Deconstructing tissue engineered trachea: Assessing the role of synthetic scaffolds, segmental replacement and cell seeding on graft performance

Abstract

The ideal construct for tracheal replacement remains elusive in the management of long segment airway defects. Tissue engineered tracheal grafts (TETG) have been limited by the development of graft stenosis or collapse, infection, or lack of an epithelial lining. We applied a mouse model of orthotopic airway surgery to assess the impact of three critical barriers encountered in clinical applications: the scaffold, the extent of intervention, and the impact of cell seeding and characterized their impact on graft performance. First, synthetic tracheal scaffolds electrospun from polyethylene terephthalate / polyurethane (PET/PU) were orthotopically implanted in anterior tracheal defects of C57BL/6 mice. Scaffolds demonstrated complete coverage with ciliated respiratory epithelium by 2 weeks. Epithelial migration was accompanied by macrophage infiltration which persisted at long term (>6 weeks) time points. We then assessed the impact of segmental tracheal implantation using syngeneic trachea as a surrogate for the ideal tracheal replacement. Graft recovery involved local upregulation of epithelial progenitor populations and there was no evidence of graft stenosis or necrosis. Implantation of electrospun synthetic tracheal scaffold for segmental replacement resulted in respiratory distress and required euthanasia at an early time point. There was limited epithelial coverage of the scaffold with and without seeded bone marrow-derived mononuclear cells (BM-MNCs). We conclude that synthetic scaffolds support re-epithelialization in orthotopic patch implantation, syngeneic graft integration occurs with focal repair mechanisms, however epithelialization in segmental synthetic scaffolds is limited and is not influenced by cell seeding. STATEMENT OF SIGNIFICANCE: The life-threatening nature of long-segment tracheal defects has led to clinical use of tissue engineered tracheal grafts in the last decade for cases of compassionate use. However, the ideal tracheal reconstruction using tissue-engineered tracheal grafts (TETG) has not been clarified. We addressed the core challenges in tissue engineered tracheal replacement (re-epithelialization and graft patency) by defining the role of cell seeding with autologous bone marrow-derived mononuclear cells, the mechanism of respiratory epithelialization and proliferation, and the role of the inflammatory immune response in regeneration. This research will facilitate comprehensive understanding of cellular regeneration and neotissue formation on TETG, which will permit targeted therapies for accelerating re-epithelialization and attenuating stenosis in tissue engineered airway replacement.

Keywords: Bone marrow mononuclear cells; Epithelialization; Syngeneic segmental tracheal transplantation; Synthetic PET/PU scaffold; Tissue engineered trachea graft.

Figure 1

Synthetic patch implantation and outcomes. A. PET/PU patch tracheoplasty: Traches exposure (A1). 1 mm × 2 mm defect (A2), patch sutured (A3); B. Axial H&E section at patch-implanted trachea at short term (1 week) (B1) and long term (> 6 weeks) (B2).

Figure 2

Synthetic patch implantation-host tissue interaction. A1. ACT IF, A2. % patch coverage with ciliated respiratory epithelium, A3. % width ACT+ patch coverage in time; B1. Representative CCSP immunofluorescent image, B2. CCSP+ density in time (* represent significant difference of cell density at 1 week, P < 0.05); C1. Representative CD68 immunofluorescent image, C2. CD68+ macrophage infiltration, C3. Quantified cell density in time (* represent significant difference of cell density at 1 week and over 6 weeks with unoperated control, P <0.05).; # represent significant difference of cell density over time between native and patch, P < 0.05).

Figure 3

K5/K14-marked basal cells in syngeneic trachea transplantation. A. Representative immunofluorescent images of K5+/K14+ basal cell markers in Proximal Host, Syngeneic graft and Distal Host of mouse tracheas at day 3 of syngeneic tracheal transplant. Scale bar = 20 μm; B. Quantification of volume of nuclei per area of basement membrane, total K5 cell mass and total K14 cell mass for each region (proximal host, donor, and distal host). Data are presented as the mean +/− standard deviation. N = 4 for all time-points except 7 days (N = 3). * represents statistical significance (P < 0.05) between compared groups tested by two-way ANOVA.

Figure 4

Expression of vascular markers in syngeneic transplanted-trachea for long term (1 year). A. Representative immunofluorescent image of CD31 expression in each 200 μm length along tracheal lumen from anastomosis junction (* marked out artefact); B. CD31 expression (% area in 200 μm) of host and graft with time. * represents significant difference between compared groups (P < 0.05).

Figure 5

BM-MNCs seeding capacity and SEM characterization of TETG. A. Seeding BM-MNCs with 1 × 10⁶, 10 × 10⁶, 100 × 10⁶ to synthetic tracheal scaffolds; B. Unseeded PET/PU scaffold morphologies; C. BM-MNCs seeded scaffold.

Figure 6

Post-operative outcomes of segmental TETG implantation. A. Survival curve (Kaplan Meier Survival Analysis); B. Representative process of grafts explantation showing no stenosis formation in the graft lumen, 4 days post-operatively. (Scale bar = 2 mm).

Figure 7

Epithelial cells coverage on scaffold at 7th day after segmental TETG implantation. A. Longitudinal H&E section; B. Quantified epithelial cell coverage percentage on graft in both seeded and unseeded groups.

Figure 8

Representative anastomosis IF images of K5/K14 basal cell markers at 7th day of syngeneic and segmental TETG (seeded and unseeded) implantation. The white dotted lines separate the donor trachea (A1 and A2) and the TETG (B1 to C2) from the host trachea and the lumen. The white arrows point out K5+/K14+basal cells. The yellow arrows point out anastomosis. Scale bar = 50 μm.

Figure 9

Expression of vascular marker, CD31, in segmental TETG implanted-trachea: A. Representative immunofluorescent images of CD31 staining of longitudinal section; B. Representative high power field image of CD31 stained endothelial cells; C. CD31 quantification (% area in 200 μm length) in 7 days (* represent significant difference between unoperated control and seeded host, graft and unseeded graft, P < 0.05). The yellow arrows point out vascular marker, CD31. The white arrows indicate the autofluorescent graft.

Figure 10

Amount of GFP+ BM-MNCs in scaffold after segmental TETG implantation at day 1, day 3, and day 7. * & ** represents significant difference of cells number between post-op day 1 and day 3, day 7, P < 0.05).