BIOPRINTING FOR SKIN - 3D Bioprinting and Nanotechnology in Tissue Engineering and Regenerative Medicine - page 280

13.3.3 EXTRUSION-BASED BIOPRINTED SKIN CELLS
Lee et al. (2009) successfully printed vital human skin fibroblasts and keratinocytes with an extrusion
printer. They printed 10 layers of collagen precursor (e.g. rat tail collagen type I, BD Biosciences, and
MA). Since this precursor is acidic, they printed it without cells. Each layer was cross-linked with
nebulized aqueous sodium bicarbonate (NaHCO 3 ). In between these layers, they printed one layer of
fibroblasts and (after six further collagen layers) one layer of keratinocytes, each suspended in cell
culture medium. Thus, they circumvented the general problem with collagen printing. As the most
abundant protein in the human skin (and in the human body), collagen has become a major area of
interest in tissue engineering. However, the collagen precursor is acidic; after neutralization with a
base, which is indispensable for cell survival, the viscosity of the collagen gel increases, hence, it can
no longer be printed with small nozzles.
On the other hand, in printing the cells, suspended in a cell medium, separately from the collagen, it
might prove difficult to achieve a tissue-like (especially epithelium-like) cell density since the intermit-
tent collagen layers serve as a scaffold but do not contain cells. Additionally, the high volume of the
cell media layers might cause displacement between the collagen layers and thus prevent the formation
of a stiff 3D hydrogel block.
A peculiarity of the work of Lee et al. is the printing in a 3D free-form mold of poly(dimethylsiloxane)
(PDMS). The intention was to replicate the surface contour of a skin wound with the mold. The single layers
of fibroblasts and keratinocytes were meant for the demonstration of the “ability to print spatially distinctive
cell layers.” However, they did not show tissue generation or the establishment of intercellular junctions.
Furthermore, the gap of about 75 m m between fibroblasts and keratinocytes did not allow the development
of an interface (basement membrane), as can be found between the dermis and epidermis in natural skin.
13.3.4 LASER-ASSISTED BIOPRINTED SKIN
13.3.4.1 Schematic of the Laser-assisted Bioprinting Setup
A further technique used by several groups is laser-assisted bioprinting. In principle, a laser-bioprinting
setup consists of a pulsed laser and two coplanar glass slides. The upper one (here called donor) is
coated with a thin layer of a laser-absorbing material, and subsequently a layer of the biomaterial that
will be printed (i.e. the cells embedded in an appropriate hydrogel). Then, this glass slide is mounted
upside-down above the second glass slide (here called collector). The distance is set from a few hundred
micrometers up to a few millimeters.
Absorption layers such as gold, titanium, and polymers like triazene ( Schiele, et al., 2010 ) or gelatin
( Schiele, Chrisey, and Corr, 2011 ) have been used. Systems with two layers ( Lin et al., 2011 ) or without
absorbing layer ( Barron et al., 2004 ) have also been described. In the latter case, the cell-containing hy-
drogel itself is used as the laser-absorbing material. A detailed description of the different realizations
and denominations of laser-bioprinting are given by Schiele et al. (2010) .
The laser pulses are focused through the donor glass slide into the absorbing layer, which is evapo-
rated locally in the focal spot. The high pressure vapor expands as a bubble into the gel layer and
propels the subjacent gel toward the collector glass slide. This bubble reaches its maximum size and
recollapses after a few microseconds, due to the vapor pressure decreasing below the outer pressure.
However, the gel underneath the bubble moves forward further on by inertia. A gel jet then forms,
which lasts for a few hundred microseconds ( Figure 13.2 ). Via this jet, between a few picoliters and
a few nanoliters of gel (with embedded cells) are transferred to the collector glass slide and remain