Biologic: H-Bot Kinematics Based Multi-Micro-Extrusion Bioprinter

Number of patients on the organ recipient list is increased over the past few years and many patients died while waiting for organs [1]. According to the American Transplant Foundation report, a new patient is being added to the waiting list every 10 min and 20 people die every day due to the lack of available organs for transplantation [2]. Besides, each organ transplantation may not be successful due to the immune system rejects [3]. Hence, evolving systems emerge to create innovative and alternative solutions. Three-dimensional (3D) bioprinting, a cutting-edge technology of medicine, has accelerated the innovative research and development of new treatment methods. Nowadays, layer by layer tissue fabrication is used to create patient specific implants including dental implants and knee/hip replacements, soft and hard tissue patches, blood vessels and noncomplex organs, and even cancer therapeutics for human use [4–6].

Tissue engineering, a multidisciplinary research field that employs both the principles of life science and engineering to improve or develop tissue and organ substitutes, was described by Langer and Vacanti in 1993 [7]. Over the last decades, limited in vitro tissue models have been developed up to its large 3D scale [8]. Although, traditional methods such as foaming, emulsification, solvent casting particulate leaching, freeze drying, and electrospinning can create 3D scaffolds to be seeded later with tissue specific cells, the tailored micro-architecture or porosity is difficult to form with these methods [9]. Moreover, the target scaffold needs to be formed in a patient-specific geometry to replace or support functions of the damaged or deficient biological system [10]. Considering these needs, 3D bioprinting may be accepted as a type of solution for mentioned cases to create the desired tissue model owing to the complex manufacturing ability.

Three-dimensional bioprinting is based on biological material deposition in a 3D workspace using specific bio-inks. The first bioprinter, cytoscribing technology, was created by modifying a typical Hewlett Packard (HP) inkjet printer in 1988 [11]. Subsequently, ink-jet print heads were filled with cells suspended in a gel and printed in desired shape [12]. Scaffold fabrication with cell-laden bio-inks was studied in the following years. Today, numerous methods and bioprinting techniques are used to form a 3D tissue model. Mainly, ink-jet, micro-extrusion, and laser assisted methods are utilized for scaffold-based bioprinting. Nowadays, these methods are used together to increase manufacturing ability and form the desired tissue models [13,14]. Moreover, print head numbers of the known systems are increased as their precise deposition ability to utilize more types of cell-laden or cell-free bio-inks according to the target complex tissue model [15,16]. Ink-jet method uses droplets generated by thermal, piezo-electric, or electromagnetic pulses that may vary from 20 to 50 microns (μm) in diameter [17]. Noncomplex vessel-free tissues like skin and cartilage can be easily created by means of spraying cell-laden hydrogel bio-inks. Ink-jet print heads have been widely used with cell-laden hydrogels and even high cell viability rate up to 90% was obtained with use of this type of heads [18]. However, high shear stress is inevitable due to the small nozzle diameter that limits the usage of this method and may also cause clogging [19]. With the micro-extrusion method porous structures and complex tissue models like vessels and heart valves can be created owing to the cheaper single nozzle based precise fabrication system [20,21]. Generally, biological material dispensing is provided by means of a pneumatic (pressurized air), a mechanical (piston or screw), or a solenoid system. Applied shear stress that directly affects the cell viability may vary according to the used needle type. Fluid dynamics of a conic type needle applies less shear stress than a cylindrical type needle [22]. The bioprinting process is also required to be performed with uniform bio-ink deposition to obtain more functional tissue substitutes [23]. Nonuniform deposition is usually caused by clogging or deposition of undissolved polymers in the needle. Hence, the polymers of each bio-ink must be well dissolved and cell distribution must be homogeneous without any bubbles in the used bio-ink to achieve uniformly bioprinted tissues [24]. High cell viability up to 97% was obtained with the micro-extrusion method [25]. Laser assisted method is widely used for implant manufacturing or scaffold fabrication [26]. Cell-laden scaffold-based bioprinting via laser assisted method may be defined as a selective bio-ink detachment from donor site to a substrate layer via laser pulses. Although cell deaths are minimized by means of a laser absorbance membrane during the bioprinting process, this method is limited by low throughput and cell viability and is also more expensive than other methods but has a high-resolution printing capability [27]. Figure 1 illustrates each bioprinting method [28].

In this study, a micro-extrusion based bioprinter, Bio-Logic, with three different print heads, namely, Universal Micro-Extrusion Module (UMM), Multi-Micro-Extrusion Module (MMM), and Ergonomic Multi-Extrusion Module (EMM) were developed to obtain a more functional low-cost bioprinting system. The print heads were tested and scaffold models were bioprinted and analyzed. Bio-Logic was compared in price with the commercially available bioprinters.

Initially, kinematics, major components, electronics, and multimicro-extrusion print head were determined as a design criteria of Bio-Logic according to the cost and functionality of the commercially available bioprinters. H-Bot mechanism-based system was preferred instead of a common Cartesian or a complex delta system in order to have a maximum precision at minimal cost [28–30]. The system was isolated from the environment to create a suitable working area. Besides a UV lamp and a glove port application was included for sterilization. In addition, carbon dioxide (CO₂), temperature, and humidity measurement were realized in real-time. Micro-extrusion based print head that allows multibio-ink usage via typical syringes was designed to form complex tissue models. A large and heatable build-plate was also included to perform large-scale bioprinting; each print head should be heated to the fixed temperature (up to 37 °C for cell-laden bio-inks and up to 300 °C for cell-free bio-ink based scaffold fabrication) with the lowest overshoot during the bioprinting process.

The design and assembly of the whole system were performed using SolidWorks 2016 (Dassault Systemes, Kocaeli, Turkey). Two-dimensional sketch drawings were converted into the 3D solid parts according to the determined components and then assembled to have a perspective view of the machine. Figure 2 illustrates the assembled design of the system. Sigma profiles were used to form the main frame. The main frame was designed as two separate cabins, namely, top and bottom chamber. Most of the electronic components including power supply, control card, and control panel were placed into the bottom chamber. Fans were utilized for cooling this chamber. The working area was separated and isolated by means of a copper layer and acrylic windows. A front door with a glove port was placed to the front side of the machine and ergonomically, attachments like carry handle and wheels were mounted externally. Front panel was designed to have USB ports for firmware updates and lighting control. All designed mounting parts and carriers were made of laser cut aluminum (3 mm). Bearings such as LM UU (smooth rod mounting), SCE UU (carrier mounting), KP (ball screw rod mounting), KFL UU (pulley mounting) were utilized to create the assembly design. Environmental measurement sensors like temperature (K type thermocouple), humidity (DHT11), and CO₂ (MG-811) sensors were placed at the interior of the ceiling of the top chamber. In the same way, UV-C (preferred for prototype manufacturing due to its low cost and can be replaced with a germicidal lamp) light sources were mounted at the edges of the ceiling at its interior part. Liquid-crystal display user control panel was fixed at the corner of the top chamber. Distal dispensing module that supports any size commercially available syringes was mounted to the top chamber. Figure 3 illustrates the assembled design of the Bio-Logic (mechanic and electronic components of Bio-Logic as illustrated in Figs. 2 and 3 are listed and explained in detail in Supplemental Table 1 available in the Supplemental Materials on the ASME Digital Collection.

There were three types of print heads (UMM, MMM, and EMM) designed and compared in functionality. UMM was designed to support only one syringe of any size that can be used as distal. Syringe locks fix the syringe parts and linear movement can be provided with a ball bearing rod that is supported with another smooth rod. The mounting apparatus and other parts were laser cut and similar bearings were used to assemble the module. A Nema stepper (NEMA 17, 0.9 deg) was mounted onto the module to perform the micro-extrusion process. MMM was designed to support three syringes (5 mL) by means of directly driven mechanisms. Previous X axis carrier was changed with a new model to acquire multi-extrusion in the same bioprinting process. In this model, extrusion was provided with a specific apparatus that was mounted between the syringe and the ball screw rod. NEMA lead screw motors were used for extrusion and an apparatus was mounted for targeted UV curing during the bioprinting process. EMM was designed to support directly driven three syringes (5 mL) that can be easily mounted and locked. Typical NEMA steppers were utilized for extrusion and the same components were mounted on the extrusion module. Each print head was designed to have heater and thermocouple mountings. Designed print heads are illustrated in Fig. 3.

After the mechanical assembly, electronic components such as power supply, control card, motors, sensors, switches, and heaters were mounted and wired. Megatronics v3 (RepRapWorld) was used due to the multiple extruder support. Planetary gearbox stepper motors (NEMA 17, 14:1) were used for 3D movement ability, while typical steppers were utilized for micro-extrusion. Each stepper was driven with DRV8825 stepper driver. K type thermocouples and heaters were mounted as well as the sensors. DHT11 temperature and humidity sensor was used for real-time measurements. Interior CO₂ concentration was measured with MG-911 CO₂ sensor. All of the electronic components were wired to the control card. Figure 4 illustrates the assembled print head and the bioprinter.

Calibration stage was performed manually and automated to have metric outputs from the system. Manual calibration included movement and extrusion validity, heaters and thermocouple measurement tests, other sensor tests (temperature, humidity, and CO₂), front panel controllers (UV lamp, UV laser, camera, and USB ports) test, liquid-crystal display functionality test and similar component tests. Autocalibration was especially performed for heaters. While minimal overshoots are acceptable for any additive manufacturing system, automatically produced proportional integral derivative settings were also manually optimized for Bio-Logic to prevent any cell deaths when cell-laden bio-inks are used (proportional integral derivative auto-tune function). Lastly, electronically erasable programmable read-only memory settings were optimized to have metric outputs (Marlin Firmware). Dry Run (Repetier Host) function was used to validate the XY axes movement. Z axis was calibrated via a dial indicator.

After calibration of the system bioprinting was tested by means of scaffold fabrication. Different calibration models (8 mm × 8 mm each) with different pore size were bioprinted. Slic3r was used to generate different infill patterns such as diagonal and honeycomb (Fig. 5). A composite bio-ink was prepared and utilized to test the printing capability of Bio-Logic and to obtain 3D scaffolds for bone tissue engineering applications.

The composite bio-ink with final concentration of 5% gelatin (Sigma, Type A, Bloom 300, w/v), 6% alginic acid (Sigma, MW: 80,000–120,000 Da, w/v), 2% agarose (Serva, low melting, w/v), and 10% glass reinforced hydroxyapatite (HA, w/v) Demirkol et al. [31] was prepared in cell culture medium (Gibco, DMEM/F-12). A heater (WiseStir MSH-D Hotplate Stirrer), fixed at 50 °C, was used to accelerate the homogeneous blend formation which helps to prevent any undesired clogging in the syringe tip during the bioprinting process. The sample was loaded into a 5-mL syringe with a 400 μm conic type blunt-end needle and bubbles of the hydrogel sample were eliminated by means of centrifugation (Rotina 380 R, Hettich). The syringe was placed in a falcon tube and centrifuged at 25 °C, 3000 rpm for 3 min. Subsequently, the bioprinting process was performed after placing the syringe on a multi-extrusion based print head. Layer height and bioprinting speed were set to 300 μm (seven layers) and 30 mm/s, respectively. Bioprinting process was performed at room temperature (20 °C). A sterile tissue culture polystyrene base was fixed on the build-plate and samples were directly printed on it (Fig. 5(c), bioprinting step). The bioprinted samples were placed into a petri dish, crosslinked for stabilization and then sterilized. The samples were first immersed into 0.3 M CaCl₂ solution for 5 min to crosslink the alginate molecules then rinsed with Hank's Balanced Salt Solution (HBSS, Gibco). In the next step, the samples were immersed into 0.1% glutaraldehyde solution for 5 min to crosslink the gelatin and then rinsed with HBSS for the second time. Finally, the samples were immersed into 70% ethanol solution for sterilization and rinsed with HBSS for the last time. The bioprinted scaffolds were stored in HBSS solution at 4 °C.

The pore size of the scaffold with diagonal infill pattern was analyzed using the Image J program (designed pore size = 0.5 mm). Bio-ink preparation, path planning, and the bioprinting process are illustrated in Fig. 5.

Total cost of Bio-Logic and commercially available bioprinters with known specifications were determined according to the supplier information and literature review (See Supplemental Table 2 available in the Supplemental Materials on the ASME Digital Collection.).

Commercially available bioprinters cannot be widely used especially in small research facilities due to their high cost, and may not be suitable for bioprinting complex tissue models (See Supplemental Table 2 available in the Supplemental Materials on the ASME Digital Collection.). Complex tissue models require multiprint heads. Most of the bioprinters have only one or dual extrusion systems. Therefore, the productivity is directly limited. Besides, most of the devices require too much workspace although their build volumes are too small. Sterilization of the workspace must be considered to prevent microbial contamination of the bioprinted construct. While some systems can be easily placed in a biological safety cabinet, some systems have too much weight and are even very difficult to move. As some bioprinters have on-board crosslink mechanisms like UV light source, it can be externally applied before, during or after the bioprinting process by means of additional upgrades. Autonomous working provides pc-independent printing and even ethernet or wireless use make possible printing from a distance. These features also provide flexibility to users.

Scaffold fabrication was successfully performed with Bio-Logic. Scaffold models with different infill pattern (diagonal and honeycomb) were bioprinted and shape fidelity determined by pore size measurement of the scaffold with the diagonal infill pattern (Fig. 6). The average pore size of the scaffold was determined as 0.37±0.04 mm (n = 20).

Bioprinters and bioprinting technologies had to be developed and widely used to accelerate research at the medical field and create alternative medical solutions. The specifications of the Bio-Logic are quite enough to fabricate 3D tissue models. Besides, the cost of the system is considerably less than any other system. Bioprinter settings are as important as the used bio-ink and the final user may need to optimize some bioprinting parameters like print speed (for each bio-ink or specific regions of the same model), temperature (dynamic changes on each layer or at specific regions), retraction, avoiding travel on model or any other parameter. Consequently, precise bioprintability optimization depends on the user skills and at the same time to the limits of the control. On the other hand, bioprinter specifications usually limit the end user. Especially complex models require multiprint heads to be able to form the desired microstructure. In addition, researchers may need to develop custom-built systems to perform the desired processes [32,33].

Although, the UMM print head of the Bio-Logic seemed to be suitable, distal extrusion may result in clogging due to the diameter of the used tubing. Since shape fidelity may require high viscosity bio-inks, the tubing may not withstand the applied pressure at that situation. The MMM print head has deficiencies in practice; especially syringe mounting takes too much time which needs to be avoided when cell-laden bio-inks are used. Besides, the specific apparatus that connects the syringe and stepper lead screw may cause nonlinear dispensing. The EMM print head eliminates all these drawbacks, but requires too much workspace that may limit the size of the bioprinted tissue model.

All of the authors declare that they have all participated in the design, execution, and analysis of the paper, and that they have approved the final version. Additionally, there are no conflicts of interest in connection with this paper, and the material described is not under publication or consideration for publication elsewhere. A part of this study was performed as an in vitro bio-ink design and bioprinting without a cell source to observe bioprintability only. Therefore, human participants and/or animals were not involved to this research. This study was supported within the scope of scientific research projects.

• Kocaeli University (Contract No. 2016/006; Funder ID: 10.13039/501100004077).

The authors attest that all data for this study are included in the paper.