Regenerative medicine approach to reconstruction of the equine upper airway

Abstract

Airway obstruction is a common cause of poor performance in horses. Structural abnormalities (insufficient length, rigidity) can be a cause for the obstruction. Currently, there are a few effective clinical options for reconstruction of the equine larynx. A regenerative medicine approach to reconstruction may provide the capability to stabilize laryngeal structures and to encourage restoration of site-appropriate, functional, and host-derived tissue. The purpose of this study was the histopathological evaluation of (1) decellularization of equine (horse) laryngeal cartilages (epiglottis and arytenoids); (2) the host response to decellularized laryngeal cartilages implanted subcutaneously in a donkey model as a test of biocompatibility; and (3) the use of decellularized laryngeal cartilages in a clinically relevant pilot study in the horse larynx. Equine laryngeal cartilages were found to be sufficiently decellularized and were subsequently implanted subcutaneously in donkeys to test biocompatibility. After 4 weeks, the implanted cartilage was harvested. In the subcutaneous model, the samples did not elicit a rejection or foreign body type reaction and were judged suitable for implantation in a clinically relevant equine model. Implants were placed in the upper airway (arytenoids and epiglottis) of one horse. At 4 weeks, the implants were observed to remodel rapidly and were replaced by dense connective tissue with signs of new hyaline cartilage formation in the arytenoids and by connective tissue containing glandular structures and an epithelial covering in the epiglottis. The results of the present study demonstrate the feasibility of a scaffold-based regenerative medicine approach to reconstruction of the equine upper airway; however, further studies investigating long-term integration, formation of new cartilage, and mechanical properties are needed.

FIG. 1.

Gross morphologic view of native (A, B), decellularized (C, D), and lyophilized (E, F) tissues. The three-dimensional structure is mostly retained. PicoGreen assay (G) and agarose gel electrophoresis (H) confirm decellularization. Color images available online at www.liebertpub.com/tea

FIG. 2.

Histologic sections of native equine epiglottis (A–C) and decellularized epiglottis (D–F). (A, D) Hematoxylin and eosin, (B, E) safranin O (red=glycosaminoglycan), and (C, F) Verhoeff's elastic stain (black=elastin). Note glandular structures (G), perichondrium (arrow), and elastic cartilage (E). Magnification=4×objective, scale bar=300 μm. Inset magnification=20× objective, scale bar=100 μm. Color images available online at www.liebertpub.com/tea

FIG. 3.

Histologic sections of decellularized epiglottis implanted subcutaneously in donkeys for 1 month. Note the maintenance of structures. A few cells were seen to invade the bulk of the scaffold; however, invasion was observed in the glands. No foreign body reaction or fibrotic encapsulation was observed. Some glycosaminoglycan and the majority of the elastin remain intact. (A) Hematoxylin and eosin, (B) safranin O (red=glycosaminoglycan), and (C) Verhoeff's elastic stain (black=elastin). Note glandular structures (G), perichondrium (arrow), and elastic cartilage (E). Magnification=4× objective, scale bar=300 μm. Inset magnification=20× objective, scale bar=100 μm. Color images available online at www.liebertpub.com/tea

FIG. 4.

Endoscopic picture of the arytenoid cartilages 4 weeks after implantation (A), ultrasound picture showing mildly enlarged arytenoid cartilage (AC) within the implant site (B), normal intrinsic laryngeal musculature (mCAL) and thyroid cartilage (TC), and post-mortem view of the mucosal covering of implant (C). Color images available online at www.liebertpub.com/tea

FIG. 5.

Histologic sections of implanted epiglottis at 1 month post-implantation. The native (N)/implant (I) interface is shown (A–C). The implanted samples were well integrated with the native tissue, and some areas of new cartilage formation were observed. Evidence of epithelialization (arrows, D, E) and glandular formation (G) were observed within the newly formed tissues. Gross morphologic view of intact (left) and bisected larynx (right) at euthanasia is shown (F). (A, D, E) Hematoxylin and eosin, (B) safranin O (red=glycosaminoglycan), and (C) Verhoeff's elastic stain (black=elastin). Magnification=4× objective, scale bar=300 μm. Inset magnification=20× objective, scale bar=100 μm. Color images available online at www.liebertpub.com/tea

FIG. 6.

Histologic sections of implanted arytenoid at 1 month post-implantation. The native (N)/implant (I) interface is shown (A, B). The implanted samples were well integrated with the native tissue, and some areas of new cartilage formation were observed. Evidence of epithelialization (arrow, C) was within the newly formed tissues. Gross morphologic view of intact (left) and bisected larynx (right) at euthanasia is shown (D). (A, C) Hematoxylin and eosin, and (B) safranin O (red=glycosaminoglycan). No positive staining was observed for Verhoeff's elastic stain (not shown). Magnification=4× objective, scale bar=300 μm. Inset magnification=20× objective, scale bar=100 μm. Color images available online at www.liebertpub.com/tea