Research Article: Regeneration of esophagus using a scaffold-free biomimetic structure created with bio-three-dimensional printing

Date Published: March 8, 2019

Publisher: Public Library of Science

Author(s): Yosuke Takeoka, Keitaro Matsumoto, Daisuke Taniguchi, Tomoshi Tsuchiya, Ryusuke Machino, Masaaki Moriyama, Shosaburo Oyama, Tomoyuki Tetsuo, Yasuaki Taura, Katsunori Takagi, Takuya Yoshida, Abdelmotagaly Elgalad, Naoto Matsuo, Masaki Kunizaki, Shuichi Tobinaga, Takashi Nonaka, Shigekazu Hidaka, Naoya Yamasaki, Koichi Nakayama, Takeshi Nagayasu, Atsushi Asakura.


Various strategies have been attempted to replace esophageal defects with natural or artificial substitutes using tissue engineering. However, these methods have not yet reached clinical application because of the high risks related to their immunogenicity or insufficient biocompatibility. In this study, we developed a scaffold-free structure with a mixture of cell types using bio-three-dimensional (3D) printing technology and assessed its characteristics in vitro and in vivo after transplantation into rats. Normal human dermal fibroblasts, human esophageal smooth muscle cells, human bone marrow-derived mesenchymal stem cells, and human umbilical vein endothelial cells were purchased and used as a cell source. After the preparation of multicellular spheroids, esophageal-like tube structures were prepared by bio-3D printing. The structures were matured in a bioreactor and transplanted into 10-12-week-old F344 male rats as esophageal grafts under general anesthesia. Mechanical and histochemical assessment of the structures were performed. Among 4 types of structures evaluated, those with the larger proportion of mesenchymal stem cells tended to show greater strength and expansion on mechanical testing and highly expressed α-smooth muscle actin and vascular endothelial growth factor on immunohistochemistry. Therefore, the structure with the larger proportion of mesenchymal stem cells was selected for transplantation. The scaffold-free structures had sufficient strength for transplantation between the esophagus and stomach using silicon stents. The structures were maintained in vivo for 30 days after transplantation. Smooth muscle cells were maintained, and flat epithelium extended and covered the inner surface of the lumen. Food had also passed through the structure. These results suggested that the esophagus-like scaffold-free tubular structures created using bio-3D printing could hold promise as a substitute for the repair of esophageal defects.

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Esophagectomy is a treatment for conditions including long-gap esophageal atresia, caustic esophagus stricture, and esophageal cancer. In this situation, reconstruction of the esophagus using portions of the more distal gastrointestinal tract, such as the stomach, colon, or intestine may be necessary [1]. However, high complication and mortality rates related to these esophageal reconstructions should be addressed. Problems related to functions of the gastrointestinal tract such as reflux, delayed esophageal transplantation, or dumping syndrome should also be addressed [2, 3]. Moreover, strictures or distensions occurring in the long term require intermittent anastomotic dilatations or re-operations [4].

At 30 days after transplantation, the boundary between the structure and the esophagus could not be clearly detected on HE staining, as the structures were completely engrafted. The esophageal mucosal epithelium extended into the lumen of the structure and covered its inner surface completely (Fig 5B, 5C, 5D and 5E). Cells on the surface of the lumen expressed pan-cytokeratin (Fig 5F, 5G and 5H). The expression of αSMA was also observed in the structure (Fig 5I, 5J and 5K). Expression of human HLA class I ABC was found only in the part of the transplanted structure, but there were no positive cells in the epithelial layer. This means that the structure made of human cells was maintained in the rats’ body and the native rat epithelium extended on the transplanted structure after transplantation (Fig 5L and 5M).

Although several auto and alloplastic esophageal substitutes have been used for esophageal reconstruction, artificial esophageal replacement remains challenging because of the high incidence of complications such as esophageal stricture [2, 4–7]. Those difficulties mainly arise from the immunogenicity and low biocompatibility of the materials [17]. In recent years, each technology has gradually developed, and good research results have come to emerged [18–21]. Among them, in the report of decellularized esophagus, Guillaume et al. reported that they had transplanted and obtained long-term survival after recellularization of decellularized porcine esophagus, and it is said to be an excellent method in terms of strength and tissue compatibility [21]. Of course, decellularization is an excellent technique, superior in maintaining tissue-specific ECM and high strength, but sacrifice is required to collect ECM. In this respect, the technology we used can make a structure from only cells, which can be a useful method against organ shortage. To overcome these issues, we focused on the novel technology of bio-3D printing and created completely biological, tissue-engineered, scaffold-free structures consisting of multicellular spheroids containing a mixture of cell types via a bio-3D-printer-based system. The present study demonstrates the feasibility of using these structures for esophageal transplantation therapy. We successfully implanted the esophageal tubular structure into rats and found that it was well engrafted with its inner luminal surface fully covered by epithelial cells.

This study is the first verification for the esophageal structure made with bio-3D printing system. The structures with four different compositions of the cells were made and compared to each other. Although the result was not sufficient compared to the native esophagus, the comparative test suggested that the structure with the highest content of MSCs was shown to be the most suitable for transplantation among the four types of structures. The structure was able to be transplanted and engrafted in the environment exposed to the gastric juice. Even in such a situation, epithelialization occurred in the inner surface of the structure. Although this work is our initial step of the study for the esophageal structure made with bio-3D printing system, it may contribute to the development of treatment for esophageal diseases that require transplantations. The structures created with bio-3D printing technology using a scaffold-free approach showed promise as a potential substitute for esophageal transplantation.




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