Introduction
Context
Three-dimensional in vitro human tissue models have the potential to act as models for disease which will not only reduce the use of animals as models for disease but may potentially increase turnover time associated with new drugs undergoing pharmaceutical trials [1]. The purchase and maintenance costs of commercially available bioprinters have meant this technology is inaccessible to most research labs whether in developed economies or developing countries [2]. There are, however, many facets of this cutting-edge technique (bioprinting) that are continuously being improved for its application, including the reduction in associated cost, design of bioinks, design of extruder systems and the ability to sustain post-printed viability of cells within the 3D structures [3,4]. General access to desktop & open-source additive manufacturing technologies has seen advancement in this field in recent years. Arguably, this has spurred the bioprinting revolution.
Extrusion based bioprinting has been largely focused on the manufacture of 3D tissue constructs or scaffold due to its many advantages over other methods, including, but not limited to; high cell viability [5], flexible geometric shapes [6], ability to incorporate multiple biomaterials and cell types [7], both homogenous and heterogeneous structures can be created [6,8]. In extrusion based bioprinting, the bioink is extruded out of a nozzle tip to form a continuous line structure driven by either pneumatic pressure or mechanical pistons. The extruded product is referred to as filaments instead of droplets [5,6,8]. A three-axis/cartesian automatic extrusion system is typically used in this type of bioprinting, equipped with a fluid dispensing nozzle [9]. This method has been extensively reported in many studies [10–16]. Most systems make use of standard plastic syringes which provide many benefits including; wide availability, low cost, aseptic, pyrogen free and compatibility with a range of needle sizes used as print nozzle heads [17]. Although the most widely employed method, it is not without its limitations. Most syringe-based extruders are designed to incorporate the fluid reservoir into the extruder carriage which is solely responsible for the high mass typically associated with this approach. A heavy extruder carriage can cause various issues during the printing process by affecting speed and resolution which may cause compromises in geometries of the printed constructs [12]. Reducing the volume of the fluid reservoir could provide a solution to the increased mass, however this will negatively impact the ability to print complex and larger constructs. Other larger volume systems have utilized the Bowden approach to minimize the weight of the extruder carriage. This approach makes use of Bowden tubing to connect the fluid reservoir (which is completely removed from the extruder carriage) to the nozzle. These systems are typically pneumatically driven which then brings in another separate set of limitations such as; poor retraction, the need for vacuum during printing and unstable extrusion pressures [12]. The ability of the printer to retract is important during the printing process as this prevents material from dripping during non-extruding moves which in turn reduces printing fidelity of the construct [12]. Pusch et al.(2018) have addressed the two major concerns of extruder carriage weight and inability to retract using the Large Volume Extruder (LVE) design. This design utilizes the Bowden tube approach with stepper motors and a lead screw driven extruder. The use of stepper motors is suggested to achieve retraction through straightforward reversal of direction and to apply constant pressure using a standard syringe [18].
The RepRapPro (RRP) Paste extruder utilized in this study also uses a Bowden tube approach along with a lead screw driven extruder system which makes use of stepper motors. As with the LVE approach, attaching the RRP paste extruder to the printer frame places all the weight on the printer frame and not the extruder carriage. This adds minimal payload to printer movements and should in turn maximize the print speeds and reduce any vibration of the nozzle during printing which may lead to nozzle dripping.
Design Criteria
Overall, the bioprinting process has multiple facets to consider which have been described under one of the three pillars required for successful tissue engineering – hardware, wetware and software considerations [19]. The outcome should include low operational cost, ability to use a wide variety of materials, allow for fine deposition of materials with high viscosities, maintain high cell viabilities, reduced maturation time of printed constructs and finally, minimal handling of the constructs. Here, we discuss the criteria required for the hardware development process applied in this study. The overall design and development process outlined in this study is specifically for the printing of cell free scaffolds which then provides the necessary foundation for future cell-laden bioink bioprinting.
1.2.1 Choice of Printer & RepRapPro (RRP) Paste extruder
A commercially sourced, RepRap based Delta 3D printer kit was selected for modification to include a hydrogel paste extruder in the place of the original thermoplastic extruder. The high printing precision and accuracy expected of delta printers led to the choice for eventual cell-free scaffold bioprinting [20]. The ANYCUBIC Kossel Linear Plus Delta printer used in this project was based off the popular Kossel RepRap delta printer designed by Johann C. Rocholl. All documents and development kits for the Kossel Delta are available online [L1] with many components of the model made readily available from Thingiverse [L2] . This allows for successful modification to be made to the printer at relatively low-cost. The firmware supplied with the Delta printer is the open-source Marlin Firmware (originally developed by Scott Laheine, [L3]). This firmware allows for modifications allowing for paste extrusion with the Delta hardware which allows for a relatively quick transition from thermoplastic extrusion to paste extrusion.
Delta printers contain an effector plate where the extruder nozzle sits and moves around the build volume, but the extruder stepper motor is situated outside the printing space, attached to the frame. The extruder nozzle must therefore be lightweight and secured properly onto the effector plate to prevent unwanted movement. Suspending the extruder nozzle above the bed arguably eliminates any contamination from particles caused by friction during print moves. The dimensions of the extruder carriage and the mass allowed on the axis that holds the extruder therefore dictate the maximum dimensions of the hydrogel paste extruder.
Furthermore, the kit used in this study was selected because of the linear rail-based motion control allowing for increased XYZ precision required for bioprinting. The ball bearing design of the linear rail system allows for a more precise motion control compared to the linear rod systems, typically found in desktop 3D printers. There is a considerable reduction in binding occurrences (ball bearings getting caught on the rail during a movement) which contributes significantly to a much smoother printing movement and creates a jerk-free print. The stepper motors (NEMA17 with 1.8̊ step angle) used in conjunction with the linear rails also contribute significantly to the increased precision found in this system, where micro-stepping allows for more controlled moves.
With such a highly application specific technology such as bioprinting, the commercially available bioprinters tend to be available at a very high cost. This has forced many research groups to develop their own bioprinters based on their specific application requirements whilst also maintaining a low overall build cost. This also allows for the functionality of the printer to be expanded over time, as required. As in this case, where a bioprinter initially designed for cell-free bioprinting of scaffolds may be easily modified for future cell-laden bioink printing. In this study a low cost commercially available 3D printer was modified to accommodate for bioprinting with cell-free hydrogels. This provided for majority of the printer parts at a relatively low cost. All additional parts were either 3D printed using thermoplastics or purchased at a low cost. Although the linear rails are considerably more expensive than smooth rod systems, the increase in printing precision is imperative to successful bioprinting and so outweighs the potential overall increase in build cost.