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Fabrication of Layered Gradient Brain-like Tissue by 3D Bioprinting
Native tissues and/or organs possess complex in the printed structure almost did not proliferate after the
hierarchical porous structures that confer highly-specific 7 days of culture, which may be related to the printing
cellular functions . The highly complex hierarchical inks in this research did not have the ability to make the
[14]
porous structures are commonly found in most biological cells growth rapidly. However, because of the complex
tissues such as skin [15-17] , corneal , and even bone . composition and complicated structure of the brain tissue,
[19]
[18]
The importance of such hierarchical porous structures in it is rarely used in the brain.
native tissues has been critically reviewed elsewhere [20,21] . The cerebral cortex is a layered gray matter covering
At present, 3D tissue engineering has been used to print the surface of the cerebral hemisphere. The lamellar
the hierarchical porous structures in skin, blood vessels, structure is one of the most obvious characteristics of the
esophagus, bladder, cartilage [22-28] , and other tissues. cerebral cortex . It has a typical structure with six layers
[32]
Ng et al. [17] used a two-step drop-on-demand bioprinting and each has specific neurons. The six-layer structure is
strategy to manipulate the microenvironment to fabricate composed of the molecular layer (layer I), the external
3D biomimetic hierarchical porous collagen-based granule cell layer (layer II), the external pyramidal
structures found in native skin tissue. The differences cell layer (layer III), the internal granule cell layer
between the two manufacturing methods (3D bioprinting (layer IV), the internal pyramidal cell layer (layer V),
and manual-casting) are also compared. The results and the multiform layer (layer VI). The thickness of the
show that the two-step bioprinting strategy enables the cortex in different functional areas ranges from 2 mm
homogeneous distribution of printed cells in a highly to 5.2 mm, and the thickness of each layer ranges from
controlled manner as compared to the manual casting 200 μm to 1000 μm. The researchers believe that the
approach. However, there is a transition region between gradient distribution of ECM or the soluble signal factors
layers in the layered structure obtained by the 3D is likely to be the inducement of the directional growth
bioprinting method, while the aperture size in the transition and migration of neurons. They also studied the effects
region is uncontrollable. Nam et al. [29] used the dragging of gradient distribution of matrix hardness and growth
[33]
technique based on 3D extrusion method to manufacture factors on the directional growth of neurons. Various
[34]
the multi-layered hierarchical structure of the esophagus, characteristic parameters of the natural cerebral cortex
it allowed the production of tubular structures with an are the important basis to determine the target parameters
adjustable line width and pore size. Moreover, their study of the brain-like cortex model with a layered gradient
also proved that porous structure can provide a more structure which we constructed in this study. Brain tissue
favorable environment for cell proliferation. However, has obvious biophysical characteristics, with the modulus
the stretching properties vary depending on the viscosity of much lower than that of the heart, cartilage, etc. (the
of the material, and there are differences and limitations modulus of newborn brain tissue is about 110 Pa, and that
in pore size control for each self-supporting material. Sun of an adult brain tissue is about 500 – 1000 Pa) . The
[35]
et al. [30] incorporated biochemical stimulus with different porosity of the cerebral matrix has a greater influence
growth factor releasing and biomechanical stimulus on cell migration and metabolism [36-41] . Designing and
with small pore sizes to induce better chondrogenesis to manufacturing the brain-like model with similar pore size
create the dual-factor releasing and gradient-structured to natural tissues are more conducive to cell migration and
cartilage construct. The results indicated that the gradient nutrient exchange. Therefore, the manufacturing targets
scaffold group showed better chondroprotective effects of the layered gradient structure that imitates the cerebral
with a significantly higher histological grading compared cortex are: (i) The bio-inks containing components with
with the nongradient groups over the 24 weeks in vivo. different concentration, (ii) the modulus of the printed
However, the change of the pore size in this study was structure which is as close as possible to 1000 Pa, and
obtained by changing the spacing between the printed (iii) the appropriate internal pore size (30 – 150 μm) of
lines, which did not change the pore size in the printed the printed structure.
structures. In this study, we have designed and built a set of
Xu et al. utilized a single type of hydrogel by integrated equipment for cell printing/culture, which can
[31]
changing the weight/volume ratio of Gelatin-methacryloyl realize the target of printing a structure with multiple cells
(GelMA) to bioprint the bilayer tubular construct which and multiple materials. This equipment can provide a
has smaller pores in the inner layers (6% GelMA) and suitable environment during the printing process for the
larger pores in the outer layers (4% GelMA). The results cells to survive. Furthermore, this equipment also provides
also indicated that the difference in pore sizes may have a long-term cultivation environment for the printed
helped prevent each of these cell types from crossing their structure, which can solve the problem of separating
respective layers. Although the cells had a high survival the printed structure from the printing process to culture
rate after printing, the 3-(4,5-dimethylthiazol-2-yl)-2,5- process in the traditional 3D printing method and can
diphenyltetrazolium bromide results showed that the cells reduce the risk of cell contamination during the process

72 International Journal of Bioprinting (2021)–Volume 7, Issue 3