BIOPRINTING FOR SKIN - 3D Bioprinting and Nanotechnology in Tissue Engineering and Regenerative Medicine - page 286

into neighboring cells and further cells, which is only possible through functional gap junctions. The
LY went through up to ten cells. This demonstrated that the printed skin grafts possessed epithelium-
specific functions with respect to adherens and gap junctions ( Koch et al., 2012 ).
13.3.4.5 Tissue Formation In Vitro (Air-Liquid Interface Culture)
The production of skin equivalents via laser-assisted bioprinting and their culture under submerged
conditions has been described. As a first step, this served to assess the printed skin constructs as well
as the printing process. As a next step, keratinocyte differentiation, which is crucial for the forma-
tion of a multikeratinized epidermis, was induced by different stimuli. First, the skin constructs were
raised to the air-liquid-interface ( Michael et al., 2013b ). This means that the skin equivalents were
supplied with nutrients and liquid from below, but had contact to the air from above—like real skin
in our bodies. Second, differentiation supporting substances were added (10 − 7 mM hydrocortisone,
10 − 7 mM isoprenaline hydrochloride, and 10 -7 mM insulin) and the calcium concentration was raised.
Skin constructs were cultured for 11 days and examined on day 0 (day after printing, when raised to the
interface), day 5, and day 11. The used cell lines were stably transduced with genes for fluorescent mol-
ecules so that the cells could easily be detected via fluorescent microscopy at the end of the experiments.
On day 0, the cells were mostly rounded and still embedded in the collagen, which had served
as the printing matrix ( Figure 13.5 ). On days 5 and 10, however, the keratinocytes were connected,
forming an epidermis-like tissue. Analogously to the previous experiments, e-cadherin as a marker for
adherens junctions could be detected in the epidermis-like layer on days 5 and 10, thereby confirming
the formation of an epithelium.
In contrast to the keratinocytes, the printed fibroblasts partly migrated into the underlying Matri-
derm™, following its fibers closely. Part of the cells stayed at the interface between the Matriderm™
and the keratinocytes, though. This is very promising for future in vivo applications.
In the Masson's trichrome staining, inclusions of collagen could be seen in the epidermis, espe-
cially directly above the Matriderm™. Probably, the printed cells—especially the fibroblasts—did not
completely digest the collagen with which they were printed, but instead migrated out of it, leaving the
collagen behind.
In summary, a bilayered skin construct was successfully created by laser-assisted bioprinting and
subsequent in vitro culture.
13.3.4.6 Laser-assisted Printing of Skin Tissue - In Vivo Culture
On their way to clinical applications, new therapies need to be tested in animal experiments to en-
sure maximum safety. In the first step, a rodent model is often used since these animals are small,
simple to keep and breed, and inexpensive. Different genetically altered strains are also available. As
Michael et al. used xenogeneic (keratinocytes) and allogeneic (fibroblasts) cells, they needed to use
T-cell-deficient mice to avoid a rejection of the skin transplants ( Michael et al., 2013b ). Analogously to
the in vitro experiments, the used cell lines were stably transduced with genes for fluorescent molecules
to enable direct detection of the printed cells.
For the in vivo evaluation the dorsal skin fold chamber in mice was used. During the preparation of
the chamber, the dorsal skin of the anaesthetized mouse was lifted and fixed via two titanium frames
(chamber), resulting in a sandwich-like construction. In one side a full-thickness wound was cut, while
the opposite skin remained unharmed. The skin construct was then inserted into the wound. The cham-
ber was closed by insertion of a glass cover slip that was fixed with a snap ring. In contrast to humans