Page 482 - IJB-9-6

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

bonds between protein molecules, resulting in the of their sides so as to see if they could be readily handled
formation of a stable hydrogel structure; however, some for additional treatments like in vivo implantation; all of
chemical crosslinking agents can be toxic to cells and them except for the ones crosslinked with 0.1 mM genipin
adversely affect cellular behaviors. Enzymatic crosslinking showed adequate controllability without the deformation
involves the employment of enzymes to catalyze the of structure.
formation of crosslinks within the hydrogel, an approach Among these constructs, only scaffolds that were
that is biocompatible and offers more control over the crosslinked with genipin having 1 mM concentration
crosslinking process. Nonetheless, enzymatic crosslinking and crosslinking time of 1 h possessed an average cell
requires longer incubation times, and the used enzymes survival rate of 84%; as a result, these conditions were
need to be carefully handled to maintain cell survival. opted as the optimal crosslinking circumstances. Within a
To be more specific, the chemically crosslinked network
providing physical support impacts the stiffness and pioneering investigation that employed extrusion printing,
[133]
mechanical characteristics of the hydrogel; thus, breaking scientists reported the impacts of the tannic acid
the chemical crosslinking bonds makes the PBHs more (TA), as chemical crosslinking agent used post-printing,
deformable and softer. Since embedded cells are able to with various concentrations (0.1%, 0.25%, 0.5%, 1%,
sense and respond to the surrounding environment’s and 3% wt) in the MC3T3-E1 preosteoblasts-embedded
mechanical properties, they experience alterations in porcine tendon-derived collagen type I scaffolds (5%
6
their behaviors [129] . In the case of physical crosslinking, wt of collagen) (cell density: 5 × 10 cells/mL) on their
the crosslinks are usually reversible and can be broken physical characteristics and cellular activities. They set the
via altering the pH, temperature, or ionic strength of crosslinking time to 10 min in order to impede high cell
the environment. Consequently, breaking these formed fixation which could disturb cellular activities. The scaffolds
bonds can result in the dissolution of hydrogel or the crosslinked with 0.1% and 0.25% wt crosslinker did not
loss of its structure; these phenomena exert effects on possess sufficient mechanical stability; indeed, they were
the encapsulated cells’ organization, distribution, and not capable of preserving their original shape and readily
survival [130–132] . Koo et al. [121] assessed the concentration bending around a steel rod. However, the concentration
and time effects of genipin, as a chemical crosslinker, on above 0.5% wt considerably enhanced the constructs’
cell-laden collagen-based printed scaffolds’ mechanical stability, and the structures could maintain their flat shape
features and encapsulated rabbit articular chondrocytes’ with mechanical strength. Compared with the cell-laden
behaviors. In this regard, these scaffolds bioprinted by collagen constructs without crosslinking, the crosslinked
extrusion printing were dipped into the genipin solution ones had significantly improved mechanical properties.
having concentrations of 0.1, 1, and 5 mM in the culture Furthermore, from concentration of 0.5% wt and above,
medium for various times (0.5, 1, and 6 h). It was revealed the mechanical strength exponentially augmented
that the cell-laden construct crosslinked with a higher (Young’s modulus = 9.81 ± 0.88, 23.34 ± 1.84, and 67.13 ±
concentration of genipin and longer crosslinking time 6.52 kPa for TA-0.5, TA-1, and TA-3 samples, respectively).
displayed higher compressive modulus. Moreover, the Interestingly, non-crosslinked scaffolds had nearly 97% of
compressive modulus enhanced with the increment of degradation rate in 6 h, whereas the other crosslinked ones
crosslinking agent’s concentration and crosslinking time. displayed relatively slow rate of degradation. During 10
At the crosslinker concentration of 0.1 mM, all of the days of observation, the degradation rate of the structures
cellular survival values at the crosslinking times of 0.5, 1, diminished with the increase of TA concentration as
and 6 h were > 90% after 1 day of cultivation, and they anticipated. In the case of cell behaviors, a minor decrease
even augmented up to 98 ± 0.4% after 7 days. At 1 mM in cellular viability was seen from TA concentrations above
concentration of genipin, the cellular viability gradually 1% wt. Nevertheless, all scaffolds preserved great survival
diminished by the crosslinking time’s extension from of cells (approximately 95–96%); to elucidate, the high
0.5 to 6 h, and the percentages of cell survival with the viability with different TA concentrations demonstrated
crosslinking time of 1 h were 88 ± 2.0% and 80 ± 0.5% at that the TA crosslinking process was totally safe for the
1 and 7 days of cultivation, respectively. Nonetheless, at MC3T3-E1 preosteoblasts. Cells initiated proliferation on
the higher genipin concentration of 5 mM, the viability T-0.1 and T-0.25 structures from day 4, and those on the
of cells was decreased dramatically after 7 days due to the T-0.5 scaffold could start at day 7. Thereafter, each of these
high genipin’s concentration toxic effect or the excessively systems exhibited significantly enhanced DNA expression.
high compressive modulus via the immoderate cross- However, the T-1 scaffold did not display a significant
linking, which could fix the cells within the collagen strut difference in the values of DNA expression during 14 days
and ultimately interrupt their metabolic activities. The of cultivation. The release of cells was noticeably high for
scaffolds’ controllability was also specified by lifting one the T-0.1 construct because of the low TA concentration

Volume 9 Issue 6 (2023) 474 https://doi.org/10.36922/ijb.1089