BIOPRINTING FOR SKIN - 3D Bioprinting and Nanotechnology in Tissue Engineering and Regenerative Medicine - page 283

13.3.4.3 Laser-assisted Printing of Skin Tissue - in vitro Culture
For printing 3D skin tissue, mouse NIH3T3 Swiss albino fibroblast (DSMZ Braunschweig, Germany)
and human immortalized HaCaT (DKFZ, Heidelberg, Germany) keratinocyte cell lines were used
( Koch et al., 2012; Michael et al., 2013b ). These well-established cell lines have been combined in
other studies ( Bigelow et al., 2005; Delehedde et al., 2001 ). 3T3 fibroblast cells are often used in
keratinocyte cultivation because of secreting growth factors favorable for keratinocytes ( Boehnke
et al., 2007; Linge, 2004; Schoop et al., 1999 ).
To approximate native skin as far as possible, collagen type I from rat tail was applied as hydrogel
for embedding the cells for the printing process and as ECM afterwards, since this is the main ECM
protein in skin ( Koch et al., 2012 ).
As the basis for the printed skin tissue, a 1 mm thick sheet of a collagen-elastin matrix (Matriderm™,
MedSkin Solutions Dr. Suwelack AG Billerbeck, Germany) was used for providing mechanical strength
directly after printing. Matriderm™ is porous and permeable for cell culture media ( Golinski et al.,
2009 ). Successively, 20 layers of NIH3T3 fibroblasts and 20 layers of HaCaT keratinocytes, both embed-
ded in collagen, were printed onto the Matriderm™ substrate. After printing, the skin constructs were
cultivated for 10 days submerged in cell media. Histologic sections, depicted in Figure 13.3 , showed a
tissue-like cell pattern. The printed cells remained in the bilayered 3D structure. Figure 13.3 also depicts
the formation of a basal lamina as part of a basement membrane at the interface between keratinocytes
and fibroblasts, as it exists between epidermis and dermis in natural skin. Basal laminae are established
by epithelial cells like keratinocytes and consist mainly of the proteins collagen type IV and laminin,
which is connected to the epithelial cell membranes.
Directly after printing, the 40 printed layers had a total thickness (without the Matriderm™) of
about 500 m m. This thickness decreased to 250 m m or 50% within 24 h due to the fibroblasts contract-
ing the surrounding collagen. This shrinkage is well known from literature ( Bellows et al., 1982 ). The
remaining thickness of 250 m m is small enough to supply the cells by diffusion.
13.3.4.4 Tissue Formation In Vitro (Submerged Culture)
Tissue formation is determined by intercellular junctions ( Ko et al., 2000 ), which can be found as
cell-cell and cell-matrix connections in all kinds of tissue, abundantly in epithelium like the epidermis.
Adherens junctions occur in epithelium often as bands that encircle the cell (zonula adherens).
In other tissues, only punctuate junctions and spots of adhesion can be seen. Adherens junctions are
composed mainly of cadherins (calcium-dependent adherent proteins), which are connected to the actin
cytoskeleton of the cells via other proteins. At the extracellular side of the cell membrane, cadherins
connect with cadherins of adjacent cells by forming homodimers. Adherens junctions are essential for
tissue morphogenesis and cohesion ( Gumbiner, 1996; Niessen, 2007 ).
Gap junctions are clusters of cell-cell channels crossing the cell membranes of two adjacent cells
and connecting their cytoplasm directly. They enable intercellular communication between neighbor-
ing cells by exchanging small molecules ( ≤ 1000 Dalton) by diffusion and synchronizing electrical
as well as physiological activities ( Mese et al., 2007; Simon and Goodenough, 1998 ). Gap junctions
are established by two cells, each forming one semichannel (connexon). Connexons consist of nor-
mally six connexins ( Richard, 2000 ) in a hexagonal pattern with a free channel in the middle. The
connexons of two adjacent cells connect, forming one intercellular channel. Gap junctions play a
fundamental role in differentiation, cell cycle progression, and cell survival ( Fitzgerald et al., 1994;
Schlie et al., 2010 ).