Development of a high-performance open-source 3D bioprinter

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). First, either the electronic and control system for the plastic printer need to adapt to bioprinting by modification or be replaced with an alternate. FlashForge’s motion control board, Fig. 2A (green rectangle) has been replaced by the open-source Duet 2 WIFI motion control board (Fig. 2B, blue rectangle).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. These instructions are for FlashForge Finder. They can also be adapted to most desktop 3D printers. Details of the supplemental assembly guide and 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. The majority of parts of the Replistruder 4 can be 3D printed from plastic, and then assembled with commonly-available hardware (Fig. 2C). This carriage platform can be mounted on to the linear motion parts of your printer. The Xaxis carriage includes pockets to mount the bearings on the linear rails of the X axis, as well as channels that allow routing the belt along the axis and keeping it in place. 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). The Replistruder 4 is ready for use after these steps are completed (Fig. The Replistruder 4 (Fig. 2G yellow an arrow, and motors connected to Duet 2 WIFI. This is located in the back of the bioprinter’s cabinet (Fig. 2H, green arrow). The FlashForge Finder can be transformed with these modifications into an open source bioprinter that features a high performance extruder, motion control system and a flashforge Finder.

[****]The steps involved in changing a plastic printer into a bioprinter. The plastic extruder printhead and the motion control board of the plastic printer are switched to a syringe pump extruder and Duet2 WiFi control board, respectively. After that, Duet2 WiFi can be configured to operate a bioprinter. To bioprint, the desired 3D model is sliced into machine pathing (G-code generated with Cura Ultimaker software) and then Duet Web Control executes the print using the desired bioink. BioRender.com was used to create the figure.

[****]The FlashForge Finder can be converted into a bioprinter. () The original motion control board and wiring (green rectangle). (B) The motion control board is replaced with the Duet 2 WiFi motion control board (blue rectangle). (C) The Replistruder 4 syringe pump extruder is printed and assembled. (D) The X-axis carriage for the FlashForge Finder is 3D printed and the Replistruder 4 is mounted to it. (E) Top-down view of the Finder’s plastic extruder printhead that is to be removed. (FAfter it has been mounted into the printer, a top-down view shows the Replistruder 4. (G) Additional view showing that the plastic extruder printhead has been replaced with a Replistruder 4 syringe pump extruder (blue arrow) mounted to a custom X-axis carriage (yellow arrow). (H) The Duet 2 WiFi is mounted in a 3D printed case covered in the back cabinet of the Finder (green arrow).

[****]Duet 2 WiFi has several major advantages over other stock motion control circuit boards such as those found in FlashForge Finder, 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 contrasts with most 3D printers where editing the configuration requires the use of 3rd-party software to flash the motion control board firmware. 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. The Duet 2 WiFi setup guide and documentation are thorough and regularly updated. A user forum is also available. The Duet2 WiFi hardware setup guide and the basic conversion are provided. For those who wish to modify the Duet3D, the official documentation is available. These features combine to provide precise motion control, extensive expandability and a web interface that allows for rapid customization. This makes Duet 2 WiFi more efficient than 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 stepper motor in order to precisely move each axis by one millimeter. The formula below is applicable to the belt-driven X/Y axes. (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.

[****]Measuring and correcting printer travel. () The X-axis travel is 105 mm, the Y-axis travel is 150 mm, and the Z-axis travel is 50 mm. (B) The error of travel for the X-axis across a 10 mm window before correction (red) and after correction (blue). (C) The error of travel for the Y-axis across a 10 mm window before correction (red) and after correction (blue). (D) The error of travel for the Z-axis across a 10 mm window before correction (red) and after correction (blue).

[****]There are several specifications that you need to take into account when comparing 3D printer performance. These specs relate to print quality. The majority of thermoplastic 3D printers have specifications regarding resolution. This is the smallest possible step that the printer can make in any given direction. Table 1 shows the numbers reported for both FlashForge Finder (and other popular bioprinters) and can be found here. The table below contains further details about the motion system. Positional error is the absolute deviation at the moment of the FlashForge Finder from its intended location. Repeatability is the maximal absolute deviation at the measured position relative to the average measurement position, when trying to achieve that position repeatedly. Bioprinting is not able to provide these more complex metrics. They can differ between printers due to differences in mechanical components and assembly accuracy. The resolutions provided by bioprinter specifications is generally based on the nominal dimensions and the screws that are used to assemble the system. The measurements provided by the manufacturers mentioned above do not include actual resolution. This is the error over the entire distance traveled or repeatability. These measurements can be obtained with high-end motion platforms, such as Aerotech and Physik Instrumente.^28,29. Measure the travel using an external instrument to optimize and determine real-world performance of low cost 3D printing 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. On the X axis there was a systematic over-travel with the nominal steps/mm. (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 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. Finaly, the Z-axis was subject to systematic undertravel according to the nominal steps/mm. (Fig. 3D, red curve) and after correction was reduced to 32.3 µm (Fig. 3D, blue curvature. 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. FRESH is one of the most recent advances in embedded bioprinting technologies.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 test the accuracy of FRESH printing from collagen type I bioink. 4B). We used optical coherencetomography (OCT), to capture a 3D volumetric picture (Fig. 4C)³⁰This revealed a good agreement between the measured and designed dimensions (Fig. 4D). Following this was an even more complicated design, based upon a 3D scan (Fig. 4E). 4E). This model was printed with collagen (Fig. 4F). 3D Volumetric images of printed ears were taken using OCT (Fig. 4G. Supplementary Fig. 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 cases together demonstrate that both the standard deviation and average error of printed scaffolds were within the limitations of our bioprinter and comparable to commercial bioprinters.³¹.

[****]The printing of dimensionally precise grids and organic forms. () Model of a gridded scaffold with 500 µm and 1000 µm grids. (BThe gridded scaffold is printed in collagen type 1. (CA OCT image of the grid-scaffold print. (D) Analysis of the accuracy of the gridded scaffold print (mean ± STD.; n = 11 measurements for 1000 µm grid, n = 26 measurements for 500 µm grid, p < 0.0001 [****] for 1000 µm compared to 500 µm by Student’s two-tailed, unpaired t-test). (EAn ear 3D model. (F(A photograph of an ear that has been printed with collagen type I. (G( An OCT volumetric view of the printed ear. (H(Quantitative measurement of the earprint against the 3D original model.