The Application of Ultrasound in 3D Bio-Printing

Abstract

Three-dimensional (3D) bioprinting is an emerging and promising technology in tissue engineering to construct tissues and organs for implantation. Alignment of self-assembly cell spheroids that are used as bioink could be very accurate after droplet ejection from bioprinter. Complex and heterogeneous tissue structures could be built using rapid additive manufacture technology and multiple cell lines. Effective vascularization in the engineered tissue samples is critical in any clinical application. In this review paper, the current technologies and processing steps (such as printing, preparation of bioink, cross-linking, tissue fusion and maturation) in 3D bio-printing are introduced, and their specifications are compared with each other. In addition, the application of ultrasound in this novel field is also introduced. Cells experience acoustic radiation force in ultrasound standing wave field (USWF) and then accumulate at the pressure node at low acoustic pressure. Formation of cell spheroids by this method is within minutes with uniform size and homogeneous cell distribution. Neovessel formation from USWF-induced endothelial cell spheroids is significant. Low-intensity ultrasound could enhance the proliferation and differentiation of stem cells. Its use is at low cost and compatible with current bioreactor. In summary, ultrasound application in 3D bio-printing may solve some challenges and enhance the outcomes.

Keywords: bioink; bioreactor; cell spheroids; low-intensity ultrasound; three-dimensional bio-printing; ultrasound standing wave field.

Figure 1

Typical six processes for 3D bioprinting: (1) imaging the damaged tissue and its environment to guide the design of bioprinted tissues/organs; (2) design approaches of biomimicry, tissue self-assembly and mini-tissue building blocks are sed singly and in combination; (3) the choice of materials (synthetic or natural polymers and decellularized ECM) and (4) cell source (allogeneic or autologous) is essential and specific to the tissue form and function; (5) bioprinting systems such as inkjet, microextrusion or laser-assisted printers; (6) tissue maturation in a bioreactor before transplantation or in vitro applications, courtesy of [24].

Figure 2

Schematic diagram of (a) thermal inkjet printers electrically heating the printhead to produce air-pressure pulses that force droplets from the nozzle, whereas acoustic printers using pulses formed by ultrasound pressure from piezoelectric element; (b) microextrusion printers using pneumatic or mechanical (piston or screw) dispensing systems to extrude bioink beads; and (c) laser-assisted printers utilizing focused laser beams on an absorbing substrate to propel bioink onto a collector substrate, courtesy of [24].

Figure 3

The processes of the spheroid formation: (1) formation of loose cell aggregates via integrin-ECM binding; (2) a delay period for cadherin expression and accumulation; (3) formation of compact spheroids through hemophilic cadherin-cadherin interactions, courtesy of [46].

Figure 4

Cell aggregation under ultrasound standing wave and view normal to sound propagation (a) column of discoid aggregates in a plane cylindrical resonator; (b) initial pattern in tubular resonator-concentric cylinders; (c) aggregates of different sizes in a tubular resonator; (d) in plane and tubular resonator; (e) in a pair of two transducers perpendicular to each other; and (f) view in the direction of sound propagation of 3D RBC aggregate in the center of half-wavelength resonator, courtesy of [71].

Figure 5

(A) Epifluorescence micrograph of 3D HepG2 aggregates by ultrasound standing wave and F-actin stained with Phalloidin-Alexa 488 with junctional F-actin marked with arrowheads (bar 25 μm) and an unsonicated single cell shown in inset (bar 10 μm); (B) aggregate maintained on a P-HEMA-coated surface after 1 day under light microscope, confocal micrograph of F-actin staining after 1 day (C) and 18 days (D); and confocal micrograph of F-actin staining in gyrotatory-produced aggregates after (E) 2 h; (F) 1 day; (G) 3 days; (H) 9 days and (I) 18 days (B–I, bar 50 μm), courtesy of [77].

Figure 6

Bioprinting of segments of intraorgan branched vascular tree using uni-lumenal vascular tissue spheroids in hanging drop after tissue fusion, courtesy of [20].

Figure 7

Phase-contrast images of neovessel formation and sprout in suspended endothelial cells in an unpolymerized collagen type-I solution following ultrasound standing wave field (USWF) exposure (A,C,E,G,I) shown by white arrow and sham-exposed (B,D,F,H,J) in scale bar of 100 μm, courtesy of [76].

Figure 8

Intrinsic cellular autofluorescence in second-harmonic generation microscopy of endothelial cell bands (green) suspended in unpolymerized collagen type-I fibers (red) on (A,B) day 1; (C,D) day 4; and (E,F) day 10 incubation treated by ultrasound standing wave field (USWF) or sham-exposure, arrows show endothelial cell sprouts in scale bar of 50 μm, and the histograms of the occurrence frequency of collagen fiber angles in (I) USWF- and (J) sham-exposed constructs. collagen gels, courtesy of [76].

Figure 9

Effect of low-intensity ultrasound (LIUS) on the chondrogenesis of rabbit MSCs seeded in fibrin-HA and cultured in chondrogenic-defined medium untreated, treated with TGF-β3 (10 ng/mL), or treated with LIUS (100 mW/cm²) for 1 and 4 weeks, (A) the gross images of the constructs at 0, 1, and 4 weeks in scale bar of 1 mm; and (B) images of safranin-O/fast green staining in scale bar of 1 mm for (a–c,g–i), 200 mm for (d–f,j–l), courtesy of [120].