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International Journal of Bioprinting Single-step bioink deposition and maturation of human epidermis

between the body and its surroundings. It not only protects of application, which is a challenge we are trying to solve
us from pathogens and harmful substances, but also in the first place.
prevents fluid loss to the environment. For a burn patient, Different biomaterial mixtures have been explored
restoration of this barrier function is often through the use in the literature for epidermis bioink [10,15,16,20,21,25,41] . The
of STSG , though in the most severe cases – precisely the formulations typically contain natural polymers like
[2]
patients most at risk – the lack of donor sites can preclude fibrinogen [10,15,16,21] and collagen [16,20] which provide
its use . CEA is a relatively well-established alternative, cell adhesion moieties necessary for cell viability.
[3]
though the absence or delayed development of BM between The bioink may additionally contain hyaluronic acid,
the epidermis and dermis contributes to its reputation pectin, gelatin, and glycerol, among others, to improve
for fragility, and remains a significant problem [6-8] . While printability [10,15,20,21,41] . Previously-reported in situ
“spray-on” skin has shown some encouraging results [8,9,36-38] , epidermis printing utilized fibrinogen-based bioink [10,15,16] .
protocols vary widely [8,38] . Furthermore, some usability In each report, the final concentration of biomaterial in the
considerations such as determining where the cells have bioink is around 2–3% w/v, which, though not too high,
been applied can be surprisingly challenging, since the
low-viscosity suspension is not easily visible, and can flow may be sufficient to affect the compact organization of
[10,15,16]
away from a contoured application site . the epidermis . Furthermore, while fibrinogen is a
[10]
popular biomaterial, heterologous sources of the protein
In recent years, bioprinting has emerged as a promising may cause severe immune reaction . For our bioink
[41]
approach to restoring tissue function. Compared with formulation, we chose to use bovine collagen, because it is
other tissues, skin is a lot more accessible, and existing very well-established [27,41] , and because the first U.S. Food
protocols for forming RHEs suggest that self-organization and Drug Administration (FDA)-approved skin substitute
of keratinocytes under the right conditions can yield a also contains bovine collagen . Perhaps because it is
[27]
functional epidermis [28,39,40] . There are numerous reports highly conserved cross-species, even heterologous collagen
that study skin bioprinting [10,15-24] . Most of these studies causes minimal immunological reactions . To minimize
[41]
are concerned with in vitro bioprinting [17-24] , where there the potential regulatory barrier for clinical deployment,
is greater freedom to manipulate the printed constructs, we have also kept the composition as simple as possible,
which may or may not be intended for implantation. avoiding the use of other biomaterials. Since natural
On the other hand, few recent works have identified epidermis does not contain large amounts of collagen
in situ printing as a possible way to treat wounds, such type I, the collagen concentration of our bioink is kept
as burns [10,15,16,26,27] (Incidentally, CEA and “spray-on” skin at a very low concentration of just 0.05% w/v, which was
can be considered to be rudimentary in vitro and in situ still sufficient to form a hydrogel, but is unlikely to affect
bioprinting, respectively.) epidermis formation.
Regardless of the type of bioprinting, the cells are In general, the previous bioprinting studies had a
delivered using a bioink, which typically consists of a strong emphasis on the printing techniques. As a result,
suitable media or buffer, and may also contain biomaterials examination of the printed skin was often limited to simple
that can form hydrogel structures. Unlike the dermis, which H&E staining and observation of morphological structures.
has ample extracellular matrix, the epidermis is almost However, in our experience, care must be taken to analyze
entirely made up of cell bodies. Consequently, epidermis the molecular profile of the printed skin, since simple
bioprinting is frequently accomplished by depositing cells histology can miss out on important features. Markers
suspended in media, without any additional biomaterials, were therefore chosen to confirm that the printed skin
directly onto a dermis or dermal template [17-19,22,23] . This recapitulates the features of the natural tissue, namely skin
process is akin to the formation of RHE described in the self-renewal (p63), active growth (Ki67 and K14), barrier
previous paragraph, and in most cases, the keratinocytes function (FLG and K10), and mechanical robustness (BM
are cultured submerged in a pro-proliferation media for protein ColIV).
a period of time, before changing to a pro-differentiation
media and exposing the top surface to air, that is, airlifted. Since the epidermis is a mostly-flat structure, bioprinting
This approach is not ideal for in situ printing for the can be accomplished by a “2.5D” approach – depositing a
following reasons. First, changing of media is difficult layer of bioink with appreciable thickness, and allowing
once the bioink is printed on the patient. To address this, self-organization to accomplish stratification into the
we developed single media formulations that can support different epidermal layers – instead of the more technically-
[16]
both proliferation and differentiation (Figures 3 and 5). challenging 3D approach . This also simplifies the
Second, like “spray-on” skin, the absence of any hydrogel instrumentation and technique. In this work, our goal
material can result in the bioink flowing away from the site was thus to develop a bioink that we can envision being

Volume 9 Issue 4 (2023) 445 https://doi.org/10.18063/ijb.738