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Fabrication of Layered Gradient Brain-like Tissue by 3D Bioprinting
maintain good molding accuracy after gelation. Then, smaller internal pores size of the structure. Therefore, this
the samples were stained to characterize the alive and highlights that the change of pore size as mentioned in this
dead cells, and it was found that there were many dead paper is caused by the change of collagen concentration.
cells after 1 day of printing. Of note, the survival rate Based on the properties of collagen forming at
of the cells in the printed structure in our research was high temperature and that of gelatin forming at low
maintained at 94.5%. Vijayavenkataraman et al. temperature, a set of ink mixing schemes has been
[48]
developed a biodegradable conductive hydrogel to developed to minimize the loss of the bio-inks during
provide a conductive environment for the proliferation the mixing process as much as possible. This paper
and differentiation of neurons. However, nerve cells also focuses on the effect of the printing performance
have extremely demanding requirements for the growth of the bio-inks by adjusting the printing parameters. By
environment. The printing system constructed in this designing the orthogonal experiment, we found that the
study cannot adjust the temperature and humidity in diameter of the needle had the most significant influence
the printing environment during the printing process, on the line width of the printed structure, and the moving
thereby providing a sterile environment that is more speed has the second most significant impact. These
suitable for the growth of nerve cell. In our research, we findings can provide some guidance in the selection and
constructed a cell printing/culturing integrated operating optimization of the printing parameters in the future.
system that can provide a suitable environment for In this study, we have further studied and optimized
cell growth during the printing process. Moreover, the the printing process. In the preliminary study, we
overall modulus of the printed tissue constructed in our determined that the pre-treatment time of the bio-inks at
study is 3.7 ± 0.8 kPa, while the neurons prefer a softer low temperature was a key parameter that affected the
substrate [49] . printing performance of bio-inks. When the pre-treatment
In view of the low elastic modulus and layered time was 3 min or 6 min, the extrusion form of the bio-
structure of the natural cerebral cortex, this paper proposed ink was spherical, and the printed grid structure appeared
a method for manufacturing the layered structure with a to adhere to each other. These phenomena indicated
gradient distribution of pore size. The density and position that the bio-ink tended to be at liquid state, which was
of cells in the printed structure can be precisely controlled mainly related to the characteristics of gelatin forming at
by extrusion 3D printing technology. By controlling the low temperature. Thus, if the pre-treatment time was too
different nozzles which contain different bio-inks to short, the formation of gelatin would become incomplete,
print the structure in turn, the layered structure can be manifested by the collapse and adherence of printed
manufactured. By changing the concentration of collagen structures. When the processing time was 12 min, the
in the bio-ink, it is possible to achieve the gradient change bio-ink had a rough surface morphology, indicating that
of pore size in the printed structure. As the collagen the gelatin has been basically formed at this time and
concentration increased from 0.5 mg/ml to 1.5 mg/ml, the can be printed. However, at this time, the surface of the
pore size of the printed structure also showed a significant printed structure was too rough due to the over-molding.
increase, thereby realizing the goal of manufacturing a Based on these observations, we determined 9 min as the
layered structure with gradient pore sizes that mimic the appropriate pre-treatment time at 4℃.
cortex in vitro. At the same time, by changing the printing The main purpose of the post-processing in this
path, more complex tissue models can be printed in vitro. study was to reduce the compression modulus of the
At present, more and more studies have shown that printed structure and obtain a smaller aperture range
changing the concentration of one or several biomaterials (30 – 150 μm). The higher the concentration of Ca , the
2+
in the mixed bio-inks can change the internal pore size of greater the osmotic pressure between the printed tissue
the printed tissue so as to manufacture the gradient porous and the post-processed solution. The main reason of
structure with heterogeneity. For example, Xu et al. this phenomenon was that the increased concentration
[31]
obtained smaller internal pore size by increasing the of Ca would cause the printed tissue to be more fully
2+
concentration of GelMA in hybrid bio-inks. Ng et al. cross-linked. Therefore, as the concentration of Ca 2+
[14]
based on the principle of macroporous crowding and increased, the compressive modulus of the printed
adopted the drop-on-demand technology to print the structure increased, and the pore size decreased. The
samples, their experimental results have indicated that the total compression modulus of 3D brain-like nerve tissue
layered structures could be achieved by controlling the printed by Gu et al. was about 7.5 kPa, while that of the
[45]
number of microporous-based bio-ink drops printed on printed structure reported in this paper was about 3.7 kPa.
each printed collagen layer. In this study, all parameters The total compression modulus of the printed structure
remained unchanged, with the exception of the collagen in our study was reduced by half compared to the results
concentration in the mixed bio-ink. Fischer proved of Gu et al. , which was closer to that of natural brain
[50]
[45]
that a higher concentration of collagen can lead to a tissue.
82 International Journal of Bioprinting (2021)–Volume 7, Issue 3