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Yang, et al.
the same degree of crosslinking. Similar to the thermal Apart from printability and collagen dissolution
gelation experiment, we found that plateaued G’ test, we assessed the cell viability post-printing using
decreased with the increase of rhCol3 concentration; different bioink formulations. The LIVE/DEAD™
rhCol3-free sample (GelMA) possessed the highest G’ staining results showed homogeneous HDFs distribution
(~4.5 kPa) at its equilibrium, while the plateaued G’ of within the printed constructs and high cell viability
GelMA-rhCol3-3.2 was only ~3.0 kPa (Figure S5B). (~97%) (Figure 3E and F), which further confirmed
The compression test further confirms the effect of the feasibility of constructing the dermal layer using 3D
rhCol3 on the mechanical properties of the composite bioprinting and GelMA-rhCol3 bioinks.
bioinks after gelation. As shown in Figure 3C, the
compressive modulus decreased with the increase 3.3. Evaluating the proliferation activities of
of rhCol3 concentration in the 7.5 wt% GelMA; the HDFs and HaCaTs
compressive moduli of GelMA-rhCol3-3.2 (~5.6 kPa) To determine the effect of rhCol3 on the growth of HDFs,
and GelMA-rhCol3-1.6 (~5.7 kPa) were significantly we bioprinted HDFs-laden dermal constructs using
lower than that of plain GelMA (~7.5 kPa). Collectively, different bioink formulations and performed a CCK-
the inclusion of rhCol3 in GelMA would slightly slow 8 assay during a 14-day culture. The results indicated
down the kinetics of thermal and photogelation, as that the embedded HDFs proliferated in both GelMA
well as the mechanical properties after gelation. This is control and GelMA-rhCol3 composite bioink over time.
probably because the soluble free rhCol3 chains would All the tested groups yielded similar HDFs proliferation
interrupt the helix formation of GelMA and chain growth rates, and adding rhCol3 to the GelMA did not reduce
photopolymerization. Similar results were reported in the the proliferation activities compared to the control group
literature, where decreased G’ was observed for GelMA/ (Figure 4A).
Alginate or GelMA/collagen hydrogels after adding non- We then examined the effect of rhCol3 on the
cross-linked alginate or collagen, respectively [45,46] . growth of upper seeded HaCaT cells (Figure 4B). It
To evaluate the shape fidelity of our bioinks, we showed that the cell activities at day 3 were significantly
performed a filament fusion test using an approach adapted higher than those at day 1, indicating the growth of cells.
from the literature . Briefly, fused segment length (fs) It also showed that the proliferation was accelerated by
[30]
of parallel printed strands to their corresponded filament adding rhCol3 to the bioinks as GelMA group possessed
thickness (ft) was determined for varied filament distance the lowest optical density (OD) value at days 1 and 3.
(fd) (Figure 3D). The results indicated that the fs value After culturing for 5 days, there was no significant
increased with the decrease of fd in all tested groups. difference among all tested samples, suggesting that
Meanwhile, adding rhCol3 into the GelMA led to a slight the HaCaTs cells became confluent on top of dermal
increase of fs. As a result, fs/ft of GelMA-rhCol3-3.2 constructs (Figure 4B). We also performed an epidermal
was higher than those of other groups at selected fd. keratinocytes migration assay to assess the formation of
Using another printability assessment approach that the epidermal layer (Figure 4C). To indicate the acellular
characterizes the circularity of the micropores in a printed area, white circles were used to label the uncovered
structure , we utilized the optimized temperatures for region and images are shown in Figure S8. After 3 days
[31]
different formulations while obtaining similar printability of culture, the coverage percentage shifted to a higher
of Pr values that were close to 1. Lower printing number (~98%) with the inclusion of rhCol3 to the bioinks
temperature (~18°C) is needed for GelMA-rhCol3-3.2 than that ~80% of rhCol3-free counterpart (Figure 4D).
compared to that of GelMA (~21°C) (Figure S6). These After culturing for 5 days, the HaCaTs monolayers
findings are associated with the rheological results that imaged from all tested samples show nearly confluent
the presence of additional rhCol3 reduces the G’. status with no significant difference (Figure 4D). This is
We fluorescently labeled rhCol3 and quantified likely due to the continuous proliferation and migration of
the release of rhCol3 at different initial concentrations HaCaTs on the GelMA-based dermis layer, confirmed by
from photocrosslinked GelMA. The results (Figure S7) the literature . Nevertheless, these results demonstrated
[47]
demonstrated a burst release of rhCol3 in the beginning that rhCol3 facilitated HaCaTs adhesion, proliferation,
(~30% at day 1) and a plateau after 1 week (~70% of and migration; therefore, the rhCol3-contained composite
release). Since the soluble rhCol3 could not be properly bioinks should have the potential of supporting epidermis
cross-linked without additional chemical modification, development and regeneration.
the major retaining mechanisms are likely the physical We further evaluated the influence of rhCol3
trapping and physical interaction with GelMA network. on the HaCaTs gene expression using PCR analysis
Nevertheless, the rhCol3 is still presenting throughout (Figure 4E). We found that the mRNA level of the
the culturing period, and nearly 30% will be stably gene P63, which regulates both the proliferation and
maintained in the formulation after 2 weeks. differentiation of epidermal keratinocytes [48] , increased

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