S32 - Studying the Effect of Vibrations on Human Vocal Fold Fibroblasts in 3D Hydrogels

Introduction: : Approximately 9% of the general population has a voice abnormality during their lifespan [1-3]. One of the severe conditions affecting voice is when part of the soft and pliable mucosa is lost or replaced by dense fibrous tissue, leading to chronic vocal fold (VF) fibrosis [4, 5]. VF fibrosis can be congenital or acquired following phono-trauma or chronic inflammatory pathologies [6]. Chronic VF fibrosis can be treated locally using a subepithelial injection of VF biomaterials or biological agents [7-9]. This study aims to develop a hydrogel bioreactor model replicating the VF's mechanical properties. Using Gelatin Methacrylate (GelMA), the model provides a physiologically relevant platform to study VF fibrosis and evaluate potential therapies to restore VF functionality. We investigated whether mechanical vibration inhibits TGF-β1 induced myofibroblast differentiation in human vocal fold fibroblasts (HVOX), under both two-dimensional (2D) and three-dimensional (3D) culture conditions.

Materials and

Methods: : Human-derived VF fibroblasts (HVOX; with the courtesy of Prof. Ryan Branski, New York University, NY), cultured in both 5% gelatin methacrylate (GelMA) hydrogel at a concentration of 500K cells/ml and a well-plate at a concentration of 40K cells/ml.TGF-β1 was added to the cells at a final concentration of 10 ng/mL to induce fibroblast-to-myofibroblast differentiation at 80% cell confluency. After addition, cells were further incubated for 12 to 72 hours (Shown in Fig. 1A). Standard compression testing, swelling, and degradation test to characterize the material model were performed. The hydrogels were characterized and mechanical properties were monitored over one week to assess consistency with VF tissue. Cell viability was tracked using a metabolic activity assay (CCK8, Sigma-Aldrich), while cell phenotypes and behavior were analyzed via immunostaining for different time points. To simulate vibratory mechanical stimulation, culture plates were placed on an orbital shaker at 900 rpm for two 1-hour intervals with intervening rest period. Immunofluorescence staining was performed for α-SMA1, Phalloidin, and DAPI. High-resolution images were analyzed using ImageJ to quantify α-SMA expression levels. α-SMA immunofluorescence intensity which is the indicative of myofibroblast differentiation was compared across four conditions: 2D static, 2D vibration, 3D static, and 3D vibration in both healthy and fibrotic samples. Statistical analyses (t-tests/ANOVA) were used to evaluate differences between vibrated and non-vibrated groups, with significance set at p < 0.05.

Results, Conclusions, and Discussions:: Compressive stiffness of GelMA hydrogels (3, 5, and 7 % w/v) and hybrid GelMA/HAMA constructs (0.5 % or 1 % HAMA) was quantified. Increasing GelMA content raised the compressive modulus from ~1.8 kPa (3 % GelMA) to ~3.3 kPa (7 % GelMA). Incorporation of HAMA further elevated stiffness, with the 7 % GelMA + 1 % HAMA formulation reaching ~4.6 kPa. Equilibrium swelling ratios were slightly higher in HAMA‐containing gels (up to ~15.5) than in pure GelMA (~14), with swelling kinetics plateauing by day 3. Live/dead assays indicated reduced cell viability in GelMA/HAMA constructs compared to GelMA-only hydrogels. Overall, these tunable hydrogel constructs recapitulate the stiffness range of healthy (1.8–3.3 kPa) to fibrotic (up to 4.6 kPa) lamina propria for mechanobiology studies.
The phenotypic response of HVOX to mechanical vibration was assessed via immunofluorescent staining for α-SMA (red), F-actin (phalloidin), and nuclei (DAPI, blue) in both 3D hydrogel constructs and 2D monolayers. TGF-β1 treatment in static cultures markedly upregulated α-SMA and induced pronounced F-actin stress fibers, confirming myofibroblast differentiation. In contrast, vibrated samples exhibited significantly lower α-SMA fluorescence intensity and fewer stress fibers, adopting an elongated, spindle morphology. Quantitative image analysis confirmed statistical significance (p < 0.05) in fluorescence intensity differences between static and vibrated samples. Mechanically stimulated HVOX cells in both 2D and 3D cultures exhibited decreased fibrotic activation markers, highlighting the modulatory effect of vibration on myofibroblast differentiation within hydrogel-based models of vocal-fold fibrosis. Our findings indicate that the vibratory stimulus attenuated the myofibroblast differentiation process.

Acknowledgements and/or References (Optional):: 1. Li, L., et al., Tissue engineering-based therapeutic strategies for vocal fold repair and regeneration. Biomaterials, 2016. 108: p. 91-110.
2. Bhattacharyya, N., The prevalence of voice problems among adults in the United States. Laryngoscope, 2014. 124(10): p. 2359-62.
3. Roy, N., et al., Voice disorders in the general population: prevalence, risk factors, and occupational impact. Laryngoscope, 2005. 115(11): p. 1988-95.
4. Nakamura, R., et al., Concurrent YAP/TAZ and SMAD signaling mediate vocal fold fibrosis. Sci Rep, 2021. 11(1): p. 13484.
5. Xu, H. and G.-K. Fan, The Role of Cytokines in Modulating Vocal Fold Fibrosis: A Contemporary Review. The Laryngoscope, 2021. 131(1): p. 139-145.
6. Velier, M., et al., Paracrine Effects Of Adipose-Derived Stromal/Stem Cells And Stromal Vascular Fraction In An In Vitro Fibrogenesis Model Of Human Vocal Fold Scarring. 2020.
7. Hess, M.M. and S. Fleischer, Laryngeal framework surgery: current strategies. Curr Opin Otolaryngol Head Neck Surg, 2016. 24(6): p. 505-509.