Poster N20 - Microgel-mediated delivery of decorin promotes scarless vocal fold regeneration after injury

Introduction: Dysphonia is the most common communication disorder, with a lifetime prevalence of nearly 30% and an annual healthcare cost of approximately $1 billion. Vocal fold scarring (VFS) is the most common cause of dysphonia and is characterized by the differentiation of VF fibroblasts to contractile myofibroblasts, which secrete an excess of a collagen-rich extracellular matrix (ECM) leading to VF stiffening. The standard of care involves serial intracordal steroid injections which must be repeated every 4-6 weeks and are followed by 3 days of vocal rest. In addition to high patient burden, serial steroid injections show low clinical efficacy with scar recurrence rates reaching up to 50%. Intracordal steroid injections are also prone to off-target toxicities, causing epithelial thinning and mucosal gland atrophy. In extreme cases, injection laryngoplasty may be performed; here, physicians inject a bulking agent directly into the VFs to restore contact and improve speech. However, this approach does not promote tissue regeneration. Thus, there is a clear clinical need to develop a regenerative VFS treatment that targets specific pro-fibrotic mechanisms in a controlled manner. Herein, we identify decorin (DCN) as a novel regulator of VF fibroblast phenotype and VF regeneration. We then engineer DCN-loaded hyaluronic acid microgels (MGs) for controlled drug delivery over 21 days for application in an in vivo rat model of VFS.

Materials and

Methods: Human VF myofibroblasts were compared to VF fibroblasts (n=6) via bulk RNA sequencing to identify unique transcriptional signatures of each fibroblast phenotype. We confirmed findings by RT-qPCR and immunostaining. Results were then validated in vivo using our rat model of VFS via histology and immunostaining at 3-, 7-, and 21-days post-injury. To assess the anti-fibrotic effects of DCN, human VF fibroblasts were treated with or without TGF-β1 and DCN or bFGF (positive control). We evaluated the efficacy of DCN in preventing myofibroblast differentiation (prevention/early intervention) and also promoting myofibroblast de-differentiation (post-injury treatment). Myofibroblast differentiation was characterized by RT-qPCR, immunostaining, and contractility assays in 3D type I collagen hydrogels. To achieve controlled drug delivery, we engineered methacrylated hyaluronic acid microgels via extrusion fragmentation and passively loaded them with either FITC-BSA (model protein) or DCN. Size was determined via optical microscopy; drug release profiles were performed in PBS and 5U hyaluronidase to represent in vivo conditions and quantified via protein assay. We then confirmed that DCN-microgels retained anti-fibrotic efficacy by treating VF myofibroblasts with releasate from DCN-microgels after 3 days. To test the efficacy of DCN-microgels in vivo, we established a novel protocol for intracordal injections (directly into the VF) in rats.

Results, Conclusions, and Discussions: Bulk RNA sequencing revealed that DCN was among the most downregulated genes in the entire VF myofibroblast transcriptome, and RT-qPCR confirmed these findings (Fig. 1A). Further, immunostaining of scarred rat VFs 3-,7-, and 21-days after injury confirmed decreased DCN protein abundance in vivo (Fig. 1B-D). Human VF fibroblasts treated with TGF-β1+DCN were protected against TGFβ1-induced myofibroblast differentiation as shown by reduced α-SMA+ cells and pro-fibrotic gene expression; these findings were comparable to the positive control TGF-β1+bFGF treatment group (Fig. 1E&F). DCN also induced VF myofibroblast de-differentiation, as determined by reduced α-SMA+ cells and decreased pro-fibrotic gene expression (Fig. 1G&H). DCN was then successfully loaded into hyaluronic acid microgels with sustained drug release over approximately 3-4 weeks (Fig. 1I). Released drug retained anti-fibrotic activity (Fig. 1J&K). In addition, we successfully developed a method for intracordal injection of DCN-microgels which will be used to test in vivo efficacy of this technology. In total, we show that DCN is transcriptionally downregulated in VF myofibroblasts and decreased in scarred VFs. DCN prevents biochemically induced VF myofibroblast differentiation and promoted VF myofibroblast de-differentiation. DCN can be readily loaded into hyaluronic acid microgels for sustained drug delivery, which is being tested in a rat model of intracordal injection after VFS.

Acknowledgements (Optional):