BIOPRINTING FOR SKIN - 3D Bioprinting and Nanotechnology in Tissue Engineering and Regenerative Medicine - page 279

13.3.2 HYDROGEL
For printing living cells using one of these techniques, the cells are embedded in a medium, mostly hy-
drogel or a hydrogel precursor, also called sol, such as collagen, fibrinogen, hyaluronic acid, alginate,
and the like. They are printed as a hydrogel droplet containing between one and several hundred cells
or extruded as a hydrogel strand with embedded cells.
If 3D cell patterns or tissue are to be printed, the hydrogel (precursor) has to fulfill several require-
ments. First, it needs to be suitable for the printing process. It must have an appropriate viscosity, which
depends on the concentration, and material-specific properties. Shear thinning gels or gel precursors
are advantageous in reducing shear forces during the printing process, while shear thickening gels are
problematic for the abovementioned printing technologies, since shear forces increase disproportion-
ally with the velocity of the gel, which is accelerated during the printing process. To avoid cell damage,
lower gel viscosities are required. Alginate and hyaluronic acid are shear thinning gels that are often
used for cell printing. Just as important are the wetting characteristics of the gel (precursor) on the
printing setup's surface materials. Second, the gel should support cell survival through a convenient
environment. A neutral pH-value is therefore mandatory. Third, after printing, the gel functions as the
ECM, which in 3D patterns requires a certain degree of stiffness and mechanical strength. Fourth, the gel
must not adversely affect the cells, since in general, cells respond to environmental cues. There can be
manifold stimuli having physical, mechanical, chemical, and biological backgrounds.
To fulfill these requirements, the hydrogel consists of four components. A primary component,
offering a suitable environment with nutrients for the cells, is mixed with a secondary component
for optimizing viscosity. Depending on the application, a third component stimuli like growth factors
or agents are added. In this mixture, called hydrogel precursor or sol, the cells are suspended. This
hydrogel precursor with the embedded cells is then printed in the predefined pattern. Afterwards, a
cross-linker is printed for gelation to attain the desired stiffness as the ECM.
Examples are (i) fibrinogen mixed with hyaluronic acid (to have a suitable viscosity for print-
ing) and cross-linked with thrombin or (ii) blood plasma mixed with alginate and cross-linked with
calcium chloride. By this means, hydrogels and hydrogel precursors with a wide range of rheological
properties ( Gruene, et al., 2011a ; Lin et al., 2009 ) have been printed. The cross-linker can be printed or
sprayed onto the printed hydrogel precursor in a second step; alternatively, the sol can be printed into a
cross-linker reservoir ( Yan Huang, and Chrisey , 2013 ).
However, some gels of interest like collagen, the most abundant protein in mammals, do not allow
postprint gelling. Collagen is gelled by a change of the pH value. Therefore, a complete merging of
all gel components before printing is required, since the cells will not survive in the acidic collagen
precursor. Thus, the ability to print high-viscosity gels is needed.
However, the printing technologies have different viscosity limitations for printing living cells,
since shear forces, especially in printing nozzles may destroy or affect the cells. Thus, ink-jet print-
ing is limited to low viscosities ( < 0.1 Pa s) and low cell densities (up to a few million cells per
milliliter) to avoid shear stress in the nozzle and cell clogging ( Born et al., 1992 ). For microdis-
pensing of cells, nozzles with about 50-1000 m m diameter have been applied. With smaller nozzle
diameters, similar limitations in viscosity and cell density as with ink-jet printers exist, but these
limitations can be varied by the extrusion velocity as a further parameter. With larger extrusion
nozzle diameters, higher viscosity and cell density can be printed at the expense of resolution
( Chang et al., 2008 ).