Converting a printer made of plastic into a Bioprinter
[****]A sequence of steps is required to convert a plastic 3D printer into bioprinter. 1). First, the electronic and control systems of the plastic printer need to either be modified or replaced. The FlashForge Motion Control Circuit Board (Fig. 2A, green rectangle), is being replaced by the open-source Duet 2 wifi motion control circuitboard (Fig. 2B, blue rectangle). This is done to increase the motion control performance, enable WiFi access, and facilitate quick firmware customization via the Duet web-based interface. This process is described for FlashForge Finder. It can also be adapted for other desktop 3D printers (details are provided in the Supplementary Figs. S1–S15, and provided Duet2 WiFi configuration files). Next, we replace the thermoplastic extruder with the Replistruder 4 an open-source high-performance syringe extruder. This extruder is a modified version of the Replistruder 3.14. The majority of the parts of Replistruder 4 can be 3D printed from plastic and assembled with commonly available hardware (Fig. 2C). A carriage platform was created to fit the existing linear motion components on the printer and serve as a mounting point. The X axis carriage features pockets for the bearings mounted on the X axis linear rails. It also has channels for routing and maintaining the X axis belt. To allow the Replistruder 4 attachment to the Xaxis carriage, four mounting points have been provided with recessed M3 hexagon nuts (Fig. 2D). The thermoplastic printhead that comes pre-installed on the Finder is replaced with the X-axis carriage/Replistruder 4 assembly (Fig. 2E,F. Details in the Supplementary Assembly Guide and Supplementary Figures. S16–S23). The Replistruder 4 is ready for use after these steps are completed (Fig. The Replistruder 4 (Fig. 2G yellow an arrow) and the motors are connected with the Duet 2 WIFI, which can be found in the back of the bioprinter. 2H, green arrow). These modifications transform the FlashForge Finder into an open-source bioprinter that features a high-performance extruder with motion control and a motion control system.
[****]Duet 2 WiFi offers several key advantages over stock motion control circuit boards in the FlashForge Finder or other low-cost desktop 3D printing machines. First, the Duet’s WiFi based web interface allows for easy, in-browser access to printer movement, file storage and transfer, configuration settings, and firmware updates. This is in contrast with most 3D printers where editing configuration settings requires flashing motion control board firmware via 3rd-party software. Inexperienced users may find this daunting and difficult. This could result in accidental firmware changes and corruption. Second, the Duet 2 adds many advanced motion control improvements including (i) a 32-bit electronic controller, (ii) high performance Trinamic TMC2260 stepper controllers, (iii) improved motion control with up to 256 × microstepping for 5 axes, (iv) high motor current output of 2.8 A to generate higher power, (v) an onboard microSD card reader for firmware storage and file transfer, and (vi) expansion boards adding compatibility for 5 additional axes, servo controllers, extruder heaters, up to 16 extra I/O connections, and support for a Raspberry Pi single board computer. The Duet 2 WiFi setup guide and support documentation are comprehensive, up-to-date, and supported by an active forum. The Duet2 WiFi setup guide and the hardware details are provided. However, the official Duet3D documentation can be accessed for those who wish to modify the Duet2 WiFi. These features combine to provide precise motion control, extensive expandability, and a web interface that allows for rapid customization and better performance than standard desktop plastic printers.
Mechanical performance of a 3D bioprinter
[****]The X, Y and Z axis travel limits were calculated after the conversion to determine the 3D printer’s build volume. For the X-axis travel is 105 mm, for the Y-axis travel is 150 mm, and for the Z-axis travel is 50 mm, resulting in an overall build volume of 787.5 cm3 (Fig. 3A). The most critical control parameter for a stepper motor-driven motion system such as this 3D bioprinter or most commercial 3D printers is the steps per millimeter calibration for each of three axes. This number tells the stepper motors how many pulses or steps must be sent each axis in order to move them exactly one millimeter. The formula for the X- and Y-axes, which have belt drive, is (steps/mm=(steps/rotationtimes microsteps)/(belt pitchtimes pully teeth)). For the Finder these parameters are the nominal pitch of the driving belt (2 mm), the number of teeth in the motor’s pully (17 teeth), the number of steps in a full rotation for the motor (200 steps), and the number of microsteps that the Duet 2 WiFi interpolates between the full steps (set to 64 microsteps). The nominal steps/mm for X and Z-axes in this example are 376.5. The formula for Z-axis which uses a leadscrew is (steps/mm=(steps/rotationtimes microsteps)/(screw pitchtimes screw starts)). The finder uses a 4 start, 2 mm pitch lead screw so the nominal steps/mm for 16 × microstepping is 400 steps/mm.
[****]There are several specifications that you need to take into account when comparing 3D printer performance. These specifications directly relate to print quality. Many thermoplastic 3D printers include specifications for resolution. This refers to the smallest steps the printer can take in any given direction. Table 1 lists the reported numbers for both the FlashForge Finder as well as several commercially-available bioprinters. Additional details about the motion system can be found in Table 1. These metrics are not commonly used in bioprinting and may vary depending on the mechanical components and assembly quality. Bioprinter specifications generally provide ideal resolution based on the nominal dimensions of the pulleys, gears and screws that are used to assemble the system. None of the manufacturers mentioned above provide actual resolution measurements. This means that there is no error across the entire distance of travel or repeatability. These measurements can be obtained with high-end motion platforms, such as Aerotech or Physik Instrumente.28,29. It is important to measure the travel using an external tool in order to determine the performance of these low-cost 3D printer-based systems.
[****]To verify that the nominal steps/mm values were correct, we quantified positional error of our system along each axis near the center of travel with 2 µm precision. The nominal steps/mm value for the X axis showed a systematic over-travel (Fig. 3B, red curve. Using the maximum error at 10 mm of travel we determined the number of missed steps per mm and corrected the value, and with this corrected steps/mm the average travel error over the 10 mm window was 7.9 µm (Fig. 3B, blue curve The Y-axis was subject to systematic under-travel according to the nominal steps/mm (Fig. 3C, red curve) and after correction this was reduced to 29.1 µm (Fig. 3C, blue contour. Finaly, the Z-axis was subject to systematic under-travel according to the nominal steps/mm (Fig. 3D, red curve) and after correction was reduced to 32.3 µm (Fig. 3D, blue curve. The values also enable calculation of the unidirectional repeatability, which is the accuracy of returning to a specific position from only one side of the axis (e.g., from 0 to 5 mm) and the bidirectional repeatability, which is the accuracy of returning to a specific position from both sides of the axis (e.g., from 0 to 5 mm and from 10 to 5 mm). For the X-axis, the unidirectional repeatability was 3.9 µm, and the bidirectional repeatability was 16.4 µm. For the Y-axis, the unidirectional repeatability was 11.5 µm, and the bidirectional repeatability was 63.9 µm. For the Z-axis, the unidirectional repeatability was 8.7 µm, and the bidirectional repeatability was 38.7 µm. Together these measurements demonstrate that with calibration the travel of our converted bioprinter had a maximal error of 35 µm and repeatability in worst case situations of 65 µm. Whereas before calibration there was a linearly increasing error in position, after calibration this error is significantly decreased. This calibration, or at the very least the measurement of errors, is essential to determine whether flaws in printed structures are caused by the printer itself, or other factors.
Assessment of bioprinter printing fidelity
[****]Fidelity and resolution of printed constructs are typically not quantified for 3D bioprinters because they cannot print bioinks in a manner approaching the mechanical limitations of the systems. With the latest advances in embedded bioprinting technologies such as FRESH13, it is now possible to perform extrusion bioprinting with resolutions approaching 20 µm. Thus, to demonstrate bioprinter printing performance we generated a square-lattice scaffold design consisting of 1000 and 500 µm filament spacing (Fig. 4A) To measure the accuracy of FRESH printing using a collagen type 1 bioink (Fig. 4B). We used optical coherencetomography (OCT), to capture a 3D volumetric image (Fig. 4C)30This revealed a good agreement between the measured dimensions and those as-designed (Fig. 4D). The next step was a more complicated design that was based on 3D scanning of the adult human ear (Fig. 4E). This model was printed with collagen (Fig. 4F). We used OCT to capture a 3D volumetric image (Fig. 4G, Supplementary Figure. S24)30. The 3D reconstruction demonstrates recapitulation of the features of the model and 3D gauging analysis revealed a deviation of − 29 ± 107 µm (mean ± STD) between the FRESH printed ear and the original 3D model (Fig. 4H)30. These two examples show that the standard deviation and average error of printed scaffolds is within the limitations of the bioprinter built and are comparable to commercial bioprinters.31.
