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Introduction of 3D Printing and Different Bioprinting Methods

Asmita Biswas1, Baisakhee Saha1, Hema Bora1, Pravin Vasudeo Vaidya1,2, Krishna Dixit1, and Santanu Dhara1

1 School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, India

2 Advanced Technology Development Centre, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, India

1.1 Introduction of 3D Printing: Principles and Utility

3D printing (3DP), also known as additive manufacturing (AM), solid‐freeform (SFF), and rapid prototyping (RP), is a fabrication technique using model data, where 3D structures are fabricated using controlled layer‐by‐layer deposition [1]. It was first described by Charles Hull in 1986, followed by production and commercialization by S. Scott Crump and his company Stratasys [2]. The basic subcategories of 3DP are stereolithography, fused deposition modeling, selective laser melting, electronic beam melting, and laminated object manufacturing [3]. 3DP involves scaffold construction by material addition, with high geometric precision reducing material waste. The primary procedure comprises data acquisition and synthesis of meshed 3D computer models in computer‐aided design (CAD), followed by surface tessellation language (STL) file creation. This is followed by the slicing of mesh data into multiple 2D layer files and transferring them to a 3DP machine for fabrication [4]. Manufacture of complex designs, low cost, ease of access, and rapid and environment‐friendly procedures are some of the advantages of 3DP in industrial, research, healthcare, and biomedical sectors.

1.2 Ink Preparation and Printability

The choice of the base material as well as the recipe of its preparation to cater to the need for 3DP are of utmost importance. Bioink is the material used to produce either engineered or artificial living tissue using 3DP. It is the cell‐trapping milieu composed of a multicomponent aqueous mixture that usually forms gels. This sol‐gel transition of bioink is offered either by ionic bonds, covalent bonds, hydrogen bonds, or van der Waals interactions. Bioinks may be hydrogels, decellularized extracellular matrix, cell pellets, or tissue spheroids, of which hydrogels are the most common [5] due to their cell adhesion, growth, and proliferation capability since they absorb and retain large amounts of water. Ink for 3D bioprinting can be subdivided into two categories: cell‐laden inks called bioink and cell‐free inks called biomaterial ink. The bioinks usually consist of hydrogel precursors and are directly printed into Petri dishes filled with media and antibodies, whereas the biomaterial inks are usually utilized to print 3D scaffolds wherein the cells can be seeded on the scaffold under controlled conditions [6].

An ideal bioink should provide mechanical stability, stiffness, viscosity, surface tension, structural integrity, and biological ability – biocompatibility and biodegradability [7]. Many natural polymers like alginate, agarose, gelatin, chitosan, collagen, fibrin, and hyaluronic acid and synthetic polymers like polylactic acid (PLA), poly‐D,L‐Lactic acid (PDLLA), polylactic‐co‐glycolic acid (PLGA), polyvinyl alcohol (PVA), acrylonitrile butadiene styrene (ABS), polyethylene glycol (PEG), polyether ketone (PEEK), polycaprolactone (PCL), polybutylene terephthalate, and polyurethane (PU) [8] are used as bioinks for 3DP in the form of single or multicomponent.

• Printability: The term “printability” is the ability of a bioink to form a 3D structure with accurate fidelity and integrity as per the design and the geometry. However, the terminology modulates itself according to the printing approach. For extrusion printing, the bioink must be able to form continuous filament; for the inkjet technique, it should form well‐defined droplets while for laser printing, a prominent jet is required. The different printability indices [5] are the following:
  1. Extrudability: The minimum extrusion pressure essential for printing at the desired flow rate.
  2. Strand printability: Comparison of the diameter of printed strands with the CAD‐generated parameters.
  3. Integrity factor: Comparison of the thickness of printed scaffolds with designed geometries.
  4. Pore printability: Comparison of the printed pores with designed internal geometry.
  5. Irregularity: Comparison of outer geometry of scaffolds with designed parameters in X, Y, and Z directions.

Rheological properties and gelation kinetics determine the printability of a bioink, which is again dictated by the type of bioprinting. Low‐viscosity bioinks are preferred in inkjet bioprinting; rapidly crosslinkable, shear‐thinning bioinks are desirable for extrusion bioprinting and photo‐crosslinkable bioinks are favorable for stereolithographic printing [9]. Rheological properties like viscosity, viscoelasticity, yield stress, shear thinning, elastic recovery, and viscoelastic shear moduli affect the printability of bioinks. The rheological properties of the crosslinked bioink must facilitate scaffold remodeling to mimic the ECM environment. This process provides physicochemical cues to the cells, promoting their spreading and proper distribution. For instance, substrates that mimic the mechanical properties and Young’s modulus (~12 kPa) of native skeletal muscles offer better myogenic differentiation [10]. The key rheological parameters for a “good” bioink are described below:

• Viscosity: Viscosity is the ratio of shear stress to shear rate and is governed by the molecular weight and concentration of the polymer. High‐viscosity inks are preferable for high‐fidelity printing but may limit cell growth within the substrate due to higher shear stress. This shear stress can be overcome by either using hydrogel inks having shear‐thinning properties or using pre‐gel solutions with lower viscosities. For e.g., alginate‐based bioinks are directly extruded into calcium solution leading to ionic crosslinking. Due to higher surface tension, viscous bioinks prevent droplet formation without any merger of the columns with one another. Hence, crosslinking agents come into the picture with the caution of appropriate concentration so as to avoid phase separation and phase change [11]. Temperature‐dependent hydrogen bonding or hydrophobic interactions may be exploited, as in case of gelatin, Pluronic, etc. Colloidal‐like suspensions of densely packed microgels or jammed gels also prevent exposure of cells to high shear stress [12].
• Viscoelasticity and yield stress: Viscoelasticity is the property of retaining elastic shape while allowing viscous flow. It is guided by three parameters – storage modulus (G′), viscous modulus (G″), and yield stress. Tan (δ), the ratio between G′ and G″, gives information about the rheological characteristics of the bioink. Yield stress is the stress limit beyond which deformation occurs. The parameters of G′ and yield stress are governed by the number of crosslinks within the bioink. These crosslinks offer resistance to shape change within the yield stress. Paradoxically, though yield stress of the bioink renders shape and stiffness to the substrate, it can also deter cell encapsulation and further growth. Hence, additives like carrageenan, gellan gum, and hyaluronan are added to the bioinks to improve yield stress [13, 14]. However, in stereolithography and light‐assisted bioprinting, low‐viscosity bioink is required for easy flow and for each layer to be crosslinked with each other.
• Shear thinning: Shear thinning is the phenomenon where increase in shear rate results in the decrease in viscosity. Partially crosslinked hydrogels, colloidal suspensions, polymer melts, or polymer solutions above certain critical concentrations show shear‐thinning properties with shape preservation. Shear thinning leads to decrease in viscosity in the extrusion phase, but rise in viscosity after extrusion results in shape preservation. For e.g., shape retention in printed calcium phosphate cement is due to high zero‐shear viscosity [15]. PCL and PLA melts, used in polymer‐based fused deposition modeling, possess intrinsic shear thinning properties due to shear‐induced disentangling and alignment of long polymeric chains [16]. High resting viscoelasticity of pastes, solid suspensions, and colloidal dispersion bioinks arises due to the restoration of interaction between the suspended particles, which had been disrupted due to the shear‐thinning process [17]. Hydrogels demonstrate non‐Newtonian fluid behavior with shear‐thinning features. So, the random polymer chains align themselves in one direction under shear force and become suitable for extrusion process.
• Surface tension: Due to surface tension, there is an attraction between the liquid molecules, which ensures a contact angle between each printed strand. When the substrate has a higher surface energy than the surface tension of the bioink, the ink spreads; conversely, lower surface energy results in less spread [18]. For e.g., shape fidelity in printed constructs of ceramic slurries is reduced by both surface tension and gravity. It has been observed that...