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Yang, et al.
to a quantitative wound healing rate at each time point levels of the key genes relevant to the proliferation and
shown in Figure 6B. There was no significant difference differentiation of HaCaTs. The inclusion of rhCol3 in the
in the wound area among groups at day 3 and day 7, GelMA bioink contributed to the faster wound healing
although GelMA-rhCol3-3.2 group seemed to yield a in vivo, though no significant increase of epidermis
slightly higher healing rate at day 7. However, by day thickness was observed in vitro. Overall, rhCol3 could be
14, the healing rate of GelMA-rhCol3-3.2 group (~94%) used as a potential biomaterial to improve the skin tissue
was significantly higher than those of GelMA-only and microenvironment and thus for the general skin tissue
blank control groups (Figure 6B). H&E staining further engineering applications. Future studies will pay more
revealed the skin regeneration of the wound sites at day attention to the signaling pathways regulated by the bioink
14 (Figure 6C). Fresh hair follicles were also found in composites during skin tissue regeneration.
the regenerated dermis of GelMA-rhCol3-3.2 group. The
regenerated epidermal layers and collagen deposition Acknowledgments
within the wounds were further stained with Masson’s The authors acknowledge the Bioengineering Lab of
trichrome (Figure 6D). Compared to the native skin Nanjing University of Science and Technology for the
tissue of the SD rat, thicker regenerated epidermal layers kind gift of rhCol3. The authors also acknowledge Prof.
were found in both GelMA and GelMA-rhCol3-3.2 Ting Zhang and Prof. Jiabin Guo for the initial discussion
group, and GelMA-rhCol-3.2 group showed freshly on the project.
regenerated hair follicles located right underneath the
new epidermis. In addition, collagen depositions were Funding
found in all wounds as collagen fibers were stained in blue
by the Masson’s trichrome. Similar to our findings, it has The authors acknowledge the funding support from
been recently reported that the human collagen facilitates the National Natural Science Foundation of China
the proliferation and migration of mouse fibroblasts by (No. 52105306), Higher Education Discipline Innovation
stimulating skin regeneration relevant growth factors, Project (111 Project, No. B17026), Tsinghua University
and further influences the repairing of the mouse skin Initiative Scientific Research Program (No. 20197050024),
defects . The mechanical properties of the hydrogels and New Faculty Start-up Funding provided by Tsinghua
[7]
might also affect the in vivo outcome. Since GelMA- University (012-53330200421).
rhCol3-3.2 has lower stiffness compared to GelMA Conflict of interest
group, it might be easier for the proliferated fibroblasts to
penetrate into the softer hydrogel and replace it with the The authors declare no conflicts of interest.
newly formed tissue.
Author contributions
4. Conclusion Conceptualization: Yang Yang, Wei Sun and Liliang

Biomaterials that support wound healing and skin Ouyang
regeneration are long needed. In this study, we introduced Data curation: Yang Yang and Runze Xu
the rhCol3 as a novel bioink component and systematically Funding acquisition: Wei Sun and Liliang Ouyang
investigated its role in skin tissue engineering. We first Investigation: Yang Yang, Runze Xu, Chengjin Wang and
selected 7.5 wt% GelMA as a base bioink formulation Yuzhi Guo
based on an optimization of the 3D culture of HDFs. Project administration: Wei Sun and Liliang Ouyang
Further, we formulated a series of GelMA-rhCol3 Supervision: Wei Sun and Liliang Ouyang
composite bioinks by varying the rhCol3 concentrations Validation: Yang Yang and Runze Xu
(from 0.8 to 3.2 wt%) and achieved desirable printability Visualization: Yang Yang and Runze Xu
and cell viability (~90%) with extrusion-based bioprinting. Writing (original draft): Yang Yang and Runze Xu
The mechanical properties, rheologies, and printabilities Writing (review & editing): Yang Yang and Runze Xu
of the composite bioinks were carefully investigated, and
we found that the addition of rhCol3 to the GelMA led Ethics approval and consent to participate
to a decrease in hydrogel stiffness, storage modulus, and All experiments and procedures were approved by
likely printability. Later, we successfully built a human Institutional Animal Care and Use Committee, Tsinghua
skin equivalent (epidermis layer of around 40 μm in University (F16-00228; A5061-01).
thickness) using our bioink and bioprinting technique. The
in vitro study suggested that adding rhCol3 to the GelMA References
would enhance the proliferation of keratinocytes in the
epidermal layers at the early stage of culture. The addition 1. Kanitakis J, 2002, Anatomy, histology and
of rhCol3 was further proven to upregulate the expression immunohistochemistry of normal human skin. Eur J

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