Additive manufacturing (more commonly called three-dimensional or 3D printing) of biomaterials for biomedical applications faces many technical hurdles. For example, complex multiluminal structures, such as vascular networks, can be made with sacrificial tubes made from sugars around which a hydrogel is cast (1), but this approach achieves limited structural complexity. Limited resolution when printing biopolymers impedes mimicking the microscale complexity of native tissues. Two studies have independently addressed these concerns. Grigoryan et al. (2) unveiled a stereolithography (SLA) printing technique called SLATE (stereolithographic apparatus for tissue engineering) that used a new set of biocompatible photoabsorbers for high-fidelity generation of multivascular networks within 3D-printed hydrogels. On page 482 of this issue, Lee et al. (3) report an update of their FRESH (freeform reversible embedding of suspended hydrogels) extrusion-based printing technique (4) with substantially higher resolution and with the ability to print complex patterns of multiple bio-inks in non–photocross-linked gels (for example, such as pH-induced collagen polymerization).
Grigoryan et al. bioprinted a complex vascular network mimicking the distal lung tissue and the surrounding air space (top). Kim et al. bioprinted a replica of the heart made from collagen (bottom).
PHOTOS: (TOP TO BOTTOM) DAN SAZER, JEFF FITLOW, AND JORDAN MILLER; DANIEL SHIWARSKI, JOSHUA TASHMAN, AND T. J. HINTON
Although 3D printing was introduced in the mid-1980s, the first reports of bioprinting of cells did not appear until about 20 years later (5). High-resolution 3D bioprinting uses either SLA or extrusion-based printing (see the figure). The SLA techniques rapidly create 3D objects because millions of volume elements (voxels) can be photocross-linked simultaneously by image projection in a plane (the x-ydirection) onto the surface of a hydrogel precursor. The process repeats in the direction normal to the plane (the z direction) until the sections finish creating the object. In extrusion printing, cells and bio-inks (which can contain biomaterials such as collagen as well as bioactive molecules that can, for example, stimulate growth) are printed voxel-by-voxel from a moving printer nozzle.
Many of these hydrogel-based printing methods suffer from poor tissue integrity and fidelity (the degree to which the print can reliably replicate the material and structural features of the vasculature, respectively) and low resolution in feature size that can be printed (20 to 100 µm). In SLA methods, the x-y resolution is determined by the resolution of the projected image and the light path, but the zresolution is usually dictated by light-attenuating additives that confine the hydrogel polymerization to a thin layer near the surface. Initially, SLA bioprinting did not use light-absorbing additives and had reduced z resolution (>50 µm) (6) and reduced fidelity of the x-y image. Previous attempts to address this issue relied on either expensive and time-intensive multiphoton excitation of the crosslinkable material (7), which achieved a resolution of 0.1 to 5 µm (6), or the use of toxic or nonbiocompatible dyes such as Sudan I (8).
The SLATE technique can generate complex multivascular networks by creating functional intravascular topologies in hydrogels that are biologically compatible. Grigoryan et al. identified synthetic and natural food dyes that are potent, biocompatible photoabsorbers to improve the zresolution in printing. In particular, tartrazine (FD&C Yellow 5) has high photoabsorption, is highly biocompatible, and is easily washed out of the polymerized hydrogels after fabrication. Given the higher resolution of 50 µm, they 3D-printed a variety of complex multivascular networks in poly(ethylene glycol) diacrylate (PEGDA) hydrogels, including an axial vessel surrounded by a helical vessel, interpenetrating Hilbert curves (see the figure), a bicontinuous cubic lattice, and a torus with an interpenetrating knot.
These multiluminal networks have high fidelity in their complex 3D geometries and could also function as intravascular networks. For example, Grigoryan et al. created an entangled network in which a helical hydrogel vessel that carried red blood cells was wound around a serpentine path axial vessel that could carry gas. Deoxygenated red blood cells flowing through the helical channel could be perfused with oxygen flowing through the axial channel to achieve an oxygen saturation of ∼90% at the end of the channel. Grigoryan et al. also 3D-printed a model of an alveolus supplied by a single airway that was surrounded by a complex capillary-like network of vasculature, and were able to demonstrate that ventilation of the alveolus oxygenated the surrounding blood.
Biocompatible hydrogels beyond PEGDA can be used with SLATE, including fibrin and gelatin-methacryloyl (Gel-MA). The resulting 3D-printed constructs could be implanted and maintained function, as shown with 3D-printed albumin-producing hepatic models. Although PEGDA is considered biocompatible, the body cannot break it down, and it is unclear whether its degradation products are safe. The use of biological hydrogels like gelatin and fibrin strengthens the impact that SLATE may have on generating engineered tissue that can functionally remodel upon implantation in vivo.
The upgrading by Lee et al. of their FRESH bioprinting technique from version 1.0 (4) to 2.0 also opens new possibilities for the field. In FRESH printing, the support bed is made up of a thermally reversible, viscous gelatin slurry that offers a somewhat flexible support for the printing nozzle. The nozzle can easily penetrate the support bed without encountering resistance and can hold the printed hydrogel structure in place without it collapsing. In effect, this approach provides a printing volume instead of a printing surface, which is what is conventionally used in extrusion-based printing techniques.
After printing, the hydrogel is released from the support simply by heating to 37°C, which melts the support. Lee et al. successfully printed parts of the human heart from collagen hydrogels at a resolution of 20 µm, which exceeds the resolution of 100 to 500 µm (6) achieved with extrusion-based techniques. They reliably printed highly concentrated native-collagen filaments as small as 20 µm in diameter, compared with their previous resolution of ů250 µm. The improved resolution was achieved with 20-µm particles prepared by a coacervation technique that also reduced particle polydispersity, whereas FRESH v1.0 used gelatin microparticles with an average diameter of ∼65 µm. The improved method could print native collagen bearing no chemical modifications (unlike Gel-MA) and without the need for ultraviolet curing to polymerize the hydrogel. Collagen is the most abundant protein in the body, so a technology that can 3D-print it is immensely useful. Porosity could be modulated by altering the gelation of the collagen constructs by changing the ionic concentration and pH of the buffer used to make the gelatin slurry.
Other methods of printing vasculature (9) or printing collagen (10) have been demonstrated but did not achieve the precision or resolution of FRESH v2.0. These methods could not print multiple biological materials, such as collagen, alginate, fibrinogen, and methacrylated hyaluronic acid, as well as print these different bioinks simultaneously, as Lee et al. did by using the respective cross-linkers for each biopolymer. Their technique creates perfusable, microvasculature-like structures that substantially increase cell viability and angiogenesis. Multiple length scales were spanned; channel sizes ranged from millimeters (microvasculature in the left ventricle of a human heart) to 100 µm (a model of the neonatal rat heart complete with vasculature, trabeculae, and valves) (see the figure). When printed with relevant cells such as embryonic stem cells, these 3D-printed tissues generated functional outcomes expected of the human heart, such as synchronized beating and the opening and closing of printed valves during pulsatile flow.
Improvements in the functional outcomes obtained from the printed tissues by both techniques are still needed. Large numbers of cells are needed for printing even smaller-scale tissues and organs, and this limitation will only be exacerbated for 3D printing of large complex organs such as the entire human heart. Resolution at the 1-µm scale or smaller, with co-registered proteins over large print volumes, is still the ultimate goal to truly mimic the complexity of the extracellular matrix (ECM) of tissues. An initial step would be printing with bioinks that mimic the complex composition of organ-specific ECMs, which has been shown to be important in both tissue growth (11) and regeneration and repair (12). Despite these challenges, the technologies outlined in these two studies mobilize the prospect of creating functional organs and tissues from computer-generated models, thereby bringing on-demand organ printing closer to reality.
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