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Research Papers

Multimaterial and Multiscale Three-Dimensional Bioprinter

[+] Author and Article Information
Jennifer Campbell

Center for Manufacturing Innovation,
Fraunhofer USA,
Brookline, MA 02446
e-mail: jcampbell@fraunhofer.org

Ian McGuinness

Center for Manufacturing Innovation,
Fraunhofer USA,
Brookline, MA 02446
e-mail: imcguinness@fraunhofer.org

Holger Wirz

Center for Manufacturing Innovation,
Fraunhofer USA,
Brookline, MA 02446
e-mail: hwirz@fraunhofer.org

Andre Sharon

Mem. ASME
Center for Manufacturing Innovation,
Fraunhofer USA,
Brookline, MA 02446;
Mechanical Engineering Department,
Boston University,
Boston, MA 02215
e-mail: asharon@fraunhofer.org

Alexis F. Sauer-Budge

Center for Manufacturing Innovation,
Fraunhofer USA,
Brookline, MA 02446;
Biomedical Engineering Department,
Boston University,
Boston, MA 02215
e-mail: asauerbudge@fraunhofer.org

1Corresponding author.

Manuscript received March 20, 2015; final manuscript received August 3, 2015; published online September 29, 2015. Assoc. Editor: Ibrahim Ozbolat.

J. Nanotechnol. Eng. Med 6(2), 021005 (Sep 29, 2015) (7 pages) Paper No: NANO-15-1020; doi: 10.1115/1.4031230 History: Received March 20, 2015; Revised August 03, 2015

We have developed a three-dimensional (3D) bioprinting system capable of multimaterial and multiscale deposition to enable the next generation of “bottom-up” tissue engineering. This area of research resides at the interface of engineering and life sciences. As such, it entails the design and implementation of diverse elements: a novel hydrogel-based bioink, a 3D bioprinter, automation software, and mammalian cell culture. Our bioprinter has three components uniquely combined into a comprehensive tool: syringe pumps connected to a selector valve that allow precise application of up to five different materials with varying viscosities and chemistries, a high velocity/high-precision x–y–z stage to accommodate the most rapid speeds allowable by the printed materials, and temperature control of the bioink reservoirs, lines, and printing environment. Our custom-designed bioprinter is able to print multiple materials (or multiple cell types in the same material) concurrently with various feature sizes (100 μm–1 mm wide; 100 μm–1 cm high). One of these materials is a biocompatible, printable bioink that has been used to test for cell survival within the hydrogel following printing. Hand-printed (HP) controls show that our bioprinter does not adversely affect the viability of the printed cells. Here, we report the design and build of the 3D bioprinter, the optimization of the bioink, and the stability and viability of our printed constructs.

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Copyright © 2015 by ASME
Topics: Hydrogels , Printing
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Figures

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Fig. 1

Bioprinter features. (a) Solid model of our first generation 3D bioprinter. A water dish placed on the heater provides humidity to the sterilizable printing chamber. (b) Photograph of the assembled 3D bioprinter.

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Fig. 2

Detailed views of the custom-engineered components of the 3D bioprinter. (a) Gel loading pistons were engineered that apply pneumatic force to the reservoir plunger while squeezing its flanges. (b) A machined aluminum block inside a Delrin sleeve directs cooled or heated water around the reservoirs and syringe barrels. (c) A modified push-to-connect junction allows the hydrogel tubing (1-mm ID, PEEK) to run inside the temperature-control loop tubing (4-mm ID, polyurethane).

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Fig. 3

CAD models of the engineered printer tip. (a) Close-up external view of the printer tip. (b) Cut-away views of the printer tip and a hydrogel line showing how they interface with the selector valve.

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Fig. 4

Photograph of the printing process. In this image, the bioprinter has deposited 40 layers of 40% Pluronic F127 in a stacked ring configuration. Each ring is 100 μm tall. Inset: Photograph showing a hollow structure of printed Pluronic.

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Fig. 5

Data showing the effect of stage speed on the thickness of printed fibers of 40% Pluronic F127. Left: Photograph was taken with a Keyence BZ9000 digital microscope. (Line overlapping the center of the bottom fiber demonstrates the straightness of the printed fibers.) Right: Averages and standard deviations of thicknesses measured at various locations (n = 9) along the length of the fibers (n = 2).

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Fig. 6

A 6-mm diameter cylinder composed of 200 layers of 40% Pluronic F127, each 100 μm tall, totaling a height of 2 cm

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Fig. 7

(a) Top-down image of a HP hydrogel ring incubated at 37 °C for 3 hrs. Note that the white flocks of material are collagen fibrils that form after printing and give the gel its long-term stability. (b) Top-down image of a HP hydrogel ring incubated in media overnight at 37 °C. For all HP controls, hydrogel was printed by hand onto glass slides using a 3-mL syringe fitted with a 30G needle (ID: 160 μm).

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Fig. 8

Supplementing media with increasing concentrations of calcium improved the stiffness and the long-term stability of the BP constructs; (a) 0% calcium; (b) 0.16% calcium; (c) and (d) 0.32% calcium. Panels (a)–(c) show constructs that were incubated in media for 24 hrs. The construct in (d) was incubated for 72 hrs. Note: Not all constructs were printed with the same initial diameter.

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Fig. 9

Microscope images showing that HUVECs are viable in the hydrogel bioink. (a) Overlay of phase contrast and fluorescence images of cells embedded in a HP hydrogel ring (10× magnification). (b) Fluorescence image only; green = live, red = dead. Images were taken on an Olympus IX70 microscope 18 hrs after media addition (see online version for color images).

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Fig. 10

Printing with two cell types. (a) Photograph of a five-layer vessel construct with an inner ring of HUVECs (gel 1; r = 3 mm) and an outer ring of SMCs (gel 2; r = 4.5 mm) immediately following printing. The construct was incubated in media at 37 °C for 72 hrs and then stained with propidium iodide (red = dead) and fluorescein diacetate (green = live) prior to imaging. (b) Fluorescence image showing the viability of the printed ECs (compact, not as bright). (c) Fluorescence image showing that the SMCs can spread within the gel and fluorescence more intensely than ECs (see online version for color images).

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Fig. 11

Graph showing HUVEC viability inside HP controls and BP constructs after 24 or 48 hrs in media at 37 °C

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Fig. 12

HP versus BP vessel constructs. (a) HP gel showing the unevenness of the construct. Alginate thickness (labeled in figure as distance 1) was 640 μm. Without a Pluronic barrier, the gel began to dry out quickly. (b) Fluorescence image showing the viability of the cells at t = 0. Note the red (dead) cells at the top of the image. (c) Photograph showing a three-layer deep vessel printed with an automated program on our 3D bioprinter prior to media addition and incubation. Thicknesses of the materials are labeled 1–3 in the figure while radii are designated as 4–6. 1: 780 μm, 2: 1010 μm, 3: 700 μm, 4: 2080 μm, 5: 1225 μm, 6: 415 μm. Note that the Pluronic has been dyed green for visualization. (d) Microscope image showing the viability of the printed cells following overnight incubation in media at 37 °C. Note that the Pluronic structure has been washed into the media. Gels were imaged on a Keyence BZ9000 digital microscope (see online version for color images).

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