Movement Settings:
Axis steps per unit: These values were changed based on the
Stepper motor used.
#define DEFAULT_AXIS_STEPS_PER_UNIT { 80, 80, 80, 96 }
set to #define DEFAULT_AXIS_STEPS_PER_UNIT { 80, 80, 80,200 }
Maximum/Default/Retraction/Travel Acceleration: All movement
related script was changed to reduce the speed of the various
movements during printing.
#define DEFAULT_MAX_ACCELERATION { 3000, 3000, 3000, 3000 }
set to #define DEFAULT_MAX_ACCELERATION { 1000, 1000, 1000,
1000 }
#define DEFAULT_ACCELERATION 2000 set to #define
DEFAULT_ACCELERATION 1000#define DEFAULT_RETRACT_ACCELERATION 2000 set to #define
DEFAULT_RETRACT_ACCELERATION 1000#define DEFAULT_TRAVEL_ACCELERATION 2000 set to #define
DEFAULT_TRAVEL_ACCELERATION 1000
Default E-Jerk: The E Jerk value is the minimum speed change
that requires acceleration. This was reduced in order to create
smoother print movements whilst printing smaller and finer constructs.
#define DEFAULT_EJERK 5.0 set to #define DEFAULT_EJERK1.0
Bill of Materials
The complete bill of materials for the final Bioprinter manufactured is
listed in Table 1. The original 3D Delta printer used for modification
was assembled according to manufacturer’s instruction and all parts used
in the final bioprinter model were from the original 3d printer, unless
mentioned in the Bill of materials Table 1.
Build Instructions
Assembly of commercially available ANYCUBIC Kossel Delta 3D printer
For ease of access to build materials, an Anycubic Kossel Linear Plus
Delta 3D printer was purchased from DIYelectronics (Durban, South
Africa). All parts were provided within the build kit and assembly of
the printer was done as per supplier’s instructions. The printer
technical specifications are outlined in Table 2.
Firmware Flashing
The Tri-gorilla control board (integrated with the function of the
Mega2560+ramps 1.4; main control chip: ATMEGA256016AU), on the assembled
delta printer, required installing (i.e. flashing) software (firmware)
specifically written for controlling and monitoring the 3D printer.
Open-source Marlin Firmware (v.1.1.0) was first used for flashing the
board; available on the manufacturers’ website [L4]. Firmware
installation was performed through the Arduino Integrated Development
Environment (IDE) desktop software (v.1.8.1.0, [L5]). The printer
was first connected to a PC using a USB cable (USB 2.0-A Male to USB-B
Male cable), the Arduino.ino file was then opened using the Arduino
software environment. In the Arduino environment, Tools >
Board > Arduino MEGA 2560 serial connection was selected.
The Firmware was subjected to several changes as described in detail in
section 1.1. Within the Arduino environment, the Configuration.htab was used for the software modifications. The “upload” option was
then selected to flash the Tri-gorilla board. The final modified
firmware is available at https://doi.org/10.17605/OSF.IO/7TQKB.
Hardware modification
Based on the criteria for bioprinting, the delta printer was modified to
accommodate for tissue engineering application purposes. The methods of
modifications to the hardware are explained throughout the following
sub-sections.
Enclosure of the printer
The printer was encased to maintain sterility during future bioprinting.
All parts used were designed using CAD software and manufactured in PLA
(corners), Perspex (sides) or Glass (lid). Assembly was as follows;
Corners:
1) Printed corners were pushed onto the aluminium frame.
Sides and door:
1) Each frame was secured to the aluminium extrusions of the printer
using six M3 x 10mm cap-head screws, six M3 T-nuts and twelve M3
washers.
2) The windows, for the two sides of the printer, were secured onto the
newly attached frames using four M3 x 8mm cap-head screws, four M3 nuts
and eight M3 washers.
3) The door handle was fitted onto the door without the need for
fasteners.
4) The door was assembled onto the frame of the front facing side of the
printer using two hinges, ten M3 x 10mm cap-head screws, ten M3 nuts and
twenty M3 washers.
Lid:
- Additional aluminum extrusions were secured to each existing extrusion
using 3D printed parts and twelve M4 x 5mm screws and twelve M4
T-nuts.
- Glass was secured onto the top of the new extrusions using six M4 x 5
mm screws and six M4 T-nuts.
- The finishing pieces were added simply by sliding into the new
extrusion grooves.
- Electronic board box
The Tri-gorilla electronic board (assembled according to manufacturer’s
instruction) was moved to outside the printer. A board box was designed
and manufactured in PLA using a standard Delta 3D printer. The designed
and printed board box consists of two separate pieces: the box and the
lid. Assembly was as follows;
1) All wires were disconnected from the Tri-gorilla electronic board and
the board removed from its original fixture (under the print bed).
2) The board was secured onto the newly designed and printed board box
using two M3 x 10mm cap-head screws.
3) The board box was secured onto the aluminium extrusions of the
correct side of the printer using two M4 x 5mm cap-head screws and two
M4 T-nuts.
4) All wires were fed through the aluminium extrusions (from within the
printer) into the board box and reconnected in their respective places.
Bed platform
A print bed platform was designed and printed using a standard Delta 3D
printer using PLA as extrusion material. The platform consists of six
separate pieces; designed to hold two 220 mm diameter glass plates, with
the top plate designed to hold a standard size petri dish. The printed
pieces were secured onto the printers aluminium extrusions using two M3
x 25mm cap-head screws and two M3 T-nuts per piece.
Extruder
The original thermoplastic extruder was removed from the effector plate
and replaced with a newly designed syringe-based extruder. Assembly of
the syringe-based extruder was carried out exactly as described online
[L6]. Instructions up until attachment of the power-supply mounting
bracket were followed exactly as described. The following changes were
made from there;
Assembly onto mounting bracket and printer
1) The assembled drive block was attached to the mounting bracket using
four M3 x 35mm cap-head screws and four M3 nuts.
2) The mounting bracket with attached drive block was attached to the
printer frame aluminium extrusions using two M4 x 15mm screws with four
M4 T-nuts.
3) The NEMA17 stepping motor was connected to the E0 port of the
Tri-gorilla electronic board using a standard 4-pin extruder cable.
Assembly of Luer-lock effector plate adaptor
A “nozzle” was designed for the extrusion of biomaterials using the
syringe-based extruder system. It was designed to attach to the original
effector plate provided with the Delta printer. The nozzle was designed
to incorporate a male Luer-lock connection fitting and a Bowden tube
connection fitting. Assembly went as follows;
1) The printed adaptor piece was secured onto the effector plate using
four M3 x 8mm cap-head screws and four M3 nuts.
2) The bowden tube was pushed into the bowden tube connection fitting
and secured into place using insulating tape and silicone paste.
3) A Luer-lock syringe needle was then fastened into place using the
male Luer-lock connection fitting part of the adaptor piece.
Addition of syringe, top strengthener and tubing
1) A 5 ml Luer-lock syringe was added to the assembled and mounted
syringe extruder.
2) The printed top strengthener was fitted onto the mounted syringe
extruder using the threaded bar used to secure the motor to the frame
and the two M3 x 35mm cap-head screws positioned on the other end of the
frame.
3) The bowden tube was attached to the syringe using a Luer-lock
adaptor, silicone sealant and insulating tape. The other end of the
tubing was attached to the Luer-lock nozzle piece.
Operation Instructions
Software requirements
- The bioprinter was connected to the PC using a USB cable (USB 2.0-A
Male to USB-B Male cable)
- An 18G needle was added to the nozzle of the bioprinter (Luer-lock
fit)
- Pronterface (printrun-2015031011,3 https://github.com/kliment/Printrun
) software was initiated and used to manually count the
Z home position. This depends on the 18G needle length used. Briefly,
once the printer was connected to the software, the homing option was
initiated. The distance between the needle nozzle and the printing
platform was then calculated using the Z-axis 10 mm, 1 mm and 0.1 mm
movement arrows within the software. This was repeated several times
until the exact distance between bed platform and nozzle after homing
was determined.
- The printer was disconnected from Pronterface and the correct Marlin
firmware file was opened in the Arduino Environment.
- The firmware was changed, in the Configuration.h tab of the
Arduino environment, to include the correct Z position calculated.
- The edited firmware was uploaded onto the printer by clicking
“upload” button in the Arduino environment.
- The new Z home position was tested by, once again, connecting to
Pronterface (printrun-2015031022).
- Setting up slicing software
- Cura (v.15.04.2) software was initiated.
- The printer was manually set up for desired bioprinter parameters
starting with Machine settings. For this study, the following machine
settings were chosen: E-steps per 1mm filament = 0, Maximum width =
60, Maximum depth = 60, Maximum height = 50, Extruder count 1, No
heated bed, Machine centre 0,0 Yes, GCode Flavor Marlin/Sprinter, All
printer head size options set to 0.0, Serial port Auto, Baudrate Auto.
- The desired bioprinting parameters were set up next, such as: Layer
height = 0.1, Shell thickness = 0.4, Disable retraction, Fill density
100%, Print speed = 10, Printing temperature set to 0, No support or
platform adhesion, Filament diameter = 1 and flow = 150. In the
“Advanced” tab, all speeds were set to 5.
- The desired STL file was uploaded into Cura (v.15.04.2).
- The Gcode was saved and loaded onto an SD card which was then inserted
into the printer awaiting print.
- Cell-free Bioprinting
- The tubes and printer environment were cleaned and sterilized
thoroughly using 70 % (v/v) ethanol. The sterilizing solution was
manually fed through the tube several times using a sterile syringe.
- The cell-freebioink solution was prepared (heating, cooling, mixing
etc.)
- The bioink was then added to a 5 ml sterilized syringe and placed into
the frame of the extruder with the top strengthener added to secure it
into place.
- One end of the sterilized tube was attached to the bioink syringe, and
the other end fastened to the top of the effector plate (via the
luer-lock adaptor).
- A newly sterilized 18G Luer-lock needle was added to the bottom
section of the effector plate adaptor.
- A sterilized petri dish was added to the printing platform where the
glass plates permit.
- The bioprinter was once again connected to the PC using a USB cable
(USB 2.0-A Male to USB-B Male cable)
- Using Pronterface (printrun-2015031033,3 https://github.com/kliment/Printrun
) software, the M302 g-code command was entered to allow
for cold extrusion and the extruder motor turned on to extrude until
the bioink was fed through the sterilized tube to the tip of the
nozzle needle.
- The bioprinter was then disconnected from the PC.
- The LCD display on the bioprinter was used to navigate to the saved
GCode files on the inserted SD card.
- Printing was initiated.
- Once completed, the bioprinter door was opened using aseptic technique
and cross-linker solution was added to the construct (if required).
The construct was left to cross-link for 2 minutes followed by removal
of the cross-linker solution.