Genipin cross-linked decellularized tracheal tubular matrix for tracheal tissue engineering applications

Abstract

Decellularization techniques have been widely used as an alternative strategy for organ reconstruction. This study investigated the mechanical, pro-angiogenic and in vivo biocompatibility properties of decellularized airway matrices cross-linked with genipin. New Zealand rabbit tracheae were decellularized and cross-linked with genipin, a naturally derived agent. The results demonstrated that, a significant (p < 0.05) increase in the secant modulus was computed for the cross-linked tracheae, compared to the decellularized samples. Angiogenic assays demonstrated that decellularized tracheal scaffolds and cross-linked tracheae treated with 1% genipin induce strong in vivo angiogenic responses (CAM analysis). Seven, 15 and 30 days after implantation, decreased (p < 0.01) inflammatory reactions were observed in the xenograft models for the genipin cross-linked tracheae matrices compared with control tracheae, and no increase in the IgM or IgG content was observed in rats. In conclusion, treatment with genipin improves the mechanical properties of decellularized airway matrices without altering the pro-angiogenic properties or eliciting an in vivo inflammatory response.

Figure 1. Characterization of decellularized rabbit tracheal matrix.

Movat pentachrome staining showed that compared with the native trachea (A), after seven decellularization cycles (B), the three-dimensional architecture and composition of the matrix remained intact and was virtually unaltered. 4′-6-Diamidino-2-phenylindole staining of the native trachea (C). After seven detergent-enzymatic treatment cycles (D), the nuclei were completely removed in the noncartilaginous tissue, and a small number of nuclei remained only in the thick cartilage and cell debris retained in the lacunae. Glycosaminoglycan expression visualized by safranin O staining in native trachea (E) and trachea after seven detergent-enzymatic treatment cycles (F); glycosaminoglycan was stained red/orange. The immunolabelling of type II collagen within the cartilage matrix suggested that compared with the native trachea (G), the trachea after seven detergent-enzymatic treatment cycles (H) experienced no significant decline in collagen content. The scale bar is 25 μm.

Figure 2. Characterization of genipin-treated decellularized tracheal matrix.

Hematoxylin and eosin (A–C), Masson trichrome staining (D–F) and SEM evaluation luminal (G–I) and external surfaces (J–L) of native trachea (A,D,G,J), trachea after seven detergent-enzymatic treatment cycles (B,E,H,K), and decellularized tracheal matrix cross-linked using 1% genipin solution for 1 h (C,F,I,L). The total network of ECM fibres appeared compact, including the network of collagen, reticular and elastic fibres, which resulted in a more organized and less fragmented structure than in decellularized samples. The collagen was stained bluevia Masson trichrome staining. SEM showsirregular collagen fibres characterizing the external surface, whereas the basal lamina was maintained on the luminal surface. The scale bars are 25 μm in A-F and are 10 μm in panels (G–L).

Figure 3

In vivo pro-angiogenic properties of Gelfoam (A), decellularized (B), genipin-treated (C), and glutaraldehyde-treated tracheal matrices (D). Representative examples of chicken chorioallantoic membrane (CAM) implanted with fragments of Gelfoam, decellularized, genipin-treated and glutaraldehyde-treated tracheal matrices. Samples were placed on the CAM surface of 8-day-old embryos and photographed 4 days later. The samples induced a “spoke-wheel” pattern of new vessels. The scale bar is 1 mm. (E) Effect of Gelfoam, decellularized, genipin-treated and glutaraldehyde-treated tracheal matrices on the number of converging blood vessels 4 days post-implantation. Genipin cross-linking induces a significant (p < 0.05) increase, comparable to the control, in the number of newlybuilt blood vessels. *p < 0.05 with respect to negative control; ^#p < 0.05 with respect to glutaraldehyde-treated tracheal matrix.

Figure 4. Macroscopic appearance of an explanted scaffold.

The native trachea, decellularized trachea, genipin-crosslinked decellularized trachea, and glutaraldehyde-crosslinked decellularized trachea matrices after being implanted in rats for 0 (A), 7 (B), 15 (C), and 30 (D) days. The native trachea (E) and glutaraldehyde-crosslinked decellularized trachea matrices (H) exhibited a thick fibrous capsule with a large amount of pus and a damaged trachea 30 days after implantation. The optimal decellularized trachea (F) and genipin-crosslinked decellularizedtracheae (G) were covered by a thin capsule and neovascularized on the surface 30 days after implantation.

Figure 5. Xenotransplantation in SD rats.

Transverse sections of native trachea (A–C), decellularized trachea (D–F), genipin-crosslinked decellularized trachea (G–I), and glutaraldehyde-crosslinked decellularized trachea matrices (J–L) after being implanted in rats for 7 (A,D,G,J), 15 (B,E,H,K) and 30 (C,F,I,L) days. The tissue sections are stained with (H,E). The black arrow indicates inflammatory cells. The scale bars are 25 μm.

Figure 6

(A)Densities of inflammatory cells observed for the xenotransplantation matrices. (B,C)In vivo evaluation of IgG and IgM levels in recipient rats. S,Sham; (D), decellularized trachea; GP,genipin-crosslinked decellularized trachea; GA,glutaraldehyde-crosslinked decellularized trachea; N, native trachea; *P < 0.05 compared with decellularized trachea group at the same time point; ^#P < 0.05 compared with genipin-crosslinked decellularized trachea group at the same time point.