Decellularized Cartilage for Airway Repair

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Doctor of Philosophy (PhD)
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laryngotracheal reconstruction
progenitor cells
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Gehret, Paul

Severe subglottic stenosis develops in over 20,000 infants per year and requires laryngotracheal reconstruction (LTR) to enlarge the airway by implanting autologous cartilage from a rib graft. However, young children often lack sufficiently sized costal cartilage for this procedure, resulting in increased donor site morbidity and operative time, as well as an elevated risk for airway restenosis, necessitating revision surgery. To overcome these limitations, we created a first-of-its-kind scaffold based on porcine meniscal cartilage decellularization (MEND) by selectively digesting away the elastin and blood vessels uniquely present in the meniscus; this creates microchannels that support cellular re-invasion. Here we demonstrated that MEND can be fully recellularized in 3 days with ear-derived cartilage progenitor cells (eCPCs) and reaches structural and functional maturation suitable for implant within 3 weeks of chondrogenic differentiation, a time frame compatible with clinical translation: a first in airway tissue engineering. To further this therapy toward clinical translation, we validated the eCPCs-MEND grafts in a New Zealand white rabbit LTR model. Our results demonstrated airway expansion, graft re-epithelialization, neocartilage formation, and integration with adjacent native laryngotracheal cartilage at 3 months. Notably, MEND implants performed better in all outcomes than autologous costal cartilage, the standard of care. No instances of adverse events such as extrusion, granulation, infection, or calcification were observed in any of the 38 rabbits in our study. Next, a neonatal pig model was used to further validate acellular MEND constructs. After 3 months in vivo, MEND successfully expanded the neonatal pig’s airway, and similar to the rabbit study, significant neocartilage formations were observed within the grafted area. Lastly chondrocytes, cartilage progenitor, and perichondrium cells were extracted from a pig’s airway and their cartilage forming potential was evaluated using a pellet assay. Sonic hedgehog (SHH) dependent organization and elongation was observed in the CPC pellets suggesting its role in airway repair. Lastly, an ex vivo approach was taken by stimulating airway cartilage pieces with various growth factors associated with cartilage injury and repair. After 3 weeks, elongated cartilage regenerations were observed from the edges of the pieces, and RNA sequencing indicated enrichment of cartilage and developmental associated genes following growth factor treatment. These results demonstrate the feasibility of our translational tissue engineering approach to laryngotracheal reconstruction and suggest that targeting the natural mechanisms to airway healing with a cartilage scaffold could overcome the autograft-associated limitations in pediatric patients, decreasing the need for invasive revision surgery.

Gottardi, Riccardo
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