Aerospace, manufacturing, construction, automotive, and medical engineering have all embraced three-dimensional (3D) printing as a cutting-edge technology. Over the past two decades, 3D printing technology itself, the materials used for printing and its potential in almost all disciplines of science and engineering has marked 3D printing with a unique identity as a futuristic technology. The possibilities of 3D printing span over various fields such as fabrication of implants, bioengineered organs and in vitro testing models. 3D printers are classified as CNC machines (Computerized Numerical Control) where, the movement of the printer head and extruder is controlled by a set of alpha-numeric machine commands called 'Geometric Code’ or G-code. G##, X##, Y##, Z##, F##, and E## are the most commonly used alpha-numeric codes where # represents a numerical value.
In recent years, this technology has gained traction in the fields of tissue engineering and regenerative medicine. By accurately placing biomaterials and cells into functioning 3D tissue structures, researchers were able to generate more realistic tissue architectures . Hydrogels are employed as the printing substrate when working with cells for 3D printing because of their outstanding biocompatibility and similarity to natural extracellular matrix . Bioink is created by mixing hydrogels with cells, and bioprinting is the technique of printing cells with bioink. The three key elements of 3D bioprinting technique are computer design, bioink and 3D bioprinter.
The capacity of a bioink to produce reproducible 3D objects while maintaining the dimensional, mechanical, and functional integrity of the embedded cells is known as printability [1, 3]. However, there is a lack of knowledge about the parameters that affect printability or shape accuracy, as well as their effects on cell viability after printing [4, 5]. Many publications on printability focus on the function of rheology and bioink composition, while the effects of printing parameters and printer settings are largely under-studied [6–8]. This area of research being de novo, there exist no clear-cut protocols or approaches to effectively quantify and characterize printability of a bioink for 3D bioprinting applications.
The printability of a bioink is defined by its property to be accurately deposited with good spatial, temporal and volume control to form 3D structures with good integrity, shape fidelity and cell viability [1, 5]. The printability of a bioink depends on various parameters such as flow properties, composition/concentration and mechanical properties of the bioink. It also depends, type of printer head, printer nozzle dimension/geometry, geometry of the print structures, cross-linking chemistry, oxygen concentration and humidity of the printing environment [9–11]. The bioink for an extrusion-based bioprinter (like the one employed in this study) should possess good shear thinning qualities and yield strength to allow for easy printing and post-printing structure stability . On the other hand, in an inkjet or laser assisted bioprinters, the bioink is expected to have very good crosslinking and gelling properties to compensate for the low viscosity bioinks used [1, 12].
3D slicers are most commonly used to automatically generate these G-codes because these codes can run for multiple pages even for a simple design, making it impractical to write them manually. 3D slicers such as Cura slice a given STL file of a design to generate appropriate machine commands (G-code) for printer parameters such as spatial movement, print speed, material flow rate, nozzle diameter, filament diameter, layer height, and so on. G-code files may vary depending on the 3D printer, such as RepRap, Griffin, Marlin, Repetier, and so on [13, 14].
This study is a systematic approach to characterize various factors that affect printability using a model hybrid-hydrogel bioink composed of Sodium salt of carboxymethyl cellulose (Na-CMC) and gelatin. To determine the optimal hydrogel concentration for printing, a series of systematic experiments were carried out to effectively measure the printability of different concentrations of the hybrid hydrogel: (i) Characterizing material flow, gelation, and crosslinking properties to select suitable CMC/Gel hydrogel blends; (ii) Designing optimal 2D/3D structures to effectively evaluate and optimize printability as well as printing parameters; (iii) Optimizing G-codes and defining the optimal 3D slicer and printer settings for effective printing of the selected CMC/Gel hydrogel blends; and (iv) Quantifying printability of each CMC/Gel blend. In addition to analyzing the printability of CMC/Gel hydrogels, this study also highlights effective use of a simple FDM 3D printer that has been modified into an extrusion-based bioprinter to optimize and characterize hydrogels/bioinks for bioprinting applications.