# Cell-Laden Thermosensitive Chitosan Hydrogel Bioinks for 3D Bioprinting Applications
## 1 Introduction  
Three-dimensional (3D) printing, also known as additive manufacturing, can be used to create a solid 3D structure through a layer-by-layer process. At present, the 3D printing of biocompatible materials represents an active area of research and constitutes tools for tissue engineering and regenerative medicine [1]. In the early years of the application of 3D printing in the fields of tissue engineering and regenerative medicine, 3D printing was applied to fabricate 3D scaffolds without cells. However, the development of 3D printing technology has led to the formation of complex tissue constructs through the deposition of cells and hydrogels via a layer-by-layer process called 3D bioprinting [2]. Cells and hydrogels have been generated from printable, non-toxic bioinks [2; 3; 4]. The major technologies used for 3D bioprinting include inkjet bioprinting, laser-assisted bioprinting, digital light processing (DLP) bioprinting, and extrusion bioprinting [3]. Inkjet bioprinting is a modification of conventional two-dimensional (2D) inkjet printing, and was the first bioprinting technology to be developed. Inkjet bioprinters use thermal or piezoelectric actuators to eject liquid droplets onto substrates [5]. The advantages of inkjet bioprinters include their relatively low cost and high-speed printing, although there is a risk that cells and materials generated this way are exposed to thermal and mechanical stress and nozzle clogging. Laser-assisted bioprinting is less common than inkjet or microextrusion bioprinting. However, laser-assisted bioprinters are being used in cell printing, tissue engineering and regenerative medicine. Laser-assisted bioprinters consist of a 'ribbon' structure that contains an energy absorbing layer on the top and a layer of liquid bioink solution suspended at the bottom [6]. During printing, a focused laser pulses on the absorbing layer of the ribbon to create a high-pressure bubble that stimulates bioink flow toward the collector substrate. Laser-assisted bioprinters do not exert any mechanical stress on cells, which results in high cell viability. Moreover, nozzle-free devices could be used to circumvent the nozzle clogging problem. Despite these advantages, laser-assisted bioprinters are not used commonly owing to their high cost and complex device setup. DLP bioprinters are based on a dynamic optical projection stereolithography (DOPsL) platform that projects an optical pattern using UV light [7]. Compared to other 3D printers, the DLP bioprinter constructs a structure by continuously projecting the plane of the optical pattern onto the photopolymer vat. The DLP bioprinter has a high printing speed and cell viability, but it is not widely used owing to its high cost of operations [2; 3]. Extrusion bioprinters commonly contain pneumatic or mechanical (piston or screw) dispensing systems, which deposit biomaterials onto substrates [8]. Almost all commercially available bioprinters are based on the extrusion model. Extrusion bioprinters are suitable for a wide range of hydrogels with different viscosities that exhibit compatible photochemical and thermal crosslinking properties. The cell viability associated with extrusion bioprinters is lower than that with inkjet ones, but the former are capable of depositing very high cell densities at a reasonable cost [2]. Based on currently available technology, only extrusion bioprinters are capable of 3D printing of a wide range of materials [9], rendering their use for 3D bioprinting important. In the field of 3D bioprinting, bioinks such as hydrogels or biomaterials could be printed onto substrates to facilitate the attachment of cells. There are several types of bioinks available, including collagen, gelatin, MatrigelTM, agarose, and alginate [10]. These materials are usually amenable to printing owing to their biocompatibility, nontoxic degradation kinetics and byproducts, and structural and mechanical properties suitable for printing and material biomimicry [2].