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Research Papers

Characterization of Cell Constructs Generated With Inkjet Printing Technology Using In Vivo Magnetic Resonance Imaging

[+] Author and Article Information
Tao Xu, Weixin Zhao, Anthony Atala

Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston-Salem, NC 27157

John Olson

Center for Biomolecular Imaging, Wake Forest University Health Sciences, Winston-Salem, NC 27157

Jian-Ming Zhu

Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston-Salem, NC 27157; Department of Radiation Oncology, Wake Forest University Health Sciences, Winston-Salem, NC 27157

James J. Yoo1

Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston-Salem, NC 27157jyoo@wfubmc

1

Corresponding author.

J. Manuf. Sci. Eng 130(2), 021013 (Apr 02, 2008) (7 pages) doi:10.1115/1.2902857 History: Received December 14, 2007; Revised January 11, 2008; Published April 02, 2008

We report the use of a high resolution magnetic resonance (MR) imaging technique to monitor the development and maturation of tissue-printed constructs in vivo. Layer-by-layer inkjet printing technology was used to fabricate three different tissue constructs on alginate∕collagen gels: bovine aortic endothelial cell-printed (to represent soft tissue), human amniotic fluid-derived stem cell-printed (to represent hard tissue as they underwent osteogenic differentiation in vivo), and cell-free constructs (scaffold only). The constructs were subcutaneously implanted into athymic mice and regularly monitored using a 7T magnetic resonance imaging (MRI) scanner. The three tissue construct types showed distinct image contrast characteristics due to the different tissue microstructures and biochemical compositions at various time points. In addition, changes in tissue microvasculature were examined with dynamic perfusion MRI. These results indicate that high resolution MRI is a promising method for noninvasive, long-term monitoring of the status of cell-printed construct growth, differentiation, and vascularization.

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Copyright © 2008 by American Society of Mechanical Engineers
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Figures

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Figure 1

A schematic diagram of the three-dimensional tissue printing process. The chamber is filled with the sodium alginate and collagen solution mixture. The alginate is known to cross-link in the presence of CaCl2 to form a biodegradable hydrogel scaffold. The ink cartridge, filled with the cell print suspension in 0.1MCaCl2 solution, ejects cell-CaCl2 drops, which immediately gel upon contact with alginate. The gel droplet is placed at target sites layer-by-layer onto the platform, resulting in the construction of 3D structures. The platform is programed to submerge into the fresh uncross-linked solution that maintains hydration of the gelled constructs while providing a new interface for the next layer.

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Figure 2

Transverse MRI of nude mice implanted with the printed constructs. Representative T2-weighted images of mice implanted with EC-printed (a) and cell-free constructs (c) ten weeks postimplantation and AFSC-printed samples (b) four months postimplantation. Corresponding T1-weighted images of mice implanted with the EC-printed constructs (d), AFSC-printed samples (e), and cell-free scaffolds (f). The marks “@,” “̂,” and “Ω” were used to indicate the EC-printed samples, AFSC-printed samples, and cell-free scaffolds, respectively. (g) Quantitative comparison of relative MR T2-weighted signals of different printed constructs: EC-printed samples ten weeks postimplantation (n=4), AFSC-printed implants four months postimplantation (n=4), and cell-free constructs ten weeks postimplantation (n=5). (h) Quantitative comparison of MR T2-weighted signals of EC-printed and cell-free constructs at the fifth week (n=6) and tenth week after implantation (n=4) ( *, 0.01<P<0.05; #, 0.001<P<0.01, &, P<0.001). The scale bars represent 5mm.

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Figure 3

Representative histological images of the center areas of the printed constructs for microstructural and biochemical analyses. EC-printed samples ((a)–(f)), cell-free scaffolds ((g)–(l)), and AFSC-printed implants ((m)–(o)) were visualized with hematoxylin and eosin staining, masson trichrome staining, and toluidine blue staining, five, eight, and∕or ten week postimplantations, respectively. Mineralization of the AFSC-printed samples was examined with von Kossa’s staining after eight week implantation (p). Black staining indicates strong mineralization. The scale bars represent 50μm. Original magnifications: 200× ((a)–(o)); 400× (p).

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Figure 4

Tissue vascularization of the EC-printed constructs eight weeks after implantation. Blood vessel networks were clearly observed within retrieved samples under light microscopy in the surface area of the EC-printed implant (a); however, fewer vascular networks were seen in that of the cell-free implants (b). Immuonohistochemical analysis showed that the blood vessel structures in the center area of the EC-printed samples stained positively expressed with the endothelial cell-specific marker: vWF antibodies (c). However, no obvious staining with blood vessels was observed in the center area of the cell-free implant (control) (d). The scale bars represent 50μm. Original magnifications: 200×(a); 400×(b).

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Figure 5

Dynamic contrast-enhanced MRI of EC-printed constructs eight weeks after implantation. (a)–(d) Pre- and postcontrast MRI of the EC-printed and control (cell-free) samples. Contrast enhancement was clearly visualized within the EC-printed groups ((a) and (c)). No or little enhancement was observed within the controls ((b) and (d)). Quantitative comparison of the contrast enhancement between the EC-printed implants (n=5) and the controls (n=4) (e). The increase in T2 weighted signal of the EC-printed samples after contrast injection was significantly higher than the cell-free samples (#, P<0.005).

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