Page 493 - IJB-10-4

International Journal of Bioprinting Embedded bioprinting of cartilage

To investigate the effect of alginate on the compressive diameter. Although fibers thinner than the nozzle diameter
modulus, cubic structures (5 × 5 × 6 mm) were printed could be obtained, the rapid nozzle motion created a larger
in different support baths. After printing, the constructs disturbance, leading to position offset and stretching of
were rinsed with PBS to remove the excess microparticles fibers. Therefore, the parameters identified in the blue
and then immersed into a CaCl solution for crosslinking columns were deemed appropriate for the printing process.
2
to form IPNs among the remaining microparticles. The A nozzle moving speed of 10 mm/s and an extrusion
addition of alginate in the microparticles resulted in a pressure of 8 kPa were selected for subsequent studies.
more than three-fold increase in the compressive modulus
(Figure 4c). However, the increase in alginate concentration 3.4. Bioprinting of the zonally stratified cartilage
from 2% to 3% did not increase the compressive modulus The zonally stratified cartilage designed in this study
remarkably. Considering the biocompatibility of printed comprises three zones, namely the bottom, middle, and
constructs, 2% alginate concentration was used to prepare superficial zones (Figure 1). In the bottom zone, vertical
a support bath for subsequent bioprinting. fibers with a height of 2 mm were printed, accounting for
36.4% of the total volume of the bioprinted cartilage. The
In addition, the concentration of the support bath is middle zone contained tilted fibers with a height of 2.5
another significant factor influencing the printing quality. mm, occupying 45.5% of the total volume. The superficial
An M-shaped pattern with lines of different lengths was zone consisting of horizontal fibers was 1 mm in height,
designed to examine the impact of parameters on fiber accounting for 18.1% of the total volume. The thicknesses
formation and printing fidelity, which measures the of individual zones and their proportions in the cartilage
degree of similarity between bioprinted features and the construct were compatible with the native counterpart.
31
intended design. Support baths with higher concentrations
resulted in smaller fiber widths and higher fidelity (Figure During the bioprinting process, cell-laden Gel-
4d). For low support bath concentrations (e.g., 40%), the CDH bioink was pre-heated to 37°C in the nozzle and
fibers exhibited large widths and a rough morphology. extruded into the support bath containing OAlg and G/A
Moreover, nozzles with different sizes (22G, 25G, and microparticles. The aldehyde groups of OAlg and amino
27G) were compared for printing in a support bath with groups of Gel-CDH formed imine bonds, facilitating
80% concentration. At a constant extrusion pressure of 8 the self-assembly of fibers in situ, with support from the
kPa and nozzle moving speed of 10 mm/s, the fiber widths G/A microparticles. In the post-processing, the excess
decreased with smaller nozzle sizes (Figure 4e). A larger support material around the bioprinted construct was
nozzle extruded more solution, resulting in increased fiber gently removed, and the cartilage construct was immersed
width, a rough fiber surface, and a distorted structural in a CaCl solution. The gelatin portion was crosslinked
2
morphology, especially at the corners. with TG during the preparation of G/A microparticles
to form semi-IPN microgels, and the alginate portion of
The impact of nozzle moving speed and extrusion the remaining G/A microparticles within the bioprinted
pressure on the resolution of the printed structure was also construct was further crosslinked with Ca to create IPN
2+
studied using a support bath with 80% concentration and microgels. This two-step crosslinking process enhanced
a 27G nozzle. As the nozzle moving speed increased from the inter-fiber connections and mechanical properties
5 to 20 mm/s, the fiber widths reduced and the corners of the bioprinted construct, utilizing the residual G/A
appeared sharper, indicating improved fidelity (Figure 4f1). microparticles effectively. The bioprinted cartilage
During the printing process, the extruded solution was demonstrated great structural integrity with a well-defined
stretched when a large nozzle moving speed was applied, zonally stratified arrangement (Figure 5a).
resulting in printed fibers with small widths and smooth
surfaces. On the contrary, a small nozzle moving speed led 3.5. Evaluation of the zonally stratified cartilage
to redundant solution deposition, causing enlarged line The bioprinted cartilage constructs were cultured without
widths, rough fiber surfaces, and inferior corner features. (Group 1) and with chondrogenic differentiation (Group
Furthermore, as the pressure increased from 6 to 12 kPa, 2). Quantitatively, cartilage constructs cultured in different
the fiber widths enlarged due to increased flow rates at the conditions exhibited similar compressive modulus after 2
outlet, which was consistent with the simulation results days of culture, with no significant difference compared
obtained in Section 3.2. The effects of nozzle moving speed to the acellular controls (~40 kPa; Figure 5b). The LIVE/
and pressure are summarized in Figure 4f2. The green DEAD staining of cartilage constructs cultured for a
columns represent the resultant fiber widths that were week displayed a large number of live cells in both groups
smaller than the nozzle diameter; the blue ones represent (Figure 5c), suggesting that cells inside the constructs
fiber widths that were ~1–1.3 times the nozzle diameter; remained viable regardless of chondrogenic differentiation.
the red ones represent fiber widths >1.6 times the nozzle Quantitative analysis revealed that cell viability was close

Volume 10 Issue 4 (2024) 485 doi: 10.36922/ijb.3520