Current scaffold‐based tissue engineering approaches are subject to several limitations, such as design inflexibility, poor cytocompatibility, toxicity, and post‐transplant degradation. Thus, scaffold‐free tissue‐engineered structures can be a promising solution to overcome the issues associated with classical scaffold‐based materials in clinical transplantation. The present study seeks to optimize the culture conditions and cell combinations used to generate scaffold‐free structures using a Bio‐3D printing system. Human cartilage cells, human fibroblasts, human umbilical vein endothelial cells, and human mesenchymal stem cells from bone marrow are aggregated into spheroids and placed into a Bio‐3D printing system with dedicated needles positioned according to 3D configuration data, to develop scaffold‐free trachea‐like tubes. Culturing the Bio‐3D‐printed structures with proper flow of specific medium in a bioreactor facilitates the rearrangement and self‐organization of cells, improving physical strength and tissue function. The Bio‐3D‐printed tissue forms small‐diameter trachea‐like tubes that are implanted into rats with the support of catheters. It is confirmed that the tubes are viable in vivo and that the tracheal epithelium and capillaries proliferate. This tissue‐engineered, scaffold‐free, tubular structure can represent a significant step toward clinical application of bioengineered organs.

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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Abstract. The frequency of the allele containing three derived nonsynonymous SNPs (157C, 378M, 681M) of the gene encoding calcium permeable TRPV6 channels expressed in the intestine has been increased by positive selection in non-African populations. To understand the nature of these SNPs, we compared the properties of Ca 2+ influx of ancestral (in African populations) and derived-TRPV6 (in non-African populations) channels with electrophysiological, Ca 2+ -imaging, and morphological methods using both the Xenopus oocyte and mammalian cell expression systems. Functional electrophysiological and Ca 2+ -imaging analyses indicated that the derived-TRPV6 elicited more Ca 2+ influx than the ancestral one in TRPV6-expressing cells where both channels were equally expressed in the cells. Ca 2+ -inactivation properties in the ancestral-and derived-TRPV6 were almost the same. Furthermore, fluorescence resonance energy transfer (FRET) analysis showed that both channels have similar multimeric formation properties, suggesting that derived-TRPV6 itself could cause higher Ca 2+ influx. These findings suggest that populations having derived-TRPV6 in non-African areas may absorb higher Ca 2+ from the intestine than ancestral-TRPV6 in the African area.

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