Research ArticleBIOMEDICAL ENGINEERING

Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels

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Science Advances  23 Oct 2015:
Vol. 1, no. 9, e1500758
DOI: 10.1126/sciadv.1500758
  • Fig. 1 FRESH printing is performed by depositing a hydrogel precursor ink within the thermoreversible support bath consisting of gelatin microparticles and initiating gelling in situ through one of multiple cross-linking mechanisms.

    (A) A schematic of the FRESH process showing the hydrogel (green) being extruded and cross-linked within the gelatin slurry support bath (yellow). The 3D object is built layer by layer and, when completed, is released by heating to 37°C and melting the gelatin. (B) Images of the letters “CMU” FRESH printed in alginate in Times New Roman font (black) and released by melting the gelatin support (gray material in the petri dish). When the gelatin support melts the change in optical properties, convective currents and diffusion of black dye out of the alginate make it appear that the letters are deforming, although they are not. (C) Representative images of gelatin particles produced by blending for 30, 75, or 120 s. (D) The mean Feret diameter of gelatin particles as a function of blending time from 30 to 120 s (n > 1000 per time point; the red line is a linear fit and error bars indicate SD). (E) Rheological analysis of storage (G′) and loss (G″) modulus for gelatin support bath showing Bingham plastic behavior. Scale bars, 1 cm (B) and 1 mm (C).

  • Fig. 2 Analysis of the hydrogel filaments and structures fabricated using FRESH.

    (A) A representative alginate filament (green) embedded within the gelatin slurry support bath (red). (B) Histogram of the diameter of isolated alginate filaments within the gelatin support bath showing a range from 160 to 260 μm. (C to E) A standard square lattice pattern commonly used for infill in 3D printing FRESH printed in fluorescent alginate (green) and viewed (D) top down and (E) in 3D. (F to H) An octagonal infill pattern FRESH printed in fluorescent alginate (green) and viewed (G) top down and (H) in 3D. (I) Example of a two-material print of coaxial cylinders in red and green fluorescently labeled alginate with a continuous interface shown in top down and lateral cross sections. (J) An example of a freeform, nonplanar FRESH print of a helix shown embedded in the gelatin support bath. (K) A zoomed-in view of the helix demonstrating that FRESH can print in true freeform and is not limited to standard layer-by-layer planar fabrication. Scale bars, 1 mm (A), 500 μm (D and G), 2 mm (I), 10 mm (J), and 2.5 mm (K).

  • Fig. 3 FRESH printing of biological structures based on 3D imaging data and functional analysis of the printed parts.

    (A) A model of a human femur from 3D CT imaging data is scaled down and processed into machine code for FRESH printing. (B) The femur is FRESH printed in alginate, and after removal from the support bath, it closely resembles the model and is easily handled. (C) Uniaxial tensile testing of the printed femur demonstrates the ability to be strained up to 40% and elastically recover. (D) A model of a section of a human right coronary arterial tree from 3D MRI is processed at full scale into machine code for FRESH printing. (E) An example of the arterial tree printed in alginate (black) and embedded in the gelatin slurry support bath. (F) A section of the arterial trees printed in fluorescent alginate (green) and imaged in 3D to show the hollow lumen and multiple bifurcations. (G) A zoomed-in view of the arterial tree shows the defined vessel wall that is <1 mm thick and the well-formed lumen. (H) A dark-field image of the arterial tree mounted in a perfusion fixture to position a syringe in the root of the tree. (I) A time-lapse image of black dye perfused through the arterial tree false-colored at time points of 0 to 6 s to show flow through the lumen and not through the vessel wall. Scale bars, 4 mm (B), 10 mm (E), 2.5 mm (F), 1 mm (G), and 2.5 mm (H and I).

  • Fig. 4 FRESH printed scaffolds with complex internal and external architectures based on 3D imaging data from whole organs.

    (A) A dark-field image of an explanted embryonic chick heart. (B) A 3D image of the 5-day-old embryonic chick heart stained for fibronectin (green), nuclei (blue), and F-actin (red) and imaged with a confocal microscope. (C) A cross section of the 3D CAD model of the embryonic heart with complex internal trabeculation based on the confocal imaging data. (D) A cross section of the 3D printed heart in fluorescent alginate (green) showing recreation of the internal trabecular structure from the CAD model. The heart has been scaled up by a factor of 10 to match the resolution of the printer. (E) A dark-field image of the 3D printed heart with internal structure visible through the translucent heart wall. (F) A 3D rendering of a human brain from MRI data processed for FRESH printing. (G) A zoomed-in view of the 3D brain model showing the complex, external architecture of the white matter folds. (H) A lateral view of the brain 3D printed in alginate showing major anatomical features including the cortex and cerebellum. The brain has been scaled down to ~3 mm in length to reduce printing time and test the resolution limits of the printer. (I) A top down view of the 3D printed brain with black dye dripped on top to help visualize the white matter folds printed in high fidelity. Scale bars, 1 mm (A and B) and 1 cm (D, E, H, and I).

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/9/e1500758/DC1

    Fig. S1. Modification of an open-source 3D printer for FRESH printing.

    Fig. S2. Preparation of the gelatin slurry support bath.

    Fig. S3. Examples of 3D printed bifurcated tubes using alginate, fibrin, and collagen.

    Fig. S4. 3D printed sheets of cells and ECM.

    Fig. S5. Mechanical characterization of cast and 3D printed alginate dog bones using uniaxial tensile testing.

    Fig. S6. A comparison of the 3D model and 3D printed arterial tree to assess print fidelity.

    Fig. S7. A 3D printed perfusion fixture for the right coronary arterial tree.

    Fig. S8. Generation of a 3D model of the embryonic heart from confocal microscopy.

    Fig. S9. A comparison of the 3D model and 3D printed embryonic heart to assess print fidelity.

    Fig. S10. A comparison of the 3D model and 3D printed brain.

    Movie S1. Time lapse of FRESH printing and heated release of the “CMU” logo.

    Movie S2. FRESH printing using the dual syringe pump extruders.

    Movie S3. Out-of-plane FRESH printing of a helix.

    Movie S4. Uniaxial strain of a FRESH printed femur model showing elastic recovery.

    Movie S5. Strain to failure of a FRESH printed femur.

    Movie S6. FRESH printing of soft collagen type I constructs.

    Movie S7. Time-lapse video of a coronary arterial tree being FRESH printed.

    Movie S8. Perfusion of a FRESH printed coronary arterial tree.

    Movie S9. Visualization of the 3D structure of a FRESH printed brain model.

    Movie S10. Modification of a sub-$400 3D printer for FRESH printing.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Modification of an open-source 3D printer for FRESH printing.
    • Fig. S2. Preparation of the gelatin slurry support bath.
    • Fig. S3. Examples of 3D printed bifurcated tubes using alginate, fibrin, and collagen.
    • Fig. S4. 3D printed sheets of cells and ECM.
    • Fig. S5. Mechanical characterization of cast and 3D printed alginate dog bones using uniaxial tensile testing.
    • Fig. S6. A comparison of the 3D model and 3D printed arterial tree to assess print fidelity.
    • Fig. S7. A 3D printed perfusion fixture for the right coronary arterial tree.
    • Fig. S8. Generation of a 3D model of the embryonic heart from confocal microscopy.
    • Fig. S9. A comparison of the 3D model and 3D printed embryonic heart to assess print fidelity.
    • Fig. S10. A comparison of the 3D model and 3D printed brain.
    • Legends for movies S1 to S10

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Time lapse of FRESH printing and heated release of the "CMU" logo.
    • Movie S2 (.mp4 format). FRESH printing using the dual syringe pump extruders.
    • Movie S3 (.mp4 format). Out-of-plane FRESH printing of a helix.
    • Movie S4 (.mp4 format). Uniaxial strain of a FRESH printed femur model showing elastic recovery.
    • Movie S5 (.mp4 format). Strain to failure of a FRESH printed femur.
    • Movie S6 (.mp4 format). FRESH printing of soft collagen type I constructs.
    • Movie S7 (.mp4 format). Time-lapse video of a coronary arterial tree being FRESH printed.
    • Movie S8 (.mp4 format). Perfusion of a FRESH printed coronary arterial tree.
    • Movie S9 (.mp4 format). Visualization of the 3D structure of a FRESH printed brain model.
    • Movie S10 (.mp4 format). Modification of a sub-$400 3D printer for FRESH printing.

    Files in this Data Supplement: