Functional Reconstruction of Tracheal Defects by Protein-Loaded, Cell-Seeded, Fibrous Constructs in Rabbits

Abstract

Tracheal stenosis is a life-threatening disease and current treatments include surgical reconstruction with autologous rib cartilage and the highly complex slide tracheoplasty surgical technique. We propose using a sustainable implant, composed of a tunable, fibrous scaffold with encapsulated chondrogenic growth factor (transforming growth factor-beta3 [TGF-β3]) or seeded allogeneic rabbit bone marrow mesenchymal stromal cells (BMSCs). In vivo functionality of these constructs was determined by implanting them in induced tracheal defects in rabbits for 6 or 12 weeks. The scaffolds maintained functional airways in a majority of the cases, with the BMSC-seeded group having an improved survival rate and the Scaffold-only group having a higher occurrence of more patent airways as determined by microcomputed tomography. The BMSC group had a greater accumulation of inflammatory cells over the graft, while also exhibiting normal epithelium, subepithelium, and cartilage formation. Overall, it was concluded that a simple, acellular scaffold is a viable option for tracheal tissue engineering, with the intraoperative addition of cells being an optional variation to the scaffolds.

FIG. 1.

Overview of the study design. Three groups were tested: Scaffold-only, transforming growth factor-beta3 (TGF-β3), and bone marrow stromal cell (BMSC) seeded (*The actual sample size was reduced due to adverse events [AE] that occurred during the experiment.). Color images available online at www.liebertpub.com/tea

FIG. 2.

Surgical schematic of defect creation and scaffold implantation (a). Photographs of the surgical procedure showing induced tracheal defect and placement of scaffold (b) and intraoperative cell seeding (c). Color images available online at www.liebertpub.com/tea

FIG. 3.

Chart mapping the survival rate of subjects over the course of 12 weeks.

FIG. 4.

Representative bronchoscopic images from each experimental group at the four bronchoscopy time points (3, 6, 9, and 12 weeks). The most common type of stenosis observed was side-to-side narrowing that occurred in 84% of total subjects with stenosis. Note that the photograph orientation is such that the scaffold is at the top of the photograph in each panel. *Bronchoscopy in the Scaffold only group was performed at week 7 and 10, instead of week 6 and 9. Color images available online at www.liebertpub.com/tea

FIG. 5.

Bronchoscopy image scoring based on stenosis severity, stenosis progression, type of stenosis, and re-epithelialization (no statistically significant differences).

FIG. 6.

Illustration of the lumen quantification process in Avizo Fire. The computed tomography data were rendered into a 3D volume (trachea in yellow) (a). The lumen space was thresholded and reconstructed into a 3D volume and quantified (lumen in blue) (b–d). Color images available online at www.liebertpub.com/tea

FIG. 7.

The average cross-sectional area statistics from the Avizo Fire quantification. The volume was normalized to the trachea length (volume divided by length). The sample sizes in each group and time point were the following: Scaffold-only 6 week (n=3), Scaffold-only 12 week (n=4), Scaffold-only AE (n=2); TGF-β3 6 week (n=3), TGF-β3 12 week (n=3), TGF-β3 AE (n=3); BMSC 6 week (n=4), BMSC 12 week (n=5), BMSC AE (n=1). The BMSC group was the only group to have significantly smaller average cross-sectional area than the native tracheas, aside from the AE (# significantly larger average cross-sectional area when compared to the AE in the same group [p<0.05], @ significantly larger average cross-sectional area when compared to the BMSC-group at week 12 [p<0.05], * significantly smaller average cross-sectional area when compared to the native tracheas [p<0.05]).

FIG. 8.

An overview of histological images in all three groups at the two time points. Hematoxylin and Eosin (H&E), Alcian Blue, Safranin O, Verhoeff-Van Gieson, and Sudan Black staining was performed. Normal epithelium (i.e., pseudostratified columnar) was present more often in the BMSC groups and at later time points. Immature cartilage was observed across all groups, but with a higher occurrence in the BMSC group. Fibroblasts and collagen were present in all subjects, mainly organizing around the scaffolds and within the scaffold. Normal connective fibroblastic tissue was observed inside the BMSC group scaffolds. Transverse sections are oriented with the scaffolded region at the top of the image and remaining native trachea rings at the bottom of the section (entire circumference of trachea section omitted due to space constraints). Scale bar represents 2 mm. Color images available online at www.liebertpub.com/tea

FIG. 9.

Magnified histological images representing scoring criteria. Representative images demonstrate the appearance of mononuclear cells, heterophils, eosinophils, multi-nucleated giant cells (MNGs), and vasculature. Regions of immature cartilage and collagen are shown. The unique immature connective tissue is shown in three stains and is surrounded by the scaffold (S). Color images available online at www.liebertpub.com/tea

FIG. 10.

Histological scoring of vasculature, collagen, and residual polymer. To determine vasculature, four representative fields of inflamed tissue were examined at 40× and vessel cross sections containing erythrocytes were counted. Collagen was scored by assessing if no collagen was present (1), few collagen fibrils present (2), and many collagen fibrils present (3). The amount of polymer present was ranked from no polymer (0), 25% (1), 50% (2), 75% (3), and 100% of polymer present (4) (@ significantly higher quantity of vasculature than other groups [and AE] within the TGF-β3 group [p<0.05], # and $ significantly higher quantity of vasculature than nonfatal cases within their respective groups [p<0.05]).

FIG. 11.

Histology scoring of inflammatory component of tissue sections. Severity of inflammation across entire section was determined, and scoring of specific inflammatory cell types, including heterophils, mononuclear cells, and MNGs.