Scaffold-free biofabrication of tissue engineered trachea by cartilage microtissue assembly

Abstract

Currently, there is no ideal scaffold for tissue engineering of the trachea to treat long-segmental tracheal defects in clinical practice. Given the desirable properties of cartilage regeneration and its self-fusion nature, chondrocyte spheroids serve as promising building blocks for robust mechanical cartilaginous tissue for engineering trachea construction. Herein, a bottom-up self-assembly approach based on chondrocyte spheroids was developed to reconstruct resilient scaffold-free trachea. Employing custom-designed molds for spheroid assembly, we identified an optimal seeding density of 2000 cells per microwell that consistently produced spheroids of approximately 163.7 ± 9.0 μm, which yielded spheroids with superior cartilage matrix secretion and significantly upregulated the expression of SOX9, ACAN and COL2A1. We fabricated scaffold-free tracheas with robust mechanical integrity with a Young's modulus of 3.5 ± 0.3 MPa. Safranin O, Alcian Blue, and collagen II immunohistochemical staining analyses of the scaffold-free tracheas indicated consecutive cellular and accumulated matrix distribution inside the microtissues. After maturation in vivo for 4 weeks, the primitive spheroids had completely fused and formed mature cartilage tissue, and the fibrous arrangement of the cartilage was similar to that of native cartilage. Immunohistochemical staining for CD31 confirmed the presence of extensive vascularization within the engineered tracheal constructs, indicating successful functional integration with the host tissue. Overall, this study highlights a bottom-up self-assembly strategy using spheroids, making it possible to construct macroscopic, large-scale tracheas through a standardized, repeatable, high-flux approach.