Evaluation of the Structural Integrity and Extracellular Matrix Components of Tracheal Allografts Following Cyclical Decellularization Techniques: Comparison of Three Protocols

Abstract

Tracheal reconstruction is indicated in cases of malignancy, traumatic injury, and subglottic or tracheal stenosis. Recent progress in airway transplantation has provided renewed optimism for potential solutions for defects involving more than half of the tracheal length in adults or one-third of the tracheal length in children. Biologic scaffolds derived from decellularized tissues and organs have shown great promise in tracheal allotransplantation, and cyclical decellularization techniques have been hypothesized as abrogating the need for immunosuppressive therapy. In this study, we performed a direct comparison of three decellularization protocols (Protocols A, B, and C) previously described in the literature, two of which were described in tracheal tissue (Protocols A and B). We concentrated on the immunogenicity within the epithelium and mucosa, quantified and qualified the extracellular matrix (ECM) components, and performed compliance measurements on large circumferential decellularized tracheal scaffolds following cyclical decellularization techniques using all three protocols. Quantitative measurements of glycosaminoglycans (GAGs) showed a significant decrease in the mucosal component following 17 cycles of all 3 protocols as well as a significant decrease of GAGs in the cartilaginous component following cycles 1, 9, and 17 of Protocol A and cycle 17 of Protocol C. Compliance measurements were also shown to be different between the protocols, with grafts becoming more compliant at physiologic pressures after cyclical decellularization with Protocols A and B and slightly less compliant but remaining similar to native trachea using Protocol C. Positive staining for anti-major histocompatibility complex Class I (anti-MHCI) and anti-MHCII remained within the submucosal glandular components despite multiple cycles of decellularization using all three protocols. This study illustrated that there are significant differences in ECM composition and resultant structural integrity of decellularized tracheal scaffolds depending on the decellularization protocol. Protocol B was shown to maintain the GAGs components despite an increase in tracheal compliance, while Protocol C decreases GAGs components following multiple cycles, despite showing a tracheal compliance resembling that of the native trachea at physiologic airway pressures.

The effects of storage and sterilization on de-cellularized and re-cellularized whole lung

“…With respect to trachea and lung, a number of recent publications have comparatively assessed different de-cellularization protocols. Notably, the resulting architecture and extracellular matrix (ECM) protein composition of either trachea or lungs may differ substantially between the different regimens utilized [5–7]. Whether this will subsequently affect re-cellularization of scaffolds and therefore the generation of functional tissue suitable for transplantation, remains unresolved [4,5].…”

Fibroblast engraftment in the decellularized mouse lung occurs via a β1-integrin-dependent, FAK-dependent pathway that is mediated by ERK and opposed by AKT

“…However, in the consideration of the long-term therapeutic application of scaffold-based strategies, several limitations become apparent. First, previous studies indicate that, even in the setting of grossly preserved structure and extracellular components, the decellularized lung scaffold demonstrates a large amount of "leakiness" that may at least partially reflect damage sustained during the decellularization procedure (12,23). Second, other studies demonstrate that the decellularization process can negatively impact ECM protein integrity (1,31), which may impede its ability to support infused progenitor cells or to persist long term in vivo.…”

Tissue Engineering of the Trachea: What is the Hold-up?

“…This mechanical detergent-enzymatic protocol requires solely one cycle. Haykal et al, 25 conducted a comprehensive comparative study between the protocols and concluded that the 17cycle protocol caused a significant decrease in mucosal and cartilaginous components of the native trachea. In addition, the first and second protocols appear to cause an increase in compliance, thereby reducing the reliability on longterm maintenance of graft integrity, while tracheae treated with the third protocol, appeared more comparable to the native trachea.…”