*3.2. Gelatin*

Gelatin is a partly hydrolyzed collagen derived from di fferent animal tissues, such as bovine tendon and fish. Unlike its ancestor collagen, gelatin is a typical thermal-responsive (or thermosensitive) linear natural polymer with excellent water solubility, biocompatibility, biodegradability, and 3D printability. For example, the immunologic rejection of gelatin is much lower (i.e., zero) than that of collagen. There are no inflammation and other negative reactions when gelatin hydrogels are implanted in vivo [91]. This is an outstanding advantage of gelatin hydrogels for use as bioinks for 3D bioprinting of bioartificial organs.

The liquefaction temperature (i.e., sol–gel transition point) of gelatin solution is approximately 28 ◦C (25–30 ◦C). The unique sol–gel transition property and super biocompatibility of gelatin hydrogels have made them the preferential natural bioinks for 3D organ bioprinting. Before 3D bioprinting, cells and bioactive agents are homogeneously encapsulated in the gelatin solution to form cell-laden hydrogels. Other natural polymers, such as alginate, fibrinogen, chitosan, and hyaluronate, can be incorporated into the gelatin solution as additives to form composite bioinks [92–97]. Di fferent chemical crosslinking strategies have been employed to improve the stability of 3D printed constructs according to the properties of the additives.

During the sol–gel transition stage, physical crosslinking occurs among the gelatin molecules. This means that thermosensitive gelatin solutions are capable of gelling and being 3D printed at mild or room temperatures, such as 1–28 ◦C, in which cells are durable. However, physical crosslinking (or gelling) is a reversible thermosensitive gelation process. The bonding strength among gelatin molecules is poor, which results in breakage of the 3D printed constructs when they are put into culture medium at physiological conditions, such as 37 ◦C. Some groups have utilized this phenomenon to print monolayer cells using extrusion-based 3D printers and washed the liquefied gelatin molecules post printing [98]. Otherwise, secondary chemical crosslinking is necessary to stabilize the 3D constructs [79–81].

Since 2005, various gelatin-based composite bioinks, such as gelatin/alginate, gelatin/fibrin, gelatin/chitosan, gelatin/hyaluronate, gelatin/alginate/fibrin, and gelatin/alginate/dextron (glaycerol or dimethyl sulfoxide), have been explored in my laboratory through various extrusion-based 3D bioprinting models (Figure 7) [15,32,79–81]. In these models, the viscosity of the gelatin-based bioinks depends largely on the polymer concentration, molecular weight, and cell density. A series of two-step stabilization strategies, containing both thermosensitive physical and ionic chemical crosslinks, have been exploited for 3D printed constructs. The chemical crosslinking methods include glutaraldehyde for gelatin, CaCl2 for alginate, sodium tripolyphosphate for chitosan, and thrombin for fibrinogen. Ten years on, these classical bioink formulations and crosslinking strategies have been widely adapted by many other groups all over the world [96,97].

**Figure 7.** A large-scale 3D printed bioartificial organ with vascularized liver tissue constructed through the double-nozzle 3D bioprinter created in Prof. Wang's laboratory at Tsinghua University: (**a**) the double-nozzle 3D bioprinter; (**b**) a computer-aided design (CAD) model containing a branched vascular network; (**c**) a CAD model containing the branched vascular network; (**d**) a few layers of the 3D bioprinted construct containing both ASCs encapsulated in a gelatin/alginate/fibrin hydrogel and hepatocytes encapsulated in a gelatin/alginate/chitosan hydrogel; (**<sup>e</sup>**–**h**) 3D printing process of a semielliptical construct containing both ASCs and hepatocytes encapsulated in different hydrogels; (**i**–**l**) hepatocytes encapsulated in the gelatin-based hydrogels after 3D bioprinting and different periods of in vitro cultures; (**<sup>m</sup>**–**p**) ASCs encapsulated in the gelatin-based hydrogels after 3D bioprinting and different periods of in vitro cultures as well as growth factor inductions. Images reproduced with permission from [15,32].

When cells are embedded in gelatin-based solutions with high polymer concentration, their bioactivities are restricted to some degree after the double physical and chemical crosslinking. Meanwhile, lower concentrations of polymers facilitate higher cell viability. Nevertheless, when the concentration of gelatin-based polymers is reduced, the mechanical strength of the 3D constructs drops sharply despite chemical crosslinking. An optimized polymer concentration is necessary for a typical 3D bioprinting technology to ensure favorable cellular activity and structural stability [79–81].

Because the components of our gelatin-based composite bioinks are similar to ECMs (i.e., proteoglycans or glycoproteins), 3D printed constructs can be engineered to possess similar water content and elastic modulus as those of soft organs with excellent in vitro cell and in vivo tissue biocompatibilities. This is a fundamental breakthrough in large-scale 3D organ bioprinting. Until now, the resulting 3D printed constructs have been extended rapidly to other biomedical areas, such as high-throughput drug screening, living tissue and organ cryopreservation, metabolism model establishment, and pathological mechanism analysis [79–81].

Besides physical and chemical crosslinking strategies, some researchers have aimed to maintain laser-based 3D printed gelatin structures by methacrylation of the gelatin molecules (i.e., gelatin methacrylamide or gelatin methacryloyl, GelMA) using irreversible ultraviolet (UV) photopolymerization techniques [99]. Some researchers have blended GelMA with poly(ethylene glycol) (PEG) and other polymers. It was reported that the extruded strut size could be reduced from 1100–1300 to 350–450 μm by blending GelMA with PEG and to 150–200 μm by coaxial extrusion of GelMA with alginate using a coaxial nozzle [100–103]. Additional concerns have arisen regarding the toxicity of polymerization initiators and degradation products of synthetic polymethacrylamide.
