Three-dimensional bioprinting of gelatin methacryloyl (GelMA)
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The three-dimensional (3D) bioprinting technology has progressed tremendously over the past decade. By controlling the size, shape, and architecture of the bioprinted constructs, 3D bioprinting allows for the fabrication of tissue/organ-like constructs with strong structural–functional similarity with their in vivo counterparts at high fidelity. The bioink, a blend of biomaterials and living cells possessing both high biocompatibility and printability, is a critical component of bioprinting. In particular, gelatin methacryloyl (GelMA) has shown its potential as a viable bioink material due to its suitable biocompatibility and readily tunable physicochemical properties. Current GelMA-based bioinks and relevant bioprinting strategies for GelMA bioprinting are briefly reviewed.
The demand for organ replacement or tissue regeneration is quickly expanding, while the number of donor organs is far from sufficient [1, 2]. Tissue engineering, initially proposed approximately three decades ago, has thus emerged as an alternative strategy aiming to generate tissues and organs that are functionally relevant to their in vivo counterparts, to replace those that are damaged or diseased in the body . Besides this conventional aspect, tissue engineering has found additional applications over the past years in serving as a tool to produce biomimetic miniaturized human tissue models for the purpose of improving the accuracy of drug screening and of promoting personalized medicine [4, 5]. Nevertheless, it is still a great challenge to fabricate complex living tissues except for a few simple organs such as skin  and cartilage . Recently, the advancements in three-dimensional (3D) bioprinting seem to have brought us a step closer to realizing the ambitious aim of tissue engineering, by providing an unprecedented means to control, in precision, the deposition/patterning of cells and biomaterials in the volumetric space at high reproducibility .
While there are several bioprinting modalities commonly used for tissue fabrication, the bioink consisting of a mixture of biomaterial(s) and cell(s) is the unanimously vital component serving as the building block of bioprinted 3D tissue structures [9, 10]. Taking extrusion bioprinting as an example, the bioinks play key roles in dispersing the cells prior to bioprinting, in maintaining the integrity of the structures during bioprinting, and in supporting the adhesion, spreading, and functionality of encapsulated cells post-bioprinting [11, 12]. The bioprinted cell-laden constructs featuring arbitrary shapes and architectures finally form 3D tissue-like structures following a period of culture .
In principle, an ideal bioink should possess physicochemical properties suitable for the bioprinting process, and the bioprinted constructs should have proper biological and mechanical properties close to those of the native tissues. Hydrogel-based bioinks encapsulating living cells and bioactive components are of particular interest for bioprinting . To this end, numerous hydrogel-based bioink formulations such as gelatin methacryloyl (GelMA) [15, 16, 17], acrylate-functionalized poly(ethylene glycol) [18, 19], alginate [20, 21], agarose , and collagen  have been adopted, either used alone or in combinations, as bioinks. Among the different types of hydrogel bioinks, those based on GelMA hold good promise attributed to the superior biocompatibility, on-demand photocrosslinkability, and broadly tunable physicochemical properties of this biomacromolecule denatured from collagen . This review outlines recent advances in the development of GelMA-based bioink formulations and strategies suitable for GelMA bioprinting.
GelMA-based bioink formulations
Pure GelMA bioink
Pore-forming GelMA bioink
Alginate-templated GelMA bioink
Strategies for bioprinting GelMA-based bioinks
Several bioprinting techniques based on modifications of extrusion bioprinting, including sacrificial bioprinting and hollow tube bioprinting, have been developed in addition to direct extrusion bioprinting to generate GelMA-based tissue constructs. Besides, GelMA-based bioinks can also be applied to non-extrusion bioprinting strategies for tissue fabrication.
Alternatively, the sacrificial templates may also be physically extracted. For example, using agarose as the bioink, which forms a mechanically robust solid hydrogel at room temperature or lower, microfibers could be bioprinted and GelMA constructs containing hollow microchannels could be fabricated in a similar manner (Fig. 4B-a, b) [38, 39, 40]. Again, these hollow microchannels may be further populated with a monolayer of endothelial cells (Fig. 4B-c), and the formed endothelium was functional serving as an efficient barrier deferring the diffusion of biomolecules into the surrounding GelMA matrix (Fig. 4B-d) .
Hollow tube bioprinting
Contrarily, the hollow alginate microfibers may as well serve as structural supports for building cell-laden constructs in low-stiffness GelMA hydrogels, providing cells with soft microenvironments suitable for engineering certain tissue types . Different with the hollow GelMA-based microfibers discussed above, here a mixture of GelMA and CaCl2 solution is employed as the core while the alginate solution is extruded from the sheath (Fig. 5C-a), resulting in bioprinted 3D microfibrous constructs where the GelMA bioink is embedded within the microfibers and subjected to subsequent photocrosslinking. The encapsulated cells showed differential morphologies when the GelMA bioinks used in the cores of the microfibers had different concentrations (Fig. 5C-b). The concentration (down to 1 w/v%) of GelMA that could be bioprinted using this strategy was even lower than that possible through direct extrusion bioprinting with the GPGs .
The above-mentioned bioprinting strategies relying on direct or indirect extrusion are easy to realize, but the processes are generally slow. In comparison, a few non-extrusion-based bioprinting strategies could shorten the time needed for tissue fabrication and possibly enhance the resolution of features in the generated tissue constructs.
More recently and interestingly, an unconventional chaotic bioprinting technique was reported to control the precise positioning of microscale and nanoscale features in the bioprinted structures . The chaotic bioprinting is designed to create fine architectures by flow itself in a controllable and reproducible manner in a solid matrix, where the bioink and the matrix can be either the same material or different materials. For this, a customized Joule bearing device, which contained an outer cylinder and an inner cylinder, was constructed (Fig. 6B-a). By rotating the cylinders in opposite directions with a defined angle, the bioink droplet in the matrix could be elongated forming a pattern that was highly predictable as a function of the rotation and material parameters (Fig. 6B-b, c). As an example, cells could be embedded in the GelMA bioink droplet, in which after rotation (bioprinting) high-resolution patterns of cells in the surround GelMA matrix could be generated to mimic, for example, multiscale vascularization, revealing also favorable cell spreading (Fig. 6B-d).
Conclusion and future perspective
To date, various types of GelMA-based bioinks have been developed, allowing us to bioprint complex tissue constructs that were difficult to produce in the past. On the other hand, improved bioprinting strategies have further enabled us to fabricate complex tissue-like structures using different GelMA-based bioinks. We envision that GelMA as a cost-effective yet biocompatible and bioactive material will play an important role in bioprinting to facilitate the generation of functional tissues and biomimetic tissue models for widespread applications in tissue engineering, regenerative medicine, pharmaceutics, and precision medicine among others.
The authors gratefully acknowledge funding from the National Institutes of Health (K99CA201603, R21EB025270, R21EB026175) and Doctoral New Investigator Grant from American Chemical Society Petroleum Research Fund (56840-DNI7). G. L. Y. acknowledges Natural and Science Foundation of Hubei Province (2014CFB778).
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interests.
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