A Pilot Study of Decellularized Cartilage for Laryngotracheal Reconstruction in a Neonatal Pig Model.

OBJECTIVE: Severe subglottic stenosis develops as a response to intubation in 1% of the >200,000 neonatal intensive care unit infants per year and may require laryngotracheal reconstruction (LTR) with autologous hyaline cartilage. Although effective, LTR is limited by comorbidities, severity of stenosis, and graft integration. In children, there is a significant incidence of restenosis requiring revision surgery. Tissue engineering has been proposed to develop alterative grafting options to improve outcomes and eliminate donor-site morbidity. Our objective is to engineer a decellularized, channel-laden xenogeneic cartilage graft, that we deployed in a proof-of-concept, neonatal porcine LTR model. METHODS: Meniscal porcine cartilage was freeze-thawed and washed with pepsin/elastase to decellularize and create microchannels. A 6 × 10-mm decellularized cartilage graft was then implanted in 4 infant pigs in an anterior cricoid split. Airway patency and host response were monitored endoscopically until sacrifice at 12 weeks, when the construct phenotype, cricoid expansion, mechanics, and histomorphometry were evaluated. RESULTS: The selective digestion of meniscal components yielded decellularized cartilage with cell-size channels. After LTR with decellularized meniscus, neonatal pigs were monitored via periodic endoscopy observing re-epithelization, integration, and neocartilage formation. At 12 weeks, the graft appeared integrated and exhibited airway expansion of 4 mm in micro-CT and endoscopy. Micro-CT revealed a larger lumen compared with age-matched controls. Finally, histology showed significant neocartilage formation. CONCLUSION: Our neonatal porcine LTR model with a decellularized cartilage graft is a novel approach to tissue engineered pediatric LTR. This pilot study sets the stage for "off-the-shelf" graft procurement and future optimization of MEND for LTR. LEVEL OF EVIDENCE: NA Laryngoscope, 134:807-814, 2024.

Drug-Eluting Endotracheal Tubes for Preventing Bacterial Inflammation in Subglottic Stenosis.

OBJECTIVES/HYPOTHESIS: Subglottic stenosis (SGS) results from dysregulated extracellular matrix deposition by laryngotracheal fibroblasts causing scar tissue formation following intubation. Recent work has highlighted a relationship between this inflammatory state and imbalances in the upper airway microbiome. Herein, we engineer novel drug-eluting endotracheal (ET) tubes to deliver a model antimicrobial peptide Lasioglossin-III (Lasio) for the local modulation of the microbiome during intubation. STUDY DESIGN: Controlled in vitro study. METHODS: ET tubes were coated with a water-in-oil (w/o) emulsion of Lasio in poly(d,l-lactide-co-glycolide) (PLGA) by dipping thrice. Peptide release was quantified over 2 weeks via fluorometric peptide assays. The antibacterial activity was tested against airway microbes (Staphylococcus epidermidis, Streptococcus pneumoniae, and pooled human microbiome samples) by placing Lasio/PLGA-coated tubes and appropriate controls in 48 well plates with diluted bacteria. Bacterial inhibition and tube adhesion were tested by measuring optical density and colony formation after tube culture, respectively. Biocompatibility was tested against laryngotracheal fibroblasts and lung epithelial cells. RESULTS: We achieved a homogeneous coating of ET tubes with Lasio in a PLGA matrix that yields a prolonged, linear release over 1 week (typical timeframe before the ET tube is changed). We observed significant antibacterial activity against S. epidermidis, S. pneumoniae, and human microbiome samples, and prevention of bacterial adherence to the tube. Additionally, the released Lasio did not cause any cytotoxicity toward laryngotracheal fibroblasts or lung epithelial cells in vitro. CONCLUSION: Overall, we demonstrate the design of an effective-eluting ET tube to modulate upper-airway bacterial infections during intubation which could be deployed to help prevent SGS. LEVEL OF EVIDENCE: NA Laryngoscope, 132:1356-1363, 2022.

Drug delivery to the pediatric upper airway.

Pediatric upper airway disorders are frequently life-threatening and require precise assessment and intervention. Targeting these pathologies remains a challenge for clinicians due to the high complexity of pediatric upper airway anatomy and numerous potential etiologies; the most common treatments include systemic delivery of high dose steroids and antibiotics or complex and invasive surgeries. Furthermore, the majority of innovative airway management technologies are only designed and tested for adults, limiting their widespread implementation in the pediatric population. Here, we provide a comprehensive review of the most recent challenges of managing common pediatric upper airway disorders, describe the limitations of current clinical treatments, and elaborate on how to circumvent those limitations via local controlled drug delivery. Furthermore, we propose future advancements in the field of drug-eluting technologies to improve pediatric upper airway management outcomes.

The Investigation into the Toxic Potential of Iron Oxide Nanoparticles Utilizing Rat Pheochromocytoma and Human Neural Stem Cells.

Magnetic iron oxide (Magnetite, Fe3O4) nanoparticles are widely utilized in magnetic resonance imaging (MRI) and drug delivery applications due to their superparamagnetism. Surface coatings are often employed to change the properties of the magnetite nanoparticles or to modulate their biological responses. In this study, magnetite nanoparticles were fabricated through hydrothermal synthesis. Hydrophobicity is often increased by surface modification with oleic acid. In this study, however, hydrophobicity was introduced through surface modification with n-octyltriethoxysilane. Both the uncoated (hydrophilic) and coated (hydrophobic) individual nanoparticle sizes measured below 20 nm in diameter, a size range in which magnetite nanoparticles exhibit superparamagnetism. Both types of nanoparticles formed aggregates which were characterized by SEM, TEM, and dynamic light scattering (DLS). The coating process significantly increased both individual particle diameter and aggregate sizes. We tested the neurotoxicity of newly synthesized nanoparticles with two mammalian cell lines, PC12 (rat pheochromocytoma) and ReNcell VM (human neural stem cells). Significant differences were observed in cytotoxicity profiles, which suggests that the cell type (rodent versus human) or the presence of serum matters for nanoparticle toxicology studies. Differences in nanoparticle associations/uptake between the two cell types were observed with Prussian Blue staining. Finally, safe concentrations which did not significantly affect neuronal differentiation profiles were identified for further development of the nanoparticles.

A Novel Cell Penetrating Peptide for the Differentiation of Human Neural Stem Cells.

Retinoic acid (RA) is a bioactive lipid that has been shown to promote neural stem cell differentiation. However, the highly hydrophobic molecule needs to first solubilize and translocate across the cell membrane in order to exert a biological response. The cell entry of RA can be aided by cell penetrating peptides (CPPs), which are short amino acid sequences that are able to carry bioactive cargo past the cell membrane. In this work, a novel cell penetrating peptide was developed to deliver RA to human neural stem cells and, subsequently, promote neuronal differentiation. The novel CPP consists of a repeating sequence, whose number of repeats is proportional to the efficiency of cell penetration. Using fluorescence microscopy, the mode of translocation was determined to be related to an endocytic pathway. The levels of β-III tubulin (Tubb3) and microtubule associated protein 2 (MAP2) expression in neural stem cells treated with RA conjugated to the CPP were assessed by quantitative immunocytochemistry.