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. 2019 Aug 19;9(1):12034.
doi: 10.1038/s41598-019-48450-4.

De-epithelialization of Porcine Tracheal Allografts as an Approach for Tracheal Tissue Engineering

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Free PMC article

De-epithelialization of Porcine Tracheal Allografts as an Approach for Tracheal Tissue Engineering

Fabio G Aoki et al. Sci Rep. .
Free PMC article

Abstract

Replacement of large tracheal defects remains an unmet clinical need. While recellularization of acellular tracheal grafts appeared to be a viable pathway, evidence from the clinic suggests otherwise. In hindsight, complete removal of chondrocytes and repopulation of the tracheal chondroid matrix to achieve functional tracheal cartilage may have been unrealistic. In contrast, the concept of a hybrid graft whereby the epithelium is removed and the immune-privileged cartilage is preserved is a radically different path with initial reports indicating potential clinical success. Here, we present a novel approach using a double-chamber bioreactor to de-epithelialize tracheal grafts and subsequently repopulate the grafts with exogenous cells. A 3 h treatment with sodium dodecyl sulfate perfused through the inner chamber efficiently removes the majority of the tracheal epithelium while the outer chamber, perfused with growth media, keeps most (68.6 ± 7.3%) of the chondrocyte population viable. De-epithelialized grafts support human bronchial epithelial cell (BEAS-2B) attachment, viability and growth over 7 days. While not without limitations, our approach suggests value in the ultimate use of a chimeric allograft with intact donor cartilage re-epithelialized with recipient-derived epithelium. By adopting a brief and partial decellularization approach, specifically removing the epithelium, we avoid the need for cartilage regeneration.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Double-chamber bioreactor configuration during De-epithelialization (De-ep) and Recellularization. (a) Schematic representation of the De-ep process in De-ep bioreactor system. (b) Image of a de-epithelialized long segment porcine tracheal graft attached in the recellularization bioreactor system. (c) Schematic representation of the recellularization process in recellularization bioreactor system showing the addition of sample ports and filters, flow sensor and thermometer to the bioreactor system used for De-ep.
Figure 2
Figure 2
Scanning electron microscopy (SEM) images of tracheal lumen and cartilage cross-section of Native and De-epithelialized (De-ep) samples. (a) Representation of the trachea with the tracheal lumen en face (box insert). (bd) SEM images of the luminal surface of Native tracheal samples (n = 3). (eg) Luminal surface of De-ep tracheal samples (n = 3). (h) Schematic representation of the cartilage ring cross-section (box insert). (ik) SEM images of the cross-section of Native tracheal samples (n = 3). (ln) Cross-section of De-ep tracheal samples (n = 3).
Figure 3
Figure 3
Histological evaluation of Native and De-epithelialized (De-ep) tracheal samples. Representative microscopy images of Native and De-ep tracheal samples (n = 3) showing (a,e,i,m) haematoxylin & eosin (H&E) staining, (b,f,j,n) Alcian blue staining for glycosaminoglycans, (c,g,k,o) elastin staining for elastic fibers, and (d,h,l,p) Masson’s trichrome staining for collagen fibers. Mucosa/Submucosa samples of (ad) Native and (eh) De-ep tracheal grafts. Cartilage samples of (il) Native and (mp) De-ep tracheal grafts.
Figure 4
Figure 4
Quantification of sulfated glycoaminoglycans (sGAGs) and immunohistochemical evaluation of Native and De-epithelialized (De-ep) samples. (ac) Quantification of sGAGs in Native and De-ep samples. Quantified sGAGs (a) in Total (intact) Native (n = 7) and De-ep (n = 5) tracheal samples containing both Cartilage and Mucosa/Submucosa; (b) in isolated Cartilage from Native (n = 5) and De-ep (n = 5) tracheal samples; and (c) in isolated Mucosa/Submucosa from Native (n = 6) and De-ep (n = 6) tracheal samples. Bars represent mean ± standard deviation (ns, p > 0.05). (df) Native and (gi) De-ep samples. (di) are representative (n = 3) immunohistochemistry images for collagen type IV, laminin and collagen type II, respectively.
Figure 5
Figure 5
Mechanical analysis of Native and De-epithelialized (De-ep) samples. (a) Schematics of the pressure-volume (PV) curve experiment. (b) Area under the PV curve and (c) compliance measured every two volume-steps of fresh Native tracheal grafts (n = 5) and De-ep grafts (n = 7). (d) Schematics of the clamped cartilaginous ring. (e) Calculated energy loss from the fifth cycle of Native cartilage rings and De-ep samples (n = 3). Bars represent mean ± standard deviation (ns, p > 0.05).
Figure 6
Figure 6
Chondrocyte viability in Native and De-epithelialized (De-ep) samples. Confocal microscopy images depicting calcein-AM for live (green) and ethidium homodimer-1 for dead (red) cells in cross-sections of cartilage rings (marked as the area within the white dotted line) in (a) Native samples (n = 3; 3 sections per sample), and (b) representative regions (inlet, middle and outlet; 4 sections per region) of De-ep samples (n = 4). White asterisks mark the position of the tracheal lumen.
Figure 7
Figure 7
Recellularization of De-epithelialized (De-ep) tracheal grafts. Confocal microscopy images of peeled Mucosa/Submucosa from De-ep tracheal samples seeded with human bronchial epithelial cells (BEAS-2Bs) at (a) 6 h and (b) 24 h after cell seeding (n = 4). Live cells are depicted in green (calcein-AM) and dead cells are shown in red (ethidium homodimer-1). (c) Cell viability (n = 4; 3–7 quantified fields per sample) of BEAS-2Bs reported as percentage (%) of live cells per total cells (bars are mean ± standard deviation; ns, p > 0.05). (d) Metabolic cell activity of BEAS-2Bs in bioreactor during a 7 day culture period (n = 3; data represented as mean ± standard deviation). (e) Representative (n = 3; 3 fields per sample) confocal microscopy image of recellularized tracheal lumen following a 7 day culture period in the bioreactor. Live cells are depicted in green (calcein-AM) and dead cells are shown in red (ethidium homodimer-1). (f) Brightfield microscopy images showing haematoxylin and eosin (H&E) staining of a representative tracheal graft recellularized with BEAS-2Bs following a 7 day culture period in bioreactor (n = 3).

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