Proteomic Analysis of Surgery-induced Stress Post-Tracheal Transplantation Highlights Changes in Matrisome.

OBJECTIVE: Investigate the impact of Surgery-induced stress (SIS) on the normal airway repair process after airway reconstruction using a mouse microsurgery model, mass spectrometry (MS), and bioinformatic analysis. METHODS: Tracheal tissue from non-surgical (N = 3) and syngeneic tracheal grafts at 3 months post-replacement (N = 3) were assessed using mass spectrometry. Statistical analysis was done using MASCOT via Proteome Discoverer™. Proteins were categorized into total, dysregulated, suppressed, and evoked proteins in response to SIS. Dysregulated proteins were identified using cut-off values of -1 1 and t-test (p value <0.05). Enriched pathways were determined using STRING and Metascape. RESULTS: At the three-month post-operation mark, we noted a significant increase in submucosal cellular infiltration (14343 ± 1286 cells/mm2, p = 0.0003), despite reduced overall thickness (30 ± 3 µm, p = 0.01), compared to Native (4578 ± 723 cells/mm2; 42 ± 6 µm). Matrisome composition remained preserved, with proteomic analysis identifying 193 commonly abundant proteins, encompassing 7.2% collagens, 34.2% Extracellular matrix (ECM) glycoproteins, 6.2% proteoglycans, 33.2% ECM regulators, 14.5% Extracellular matrix-affiliated, and 4.7% secreted factors. Additionally, our analysis unveiled a unique proteomic signature of 217 "Surgery-evoked proteins" associated with SIS, revealing intricate connections among neutrophils, ECM remodeling, and vascularization through matrix metalloproteinase-9 interaction. CONCLUSIONS: Our study demonstrated the impact of SIS on the extracellular matrix, particularly MMP9, after airway reconstruction. The novel identification of MMP9 prompts further investigation into its potential role in repair. LEVEL OF EVIDENCE: NA Laryngoscope, 2024 Laryngoscope, 2024.

Assessing the Biocompatibility and Regeneration of Electrospun-Nanofiber Composite Tracheal Grafts.

OBJECTIVE: Composite tracheal grafts (CTG) combining decellularized scaffolds with external biomaterial support have been shown to support host-derived neotissue formation. In this study, we examine the biocompatibility, graft epithelialization, vascularization, and patency of three prototype CTG using a mouse microsurgical model. STUDY DESIGN: Tracheal replacement, regenerative medicine, biocompatible airway splints, animal model. METHOD: CTG electrospun splints made by combining partially decellularized tracheal grafts (PDTG) with polyglycolic acid (PGA), poly(lactide-co-ε-caprolactone) (PLCL), or PLCL/PGA were orthotopically implanted in mice (N = 10/group). Tracheas were explanted two weeks post-implantation. Micro-Computed Tomography was conducted to assess for graft patency, and histological analysis was used to assess for epithelialization and neovascularization. RESULT: Most animals (greater than 80%) survived until the planned endpoint and did not exhibit respiratory symptoms. MicroCT confirmed the preservation of graft patency. Grossly, the PDTG component of CTG remained intact. Examining the electrospun component of CTG, PGA degraded significantly, while PLCL+PDTG and PLCL/PGA + PDTG maintained their structure. Microvasculature was observed across the surface of CTG and infiltrating the pores. There were no signs of excessive cellular infiltration or encapsulation. Graft microvasculature and epithelium appear similar in all groups, suggesting that CTG did not hinder endothelialization and epithelialization. CONCLUSION: We found that all electrospun nanofiber CTGs are biocompatible and did not affect graft patency, endothelialization and epithelialization. Future directions will explore methods to accelerate graft regeneration of CTG. LEVEL OF EVIDENCE: N/A Laryngoscope, 134:1155-1162, 2024.