Tissue scaffolds are a very important part of the bioprinting process – they are there to provide structure to the 3D bioprinted cells as they develop and grow. We hear about 3D printed tissue scaffolds often as research picks up around the globe, whether it’s about different types of tissue or a better way to make them. But, according to a team of researchers from Nagasaki University in Japan, tissue scaffolds for artificial organs may not be the best option in every case. There are some minor limitations, such as irritation, and some more major problems, like infection, immunosuppression, reduced biocompatibility, and degradation over time.

However, scaffold-free approaches, with 3D bioprinted spheroids made of aggregated cells, do exist, and that’s what the researchers are working on now.

Gross macroscopic images of 3D bioprinted artificial tracheas before transplantation.

The trachea clears secretions, ventilates and keeps the airway free from foreign materials through defense mechanisms such as coughing, and warms, humidifies, and cleans air for the respiratory system. Tissue used for tracheal reconstruction must be very strong, as it has to hold up under both positive and negative pressure, and many artificial airway organs still need the help of scaffolds to maintain the necessary strength and stiffness of the airway.

The team recently published a paper, titled “Scaffold-free trachea regeneration by tissue engineering with bio-3D printing,” in the Interactive Cardiovascular and Thoracic Surgery journal. Co-authors include Daisuke Taniguchi, Keitaro Matsumoto, Tomoshi Tsuchiya, Ryusuke Machino, Yosuke Takeoka, Abdelmotagaly Elgalad, Kiyofumi Gunge, Katsunori Takagi, Yasuaki Taura, Go Hatatchi, Naoto Matsuo, Naoya Yamasaki, Koichi Nakayama, and Takeshi Nagayasu; all but Nakayama, who is from Saga University, are affiliated with Nagasaki University’s Graduate School of Biomedical Science.

The paper reads, “Currently, most of the artificial airway organs still require scaffolds; however, such scaffolds exhibit several limitations. Alternatively, the use of an autologous artificial trachea without foreign materials and immunosuppressants may solve these issues and constitute a preferred tool. The rationale of this study was to develop a new scaffold-free approach for an artificial trachea using bio-3D printing technology. Here, we assessed the circumferential tracheal replacement using scaffold-free trachea-like grafts generated from isolated cells in an inbred animal model.”

Schematic representation showing the process for artificial trachea generation.

For their experiment, the researchers isolated chondrocytes and mesenchymal stem cells from 3-week-old male F344 rats, and purchased rat lung microvessel endothelial cells. The isolated cells were evaluated, then trypsinized (a process of cell dissociation with trypsin enzyme that breaks down proteins), washed, incubated, and washed again. The cells were then placed onto plates containing growth medium, and 72 hours later, had spontaneously aggregated to form spheroids (cell balls), which were used as the 3D bioprinting materials.

Regenova [Image: Cyfuse]

In order to assemble multicellular spheroids in a tube-shaped artificial trachea with no scaffolds, the team used a Regenova 3D bioprinter from Cyfuse Biomedical, a Japanese company that developed a novel, robotic process to 3D print human tissue.

“According to the 3D design, the bio-3D printer placed spheroids in a 9 × 9 needle array in a printer (3.2 mm in length per side). The outer diameter of the needle was 0.17 mm, and the distance between each needle was 0.4 mm. Spheroids were aspirated by a robotically controlled 25-gauge nozzle from the 96-well plate and inserted into the needle array, which was made of multiple medical-grade stainless needles automatically under computer control. In total, 384 spheroids were used to generate a 3D tubular structure. After bio-3D printing, the printed artificial trachea was matured inside the bioreactor with perfusion of medium,” the paper reads.

The Regenova 3D bioprinter generated spheroids with several different types of cells in order to make 3D structures, and the resulting 3D printed, scaffold-free artificial tracheae were matured in a bioreactor, before their mechanical strength was measured and they were transplanted into a different set of rats; silicone stents kept them from collapsing until a good blood supply was obtained. Following the tracheal transplantations, the researchers kept a close eye on the rats for several weeks, measuring the strength of the scaffold-free bioprinted tracheae to see if they would hold up well.

According to the paper, no immunosuppressants were used, but acute rejection was not observed, which is good news. The team concluded that their 3D bioprinted scaffold-free tracheae “could be utilized as tracheal grafts in rats,” which of course opens up all kinds of possibilities for human tracheal regeneration.

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