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International Journal of Bioprinting Skin bioprinting: Keratinocytes and stem cells

calculating the area of the well bottom covered by living The keratinocytes seeded into hydrogels I (Alg/Gel), V
cells using Image J. HaCaTs and ADSCs were differentiated (Alg/Gel/HA), and VI (GelMA) formed fewer but larger
based on their morphology in the live/dead staining. colonies. The images of HaCaT within the different
hydrogels indicate good cell survival, i.e., without any
2.5. Statistical analysis notable dead cells.
All experiments were performed in triplicate, and results
were analyzed using Graph Pad Prism 10 (GraphPad 3.2. Material characterization of hydrogels
Software Inc., USA). Data are presented as the mean ± Characterizations of all hydrogels (listed in Table 2) are
standard deviation or box plots with whiskers. The p-value presented in this section.
was set to ≤0.05 to define statistical significance. Differences
between the groups were analyzed using a t-test and two- 3.2.1. Printability assay
way analysis of variance (ANOVA) with Tukey’s multiple As illustrated in Figure 5, hydrogels IV, V, and VI
comparisons for assessing cell viability and metabolic demonstrated good printability. The printability of
activity. For the transwell model, two-way ANOVA with hydrogel II was immeasurable as the hydrogel deliquesced
Tukey’s multiple comparisons and Fisher’s Least significant right after extrusion from the printing nozzle. Therefore,
difference (LSD) were used for analysis. The stiffness of fibrinogen was enriched with 1% (m/v) Alg and 1% (m/v)
the biofabricated constructs was analyzed with two-way HA (i.e., hydrogen III), resulting in a DCR of 0.30. For
ANOVA and three-way ANOVA with Fisher’s LSD test. hydrogels V and VI, the highest DCR was observed at 0.40
and 0.42, respectively. Hydrogels I and IV reported the
Three-way ANOVA with Fisher’s LSD test was also used lowest DCR of 0.24 and 0.29, respectively.
to analyze the degradation results. Figures 1 and 3 were
created with biorender. 3.2.2. Microstructure and material properties
The hydrogels were analyzed on day 1 using cryo-SEM
3. Results (Figure 6). No significant difference in pore size was
3.1. Cell viability of HaCaT in co-culture with ADSCs observed when comparing hydrogels I–IV. Hydrogel VI
in a transwell model exhibited smaller pores than the other samples. The pore
The metabolic activity of the cell cultures within the sizes ranged from 0.21 (hydrogel VI) to 1.03 (hydrogel
transwells was measured on days 1, 4, and 7. All samples V). Hydrogel V has a less homogenous pore distribution
(listed in Table 1) exhibited increased metabolic activity compared with the other hydrogels (Figure 6A).
over 7 days (Figure 4A). Analysis of the metabolic rate Measuring the mechanical properties of the hydrogels
suggests that time played a significant role for all groups, on day 1 revealed notable differences (Figure 7). All
except group IV (Alg/Gel/collagen/HA). hydrogels, except for hydrogel IV (Alg/Gel/collagen/HA),
Hydrogels I (Alg/Gel), II (Fib), and VI (GelMA) primarily exhibit elastic properties, with a higher storage
displayed significantly higher cell metabolic activity on modulus than loss modulus. Hydrogel IV exhibited much
day 7 compared with day 1 for the co-culture and control higher viscous properties, resulting in a less stable hydrogel
(without ADSCs) groups. Additionally, cells seeded that could barely be transferred into the rheometer.
within hydrogel II demonstrated a significant increase Hydrogel VI (GelMA) was the softest hydrogel. The other
hydrogels were in a similar range throughout the tested
in metabolic activity from days 1 to 4 for the group frequency range.
containing HaCaT and ADSC co-culture. Although the
difference in metabolic activity of hydrogel V between Hydrogels that could not be printed (I, II, and III) or
days 1 and 7 was less pronounced compared to the other unstable (IV) were not suitable for use as biofabricated
bioinks, it was still statistically significant. The metabolic constructs. However, hydrogel III, despite issues with
activity of cells within the collagen-elastin template matrix shape fidelity, was still used in the pockets of the constructs
was significantly higher on day 7 compared with day 1 due to its high cell compatibility.
for the co-culture group. Samples containing HaCaT and
ADSCs in hydrogels II (Fib) and III (collagen-elastin 3.2.3. Diffusion assay
template matrix) exhibited increased metabolic activity A diffusion assay was performed to evaluate the diffusion
in comparison to the groups without ADSCs. The weakest characteristics of the hydrogels (Figure 8). For this assay,
metabolic activity was observed for hydrogel IV (Alg/ HEK-293 cells producing a 150 kDa protein with a
Gel/collagen/HA). Hydrogel II and the collagen-elastin luciferase domain were implanted into the hydrogels.
template matrix reported the highest metabolic activity. An increase in protein concentration was measured
Within these hydrogels, cells proliferated rapidly and over 7 days in the supernatant for all hydrogels, suggesting
formed multiple small colonies, i.e., visible in Figure 4B. HEK-293 proliferation and diffusion of the protein into the

Volume 10 Issue 6 (2024) 269 doi: 10.36922/ijb.3925