Enhancement of a decellularized tracheal scaffold for tracheal tissue engineering

Abstract

Development of tracheal substitutions currently lacks a standard protocol due to the complexity of trachea structure and function. Tissue-based treatments using patient's cells grown on an extracellular matrix (ECM) from donor tissue are promising approach in tissue engineering. The aim of this study was to develop canine dECM-based tissue engineered tracheal constructs, which serve as tissue model to develop decellularization process and cell carriers. Canine tracheas were performed vacuum-assisted decellularization (VAD) to create dual cell carrier systems, which are a decellularized tracheal scaffold (dTracheal scaffold) and a dECM hydrogel. The results from histology and biochemistry assay found that dTracheal scaffolds were effectively decellularized, resulting in a significantly low level of DNA content and Major histocompatibility complex class II. However, decellularization process preserved ECM components. Scanning electron micrographs revealed a fibrous structure of ECM network. The cytotoxicity testing demonstrated the biocompatibility with epithelial cells. Moreover, hMSCs encapsulated in ECM-rich hydrogel for 3 days showed excellent viability. Furthermore, the effects of fibroblasts on epithelial tissue formation in vitro were evaluated by a co-culture system between epithelial cells and fibroblasts, cultured on the luminal surface of the dTracheal scaffold. The H&E staining demonstrated a ciliated epithelial formation on the mucosa layer after co-culture for 21 days, while an epithelial cell monoculture group could observe the epithelial cell infiltration in the submucosa layer. The tri-culture system between epithelial cells, fibroblasts, and human mesenchymal stem cells (hMSCs) exhibited a ciliated epithelial formation on the mucosa layer similar to co-culture construct. Moreover, hMSCs encapsulated in dECM hydrogel demonstrated a chondrogenic differentiation to cartilage-like tissue, which confirms by Alcian blue staining of sGAG and detection of collagen type II. The tri-culture construct found the expression of proteins associated with epithelium tissue formation, such as epithelial tight junctions, ciliation, and mucin production, as assessed by immunostaining. Furthermore, proteomics analysis identified the increase of proteins such as alpha-tubulin, beta-tubulin, vimentin, collagen type VI, and tenascin-C in the tri-culture construct. This study highlights an insightful information of structure and biochemistry of the decellularized trachea scaffold and the hydrogel, an influence of fibroblasts on epithelial layer formation and chondrogenic differentiation of hMSCs. Furthermore, this study provides a proof-of-concept for creating a well-structured trachea that can effectively function. Additionally, it contributes to the formation of knowledge and sets the stage for the future development of tracheal substitutes.