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Recombinant Human Collagen for 3D Bioprinting of Skin Equivalent
found to be critical in dermal collagen fibrillogenesis and skin models. Moreover, the typically formulated bioinks
tissue integrity. Meanwhile, type III collagen has been based on collagen usually lack proper printability due to
shown to induce the transcription of growth factors for the slow gelation kinetics at physiological conditions.
fibroblasts, including keratin growth factor, vimentin, To address these problems, we propose to formulate
and transforming growth factor beta (TGF-β) [6-8] . The recombinant human type III collagen (rhCol3) into
content and the ratio of these two types of collagens bioinks for the bioprinting of a full-thickness human tissue
were found to vary with age and injury. For instance, equivalent. In general, recombinant human collagens
the type III collagen in normal fetal skin accounts for have been designed and synthesized as alternative
approximately 34 – 65% of the total collagen, while biomaterials to the native human collagen, with the
later in the childhood and early adult life, approximately advantages of high purity, batch-to-batch consistency,
80 – 85% of the collagen in the skin are type I [9-12] . and low immunogenicity . Moreover, they have been
[23]
Meanwhile, the content ratio of type III collagen in the reported to support cellular activities , being applied to
[24]
skin hypertrophic scar tissuewas found to be from 14% to the engineering of bone and neural tissues . Recently,
[25]

28% . This content change with time has been inferred a biomaterial composed by gelatin and recombinant
[13]
to be involved in skin ECM remodeling, in support of the type III collagen has been proven to support the growth
fact that skin tissue architecture is highly dynamic and of seeded NIH-3T3 cells and promote the regeneration
heterogeneous [10,14] . The previous studies have indicated of damaged rat skin . Herein, we hypothesize that
[26]
that both epidermal cells and fibroblasts are sensitive to rhCol3 has an important role in supporting human skin
attached or embedded substrate with various physical cell growth and hence would facilitate the formation of
cues, for instance, stiffness . The integrin-binding the epidermis in vitro and wound healing in vivo since
[15]
motifs decorated to the matrices surface have also shown native human type III collagen is the second abundant
to bind epithelial progenitors and further promote their ECM structural collagen in the skin . To achieve
[27]
migration and wound healing. For instance, human skin printing convenience, we formulated a composite bioink
fibroblasts with mutated type III collagen gene were by mixing rhCol3 with gelatin methacryloyl (GelMA),
unable to organize collagens and fibronectin in the ECM a commonly used printable biomaterial . To model the
[28]
due to the downregulation of α2β1 integrin [8,16] . contents of type III collagen in native human skin tissues
Thus, it is desirable to recapitulate the dynamic and (Figure 1A) [11,29] , we developed the composite bioinks
heterogeneous human skin tissue microenvironment when with varied rhCol3 contents ranging from 0.8 to 3.2
engineering in vitro skin tissue for either pharmaceutical wt%, representing 10 – 30% ratio of matrix polymer in
or regenerative applications. The previous studies have the bioinks. We optimized the bioprinting of this novel
engineered in vitro three-dimensional (3D) human skin bioink formulation based on rheological and printability
equivalents based on human skin cells, which are both assessments. Then, we constructed an in vitro 3D human
structurally and functionally more similar to native human skin equivalent with human epidermal keratinocytes
skin than 2D culture models and animal models [17,18] . (HaCaTs) and dermal fibroblasts (HDF) using extrusion-
This field is greatly advanced by the use of innovative based 3D bioprinting. Basically, the rhCol3-based bioink
biofabrication technologies, such as 3D bioprinting, in formulations consisting of base component GelMA,
which 3D skin tissue could be pre-designed and then bioactive rhCol3, and photoinitiator (lithium phenyl-
precisely fabricated following layer-by-layer assembling 2,4,6-trimethylbenzoylphosphinate, LAP) were prepared
of cell-laden bioinks to yield spatial heterogeneity . at the predefined concentration (Figure 1B). Then,
[19]
Laser-assisted, inkjet-based, and extrusion-based we fabricated the dermal constructs by 3D bioprinting
bioprinting are three major techniques used for the of HDF-laden bioinks followed by photocrosslinking
fabrication of skin equivalents. For instance, Lee (Figure 1C). After 3 days of culture, the constructed
et al. fabricated 3D skin tissue through inkjet printing dermal layers were seeded with HaCaTs on top
multilayer constructs [20] , and Koch et al. utilized (Figure 1D) followed by submerging culture to allow for
laser-assisted bioprinting to build in vitro skin tissue HaCaTs and HDFs proliferation (Figure 1E). An air-liquid
in predefined patterns [21] . Some groups have obtained interface (ALI) culturing method was performed to obtain
3D-printed human skin equivalents using naturally the differentiated epidermal layers (Figure 1F). Based
derived biomaterials, such as collagen I, to resemble on this skin model, we comprehensively investigated
ECM environment [22] . Nonetheless, efforts are still the effects of rhCol3 on the cellular processes of skin
required to improve mimicking complex human skin cells in vitro. We also assessed the skin repair process
tissue microenvironment of the in vitro skin equivalent. based on in vivo analysis of implanting GelMA-rhCol3
For instance, the effect of significant type III collagen hydrogel onto Sprague Dawley (SD) rat dorsal wound
on skin formation is poorly understood as it has been (Figure 1G). Collectively, both in vitro skin construct
rarely involved in the engineering of in vitro human and in vivo wound healing analysis indicated that

146 International Journal of Bioprinting (2022)–Volume 8, Issue 4