Page 496 - IJB-10-4

International Journal of Bioprinting Embedded bioprinting of cartilage

culture, where fibers were obliquely oriented. Both proteins diameter decreased, the support bath became dense, and
were also distributed in other regions of constructs in the small gaps mitigated the bioink diffusion, resulting in
Group 2. Quantitatively, sGAG content for Group 2 the smooth surface of printed fibers, which was desired.
demonstrated ~2.2- and ~2.3-fold increase compared to Besides the composition of the support bath, complex
those for Group 1 on days 7 and 14, respectively (Figure interactions among the printing parameters and outcomes
6c). Meanwhile, COL-II content for Group 2 demonstrated have been evidenced by both numerical and experimental
~1.9- and ~3.0-fold increase compared to Group 1 on studies. 37–41 For example, as the extrusion pressure increases,
days 7 and 14, respectively (Figure 6d). These findings the flow rate at the nozzle outlet increases, resulting in
confirmed the up-regulated protein expression in the increased fiber width. In contrast, higher nozzle moving
chondrogenic differentiation culture.
speeds resulted in smaller fiber widths. The disturbance
4. Discussion of the support bath is another important consideration
in embedded bioprinting. Increasing the nozzle moving
In this study, zonally stratified cartilage was prepared speed aggravates liquid disturbance, as higher moving
using embedded bioprinting, incorporating the Schiff base speeds generate greater shear stress, causing the support
reaction, where Gel-CDH and OAlg combined to enable bath around the nozzle to fluidize rapidly from a solid-
rapid in situ crosslinking. The 12% Gel-CDH was selected like state. 42,43 A support bath with low concentration also
as this concentration exhibited phase transition at 25°C, increases the disturbance at high nozzle moving speed,
thereby enabling cell-laden extrusion at 37°C. The support which might otherwise cause the extruded fibers to shift
bath, consisting of OAlg solution and G/A microparticles, positions and be dragged in the support bath.
behaved as a Bingham plastic during embedded bioprinting,
exhibiting solid-like behavior under low shear stresses By applying the optimal support bath concentration
and fluid-like behavior under high shear stresses. 34,35 The (80%), nozzle size (27G), and other process parameters,
liquid-to-solid transition and a short self-healing time (i.e., we obtained zonally stratified cartilage with good printing
~2 s) of the support bath enabled the bioink to be smoothly resolution and shape fidelity. The vertical fibers at the
deposited and firmly remain in the designated position. bottom were closely arranged; fibers in the middle zone
were tilted at an angle of ~45°; and fibers in the superficial
In embedded bioprinting, the main process parameters zone were firmly attached. The total width and thickness of
affecting the fidelity of the printed structure included the the cartilage construct were ~8.5 and 5 mm, respectively,
composition and concentration of the support bath, nozzle and the approximation was about 96.5% compared with the
size, extrusion pressure, and nozzle moving speed. In this design size. This size deviation resulted from the diffusion
study, the impact of different parameters on printing fidelity of the support bath into the bioink during crosslinking,
was evaluated quantitatively by measuring the width of which left residual microparticles attached to the surfaces of
printed fibers (Figure 4d, e, and f2) and qualitatively by printed fibers. Additionally, the swelling of the crosslinked
observing the corner features of the print patterns (Figure hydrogel in the aqueous solution contributed to the size
4f1). In the support bath, agglomeration of microparticles difference. Overall, the bioprinted cartilage demonstrated
at room temperature was not observed, which confirmed the reliability of the process window established through
the effective deposition and accurate positioning of the simulation and experimentation.
bioink. The gelatin and alginate combined to form IPNs
in microparticles through a dual crosslinking process Previously, we have explored the degradation behavior
to enhance the connectivity of printed fibers and the of composite hydrogels, OAlg and Gel-CDH, revealing that
mechanical properties of the final construct. Although over 70% of the mass remained after 3 days of enzymatic
36
27
the addition of alginate in microparticles significantly degradation. This demonstrates the stability of OAlg/
increased the compressive modulus of the printed structure Gel-CDH hydrogels against degradation and ensures the
compared to the gelatin-only microparticles, there was stable degradation behavior of the bioprinted cartilage
little difference when the alginate ratio increased from during culture. In terms of the biological functionality
1% to 3%. This might be because there were few residual of the bioprinted cartilage, the results of cell viability
microparticles attached to the fibers, and the strengthening and proliferation indicated high cell survival throughout
effect of the alginate was limited. The size of microparticles bioprinting and culture. Cells were homogeneously
also determined the roughness of the printed fibers. During distributed throughout the entire constructs, and cell
the deposition and in situ crosslinking of the bioink, spreading was observed over time via cytoskeleton
the bioink diffused into the gaps among microparticles, staining (Figure 5f). In addition, the bioprinted constructs
and rough surfaces remained when microparticles were maintained their shape over 14 days of culture without
removed in the post-processing. As the microparticle visible changes in shape, swelling, or disintegration.

Volume 10 Issue 4 (2024) 488 doi: 10.36922/ijb.3520