3D biofabrication strategies for tissue engineering and regenerative medicine

Abstract

Over the past several decades, there has been an ever-increasing demand for organ transplants. However, there is a severe shortage of donor organs, and as a result of the increasing demand, the gap between supply and demand continues to widen. A potential solution to this problem is to grow or fabricate organs using biomaterial scaffolds and a person's own cells. Although the realization of this solution has been limited, the development of new biofabrication approaches has made it more realistic. This review provides an overview of natural and synthetic biomaterials that have been used for organ/tissue development. It then discusses past and current biofabrication techniques, with a brief explanation of the state of the art. Finally, the review highlights the need for combining vascularization strategies with current biofabrication techniques. Given the multitude of applications of biofabrication technologies, from organ/tissue development to drug discovery/screening to development of complex in vitro models of human diseases, these manufacturing technologies can have a significant impact on the future of medicine and health care.

Keywords: bioprinting; hydrogels; photolithography; scaffolds; stem cells; vascularization.

Figure 1

The gap between the number of people waiting for an organ transplant and the number of people receiving one continues to widen. (Data obtained from 1).

Figure 2

Overview of the tissue engineering–based approach using three-dimensional (3D) biofabrication for de novo organogenesis.

Figure 3

Chemical structures of common polymers for biofabrication. Abbreviations: DIFO3, difluorinated cyclooctyne; HA, hyaluronic acid; maPEG, multiarm PEG; NIPAAm, N-isopropyl acrylamide; PAm, poly(acrylamide); PEG, poly(ethylene glycol); PEGDA, PEG-diacrylate; PEGDMA, PEG-dimethacrylate; PHEMA, poly(2-hydroxy ethyl methacrylate).

Figure 4

Bioprinting strategies for biofabrication. (a) Schematic of droplet-based bioprinting, in which 3D structures can be rapidly constructed through the controlled deposition of polymer droplets. (b) Depiction of extrusion bioprinting, in which the polymer is deposited from direct contact of the print head with the substrate. (c) An example of droplet-based bioprinting in which aqueous droplets were released within an oil environment to build spherical structures with multiple colored droplets and to create several different 3D architectures (97). (Reprinted with permission from the American Association for the Advancement of Science.)

Figure 5

Maskless stereolithography for biofabrication. (a) Multilumen PEG hydrogel conduit with fluorescent particles having an outer diameter of 5 mm and an inner diameter of 3 mm (left, isometric view; right, top view) (142). (b) Bright-field images of neovessels formed under hydrogels containing microchannels of the following diameters: 0 μm (a-i), 300 μm (a-ii), and 500 μm (a-iii). The implant sites from the top row are magnified in the bottom row (b-i, b-ii, and b-iii) (145). (c, top) Schematic outline of the SL fabrication process. (bottom) ChAT specific activity of HNs in different culture conditions (107). (d) Schematic showing the overall process of fabricating 3D spatially patterned hydrogel constructs by combining SL with DEP (106). Abbreviations: 3D, three dimensional; ChAT, choline acetyltransferase; CM, conditioned media; DEP, dielectrophoresis; HNs, hippocampal neurons; MCs, myoblast cells; OMA, oxidized methacrylic alginate; PEG, poly(ethylene glycol); SL, stereolithography; SLA, stereolithography apparatus; UV, ultraviolet. (Reprinted with permission from Springer and John Wiley and Sons.)

Figure 6

Multiphoton-based patterning and degradation of hydrogel environments. (a) Illustration of the two photon effect—that is, the absorbance of two photons using half the energy required by a single-photon absorption event—which yields a small excitation volume (V_TPE) described by the lateral (ω_xy) and axial (ω_z) dimensions. (b) The basic equations that govern the multiphoton excitation volume (V_TPE) and its parameters (ω_xy and ω_z), where NA is the numerical aperture of the microscope objective, λ is the wavelength of the excitation light, and n is the refractive index of the material. (c) The chemical reaction mechanisms commonly employed for two-photon patterning and degradation/cleavage techniques. PI indicates that a photoinitiator molecule is required for the reaction to proceed. (d) 3D projection of two-photon patterning using acrylate-based polymerization mechanisms to incorporate fluorescently labeled acrylate-PEG-RGDS molecules within PEG hydrogels. (e) An example of two-photon photodegradation in PEG hydrogels through the incorporation and photocleavage of a nitrobenzyl ether–based compound (160). Abbreviations: 3D, three-dimensional; PEG, poly(ethylene glycol); PI, photoinitiator. (Reprinted with permission from the American Association for the Advancement of Science.)

Figure 7

Next-generation biofabrication techniques. (a,i) SEM image of the a nanoES made of alginate. The epoxy ribbons are false-colored brown for clarity. (ii) Fluorescence image of a cardiac patch. Green shows α-actin, and blue indicates cell nuclei. The dashed lines show the position of the source–drain electrodes. (iii) Monitoring capability (conductance versus time) of the nanoES sensor in a 3D cardiomyocyte mesh before (black) and after (blue) applying noradrenaline. (iv) Multiplexed simultaneous electrical recording of the extracellular field potentials from four nanowire FETs with 6.8-mm separation in a nanoES/cardiac construct (168). (b) Immunofluorescent images of sarcomeric α-actinin (green), nuclei (blue), and Cx-43 (red) on (i) pristine GelMA and (ii) CNT-GelMA. (iii) Spontaneous locomotion of a triangular swimmer at different time points and the corresponding displacement versus time plot (169). (c,i) Schematic overview of an open interconnected self-supporting glass lattice that serves as a sacrificial element for the casting of 3D vascular architectures. (ii) Primary rat hepatocytes and fibroblasts in agarose gel (slab versus channeled) after 8 days of culture stained with live/dead assay. Green indicates live cells, whereas red shows dead ones (173). Abbreviations: 3D, three-dimensional; CNT-GelMA, carbon nanotubes incorporated into GelMA; ECM, extracellular matrix; FETs, field-effect transistors; GelMA, gelatin methacrylate; nanoES, nanoelectronic scaffold. (Reprinted with permission from Nature Publishing Group and the American Chemical Society.)