Introduction
Context
Three-dimensional in vitro human tissue models have the potential
to act as models for disease which will not only reduce the use of
animals as models for disease but may potentially increase turnover time
associated with new drugs undergoing pharmaceutical trials [1]. The
purchase and maintenance costs of commercially available bioprinters
have meant this technology is inaccessible to most research labs whether
in developed economies or developing countries [2]. There are,
however, many facets of this cutting-edge technique (bioprinting) that
are continuously being improved for its application, including the
reduction in associated cost, design of bioinks, design of extruder
systems and the ability to sustain post-printed viability of cells
within the 3D structures [3,4]. General access to desktop &
open-source additive manufacturing technologies has seen advancement in
this field in recent years. Arguably, this has spurred the bioprinting
revolution.
Extrusion based bioprinting has been largely focused on the manufacture
of 3D tissue constructs or scaffold due to its many advantages over
other methods, including, but not limited to; high cell viability
[5], flexible geometric shapes [6], ability to incorporate
multiple biomaterials and cell types [7], both homogenous and
heterogeneous structures can be created [6,8]. In extrusion based
bioprinting, the bioink is extruded out of a nozzle tip to form a
continuous line structure driven by either pneumatic pressure or
mechanical pistons. The extruded product is referred to as filaments
instead of droplets [5,6,8]. A three-axis/cartesian automatic
extrusion system is typically used in this type of bioprinting, equipped
with a fluid dispensing nozzle [9]. This method has been extensively
reported in many studies [10–16]. Most systems make use of standard
plastic syringes which provide many benefits including; wide
availability, low cost, aseptic, pyrogen free and compatibility with a
range of needle sizes used as print nozzle heads [17]. Although the
most widely employed method, it is not without its limitations. Most
syringe-based extruders are designed to incorporate the fluid reservoir
into the extruder carriage which is solely responsible for the high mass
typically associated with this approach. A heavy extruder carriage can
cause various issues during the printing process by affecting speed and
resolution which may cause compromises in geometries of the printed
constructs [12]. Reducing the volume of the fluid reservoir could
provide a solution to the increased mass, however this will negatively
impact the ability to print complex and larger constructs. Other larger
volume systems have utilized the Bowden approach to minimize the weight
of the extruder carriage. This approach makes use of Bowden tubing to
connect the fluid reservoir (which is completely removed from the
extruder carriage) to the nozzle. These systems are typically
pneumatically driven which then brings in another separate set of
limitations such as; poor retraction, the need for vacuum during
printing and unstable extrusion pressures [12]. The ability of the
printer to retract is important during the printing process as this
prevents material from dripping during non-extruding moves which in turn
reduces printing fidelity of the construct [12]. Pusch et al.(2018) have addressed the two major concerns of extruder carriage weight
and inability to retract using the Large Volume Extruder (LVE) design.
This design utilizes the Bowden tube approach with stepper motors and a
lead screw driven extruder. The use of stepper motors is suggested to
achieve retraction through straightforward reversal of direction and to
apply constant pressure using a standard syringe [18].
The RepRapPro (RRP) Paste extruder
utilized in this study also uses a Bowden tube approach along with a
lead screw driven extruder system which makes use of stepper motors. As
with the LVE approach, attaching the RRP paste extruder to the printer
frame places all the weight on the printer frame and not the extruder
carriage. This adds minimal payload to printer movements and should in
turn maximize the print speeds and reduce any vibration of the nozzle
during printing which may lead to nozzle dripping.
Design Criteria
Overall, the bioprinting process has multiple facets to consider which
have been described under one of the three pillars required for
successful tissue engineering – hardware, wetware and software
considerations [19]. The outcome should include low operational
cost, ability to use a wide variety of materials, allow for fine
deposition of materials with high viscosities, maintain high cell
viabilities, reduced maturation time of printed constructs and finally,
minimal handling of the constructs. Here, we discuss the criteria
required for the hardware development process applied in this study. The
overall design and development process outlined in this study is
specifically for the printing of cell free scaffolds which then provides
the necessary foundation for future cell-laden bioink bioprinting.
1.2.1 Choice of Printer & RepRapPro (RRP) Paste extruder
A commercially sourced, RepRap based Delta 3D printer kit was selected
for modification to include a hydrogel paste extruder in the place of
the original thermoplastic extruder. The high printing precision and
accuracy expected of delta printers led to the choice for eventual
cell-free scaffold bioprinting [20]. The ANYCUBIC Kossel Linear Plus
Delta printer used in this project was based off the popular Kossel
RepRap delta printer designed by Johann C. Rocholl. All documents and
development kits for the Kossel Delta are available online [L1] with
many components of the model made readily available from Thingiverse
[L2] . This allows for successful modification to be made to the
printer at relatively low-cost. The firmware supplied with the Delta
printer is the open-source Marlin Firmware (originally developed by
Scott Laheine, [L3]). This firmware allows for modifications
allowing for paste extrusion with the Delta hardware which allows for a
relatively quick transition from thermoplastic extrusion to paste
extrusion.
Delta printers contain an effector plate where the extruder nozzle sits
and moves around the build volume, but the extruder stepper motor is
situated outside the printing space, attached to the frame. The extruder
nozzle must therefore be lightweight and secured properly onto the
effector plate to prevent unwanted movement. Suspending the extruder
nozzle above the bed arguably eliminates any contamination from
particles caused by friction during print moves. The dimensions of the
extruder carriage and the mass allowed on the axis that holds the
extruder therefore dictate the maximum dimensions of the hydrogel paste
extruder.
Furthermore, the kit used in this study was selected because of the
linear rail-based motion control allowing for increased XYZ precision
required for bioprinting. The ball bearing design of the linear rail
system allows for a more precise motion control compared to the linear
rod systems, typically found in desktop 3D printers. There is a
considerable reduction in binding occurrences (ball bearings getting
caught on the rail during a movement) which contributes significantly to
a much smoother printing movement and creates a jerk-free print. The
stepper motors (NEMA17 with 1.8̊ step angle) used in conjunction with the
linear rails also contribute significantly to the increased precision
found in this system, where micro-stepping allows for more controlled
moves.
With such a highly application specific technology such as bioprinting,
the commercially available bioprinters tend to be available at a very
high cost. This has forced many research groups to develop their own
bioprinters based on their specific application requirements whilst also
maintaining a low overall build cost. This also allows for the
functionality of the printer to be expanded over time, as required. As
in this case, where a bioprinter initially designed for cell-free
bioprinting of scaffolds may be easily modified for future cell-laden
bioink printing. In this study a low cost commercially available 3D
printer was modified to accommodate for bioprinting with cell-free
hydrogels. This provided for majority of the printer parts at a
relatively low cost. All additional parts were either 3D printed using
thermoplastics or purchased at a low cost. Although the linear rails are
considerably more expensive than smooth rod systems, the increase in
printing precision is imperative to successful bioprinting and so
outweighs the potential overall increase in build cost.