CARTILAGE 3D PRINTING - 3D Bioprinting and Nanotechnology in Tissue Engineering and Regenerative Medicine - page 262

In some bio-inks, cells are not simply suspended, they are clustered into spheroids or other micro-
structures ( Mironov et al., 2009; Norotte et al., 2009 ). The goal in these cases is to print with small,
preassembled tissue-like constructs and decrease the time necessary for cells to self-organize after
printing.
Of the various 3D printing technologies, the earliest was stereolithography ( Billiet et al., 2012 ).
While it revolutionized the prototyping process for some industries, it has not been as effective in the
biomedical sciences. In stereolithography, a laser is rastered over a reservoir of photocrosslinkable
polymer, solidifying the material ( Figure 12.3 A). After each layer, the printing substrate is lowered or
the reservoir depth is increased, causing the solidified material to be covered in uncrosslinked polymer.
The process is repeated to form each layer in the object. This can be easily adapted to photocrosslink-
able biomaterials, and has been used for simple implant and scaffold creation ( Billiet et al., 2012;
Grogan et al., 2013b; Soman et al., 2013 ). Bio-inks have been made possible by the development of
photocrosslinkable hydrogels. If cells are suspended in the bioink, they are trapped within the hydrogel
matrix at the time of cross-linking. It has been demonstrated that cells can survive the relatively brief
UV laser exposure during printing ( Lin et al., 2013 ). Incorporating 2D projection techniques, for ex-
ample with digital micromirrors, has dramatically increased the print speed while preserving resolution
and limiting cell exposure to UV light ( Beke et al., 2012 ). Despite these advantages, stereolithography
requires a large amount of bioink in the reservoir in which layers can be constructed, and the feasibility
of multi-ink printing is limited ( Melchels et al., 2012 ).
Extrusion printing is the most familiar 3D printing technology, bearing a strong resemblance to
consumer-grade plastic 3D printers. In the consumer systems, a thermoplastic is heated above the
glass transition temperature and is extruded from a nozzle, building an object one layer at a time
( Figure 12.3 B). Early uses of 3D printing for biomedical applications used this process directly; parts
were made from biocompatible polymers, either for implantation or for use as a tissue-engineering
scaffold ( Fedorovich et al., 2012; Seyednejad et al., 2011 ).
For bioprinting applications, the cells suspended in a bioink are loaded into a delivery system and
extruded to build layers. The delivery system can be as simple as a syringe or a capillary tube with a
fine plunger ( Skardal et al., 2010 ). The relatively large nozzle sizes and high extrusion pressure allow
extrusion of viscous inks with high cell density. Bioinks can be developed with some complex struc-
tures and properties, such as tissue spheroids. This advantage can also be seen as a disadvantage: large
nozzle sizes and bulk extrusion volumes limit the minimum resolution. Extrusion printing requires
contact with the print substrate, limiting potential in situ applications and increasing the possibility of
contamination. Organovo (San Diego, CA) has commercialized extrusion printing for the pharmaceuti-
cal industry. Organovo's bioprinter is capable of placing cell aggregates, such as spheroids, with high
accuracy, although resolution of the actual printed construct is still limited by the size of the aggregates
( Khatiwala et al., 2012 ). Organovo's approach generates analogs of human tissue, such as liver, that
can be used for drug discovery.
Ink-jet bioprinting draws on the same technology as ink-jet document printing. A low viscosity fluid
is ejected from a nozzle toward the printing substrate. Generally, the necessary pressure is achieved
by a thermal bubble or a piezoelectric effect ( Figure 12.3 C). Like the other 3D printing techniques,
this method has been used to create scaffolds for tissue engineering purposes ( Butscher et al., 2011;
Lee et al., 2005 ). Both thermal and piezoelectric ink-jets are capable of printing viable cells ( Cui
et al., 2012a, 2012b; Saunders et al., 2008; Tirella et al., 2011 ). Ink-jet does not require contact with the
print substrate or previous printed layers, which can be an advantage in certain medical applications. A