Serial Analysis of Tracheal Restenosis After 3D-Printed Scaffold Implantation: Recruited Inflammatory Cells and Associated Tissue Changes

Abstract

Tracheal restenosis is a major obstacle to successful tracheal replacement, and remains the greatest challenge in tracheal regeneration. However, there have been no detailed investigations of restenosis. The present study was performed to analyze the serial changes in recruited inflammatory cells and associated histological changes after tracheal scaffold implantation. Asymmetrically porous scaffolds, which successfully prevented tracheal stenosis in a partial trachea defect model, designed with a tubular shape by electrospinning and reinforced by 3D-printing to reconstruct 2-cm circumferential tracheal defect. Serial rigid bronchoscopy, micro-computed tomography, and histology [H&E, Masson's Trichrome, IHC against α-smooth muscle actin (α-SMA)] were performed 1, 4, and 8 weeks after transplantation. Progressive stenosis developed especially at the site of anastomosis. Neutrophils were the main inflammatory cells recruited in the early stage, while macrophage infiltration increased with time. Recruitment of fibroblasts peaked at 4 weeks and deposition of α-SMA increased from 4 weeks and was maintained through 8 weeks. During the first 8 weeks post-transplantation, neutrophils and macrophages played significant roles in restenosis of the trachea. Antagonists to these would be ideal targets to reduce restenosis and thus play a pivotal role in successful tracheal regeneration.

Keywords: 3D-printing; Inflammation; Restenosis; Scaffold; Trachea.

Fig. 1

A A 3D reconstructed image (left) and axial images of micro-computed tomography (µCT) at the level of scaffold transplantation. B A 3D reconstructed image (left) and axial µCT images of normal trachea

Fig. 2

A Hematoxylin-eosin (H&E) and B Masson’s trichrome staining results of cross-sections of specimens taken 1, 4, and 8 weeks after implantation. A normal pseudostratified columnar ciliated epithelium was observed in the control group. The presence of inflammatory cells, predominantly neutrophils, within and around the implant at week 1. Histological examination showed inflammation around the implant with submucosal hyperplasia caused by proliferation of fibroblasts at week 4. At week 8, the outer layer of the implant was consistently covered with inflammatory cells and fibrosis (arrows)

Fig. 3

A Representative images of tissue specimens showing histological evidence of a dense inflammatory infiltrate taken 1, 4 and 8 weeks after implantation (H&E staining). B Assessment of the cellular response to the tracheal scaffold revealed that acute inflammatory cells (neutrophils: arrow in week 1) and chronic inflammatory cells (mononucleated cells: arrows in week 4 and 8) were present around the implant according to time interval. Panel B represents a higher magnification of A. Scale bars in A and B = 100 μm

Fig. 4

A Immunohistochemical detection of macrophages and B, C evaluation of inflammatory cell quantity. Representative photomicrographs of tissue sections demonstrating increased macrophage infiltration at 4 and 8 weeks after implantation. Macrophage-positive cells are indicated by arrows and quantified per high-powered field (HPF; A–C). Data are expressed as means ± SD. *p < 0.05, compared to the control group; #p < 0.05 compared to week 1; Mann–Whitney U test. Scale bar = 100 μm

Fig. 5

A Masson’s trichrome staining showed fibroblast infiltration at week 4 and increased collagen deposition at week 8. Fibroblasts and collagen were present in all animals, mainly organizing around the tracheal scaffold over time. A significant fibroproliferative response with deposition of fibroblasts (arrows) was seen at week 4. The black box indicates the magnified area. B Average number of fibroblasts per high-powered field (HPF) was quantified. Fibroblast count was performed in 5 HPF per tissue section (400x). Data are expressed as means ± SD. *p < 0.05, compared to the control group; #p < 0.05 compared to 4 weeks; Mann–Whitney U test. Scale bar = 100 μm

Fig. 6

Evaluation of the degree of α-smooth muscle actin (α-SMA) deposition. A Expression of α-SMA visualized by immunohistochemistry at 1, 4 and 8 weeks after implantation (arrows). B There was no difference in the intensity of immunohistochemical staining between week 4 and week 8. Data are expressed as means ± SD. *p < 0.05, compared to the control group; #p < 0.05 compared to week 1; Mann–Whitney U test. Scale bar = 100 μm