300°C hMSCs-encapsulated alginate/gelatin bioinks (4.1% w/v
are not greatly harmed via short exposures of 2 µm during for gelatin and 0.8% w/v for alginate) (cell density: 1.67 ×
the printing process [219,220] ; in contrast, the vibrations and 10 , 5 × 10 , and 15 × 10 cells/mL) for bone TE illustrated
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wave frequencies of piezoelectric bioprinting can disrupt that after 21 days of culture, the cells could spread and form
cell membranes as well as unfold protein structures [221] . a 3D interconnecting network in all groups, particularly
Moreover, in laser-assisted bioprinting, the encapsulated in the 15 × 10 cells/mL bioink (Figure 5B) [114] . The
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living cells and/or proteins are coated with hydrogel above-mentioned investigation on developing arch-like
beforehand, and the absorption layer is transparent to bioprinted structures using GelMA and silk fibroin/gelatin

Volume 9 Issue 6 (2023) 485 https://doi.org/10.36922/ijb.1089