A Decellularized Cartilage Biomaterials Approach to Pediatric Airway Reconstruction

<b>Introduction: </b>Severe subglottic stenosis (SGS), the narrowing of the airway just below the vocal folds, develops in 8.3% of ICU neonates and directly leads to debilitating comorbidities [1,2]. Pediatric SGS is the result of prolonged intubation and dramatically lowers the child’s long-term quality of life, as they often suffer speech and cognitive delays. In the most severe cases, laryngotracheal reconstruction (LTR) surgery is required to expand the airway and restore airflow [1,2]. During LTR, autologous costal cartilage is harvested, shaped, and implanted into the child’s airway. However, a major obstacle is that young children often lack sufficient costal cartilage to ensure a successful graft surgery, leading to donor site morbidity and high risk for restenosis, necessitating revision surgery3. Our objective is to utilize xenogeneic decellularized meniscal cartilage (MEND) to engineer a cartilage implant populated with patients’ cells to circumvent the limitations of pediatric autologous cartilage.<br/><b>Materials and Methods: </b><i>Meniscus decellularization and channel formation:</i> Meniscus cross-sections were devitalized with 4 freeze/thaw cycles then subjected to pepsin and acetic followed by treatment with elastase. 6 mm cylinders were created with a biopsy punch from the posterior half of the digested cross-sections to ensure the maximum number of channels. <i>Decellularized Meniscus Reinvasion:</i> Sterile decellularized, digested meniscus (MEND) cylinders were placed on top of the membrane insert of a transwell plate and cartilage progenitor cells (CPC) were seeded on top of the MEND cylinders. After 3 days, the MEND cylinders were fixed, sectioned, and stained. <i>In Vitro Differentiation: </i>CPC-invaded MEND were cultured in chondrogenic medium for 3 weeks and media was renewed twice/week. The differentiated construct were examined for gene expression, compression testing, and histology. <i>Rabbit Laryngotracheal Reconstructions:</i> Acellular, re-cellularized, or differentiated decellularized cartilage was implanted in an anterior cricoid split LTR. After 3 months, the rabbits were euthanized and the construct phenotype, airway expansion, and histomorphometry were assessed.<br/><b>Results and Discussion:</b> After enzymatic treatment, H&E images of circumferential sections showed that clearly aligned channels are visible post-treatment. Following a serum gradient (transwell) migration, cartilage progenitor cells (blue DAPI nuclei) penetrated in 3 days through the channels the entire depth of the construct (collagen red auto-fluorescence) reaching a density similar to that of native cartilage. After 3 weeks of differentiation, cells were arranged into lacunae with columnar structures resembling the organization of native cartilage and secreted a substantial amount of glycosaminoglycans (GAGs). Importantly, the constructs increased in both bulk mechanical strength and GAG content. Finally, a 38 rabbit study was conducted and MEND was implanted in a rabbit LTR. MEND successfully expanded the airway over the course of the 3 months and integrated with the existing cartilage as shown by the increase of GAGs within the construct and the neo-chondrification protruding from the cartilage rings. We established how decellularized meniscal cartilage reinvaded with progenitor cells can significantly improve outcomes for pediatric LTR.<br/><b>Ethical Statement: </b>All animal studies were conducted with IACUC approval.<br/><b>References: [1] </b>Ghavimi S et. Al Adv Drug Deliv Rev. 2021, <b>[2] </b>Sherman et. al J. Pediatr1986 <b>[3] </b>Choi et. al. Head Neck Surg 199.<br/><b>Acknowledgements: </b>Support from the Ri.MED Foundation, the Children’s Hospital of Philadelphia Research Institute, the Frontier Program in Airway Disorders of the Children’s Hospital of Philadelphia, The American Society of Pediatric Otolaryngology Research Grant 2020, The National Institute of Health P30 AR069619, R21HL159521, R56HL16453, and the National Science Foundation Graduate Research Fellowship.