3D BIOPRINTING TECHNIQUES - 3D Bioprinting and Nanotechnology in Tissue Engineering and Regenerative Medicine - page 62

since the resulting ink leakage and mist formation during printing would affect the spatial and feature
resolution ( Cui et al., 2012; Xu et al., 2005 ). However, it should be noted that in recent years, other re-
search groups have successfully demonstrated more than 90% viability for piezoelectrically deposited
mammalian cells including human osteoblasts, fibroblasts, and bovine chondrocyte cells ( Saunders
et al., 2005; Saunders et al., 2008; Saunders et al., 2004 ). Nonetheless, the original investigations into
the feasibility of ink-jet bioprinting of live cells were performed on a piezoelectric system. ( Wilson
and Boland, 2003 ) used a bioprinter derived from commercially available HP 660C piezoelectric ink-
jet printer and custom designed piezoelectric printheads to print viable line patterns (2D) of bovine
aortal endothelial cells (BAEC) and smooth muscle cells. In a subsequent study, they printed BAEC
aggregates layer-by-layer (3D) in thermosensitive gels with the same printer, and demonstrated that
the closely placed cell aggregates could fuse together, which is critical for tissue formation ( Boland
et al., 2003 ). Later, they also became the first to use a commercial thermal ink-jet printer (HP 550C
and a modified HP 51626a ink cartridge) to create viable patterns of mammalian cells (Chinese Ham-
ster Ovary (CHO) and embryonic motoneuron cells) onto gel substrates ( Cui et al., 2010 ). The viability
of printed mammalian cells was found to be in the range of 85-95% for different cell concentrations.
The difference in apoptosis ratio and heat shock protein expression level between printed and non-
printed cells was reported to be not statistically significant. Other studies have further investigated
the fundamental effects of ink-jet process parameters on viability of different types of cells, and also
developed multimaterial composite strategies that combine cells with other biomaterials including pro-
teins, growth factors, and scaffolding polymers ( Pepper et al., 2012a; Pepper et al., 2012b; Cui and
Boland, 2009; Xu et al., 2006; Chahal et al., 2012 ). Binder et al. (2011) have provided a primer on
DOD ink-jet bioprinting for the research community. Cui et al. (2012) have discussed thermal ink-jet
printing from the tissue engineering and regenerative medicine perspective. Derby (2008 ) has provided
a detailed review of ink-jet bioprinting of proteins and hybrid cell-based biomaterials.
3.3.2 PRESSURE-ASSISTED BIOPRINTING
Pressure-assisted bioprinting (PAB) refers to a set of extrusion-based layered manufacturing processes
capable of creating digitally controlled 3D patterns and constructs. Biomaterials including polymers and
ceramics, proteins and biomolecules, living cells, and growth factors as well as their hybrid structures
can be printed using PAB. For printing cells, the bioink is essentially a cell-laden hydrogel of the ap-
propriate viscosity capable of being extruded under pressure through a microscale nozzle orifice or a
microneedle at temperatures around 37°C to maintain cell viability. The mechanical integrity of the ex-
truded structures can be controlled through thermal or chemical cross-linking, or multimaterial channel
approaches postdeposition. During the process, the biomaterial is contained in a temperature controlled
cartridge inside a three axis robotic printhead with a nozzle or microneedle. Deposition takes place by
pneumatic pressure, plunger or screw-based extrusion of the material as a continuous filament through
the nozzle or microneedle orifice onto a substrate. The substrate can be solid (e.g. culture dish), liquid
(e.g. growth media) or a gel based substrate material. The substrate as well as the deposition setup can be
contained within a sterile and climate-controlled environment further enabling the use of temperature-
sensitive cells and biomaterials. The printhead trajectory is guided by layered data obtained from the
digital model of the construct to be laid out. A schematic of the PAB setup is presented in Figure 3.3 . The
rheological properties of the biomaterial, extrusion temperature, nozzle type used, and applied pressure
are the critical parameters that affect the physical and biological characteristics of the printed construct.