Multi-Tissue Integrated Tissue-Engineered Trachea Regeneration Based on 3D Printed Bioelastomer Scaffolds

Abstract

Functional segmental trachea reconstruction is a critical concern in thoracic surgery, and tissue-engineered trachea (TET) holds promise as a potential solution. However, current TET falls short in fully restoring physiological function due to the lack of the intricate multi-tissue structure found in natural trachea. In this research, a multi-tissue integrated tissue-engineered trachea (MI-TET) is successfully developed by orderly assembling various cells (chondrocytes, fibroblasts and epithelial cells) on 3D-printed PGS bioelastomer scaffolds. The MI-TET closely resembles the complex structures of natural trachea and achieves the integrated regeneration of four essential tracheal components: C-shaped cartilage ring, O-shaped vascularized fiber ring, axial fiber bundle, and airway epithelium. Overall, the MI-TET demonstrates highly similar multi-tissue structures and physiological functions to natural trachea, showing promise for future clinical advancements in functional TETs.

Keywords: 3D printing; bioelastomer; cartilage; tissue engineering; trachea regeneration.

Figure 1

The overall research design and characterization of the PGS/PCL‐Gelatin (PPG) scaffolds. A) Schematic illustration for the design of MI‐TET. B) Fabrication process of the PPG scaffolds. 3D‐printed and thermo‐cured PGS/PCL scaffolds were composited with gelatin fibrous network to fabricate PPG scaffolds. C) Optical microscope images of the PPG scaffolds. Green and red arrows represent PGS/PCL framework and gelatin fibrous network, respectively. Scale bars: 1 mm. D) Gross images of 3D‐printed PGS/PCL scaffolds and PPG scaffolds. E,F) SEM images of the two scaffolds at different magnifications in top view and section view. Scale bars: 1 mm, 500 µm, 200 µm, 100 µm. G) Macropore size of the PGS/PCL framework and gelatin fibrous network, *p < 0.05. H) Micropore size inside the filaments. I) Typical compressive stress–strain curves resulting from uniaxial compression tests. J) Cyclic compression tests for ten cycles with maximum strain of 40%. K) Comparison of compressive modulus, *p < 0.05. L) Water absorption rate, *p < 0.05. M) FTIR spectroscopy analysis. N) Dynamic water contact angle changing curve with time.

Figure 2

2D‐patterned construction of the MI‐TET in vitro. A) Schematic representation of the 2D‐patterned construction process in vitro using Post‐Occupancy Sacrifice (POS) strategy. B) The state of thermosensitive hydrogel (Pluronic F127) at 4 and 37 °C. C) The precise patterned distribution of Pluronic F127 on PPG scaffolds. Scale bars: 1 cm. D) The process of multi‐cellular patterned construction on PPG bioactive scaffolds using the POS strategy. Scale bars: 1 cm. E) Optical images during the process of cell patterned seeding. Scale bars: 500 µm. F) Distribution map of tracheal derived and “SJTU” abbreviation patterns, which were taken under confocal microscope after fluorescent live cell staining of cells. Green region represents DiO‐labeled chondrocytes, red region represents Dil‐labeled fibroblasts, and black region represents filaments of PPG scaffolds. Scale bars: 5 mm. G,H) Histological staining results of patterned multicellular scaffold complex after 4 weeks in vitro culture (HE and SO/FG staining on the horizontal and vertical section of regenerated tissue at 4 weeks in vitro). Scale bars: 1 mm, 100 µm.

Figure 3

Regeneration of the MI‐TET in nude mice for 8 and 12 weeks. A) Schematic illustration of the MI‐TET regeneration via 3D assembly strategy. B) General morphology of the MI‐TET before and during subcutaneous implantation in nude mice. C) General morphology of the MI‐TET after subcutaneous culture in nude mice. D–G) Results of SO/FG and vWF and α‐SMA staining of 8/12w‐in vivo regenerated trachea in four sections: C ring section and F ring section in radial direction; C/F band and F bundle section in axial direction. I/II represents the SO/FG staining results corresponding to 8 and 12 weeks. III/IV represents the vWF (Red) and α‐SMA (Green) staining results corresponding to 8 and 12 weeks. Red rectangles represent cartilage regions; green rectangles represent vascularized fibrous tissue regions; Scale bar: 1 mm, 100 µm. Statistical map of H) averaged thickness, J) cartilage proportion and K) vascularization proportion of C ring, F ring and F bundle at 8/12 weeks, *p < 0.05.

Figure 4

Airway basal cells culture and epithelium construction of MI‐TET. A) Schematic illustration for the construction process of airway epithelium regeneration. B) Optical microscope images and C) immunofluorescence images of PCK and CK‐5 for airway basal cells. Scale bar: 500, 200, 100 µm. D) Histological examinations of regenerated airway epithelium (Orange arrows). Red arrows show cartilage, green arrows show fiber texture, black arrows show airway basal cells. Scale bar: 500, 50 µm. E) Immunofluorescence staining of PCK and CK‐5 for regenerated airway epithelium. White arrows show airway basal cells. Scale bar: 100 µm.

Figure 5

Comprehensive evaluation of the MI‐TET. A‐C) Quantitative analyses of the DNA contents, GAG contents, COL II among 8/12 week‐regenerated trachea and natural trachea, n = 4, *p < 0.05, ns: no significance. D,E) Morphological comparison of MI‐TET in original and compressed state from side and top view. Scale bar: 1 cm. F) Stress–strain curves resulting from uniaxial compression tests. G) Column diagram of modulus corresponding to compressive strain during 0–10% and 20–30%. H) Cyclic compression tests with maximum strain of 40%. I) The elastic recovery rate curve with the number of cyclic compressions in the first ten cycles. Elastic recovery rate is the ratio of rebound stress to compressive stress in the same compression test. J) A comprehensive evaluation chart from five dimensions of DNA, GAG, Col II, elastic modulus and elastic recovery rate. K) Scatter plot of TET regeneration studies in past decade based on functional and structural reconstruction effect of regenerated trachea. The data used are summarized in Table S1 (Supporting Information).