TEMPLATE
Three dimensional inkjet biofabrication based on the designed images
Kenichi Arai, Shintaroh Iwanaga, Hideki Toda, Genzi Capi, Makoto Nakamura
Graduate School of Science and Engineering, University of Toyama
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TEMPLATE
Introduction:
It is the ultimate theme for tissue engineering to provide artificially-fabricated functional tissues or organs for medical therapeutics. At present, engineered skin and cornea are successfully used clinically, both of which have simple structures with only a few layers of cells. However the organs such as heart or liver are consisted of thick and complicated tissues, which are composed of various types of cells and have micro-scaled structures. Thus, the effective technologies to manufacture such thick and complicated tissues with micro-structures using several types of cells have been desired for a long time.
Then, we have ever focused on tissue engineering applying computer and machine technologies. The computer-assisted fabrication machines must be able to realize extremely delicate structures beyond human hand works. Among many kinds of machine technologies, we selected inkjet printing technique. The inkjet printer has a variety of abilities such as direct printing with multi color inks and ejecting the extremely small-sized droplets. And the first version 3D bioprinter applied inkjet technology was developed at Kanagawa Academy of Science and Technology (KAST). Using this, we have ever fabricated and demonstrated the 3D gel tubes and 3D laminated gel sheets including living cells together [1].
At the next step, we aimed to fabricate more complicated structures, and started to develop the second version 3D bioprinter during the KAST project. No matter how complex the 3D structures are, the original 3D structures can be reconstructed very closely with laminating each laminagram, as is known that the 3D virtual images are reconstructed by laminating many serial sectional images taken by X-ray CT scanner. Therefore, the second version bioprinter was developed, being improved to be suitable for laminating printing based on printing 2D images.
In this paper, we will introduce our developments in fabricating the complicated 2D and 3D structures with our novel biofabrication system.
Materials and Methods:
As for gel materials, sodium alginate has been used as a gel precursor, while CaCl2 solution as a gelling agent. Sodium alginate solution is ejected by inkjet into CaCl2 solution. Ejected inkjet droplets gelate at once, and 3D gel structures are fabricated into the CaCl2 solution. To receive the fabricated 3D structures, the viscosity of CaCl2 solution was adjusted with sodium hyaluronate.
To examine the capacity of the new system in fabricating complicated 3D structures, we studied as followings. At first, we prepared the 2D bitmap image of the symbol mark of the University of Toyama as a sample of a complicated 2D image. Then that image was printed and laminated to a few layers. Next, we prepared 95 different bitmap images of multi-sized squares for fabrication of 3D pyramid structures. It was because different 2D images should be printed out and laminated to construct complex 3D structures. Then, 2D and 3D computer models were reconstructed based on the designed images, and next we challenged to fabricate those structures using 2nd version Bioprinter. At last, we fabricated those structures including living cells. To evaluate the cell viability in these structures, cells were stained with fluorescent dyes of calcein-AM and propidium iodide, and observed.
Results:
The symbol mark of the University of Toyama was successfully fabricated (Figure.1). Each character of about 1 mm wide could be clearly printed out. The 3D pyramid gel structures were also successfully fabricated, printing and laminating 95 different sized images automatically (Figure.2). Moreover, those structures were successfully fabricated including living cells, too, and the viability of the cells in them were also confirmed almost all alive.
Figure 1. The symbol mark of the University of Toyama (left) and the fabricated gel structure (right).
Figure 2. The 3D computer mode (left) and the fabricated pyramid structure laminated with 90 layers (right).
Conclusions:
These results indicate that our 2nd version 3D bioprinter has a good potential to fabricate several complicated 2D and 3D structures by direct cell printing and laminating printing of multi-different bit map images. We expect and hope that the more complicated structures will be fabricated in near future and the functional 3D tissues or organs will be manufactured with biofabrication technique.
References:
[1] Y. Nishiyama et al., J. Biomech. Eng. 2009;131: 035001-6