In Vitro Investigation of Vocal Fold Cellular Response to Variations in Hydrogel Porosity and Elasticity

Abstract

Tissue regeneration is intricately influenced by the dynamic interplay between the physical attributes of tissue engineering scaffolds and the resulting biological responses. A tunable microporous hydrogel system was engineered using gelatin methacryloyl (GelMA) and polyethylene glycol diacrylate (PEGDA), with polyethylene glycol (PEG) serving as a porogen. Through systematic variation of PEGDA molecular weights, hydrogels with varying mechanical and architectural properties were obtained. The objective of the present study was to elucidate the impact of substrate mechanics and architecture on the immunological and reparative activities of vocal fold tissues. Mechanical characterization of the hydrogels was performed using tensile strength measurements and rheometry. Their morphological properties were investigated using scanning electron microscopy (SEM) and confocal microscopy. A series of biological assays were conducted. Cellular morphology, differentiation, and collagen synthesis of human vocal fold fibroblasts (hVFFs) were evaluated using immunostaining. Fibroblast proliferation was studied using the WST-1 assay, and cell migration was investigated via the Boyden chamber assay. Macrophage polarization and secretions were also examined using immunostaining and ELISA. The results revealed that increasing the molecular weight of PEGDA from 700 Da to 10,000 Da resulted in decreased hydrogel stiffness, from 62.6 to 8.8 kPa, and increased pore dimensions from approximately 64.9 to 137.4 μm. Biological evaluations revealed that hydrogels with a higher stiffness promoted fibroblast proliferation and spreading, albeit with an increased propensity for fibrosis, as indicated by a surge in myofibroblast differentiation and collagen synthesis. In contrast, hydrogels with greater molecular weights had a softer matrix with expanded pores, enhancing cellular migration and promoting an M2 macrophage phenotype conducive to tissue healing. The findings show that the hydrogels formulated with a PEGDA molecular weight of 6000 Da are best among the hydrogels considered for vocal fold repair. The microporous hydrogels could be tuned to serve in other tissue engineering applications.