Decellularized Tracheal Matrices For Tracheal Tissue Engineering

Objective:Reconstruction of long circumferential tracheal defects remains a major clinical problem owing to the lack of effective and reliable reconstructive techniques. Tissue engineering has prompted exploration into tracheal substitutes that may provide replacement options. The aims of this study were to first produce a rat tracheal matrix by decellularization, and evaluate the decellularization efficacy and the impacts of decellularization on extracellular matrix (ECM) integrity and scaffold recellularization in vitro; then to assess the host response to the decellularized tracheal matrix and matrix remodeling in vivo; finally to recellularize the matrix with syngenic cells to generate a tissue-engineered trachea in vitro and examine the cartilage and epithelial tissue regeneration of the constructs in vivo.Methods:Tracheae were harvested from Brown Norway (BN) and Lewis rats weighing approximately 300g and were decellularized by exposing the tracheae to detergent-enzymatic treatment cycles. In vitro, the efficacies of cell and alloantigen removal were assessed by 4',6-diamidino-2-phenylindole (DAPI) staining, DNA quantification and immunohistochemical analysis of class I and II major histocompatibility complex (MHC I and II) antigens. Histological and biochemical analyses were used to evaluate desirable extracellular matrix components (glycosaminoglycans and collagen type II) retention. Matrix histoarchitecture and surface microstructure were characterized by histology and scanning electron microscope (SEM) examination. Biomechanical properties of the matrices were determined by compressive and tensile test. Contact and extract cytotoxicity tests were used to evaluate biocompatibility of the decellualrizaed tracheal matrices. Bone marrow stem cell derived chondrocytes and tracheal epithelial cells were seeded onto the matrix and SEM examination was performed to evaluate cell-matrix interaction. For the in vivo study, the bone marrow stem cell derived chondrocytes were seeded on the outer surface and freshly isolated tracheal epithelial cells were seeded to luminal surface of the acellular tracheal matrices to engineer a cell-seeded construct. The engineered tracheae (n=6) and the acellular tracheal matrices of both BN (n=6) and Lewis rats (n=6) were implanted in subcutaneous pockets in Lewis rat. In addition, fresh tracheae harvested from BN (allograft) and Lewis (isograft) rats were implanted in Lewis recipients to serve as positive (n=6) and negative (n=6) control. Host response to those different grafts and tissue remodeling were evaluated by histological analysis at 4 weeks.Results:The major alloantigens were completely removed after 5-cycle detergent-enzymatic treatment but small amount of nuclear components and cell debris remained in the decellularized matrices. The decellularized tissue preserved the collagen type II components, but there was a significant loss of glycosaminoglycans (GAG) content. Histology and SEM showed an intact histoarchitecture with various degrees of local surface topography disturbance. Both compressive and tensile strength decreased following decellularization. Decellularized tissue and extracts were not toxic to cells. In addition, the matrices were able to support adhesion and proliferation of both stem cell derived chondrocytes and epithelial cells. Following one month in vivo implantation, acellular tracheal matrices grafts resulted in no obvious allorejection or chronic inflammation. The tubular structure of tracheal matices was well maintained. Cartilage calcification and granulation tissue growth in the lumen causing obstruction obviously due to the lack of epithelial lining were observed. The SEM examination of cell seeded matrix showed relatively low cell seeding efficacy in some engineered constructs. There was no obvious repopulation in cartilaginous portion. However, the development of ciliated cells and efficient epithelial lining were observed in tissue-engineered grafts, thus preserving luminal patency.Conclusions:The detergent-enzymatic treatment was efficient in antigen removal but was not as efficient to eliminate cell components. The decellularization process reduced GAG components but retained collagen and major histoarchitecture of the native trachea. The biomechanical properties changed after decellularization, but the resulted matrices maintained sufficient mechanical integrity withstanding physiologic pressures in the short-term. Combined with good cell and tissue compatibility and cell adhesion properties, these decellualrized matices can be excellent scaffolds for tissue engineering of trachea. However, decellualrized matrix scaffold alone is not appropriate for tracheal reconstruction due to the lack of epithelial cells and chondrocytes. Therefore, cell seeding is required to further engineer a tracheal construct. Implantation of such cell-seeded tracheal constructs in vivo showed good epithelial cell survival and differentiation into ciliated cells, although stem cell failed to repopulate the cartilaginous matrices most likely due to static cell seeding and culture technique. A bioreactor may be needed to facilitate cell seeding and recellularization.