Transforming a plastic printer to a Bioprinter
[****]There are several steps involved in converting a plastic 3D printing machine into a Bioprinter. These steps generally follow the same sequence (Fig. 1). The electronics and control systems of the plastic printer must be modified to allow bioprinting. FlashForge’s motion control board, Fig. 2A (green rectangle) has been replaced by the open-source Duet 2 WIFI motion control board (Fig. 2B, blue rectangle). It is used to increase the performance of the motion controller, enable WiFi access and facilitate quick firmware customization via the Duet web interface. This process is described for FlashForge Finder. It can be adapted by most desktop 3D printing machines (details in Supplementary Figs). S1–S15, and provided Duet2 WiFi configuration files). We have now replaced the original thermoplastic extruder with the Replistruder 4. This is an open-source high-performance, high-performance, syringe extruder.14. Replistruder 4’s majority are easily 3D-printed from plastic. They can also be assembled with common hardware (Fig. 2C). This carriage platform can be mounted on to the linear motion parts of your printer. These pockets are for bearings that have been mounted to the X axis rails. There is also a channel for routing and maintaining the X axis belt. Four mounting points are provided with M3-hex nuts that can be used to attach the Replistruder 4 (Fig. 2D). The thermoplastic printhead that comes pre-installed on the Finder is replaced with the X-axis carriage/Replistruder 4 assembly (Fig. 2E and F, see Supplementary Figs. S16–S23). After these steps have been completed, the Replistruder 4(Fig. The Replistruder 4 (Fig. 2G, yellow arrow) is mounted on the X-axis of the printer in the X-axis carriage (Fig. 2H, green arrow). These modifications transform the FlashForge Finder into an open-source, free-standing bioprinter equipped with a motion control and high-performance extruder.
[****]Duet 2 WiFi offers several advantages over stock motion control boards in FlashForge Finder or other desktop 3D printers. 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. Contrary to other 3D printers that require you flash the firmware of the motion controller board using third-party software, editing configuration settings on Duet is possible with this interface. For inexperienced users, this can prove difficult and daunting. Incorrect changes and firmware corruption could result. 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. You can also access the Duet 2 WiFi support and setup documentation online. It is regularly up-to-date and supported by an active forum. We provide an easy-to-follow guide to help you with the Duet 2 WiFi setup and hardware conversion. However, we also have the Duet3D documentation that can be used to assist in further modifications. The combination of these features provides high precision motion control with extensive expandability. It also includes an intuitive web interface which allows rapid customization and improves performance over standard desktop plastic printers.
Mechanical performance of a 3D bioprinter
[****]The X-, Y, & Z axis travel limit limits were determined after conversion to calculate the 3D printer’s 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 the 3D bioprinter or most commercial 3D printers is the calibration steps per mm for each of three axes. This determines the amount of pulses (or steps) that need to be sent to each axis’ stepper motors in order to move it precisely one millimeter per. The formula below is applicable to the belt-driven axes X, Y, and Z. (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). For the X, Y and Z-axis the nominal steps/mm are 376.5. The formula for the Z-axis 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 specs relate to print quality. Many thermoplastic 3D printers include specifications on resolution. It is the smallest distance the printer can travel in any direction. Table 1 lists the numbers reported for the FlashForge Finder, as well several other popular bioprinters. Table 1 contains additional specifications about the motion system. These include positional error which refers to absolute deviation of current printhead location from intended location and repeatability which refers to absolute deviation from average position for multiple attempts to get to that location. The bioprinting industry is lacking these advanced metrics. These can be varied between different printers depending on the mechanical components used and the accuracy of assembly. The resolutions provided by bioprinter specifications is generally based on the nominal dimensions and the screws that are used to assemble the system. All of these manufacturers do not provide actual resolution measurements. That is, errors across all distances traveled, and repeatability. This measurement is often provided by ultra-high end motion platforms like those offered by Aerotech or Physik Instrumente.28,29. It is important to use an external measurement tool in order to determine the performance and optimize these 3D printer-based low-cost 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. We found a systematic under-travel on the X axis using nominal steps/mm (Fig. 3B, the 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. Blue curve, 3B. The Y-axis was subject to systematic undertravel according to the nominal steps/mm. (Fig. 3C, red curve) and after correction this was reduced to 29.1 µm (Fig. 3C is the blue curve. Finally, there was under-travel on the Z-axis using nominal steps/mm (Fig. 3D, red curve) and after correction was reduced to 32.3 µm (Fig. 3D, blue contour. 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. It would not be possible to tell if defects in printed constructions are due to flaws within the printer or to other factors that impact print quality.
Bioprinter printer 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. However, advances in embedded bioprinting techniques like FRESH have made it possible to print bioinks with greater precision and resolution.13, 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 precision of FRESH-printed collagen type 1 bioink (Fig. 4B). 3D Volumetric images using optical coherence imaging (OCT) were taken to measure grid spacing (Fig. 4C)30This revealed a good agreement between the measured and designed dimensions (Fig. 4D). The next step was a complex design that used a 3D scan to examine an adult’s ear. (Fig. 4E). 4E). This model was printed with collagen (Fig. 4F). We used OCT to capture a 3D volumetric picture of each printed ear. 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. The two illustrations together show that the error average and standard deviation for printed scaffolds is within the limits of the bioprinter built and are comparable with commercial bioprinters.31.
