Long-term observation of airway reconstruction using decellularized tracheal allografts in micro-miniature pigs at growing stage

Abstract

Introduction: Decellularized tissue exhibits cell matrix-like properties, along with reduced antigenicity. We explored the potential of decellularized allogeneic trachea to restore the upper respiratory tract, focusing on pediatric application. This study specifically aimed at long-term observation of tissue regeneration using a micro-miniature pig model.

Methods: Artificial defects (15 × 15 mm) in the subglottis and trachea of micro-miniature pigs were repaired by transplantation of either allogeneic decellularized or fresh (control) tracheal patches. Pigs were evaluated in situ, by bronchoscopy, every three months, and sacrificed for histological examination at six and twelve months after transplantation.

Results: No airway symptom was observed in any pig during the observation period. Bronchoscopy revealed the tracheal lumen to be restored by fresh grafts, showing an irregular surface with remarkable longitudinal compression; these changes were mild after restoration with decellularized grafts. Histologically, while fresh graft patches were denatured and replaced by calcified tissue, decellularized patches remained unchanged throughout the observation period. There were regeneration foci of cartilage adjacent to the grafts, and some foci joined the decellularized graft uniformly, suggesting the induction of tracheal reconstitution.

Conclusion: Allogeneic decellularized tracheal tissue could serve as a promising biomaterial for tracheal restoration, especially for pediatric patients at the growing stage.

Keywords: Decellularized tracheal tissue; HE, Hematoxylin and Eosin; High hydrostatic pressure; Pig; Regeneration; Tissue-engineering; Tracheal restoration.

Fig. 1

Fresh (a) and decellularized (b) tracheal grafts and outline of the surgical procedure (c, d). Tracheas (a) were purchased from Tokyo Shibaura Zouki and decellularized using an ultra-high pressure protocol. The decellularized tissue has a characteristic white appearance (b). After dissection of approximately 1/2 of the tracheal trunk (c, arrowhead indicates intubation), the defect was restored with either fresh or decellularized allogeneic tracheal patch of appropriate size (d). Orientation of recipients was indicated by arrows. Bars: 10 mm.

Fig. 2

Bronchoscopic observation of the engrafted section of recipient trachea. At 6 months after transplantation of fresh (a) and decellularized (b) allogeneic tracheal patches, and 12 months after the transplantation of fresh (c) and decellularized (d) allogeneic tracheal patches. In all cases, the inner surfaces were covered well with tracheal mucosa, and there was no breathing abnormality, including in- and outflow noises. Some wall irregularities and longitudinal compressions were slightly more severe in the fresh tracheal graft than in the decellularized graft. Bars: 10 mm.

Fig. 3

Appearance of exposed trachea 12 months after the transplantation of fresh (a) and decellularized (b) tracheal patches. Macroscopic views of the cross sections of autopsy tracheas, harvested at 6 and 12 months after the transplantation of fresh (c, e) and decellularized (d, f) grafts. Arrowheads in a and e indicate inflammatory granulation and large lymph nodes. Bars: 10 mm.

Fig. 4

Microscopic observation of tracheal restoration of decellularized tracheal patches. Microscopic observation of tracheal restoration with decellularized tracheal patches, 12 months after transplantation, using hematoxylin and eosin (a–d), toluidine blue (e), and safranin-O (f) staining. Arrowheads indicate decellularized tracheal grafts. b and d indicate magnified views of the framed area in a. c indicates a magnified view of the framed area in b. In c, asterisk and arrows indicate membranous and nodulous structure, respectively. Bars: 500 μm in a, e, f, 50 μm in b, c, d.

Fig. 5

Microscopic observation of tracheal restoration of fresh tracheal patch. Microscopic observation of tracheal restoration 12 months after transplantation of fresh tracheal patch (a; hematoxylin and eosin staining, b; alizarin red staining). Arrowheads indicate degradation and calcification of fresh tracheal graft. c and d show magnified views of the framed areas in a (upper; B, lower; C). Arrows in a and d indicate the cartilage that appeared to be regenerated. Bars: 500 μm in a, b and 50 μm in c, d.