Page 285 - IJB-10-6

International Journal of Bioprinting Skin bioprinting: Keratinocytes and stem cells

the bioprinted constructs at a constant deformation of 1% Mechanical characterization of the hydrogels revealed
(0.15 N pre-load) and an increasing frequency in the range that hydrogel IV (Alg/Gel/collagen/HA) had much higher
of 0.3–100 rad/s on days 1, 7, and 14. By comparing the viscous characteristics than the others (that exhibit
samples made of Alg/HA/Gel with those made of GelMA elastic properties). Hence, hydrogel IV is less suitable for
at a frequency of 7.5 rad/s, two-way ANOVA revealed a constructs that require long-term stability.
significant difference between the hydrogels. Alg/HA/Gel The reporter protein TNFR2-Fc-GpL expressed by
constructs had a significantly higher storage modulus and encapsulated HEK293 cells was detected in the culture
value of complex modulus than GelMA constructs. medium. Since TNFR2-Fc-GpL has a relatively high
On day 1, the biofabricated Alg/HA/Gel construct molecular weight (~150 kDa), the secreted proteins can
displayed significantly higher storage modulus and complex diffuse through the hydrogels and stimulate the cells in co-
modulus compared to GelMA constructs, regardless of cell culture. Alg/HA/Gel and GelMA demonstrated adequate
presence. Considering the storage modulus, this significant protein diffusion into the medium.
difference between the hydrogels was also observed on day Both Alg/HA/Gel and GelMA hydrogels were chosen
14 for the constructs without cells. for the bioprinted 3D model due to stable cell viability
inside the hydrogels and good bioink printability. The
4. Discussion excellent cell metabolic activity within the Fib hydrogel in

The treatment of large-scale burn wounds and skin defects the transwell model led us to integrate the hydrogel into the
remains a major challenge in reconstructive surgery due 3D model. Since printing the original Fib concentration
to the limited availability of autologous skin grafts and from the transwell experiments proved unfeasible, as
demonstrated by the printability assay, we opted to add 1%
the resulting donor-site morbidity. 1,2,8 Given the urgency Alg and 1% HA to the hydrogel to enable bioprinting.
of closing large burn wounds, biofabrication offers new
possibilities by enabling automated and precise skin During the experimental period of the bioprinted 3D
substitute production in large quantities. Bioengineered model, an increase in metabolic activity in all experimental
skin substitutes in combination with autologous stem groups was demonstrated. Within the constructs of both
cells could represent a promising therapeutic alternative hydrogels, the metabolic activity was significantly higher
to the transplantation of autologous tissue. 1,6,25 Within this on day 14 compared to days 1 and 4. The live/dead assay
study, the influence of ADSCs on the growth of HaCaT indicated an increasing cell count for the co-cultured
keratinocytes was evaluated in a transwell model, as well groups over the experimental period. The biofabricated
as in a bioprinted 3D model composed of two different GelMA constructs demonstrated nine-fold higher cell
hydrogel combinations. This study aimed to identify a viability on day 14 compared to day 1, consistent with
bioink that facilitates optimal cell survival and elucidate the findings of Zhao et al., whereby GelMA hydrogels
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the synergistic influence of ADSCs on HaCaT keratinocyte reportedly support the growth of HaCaT. The Alg/Ha/Gel
constructs also displayed a steady increase in cell viability
viability and growth.
and count. Alg- and HA-based bioinks are widely used
In this study, we initially employed a transwell model to in tissue engineering due to their good biocompatibility
compare the cell metabolic activity of HaCaT keratinocytes and biodegradability. Several studies reported a high cell
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within different hydrogels. The cells displayed a significant viability in Alg-, Gel-, or HA-containing hydrogels. 10,27–30
increase in metabolic activity from days 1 to 7 when seeded In this study, we incorporated ADSCs into biofabricated
into or onto different hydrogels composed of Alg/Gel, Fib, constructs. It has been proposed that HA/Gel-based
collagen/elastin, Alg/HA/Gel, or GelMA. The keratinocytes hydrogels enhance the cell differentiation and viability
within the Fib hydrogel demonstrated excellent metabolic of ADSCs. ADSCs play a major role in activating tissue
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activity with significantly increasing metabolic activity regeneration via paracrine signaling and can differentiate
within 7 days, despite Fib hydrogels possessing weak shape into skin cells. 31–33 Seo et al. examined human ADSCs
fidelity (confirmed via bioprintability assay), which is and human keratinocytes in a transwell model and
consistent with other studies. Hydrogels VI (GelMA) and demonstrated that, when co-cultured with keratinocytes,
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V (Alg/HA/Gel) exhibited precise printing properties and ADSCs express increased amounts of keratinocyte-specific
shape fidelity. For GelMA, the highest DCR was observed markers and may differentiate into keratinocyte lineage.
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at 0.42, consistent with the findings of Schulik et al. There Previous 2D and transwell studies described how HaCaT
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was no significant difference in pore size between hydrogels and ADSCs could positively influence each other. 33–36
I and IV. GelMA (hydrogel VI) resulted in significantly Treating HaCaT keratinocytes with a 50% conditioned
smaller pores in SEM than the other samples. medium of ADSCs increased the proliferation of HaCaT

Volume 10 Issue 6 (2024) 277 doi: 10.36922/ijb.3925