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Research Papers

Biofabrication of Multimaterial Three-Dimensional Constructs Embedded With Patterned Alginate Strands Encapsulating PC12 Neural Cell Lines

[+] Author and Article Information
Rachel Dreher

University of Oklahoma Bioengineering Center,
Norman, OK 73019
e-mail: rachel.e.dreher@gmail.com

Binil Starly

Edward P. Fitts Department of Industrial
and Systems Engineering,
North Carolina State University,
Raleigh, NC 27695
e-mail: bstarly@ncsu.edu

1Corresponding author.

Manuscript received April 6, 2015; final manuscript received July 21, 2015; published online September 29, 2015. Assoc. Editor: Ibrahim Ozbolat.

J. Nanotechnol. Eng. Med 6(2), 021004 (Sep 29, 2015) (8 pages) Paper No: NANO-15-1026; doi: 10.1115/1.4031173 History: Received April 06, 2015; Revised July 21, 2015

In this study, we report the bioprinting of a three-dimensional (3D) heterogeneous conduit structure encapsulating PC12 neural cells. A core–shell-based hybrid construct is fabricated by combining electrospinning, polymer extrusion, and cell-based bioprinting processes to create a multiscale and multimaterial conduit structure. PC12 nerve cells were shown to be printed with high cell viability (>95%) and to proliferate within the rolled construct at a rate consistent with traditional two-dimensional (2D) culture. Light microscopy and scanning electron microscopy (SEM) have also shown encapsulation of cells within the printed alginate gel and an even cell distribution throughout the heterogeneous cellular construct.

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Figures

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Fig. 1

Fabrication stages for a rolled multimaterial 3D neural construct. (a) Printed strands of PCL fibers using a polymer extrusion system; (b) deposition of PCL nanofibers on the prefabricated porous layers; (c) bioprinting alginate hydrogel strands with encapsulated PC12 cells on the multiscale PCL structure; and (d) the combined hybrid construct is rolled to form a conduit.

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Fig. 2

(a) PED process and (b) electrospinning setup with a gap between grounded substrate and nozzle tip at approximate 15 cm gap. Submicrometer PCL fibers were electrospun onto the previously fabricated pattern.

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Fig. 3

(a) Microscale scaffold created using PED technique. The 2 cm × 1 cm grid patterns with the electrospun PCL fiber mesh on the scaffold pattern. Scale bar is 2 mm. (b) Rolled construct with regular red sewing thread to show contrast. In our actual constructs, sterile black suture threads were used to integrate the construct. Scale bar is 200 μm. (c) Scale-up view of the porous PCL scaffold prior to laying down the electrospun mesh produced with 150 μm fiber diameter strands spaced 250 μm apart. Scale bar is 5 mm.

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Fig. 4

SEM images of electrospun fibers with varying PCL concentrations; representative images of scaffolds made from 30% PCL ((a) and (b)), 33% PCL ((c) and (d)), 35% PCL ((e) and (f)), and 37% PCL ((g) and (h)) at 50 × and 5000 × magnifications

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Fig. 5

Light microscopy images of printed alginate strands: (a) 4 × magnification and (b) 10 × magnification. Scale bar is 200 μm. Red arrows indicate cluster of encapsulated cells.

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Fig. 6

Proliferation rate of printed cells on glass slides over one week. Intensity levels are significantly different (*p < 0.01) between days 3 and 5.

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Fig. 7

SEM images of final construct: (a) macroscopic cross-sectional view of PCL shell support structure. Two layers of the first stage are clearly seen in the image. The inner layer also shows the electrospun PCL mesh (scale bar 200 μm). (b) This image shows a top view of the inner layer. The electrospun PCL mesh is seen spread on the extruded strands. The inset block shows a printed alginate strand (scale bar 100 μm). (c) PC12 cells caught in alginate gel inside of the printed strand is visible (scale bar 2 μm).

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Fig. 8

SEM images of cells within alginate: (a) PC12 cells within the alginate hydrogel; (b) image of a cluster of PC12 cells within the hydrogel pores. The arrows indicate cluster of cells within the alginate hydrogel. The scale bars indicate 10 μm.

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Fig. 9

Viability of printed cells on glass slides and rolled constructs as well as manually pipetted cells over one week. Statistical significance between each group was evaluated using a Student t-test. # indicates significant difference between days 1 and 7 intensities for manually pipetted strands. * indicates statistically significant difference between all days 1, 4, and 7 for the strands bioprinted on glass slides. % indicates significance between days 1 and 7 for the rolled constructs, and indicates difference between days 4 and 7 for the rolled constructs (N = 4, significance determined at p < 0.05).

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Fig. 10

Cell viability on rolled versus unrolled constructs. * indicates significance in intensity levels between days 1 and 7 for the unrolled flatted construct. % indicates significance between days 4 and 7 for the rolled construct. indicates significance between days 1 and 7 for the rolled construct (N = 4, p < 0.05).

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