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Research Papers

Evaluation of Cell Viability and Functionality in Vessel-like Bioprintable Cell-Laden Tubular Channels

[+] Author and Article Information
Yin Yu

BioMfG Laboratory,
Center for Computer-Aided Design,
The University of Iowa,
Iowa City, IA, 52242;
Department of Biomedical Engineering,
The University of Iowa,
Iowa City, IA, 52242;
Department of Orthopaedics and Rehabilitation,
The University of Iowa,
Iowa City, IA, 52242

Yahui Zhang

BioMfG Laboratory,
Center for Computer-Aided Design,
The University of Iowa,
Iowa City, IA, 52242;
Department of Mechanical and Industrial Engineering,
The University of Iowa,
Iowa City, IA, 52242

James A. Martin

Department of Orthopaedics and Rehabilitation,
The University of Iowa,
Iowa City, IA, 52242

Ibrahim T. Ozbolat

BioMfG Laboratory,
Center for Computer-Aided Design,
The University of Iowa,
Iowa City, IA, 52242;
Department of Mechanical and Industrial Engineering,
The University of Iowa,
Iowa City, IA, 52242
e-mail: ibrahim-ozbolat@uiowa.edu

1Corresponding author.

Contributed by the Bioengineering Division of ASME for publication in the Journal of Biomechanical Engineering. Manuscript received November 7, 2012; final manuscript received May 1, 2013; accepted manuscript posted May 15, 2013; published online July 11, 2013. Assoc. Editor: Dror Seliktar.

J Biomech Eng 135(9), 091011 (Jul 11, 2013) (9 pages) Paper No: BIO-12-1543; doi: 10.1115/1.4024575 History: Received November 07, 2012; Revised May 01, 2013; Accepted May 06, 2013

Organ printing is a novel concept recently introduced in developing artificial three-dimensional organs to bridge the gap between transplantation needs and organ shortage. One of the major challenges is inclusion of blood-vessellike channels between layers to support cell viability, postprinting functionality in terms of nutrient transport, and waste removal. In this research, we developed a novel and effective method to print tubular channels encapsulating cells in alginate to mimic the natural vascular system. An experimental investigation into the influence on cartilage progenitor cell (CPCs) survival, and the function of printing parameters during and after the printing process were presented. CPC functionality was evaluated by checking tissue-specific genetic marker expression and extracellular matrix production. Our results demonstrated the capability of direct fabrication of cell-laden tubular channels by our newly designed coaxial nozzle assembly and revealed that the bioprinting process could induce quantifiable cell death due to changes in dispensing pressure, coaxial nozzle geometry, and biomaterial concentration. Cells were able to recover during incubation, as well as to undergo differentiation with high-level cartilage-associated gene expression. These findings may not only help optimize our system but also can be applied to biomanufacturing of 3D functional cellular tissue engineering constructs for various organ systems.

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Figures

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Fig. 1

Robotic bioprinting system and the coaxial nozzle assembly: single-arm robotic bioprinter with (a) a syringe pump, (b) a motion unit, (c) a pressure regulator, and (d) a physical coaxial nozzle system

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Fig. 2

Coaxial nozzle assembly and associated mechanical forces: (a) coaxial nozzle design for tubular channel manufacturing; (b) shear stress generated by coaxial nozzle system

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Fig. 3

Bioprinted cell-laden tubular constructs: (a) tubular channels were printed into zigzag orientation with perfused cell-type media, (b) bubble inclusion in tubular center demonstrating its hollow feature and microscopy images showing cell encapsulation in the wall of cellular channels with relatively uniform distribution of cells, and (c) an 1 week cultured cell-laden tubular channel showing promising mechanical and structural integrity

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Fig. 4

Dispensing rheology (I23GO16G): (a) effect of alginate pressure rate on volume flow rate of 4% alginate solution, (b) effect of pressure rate on − ΔP/L, (c) shear stress distribution in coaxial nozzles, and (d) maximum shear stress with varying alginate dispensing pressure

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Fig. 5

Quantitative cell viability for various cell densities and alginate concentrations: (a) effect of cell density on cell viability at different alginate dispensing pressures, (b) effect of sodium alginate concentration on cell viability at 5 psi (35 kPa) with cell density of 2 × 106 cells/ml (data are mean ± SD; p < 0.05)

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Fig. 6

Laser confocal imaging for live/dead staining of the printed structure at 5 psi (35 kPa) with I23GO16G nozzle: CPCs labeled with calcein AM and ethidium homodimer after cell encapsulation and imaged with confocal laser scanning microscope: (a) quantifiable dead cells were present, while most of cells were viable; (b) zoom-in image shows live and dead cells with fluorescence green and red, respectively

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Fig. 7

Effect of bioprinting parameters on cell viability for 72 hr postbioprinting: (a) effect of alginate dispensing pressure (psi) on cell viability, (b) cell viability for different-sized coaxial nozzle assemblies (data are mean ± SD; p < 0.05)

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Fig. 8

Experimental and predicted cell viability (E: experimental; P: predicted): (a) for nozzle I23GO16G; (b) for nozzle I26GO16G

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Fig. 9

Laser confocal imaging for live/dead staining of the same sample at different time points: (a) 12 h postprinting, massive cell death (red fluorescent) was observed all over the printed structure; (b) after 72 h incubation, a few dead cells were scattered among an increasing number of green fluorescent live cells

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Fig. 10

Cell recovery in the printed structure using I26GO16G during postprinting incubation for a 72 h period. Increased cell viability is observed from 12 h postbioprinting to 72 h incubation at different alginate dispensing pressures (data are mean ± SD; p < 0.05).

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Fig. 11

Real-time PCR revealed significantly higher expression of cartilage-specific markers; PRG-4, Sox-9, and COL-2 all showed over a twofold upregulation in alginate tubular channel encapsulated CPCs compared with CPCs in the monolayer culture after bioprinting. ACAN showed over a 12-fold higher expression level (data are mean ± SD; (*: p < 0.05; **: p < 0.01)).

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Fig. 12

Cell viability under varying maximum shear stress

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