3D Bioprinter for Scaffold Design In Tissue Engineering

Definition and Principles of 3D Bioprinting

3D Bioprinting could be defined as a computer-aided transfer processes for patterning and
assembling living and non-living materials with a prescribed layer-by-layer stacking
organization in order to produce bio-engineered structures serving in regenerative medicine
and other biological studies.
Bioprinting is the process of automated deposition of biological molecules on a substrate to
form a 3D heterogeneous functional structure with data derived from a digital model. The
"print" material used in bioprinting techniques, also known as bioinks, often include a
judicious combination of living biological cells, polymers, chemical factors, and
biomolecules to form a physical and functional 3D living structure. The substrate is typically
planar solid surfaces such as those of Petri dishes, glass slides, or wells of culture plates.
Living biological cells can be mammalian, insect, and plant-based as well as viruses and
bacteria. 3D bioprinting has its roots in the conventional "ink-jet" process developed in the
early 1950s that reproduces text and images from a computer file through droplets of ink
deposited on a substrate such as paper. Much of the 3D bioprinting techniques have also
grown from conventional additive manufacturing (AM) or layered manufacturing (LM)
approaches. The complexity of the 3D bioprinting techniques, when compared to AM-based
methods of scaffold fabrication, is attributed to the direct involvement of biological living
materials during the fabrication process. Lipson had highlights 10 principles of 3D printing as
guiding beacons to disrupt the current notion of manufacturing by reducing key barriers of
time, cost, and skill level. These 10 principles of 3D bioprinting that will help shape the
future of printing living tissue and organs for applications in regenerative medicine and tissue
engineering.
Principle 1: Physical replication of the living construct from a digital blueprint file.
Bioprinting machines receive initial manufacturing process data input from a digital model of
the construct. This digital model must serve as a repository of information that informs
upstream and drives downstream manufacturing process activities.
Principle 2: Product customization with high degree of feature variety
Decision-makers and end-users of the printed construct must have the freedom to specify
feature sets and functionalities of the construct with minimal complexity in manufacturing
process activities.
Principle 3: Structurally heterogeneous product spanning more than two dimensional
scales
Bioprinting processes must enable the realization of constructs with structural properties that
vary in more than two dimensions. This is necessary to mimic the complexity of nature's own
tissue and organ architecture. Processes must take advantage of repeating functional units
often seen in complex organs, such as the liver and kidney.

Principle 4: Precise spatial patterning of "bio-ink" materials

The drop-on-demand and the continuous print capability combined with robotic automation
in bio-printing processes enable the precise spatial patterning of biological entities in two and
three dimensions.
Principle 5: Minimal handling and manipulation of living cells
All bioprinting processes will need to ensure minimal mechanical and environmental stress
on non-living biological molecules, chemical factors, and living biological cells. This is to
ensure protein and growth factor stability and maximum cellular viability during fabrication.
Principle 6: Conducive microenvironment for functional cells before and after printing
Processes must provide a suitable microenvironment for cells to sustain themselves both prior
to and post-printing. Both viability and functionality of cells must be minimally altered
during the printing process.
Principle 7: Construct must be functionally stable for downstream operations.
Any printed construct that involves biomaterials and cells must be mechanically, chemically,
and/or biologically stable for use in downstream applications. Structurally weak constructs
cannot be handled by either humans or automated equipment. Chemically unstable structures
will lead to disintegration of properties. while biologically unstable constructs simply cannot
be used reliably for downstream processes.
Principle 8: Plug-n-Play bioprinting machines with minimal operator assistance
The machines should be relatively easy to operate with minimal input from operators and
should be capable of being installed at any qualified manufacturing facility or hospital. This
capability is essential to economically produce the final construct at a price acceptable to the
end user. If the ma-chines require very high skilled operation with labour intensive
monitoring, manufacturing costs will be untenable.
Principle 9: Cellular construct, engineered tissue and organs on demand
The digitally enabled technology provides the capability for decision-makers and end-users to
custom specify features of the product and request them on demand, reducing the need for
large inventory stock-up of biomaterials and final product storage solutions.
Principle 10: Repeatable and assured functional quality of the bioprinted structure
Bioprinting processes must be accurate to initial design specifications, precise in terms of
structural/biological variability, and repeatable in terms of its operation. This is an essential
requirement for any process to be qualified by regulatory agencies for any targeted
biomedical application.
Several bioprinting processes have achieved varying degrees of adherence to each of the 10
listed principles. The processes themselves are continually being improved by several
research groups through expanded biomaterial selection, improved accuracy of spatial
arrangement, degree of automation and complexity of structures generated. Processes
successfully meeting all 10 principles will yield commercially viable fabrication processes

for the scale-up manufacturing of engineered tissue and organoid systems. If developed well,
processes will also find limited challenges to being integrated into any upstream operation
that would define the entire manufacturing process cycle for TERM. In the following
sections, we will go through some of the most widely investigated 3D bioprinting processes.
their fundamental working principles, their application toward the fabrication of cellular
constructs, and differences between each process.
BIOPRINTING TECHNOLOGIES
Scaffold-based regenerative medicine therapies involve the fabrication of scaffolds followed
by seeding them with cells, preconditioning them inside an incubator, and then conditioning
the construct inside a bioreactor to achieve adequate cell proliferation and function.
Traditionally both scaffold fabrication and cell seeding are two mutually separate and distinct
steps in the process cycle. 3D Bioprinting techniques essentially combine both the scaffold
fabrication and cell placement steps in an insitu layered manufacturing process step. The
layer by layer bioprinting process enables the direct realization of scaffolding biomaterials,
chemical molecules, and living cells in a desired spatial pattern to form the heterogeneous 3D
construct. This feature was previously impossible to achieve with any of the conventionally
produced tissue scaffolds or even with bio patterning approaches such as UV
photo-polymerization. The fundamental ability to "bioprint" essentially means that it is
possible to accurately control the amount of "bioink" ejected out of the printhead or delivered
on to a substrate.
A general framework for the application of rapid prototyping in the area of tissue engineering
is shown in Figure 1.

Figure 1: A framework of biomedical RP

A specific area of the patient is scanned by computer tomography or magnetic resonance and
the data are imported into CAD software. The scaffold is designed according to the individual
requirements using the CAD software and post processed data for the fabrication of the
scaffold is then transferred to a RP system to produce the scaffold with a biocompatible and
biodegradable material. Living cells are seeded onto the surface of the scaffold after or during
the RP process. When the cell number increases following cell culture treatment, the scaffold
is implanted into the human body and eventually replaced by natural tissue.
Figure 2 gives the generic architecture for a bioprinting machine with information and
material flow paths highlighted among the entities contained with any system.

3D Bioprinting processes begin with a digital model definition of the cellular and tissue
construct architecture to be fabricated. In the case of 2D patterns, the digital files can be
directly coded into the process control interface to drive the motion of print heads to help
physically reproduce the architecture. This digital modelling of a complex 3D construct is
typically performed in a computer-aided design (CAD) software environment. Both the
external and internal architecture for the 3D construct can be designed with initial data
obtained from CT/MRI images of the patient in need of a tissue replacement strategy. Imagebased 3D reconstruction procedures can be carried out to help define the 3D digital model of
the tissue replacement construct. Tools available in the software environment can help
identify different material regions which specify the placement of the biomaterial matrix,
biological molecules, and living cells. Process algorithms are written to convert the digital

model to print head path instructions necessary to drive the hardware systems. The exact
format for machine instructions in such computer-aided manufacturing (CAM) depends on
the printing technique and hardware configuration utilized. Once the signal for printing is
activated, the process control system drives the bioprinting system hardware components for
the physical realization of the printed construct. Complex engineered tissue and cellular
construct-based products will be made from spatially patterned cellular layers which
ultimately will become large aggregates for a specialized tissue function. The entire operation
must take place in sterile conditions to limit contamination of both source raw material and
the final construct. If cells are involved in the fabrication process, the total time needed to
produce a construct can be critical. The amount of time available is dependent on the cell
type used. Unless printing conditions are well suited for cell maintenance, time to fabricate
constructs should not exceed an hour. Longer times will result in reduced cellular viability
and abnormally higher cellular stress, which will lead to degraded function. We describe the
main bioprinting techniques used by the research community to print biomaterials including
cells and biomolecules to form 3D constructs. These 3D constructs can be used as tissue
models for drug screening, as disease models to study cancer, and as constructs meant for
animal or human implantation. Due to the size scale of cells, which are generally in the
5-20 m size range, all bioprinting processes work at dimensional scale levels larger than the
cell type utilized.
Following are different types of 3D bio-printers
1. INK-JET-BASED BIOPRINTING
Ink jet bioprinting is a noncontact printing process involving the precise deposition of
picoliter to nano-liter droplets of "bioink" (a low-viscosity suspension of living cells,
biomolecules, growth factors, etc.) onto a "biopaper" (a hydrogel substrate, culture dish, etc.)
in a digitally controlled pattern. It is a direct adaptation of the conventional ink jet printing
process and a majority of current ink jet bioprinting activities continue to be conducted using
partially modified commercially available desktop ink-jet printers. There are two
fundamental approaches to ink-jet printing: continuous (CU) and drop-on-demand (DOD).
In the CU approach, an uninterrupted stream of droplets is produced by forcing the ink
through a microscopic nozzle orifice under pressure and deflecting it onto the substrate using
an electrostatic field. Where droplet deposition is not required in the digital pattern, the
droplets are steered into a gutter and collected for reuse. In the DOD approach, the ink
droplets are ejected through the nozzle orifice by creating a pressure pulse inside a
microfluidic chamber only when required. The DOD approach is of primary interest in
bioprinting due to the pulsed nature of printing. The CU approach is not well suited to
bioprinting due to the need for conductive ink formulations and the risk of contamination due
to ink recirculation. among other reasons.

The DOD approach can be further categorized into thermal (heat) or piezoelectric
(mechanical compression) based on the droplet actuation mechanism. A schematic of DOD
ink-jet printing based on both mechanisms is presented in Figure 3. In thermal DOD, an
electric current pulse applied to the heating element (thin film resistor) rapidly vaporizes a
small pocket of ink in the microfluidic chamber. The resulting vapour bubble creates the
pressure pulse that propels the ink droplet through the nozzle orifice and onto the substrate. In
piezoelectric DOD, a microfluidic chamber above the nozzle contains a piezoelectric
transducer for droplet actuation instead of a heating element. A voltage pulse applied to the
transducer causes it to expand, creating the transient pressure that results in droplet ejection.
For both forms of DOD, the rheological and surface tension properties of the ink govern their
ability to be printed. The ink viscosity requirements vary from system to system, but a typical
threshold is around 30 mPa/s. In addition to the ink characteristics, the orifice size, the
distance between nozzle and substrate, the frequency of the current pulse and resulting
temperature gradient (thermal DOD), and the frequency of the voltage pulse and piezodeformation characteristics of the transducer (piezoelectric DOD) have an effect on the
ejected droplet size and spatial resolution in ink-jet printing.
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-loaded hydrogel of the appropriate viscosity capable of being
extruded under pressure through a micro scale nozzle orifice or a microneedle at temperatures
around 37C to maintain cell viability. The mechanical integrity of the extruded structures
can be controlled through thermal or chemical cross-linking, or multi-material channel
approaches post deposition. 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 Theological 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.

3. LASER-BASED BIOPRINTING
Laser-based direct writing (LDW) is one of the leading methods in laser-based bioprinting
techniques. In LDW, a laser pulse guides an individual cell from a source to a substrate. The
laser pulse is used to transfer the suspended cells in a solution from a donor slide to a
collector slide. The laser pulse creates a bubble, and shock waves generated by the bubble
formation eventually force cells to transfer toward the collector substrate. The most popular
techniques in LDW are laser-induced forward transfer (LIFT) and matrix-assisted pulsed
laser evaporation direct writing (MAPLE DW). MAPLE DW is schematically similar to
LIFT; however, MAPLE DW utilizes a lower powered pulsed laser compared to LIFT. These
techniques allow precise deposition of cells in relatively small 3D structures.

4. STEREOLITHOGRAPHY
Stereolithography is a fabrication process using a liquid photosensitive polymer that can be
solidified by exposure to UV or laser light, which cures the pattern traced on the resin and
adheres it to the layer below. After one layer has been solidified, the next layer of liquid
polymer is applied and patterned with the laser. When a complete 3D part is formed the
constructs are cleaned of excess resin and cured in a UV oven. For the construction of 3D
biological constructs with stereolithography, cells can be encapsulated in photopolymerizable
hydrogels and gelation of the cell-hydrogel-construct can again be induced via laser or UV
light. In general, the laser has the power to crosslink a special pattern within one layer depth.
Layer-by-layer the hydrogel-cell mixture is applied and subsequently crosslinked according
to the predefined design. Several studies with different cell compatible photopolymerizable
hydrogels have been performed, where the hydrogel precursor prevents the cells from
damage.