Spatial and Temporal Analysis of Host Cells in Tracheal Graft Implantation

Abstract

Objectives/Hypothesis The ideal trachea replacement would be a living graft that is genetically identical to the host, avoiding the need for immunosuppression. We have developed a mouse model of syngeneic tracheal transplant that results in long‐term survival without graft stenosis or delayed healing. To understand how host cells contribute to tracheal transplant integration, we quantified the populations of host cells in the graft and native trachea following implant. Study Design: Tracheal transplant, tracheal replacement, regenerative medicine, animal model. Methods Tracheal grafts were obtained from female C57BL/6 mice and orthotopically transplanted into syngeneic male recipients. Cohorts were euthanized on day 14, day 45, and day 90 post‐transplantation. Host and graft tracheas were explanted and analyzed by histology. Male host cells were quantified using fluorescence in situ hybridization, and macrophages were quantified with immunofluorescence. Results Evidence of host‐derived cells was found in the midgraft at the earliest time point (14 days). Host‐derived cells transiently increased in the graft on day 45 and were predominantly found in the submucosa. By day 90, the population of host‐derived cells population declined to a similar level on day 14. Macrophage infiltration of host and graft tissue was observed at all time points and was greatest on day 90. Conclusions Tracheal graft integration occurs by way of subacute transient host‐cell infiltration and is primarily inflammatory in nature. Host‐cell contribution to the graft epithelium is limited. These data indicate that creation of living, nonimmunogenic tracheal graft could serve as a viable solution for long‐segment tracheal defects. Level of Evidence 3 Laryngoscope, 131:E340–E345, 2021

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

“…Tracheal grafts were harvested from 6 to 8-week-old C57BL/6J female mice as previously described. 19 , 20 Proximal tracheas were dissected, and a 5 mm tracheal segment was harvested and immersed in phosphate-buffered saline (PBS, Gibco, Thermo Fisher Scientific, Waltham, WA) before implantation. STG were implanted following harvest without additional processing.…”

“…STG were implanted into C57BL/6J mice as previously described. 20 Briefly, the airway was dissected free from adjacent structures. A 3–4 mm segment was resected and orthotopically implanted.…”

“…PDTG were implanted following previously published methods. 20 , 24 A 3D-printed tracheal splint was then implanted on PDTG, creating a composite tracheal graft (CTG) as described in Section 2.6. Radiopaque markers were secured at the distal and proximal anastomoses to identify the boundaries of the graft during micro-computed tomography (microCT).…”

“…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.…”

A Multimodal Approach to Quantify Chondrocyte Viability for Airway Tissue Engineering

“…Tracheal segments were harvested from 6 to 8-week-old female C57BL/6 mice as previously described. 19,20 The number of animals assigned for experimental and control groups was determined based on anticipated drops from previous surgical morbidity and mortality. 10,19 Proximal tracheas were dissected free and 5 mm segmental tracheal grafts were explanted from donors.…”