Tracheal Macrophages During Regeneration and Repair of Long‐Segment Airway Defects

Abstract

Objectives/Hypothesis: Tissue-engineered tracheal grafts (TETGs) offer a potential solution for repair of long-segment airway defects. However, preclinical and clinical TETGs have been associated with chronic inflammation and macrophage infiltration. Macrophages express great phenotypic heterogeneity (generally characterized as classically activated [M1] vs. alternatively activated [M2]) and can influence tracheal repair and regeneration. We quantified and characterized infiltrating host macrophages using mouse microsurgical tracheal replacement models.Study Design: Translational research, animal model. Methods:We assessed macrophage infiltration and phenotype in animals implanted with syngeneic tracheal grafts, synthetic TETGs, or partially decellularized tracheal scaffolds (DTSs).Results: Macrophage infiltration was observed following tracheal replacement with syngeneic trachea. Both M1 and M2 macrophages were present in native trachea and increased during early tracheal repair (P = .014), with an M1/M2 ratio of 0.48 AE 0.15. In contrast, orthotopic implantation of synthetic TETGs resulted in a shift to M1 predominant macrophage phenotype with an increased M1/M2 ratio of 1.35 AE 0.41 by 6 weeks following implant (P = .035). Modulation of the synthetic scaffold with the addition of polyglycolic acid (PGA) resulted in a reduction of M1/M2 ratio due to an increase in M2 macrophages (P = .006). Using systemic macrophage depletion, the M1/M2 ratio reverted to native values in synthetic TETG recipients and was associated with an increase in graft epithelialization. Macrophage ratios seen in DTSs were similar to native values.Conclusions: M1 and M2 macrophages are present during tracheal repair. Poor epithelialization with synthetic TETG is associated with an elevation of the M1/M2 ratio. Macrophage phenotype can be altered with scaffold composition and hostdirected systemic therapies. DTSs exhibit M1/M2 ratios similar to those seen in native trachea and syngeneic tracheal replacement.

Tissue-engineered composite tracheal grafts create mechanically stable and biocompatible airway replacements

“…Several factors contributed to respiratory distress that required early euthanasia. First, the mouse model of orthotopic tracheal replacement has inherent challenges and is associated with perioperative morbidity 7 , 19 , 25 , 33 Additionally, a reduction in graft diameter leads to airway obstruction and respiratory symptoms. However, the specific histologic factors identified in the early euthanasia group were diverse, including both cartilaginous collapse and intraluminal stenosis.…”

“…48,49 The splint did not increase macrophage infiltration, change macrophage phenotype, or attenuate graft epithelialization and endothelialization, resulting in similar submucosal thickening to syngeneic controls. 19,25 The effect of our splint on submucosal thickness could be attributed to changes in the micromechanical environment, limiting cell infiltration. 24,50,51 Further study of inflammatory cell types and populations in the lamina propria and their roles in submucosal thickening will be characterized based on ongoing work using single-cell RNA sequencing.…”

“…Previous work has demonstrated host-derived myeloid cells as the major population infiltrating the lamina propria and mediating the chronic inflammatory response in tracheal grafts resulting in stenosis. 19 , 20 , 25 – 30 Immunohistochemistry (IHC) was performed to examine macrophage (pan, M1, and M2) distribution (CD68, CD206, iNOS respectively) using methods described previously as this is one of the most prevalent myeloid cell type seen in implanted tracheal grafts. 31 , 32 Immunofluorescent (IF) staining for epithelial (ACT, CCSP, K5/K14) and endothelial (CD31) biomarkers was completed using previously described methods.…”

Preclinical and clinical orthotopic transplantation of decellularized/engineered tracheal scaffolds: A systematic literature review

“…Chemical treatments adopt detergents/chemicals to induce cellular bonds disruption up to cellular components removal. Considering tracheal tissue, the resort to chemicals alone (no enzymes) was attempted by Dang et al, 3 , 17 Kutten et al, 20 Liu et al, 22 Wood et al, 50 Hung et al, 51 and Tan et al 52 Specifically, the solutions consisted in detergents (sodium dodecyl sulfate (SDS) (ionic) and Triton X-100 (non-ionic)), hypertonic solutions (sodium chloride (NaCl)), acids (peracetic acid), bases (ammonium hydroxide (NH 4 OH)) and organic solvents (ethanol), alone or combined within more complex protocols. Regarding the physical treatments, agitation (broadly recommended by the Authors) was also eventually associated with sonication/ultrasonication, 3 , 17 , 51 freeze-drying, 51 freeze-thawing, 20 , 50 and vacuum.…”

“…The methods described, free from enzymes presence, allowed to set up only segmental, partially decellularized substitutes still showing chondrocytes within the lacunae; no repopulation of the samples was performed before graft positioning. 20 , 22 , 52 …”