Decellularized Meniscus Scaffolds for Pediatric Laryngotracheal Reconstruction in a Porcine Model

Introduction:: Pediatric patients who undergo prolonged periods of intubation commonly develop severe subglottic stenosis (SGS), a disease characterized by the narrowing of the airway below the vocal folds. SGS occurs in nearly 20,000 neonates each year and can result in significant comorbidities such as cognitive and speech delays1,2. The current standard of care, laryngotracheal reconstruction (LTR), is a highly invasive surgery to widen the airway using autologous rib cartilage. However, this approach is limited by availability of sufficient cartilage in pediatric patients and may lead to restenosis necessitating revision surgery and donor site morbidity3. Tissue engineering provides an alternative to autologous cartilage grafts to alleviate these comorbidities. Our objective is to utilize our newly developed cartilaginous scaffold based on decellularized meniscus (MEND) as a graft for LTR in our novel pediatric porcine model.


Materials and Methods:: Meniscus decellularization and channel formation: Meniscus cross-sections were devitalized by four freeze/thaw cycles (-20ºC to RT), then incubated for 24h at 150rpm and 37ºC in a solution of pepsin and acetic acid, before treating with elastase for 24 hrs to create channels. Biopsy punches were used to create 6mm cylinders at the posterior end of the decellularized cross sections, the area with the highest number of created channels. Cartilage Progenitor Cells: Decellularized meniscus scaffolds (MEND) were placed in a transwell insert for chemotactic cellular re-invasion by a serum gradient across the insert driving cell infiltration. 600µL of 20% FBS was added to the well below the inserts whilst 100 µL of cartilage progenitor cells (CPC) at 3x106 cell/ml in DMEM with 1% fetal bovine serum (FBS) were seeded on top of MEND and cultured for three days to achieve full re-invasion. CPC Differentiation:Re-cellularized constructs were cultured for three weeks in chondrogenic medium on an orbital shaker (150 rpm), with medium renewed twice/week. Differentiated CPC-MEND constructs were then analyzed by histology, mechanical testing, and RT-qPCR. Laryngotracheal Reconstructions: Acellular MEND scaffolds were used as grafts in an anterior cricoid split in 1-month-old Yucatan Miniature Pigs as a pilot study to determine acellular MEND integration and regeneration after airway expansion, before exploring the effectiveness of cellularized grafts. Airway expansion was assessed periodically by endoscopy until sacrifice after 3 months, when graft integration and remodeling was assessed by contrast-enhanced microCT, histology, immunohistochemistry, and mechanical testing.


Results, Conclusions, and Discussions:: Results & Discussion: Following enzymatic treatment well-defined channels are visible in MEND constructs (Fig 1. A&B). These channels significantly increased porosity compared to the native tissue(1C). Meniscus digestion successfully decellularized tissue, demonstrated by DNA content below 50 ng/mg dry tissue (Fig 1D). Following cell seeding, CPCs penetrated the decellularized constructs after three days throughout the depth of the scaffold (Fig. 2A). Chondrogenic differentiation resulted in substantive secretion of glycosaminoglycans (GAGs), and an increase in bulk mechanical properties. (Fig. 2B, C&D). Lastly, following porcine LTR (Fig.3A) acellular MEND integrate well with native cricoid cartilage as seen by µCT and endoscopic imaging (Fig. 3B, C&D). Furthermore, histology evidences how MEND LTR resulted in graft integration, remodeling, invasion of MEND by host cells and secretion of glycosaminoglycans in vivo resulting in improved mechanics (Fig.3E, F).

Conclusion: MEND integrated with native tissue, mechanically supported and expanded pig airways. These factors demonstrate feasibility of this biomaterials solution for pediatric LTR.


Acknowledgements (Optional): : Support from: NIH (1R56HL164536-01), Children’s Hospital of Philadelphia Research Institute, Frontier Program in Airway Disorders of the Children’s Hospital of Philadelphia, Ri.MED Foundation (to RG), National Science Foundation Graduate Research Fellowship (to PMG), and Penn Undergraduate Research Mentoring Program and University Scholars (to AAD).


References (Optional): :
[1] Ghavimi S et. Al Adv Drug Deliv Rev. 2021, [2] Sherman et. al J. Pediatr1986 [3] Choi et. al. Head Neck Surg 1999