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

Converting a plastic printer into a bioprinter[****]

When converting a plastic 3D printer into a bioprinter there are a sequence of steps that generally occur in the same order (Fig. 1). First, the electronics and control system of the plastic printer either need to be adapted to bioprinting through modification, or they need to be replaced with an alternative. The proprietary FlashForge Finder motion control circuit board (Fig. 2A, green rectangle) is replaced with the open-source Duet 2 WiFi motion control circuit board (Fig. 2B, blue rectangle). This is done to improve the motion control performance, provide WiFi access, and to facilitate rapid firmware customization through the Duet web-based interface without needing to use additional software. The step-by-step instructions for this process are laid out for the FlashForge Finder and can be adapted for most desktop 3D printers (details provided in the supplemental assembly guide, Supplementary Figs. S1S15, and provided Duet2 WiFi configuration files). Next, the thermoplastic extruder that came with the printer is replaced with the Replistruder 4, an open-source, high-performance syringe pump extruder we have previously developed14. Most parts of the Replistruder 4 are readily 3D printed out of plastic and assembled using commonly available hardware (Fig. 2C). A carriage platform was designed to fit on the existing linear motion components of the printer and provide a mounting point for the Replistruder 4. This X-axis carriage has pockets for the bearings already mounted on the X-axis linear rails as well as channels for routing and retaining the X-axis belt that drives motion along the axis. Additionally, four mounting points with recessed M3 hex nuts are incorporated to allow the Replistruder 4 to be attached to the X-axis carriage (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 supplemental assembly guide, and Supplementary Figs. S16S23). At the completion of these steps, the Replistruder 4 (Fig. 2G, blue arrow) is mounted on the X-axis of the printer in the X-axis carriage (Fig. 2G yellow arrow), and the motors are connected to the Duet 2 WiFi, which is positioned in the back cabinet of the bioprinter (Fig. 2H, green arrow). With these modifications the FlashForge Finder is transformed into an open-source bioprinter with a high-performance extruder and motion control system.

Figure 1
figure 1

Steps in converting a plastic printer to 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. The Duet2 WiFi is then configured to run 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. Figure created in part with BioRender.com.

Figure 2
figure 2

Converting the FlashForge finder into a bioprinter. (A) 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. (F) Top-down view of the Replistruder 4 after it is mounted in the printer. (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).

The Duet 2 WiFi provides several key advantages over the stock motion control circuit boards found in the FlashForge Finder and other low-cost 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. This is in contrast to most 3D printers, where the process of editing configuration settings requires flashing the motion control board firmware using 3rd party software. This can be challenging and intimidating for an inexperienced user and may lead to accidental changes or firmware 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. Finally, the online Duet 2 WiFi setup and support documentation is thorough, regularly updated, and backed up by an active user forum. While we provide a step-by-step guide for the hardware aspects of this conversion and basic setup of the Duet 2 WiFi, those looking to make additional modification are referred to the official Duet3D documentation. Together, these features provide high precision motion control and extensive expandability with an easy-to-use web interface that enables rapid customization and improved performance beyond standard desktop plastic printers.

3D bioprinter mechanical performance[****]

After conversion, the X, Y, and Z axis travel limits were measured to determine the build volume of the 3D bioprinter. 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). For a stepper motor driven motion system, such as this 3D bioprinter and most commercial 3D printers, the most important control parameter affecting performance is the steps per mm calibration for each of the three axes. This number determines how many pulses, or steps, must be sent to the stepper motors that drive each axis to move them precisely one millimeter each. For the X and Y axes, which are belt driven, the formula for this is \(steps/mm=(steps/rotation\times microsteps)/(belt pitch\times pully teeth)\). 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). In this case the nominal steps/mm for the X and Y-axis is 376.5. For the Z-axis, which uses a leadscrew, the formula is \(steps/mm=(steps/rotation\times microsteps)/(screw pitch\times screw starts)\). The finder uses a 4 start, 2 mm pitch lead screw so the nominal steps/mm for 16 × microstepping is 400 steps/mm.

Figure 3
figure 3

Measurement and correction of printer travel. (A) 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).

When comparing 3D bioprinter performance there are several key specifications to consider that relate directly to print quality. Most thermoplastic 3D printers provide specifications for resolution, which is defined as the smallest step the printer can take in any direction. The reported numbers for the FlashForge Finder and several popular commercially available bioprinters can be found in Table 1. Additional specifications related to the motion system are positional error, which is defined as the absolute deviation of the current location of the printhead from the intended location, and repeatability, which is defined as the maximum absolute deviation in measured position from the average measured position when attempting to reach that position multiple times. These more sophisticated metrics are largely absent in the bioprinting space and can vary between individual printers based on mechanical components and accuracy of assembly. Additionally, the resolution provided in bioprinter specifications are generally ideals based off of the nominal dimensions of the gears, pulleys, and screws used to assemble the motion system. None of the previously mentioned manufacturers provide measurements of actual resolution, that is error across the full distance of travel, or repeatability. These measurements are commonly provided with ultra-high-end motion platforms such as those from Aerotech and Physik Instrumente28,29. To determine and then optimize the real world performance of these low cost 3D printer based systems it is necessary to measure the travel with an external tool.

Table 1 Reported resolutions of FlashForge Finder and commercial bioprinters.

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. For the X-axis, there was a systematic under-travel using 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). For the Y-axis there was systematic under-travel using the nominal steps/mm (Fig. 3C, red curve) and after correction this was reduced to 29.1 µm (Fig. 3C, blue curve). Finally, for the Z-axis there was systematic under-travel using 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. Without this calibration, or at least measurement of the errors, it would be impossible to determine if flaws in printed constructs were due to the printer itself or other factors impacting print quality.

Assessing 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. However, with the recent advancements made in embedded bioprinting techniques 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 accuracy when FRESH printed from a collagen type I bioink (Fig. 4B). To measure the grid spacing we captured a 3D volumetric image using optical coherence tomography (OCT) (Fig. 4C)30, which revealed close agreement between the as-designed and measured dimensions (Fig. 4D). This was followed by a more complex design based on a 3D scan of an adult human ear (Fig. 4E). This model was printed using collagen (Fig. 4F). To analyze the accuracy we captured a 3D volumetric image of the printed ear 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. Together, these two examples demonstrate that the average error and standard deviation of the printed scaffolds are within the mechanical limitations of the bioprinter we built, and on par with commercial bioprinters31.

Figure 4
figure 4

Printing dimensionally accurate grids and organic shapes. (A) Model of a gridded scaffold with 500 µm and 1000 µm grids. (B) Photograph of the gridded scaffold printed in collagen type I. (C) An OCT image of the gridded 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). (E) A 3D model of an ear. (F) A photograph of the ear printed in collagen type I. (G) An OCT volumetric image of the printed ear. (H) Quantitative gauging of the ear print against the original 3D model.

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