**3. Bioinks**

Bioink is a term given to the precursor material used during printing. The main component of the bioink usually consists of a hydrogel. Hydrogels are used as they are highly hydrated, enable cell encapsulation and provide a cushioning effect during fabrication. Furthermore, hydrogels have adaptable rheological properties making them suitable for processing and printing [15]. Hydrogels used in tissue engineering applications are either naturally derived or synthetically derived polymers.

#### **3.1 Naturally derived hydrogels**

Collagen, gelatin (denatured collagen), silk, alginate and chitosan are the most commonly used naturally derived polymers in bioprinting with collagen being used in 26% of the bioprinting-relevant literature [26]. Natural polymers provide great bioactivity and high adhesiveness, resulting in high cell viability and proliferation. However, a lack of mechanical competence and reproducibility limit their use in a non-composite form.

#### **3.2 Synthetically derived hydrogels**

Aliphatic polyesters and poly(ethylene glycol) are the most commonly used synthetic polymers in bioprinting [27]. Synthetic polymers have a high printing fidelity and controllable degradation kinetics and mechanical properties. However, their inert properties result in low cell viability and proliferation rate [28]. Aliphatic polyesters used in tissue engineering are usually poly(lactic acid), poly(glycolic acid) and poly(caprolactone). Refer to **Table 1** for a comparison of their different properties.

#### **3.3 Characteristics**

When processing bioinks, three main parameters must be considered: viscosity of the precursor hydrogel, gelation and network stiffness post-processing [30].


#### **Table 1.**

*Correlation between water affinity and degradation rate for aliphatic polyesters.* 

The latter two points can affect cell viability, proliferation, migration and even differentiation. The three parameters are mainly affected by the type of polymer, its concentration and molecular weight. Other factors that can have an adverse effect on cells are the shear stress induced during printing and the time period during which the cells are exposed to non-physiological conditions (fabrication time).

Rheology of the bioink, particularly viscosity, is an important consideration for printing. Viscosity affects printing fidelity and also influences the shear stresses induced during printing. High shear stresses during printing can damage cells in nozzle-based techniques leading to higher cell apoptotic activity. Each printing technique requires a different viscosity range; inkjet-based printers require low viscosity solutions [31], extrusion-based printers require a high viscosity [9], while laser-assisted bioprinting requires a medium viscosity [5]. However, stereolithographic techniques are not as limited by hydrogel viscosity ranges. Recent research has shown that the sheer stresses induced by the applied pressure in extrusion printing can be alleviated if the bioinks are prepared as isotonic solutions [32].

The objective to have a 3D structure laden with cells that are viable, proliferative and differentiative, along with a structure that exhibits mechanical competence, stability and biologically relevant complexity is dependent on the bioink and the printing process. Ultimately, the type of polymer, its chemical properties, viscosity, gelation, stiffness and fabrication time will affect the cells' status and the print fidelity.
