H20 - Towards an Inhalable Tissue-Specific Extracellular Matrix-based Substrate for Targeted Laryngeal Tracheal Regeneration.

Introduction: : Each year, an estimated 28 million Americans experience dysphonia-related pathologies. VF fibrosis, a significant contributor to dysphonia, leads to VF scarring and is often triggered by procedures such as endotracheal intubation. The severity of the lesions may require laryngeal surgery, which often results in further vocal fold scarring, worsening the patient's condition. Current technologies for VF regeneration are limited. Clinicians enhance the voice fold (VF) with various biomaterials to achieve glottic closure, but with minimal long-term success. Our laboratory has developed an injectable, porcine-derived vocal fold lamina propria extracellular matrix hydrogel (VFLP-ECMh) that preserves native proteins and peptides, demonstrating anti-fibrotic properties in vitro. While this is promising, the injection of these materials requires repeated invasive procedures, implant monitoring, and potentially multiple doses. The objective of this study was to develop a minimally invasive alternative to VFLP-ECM that can be nebulized at therapeutic doses while maintaining its bioactive properties.

Materials and

Methods: : Nebulizable ECM (NebECM) formulation was developed from VFLP-ECM, which can be delivered directly to the larynx using a commercially available ultrasonic mesh nebulizer. The particle size and delivery dose of NebECM were quantified utilizing dynamic light scattering and ultraviolet-visible spectroscopy techniques, respectively. To evaluate the preservation of ECM-related proteins in the NebECM, we conducted discovery proteomics analysis. To assess the cytocompatibility of the NebECM, we cultured Human Vocal Fibroblasts (HVOX) with Neb-ECM and growth medium for 24 hours. Subsequently, we examined changes in cell metabolic activity and viability using the MTT, PicoGreen, and LIVE/DEAD assays. Additionally, we investigated the impact of NebECM on the TGF-β1-mediated activation of HVOX into myofibroblasts and initial gene expression using Nanostring gene expression assays.

Results, Conclusions, and Discussions:: The final NebECM, before nebulization, was shown to contain particles with diameters of approximately 1 micrometer. Cytotoxicity studies demonstrated that the NebECM does not decrease cell metabolism or alter growth rates. We observed a reduction in ACTA2 gene expression, a key indicator of fibrosis, in HVOX treated with NebECM in the presence of TGF-β1. We’ll evaluate proteomics analysis data to determine whether proteins interacting with TGF- β1 are retained after nebulization.

Acknowledgements and/or References (Optional)::