The physical properties of a biomaterial play an essential role in regulating immune and reparative activities within the host tissue. This study aimed to evaluate the immunological impact of material stiffness of a glycol‐chitosan hydrogel designed for vocal fold tissue engineering. Hydrogel stiffness was varied via the concentration of glyoxal cross‐linker applied. Hydrogel mechanical properties were characterized through atomic force microscopy and shear plate rheometry. Using a transwell setup, macrophages were co‐cultured with human vocal fold fibroblasts that were embedded within the hydrogel. Macrophage viability and cytokine secretion were evaluated at 3, 24, and 72 hr of culture. Flow cytometry was applied to evaluate macrophage cell surface markers after 72 hr of cell culture. Results indicated that increasing hydrogel stiffness was associated with increased anti‐inflammatory activity compared to relevant controls. In addition, increased anti‐inflammatory activity was observed in hydrogel co‐cultures. This study highlighted the importance of hydrogel stiffness from an immunological viewpoint when designing novel vocal fold hydrogels.

The vocal folds are functional organs located in the larynx that play a critical role in our daily breathing, speech, and swallowing functions. A key structural feature of the vocal folds is their delicate mucosa, which consists of a thin, layered epithelium and underlying extracellular matrix (ECM)-rich lamina propria. [1,2] Irreversible changes to the vocal fold mucosa, such as scarring, atrophy, and sulcus vocalis, require regenerative technologies to stimulate a controlled regrowth of the tissue. An increasing number of biomaterial systems have been proposed with the concurrent capability of refilling and regenerating severely damaged or absent vocal fold mucosae in situ (see review on public health significance in the study by Green et al. [3] ).Notably, as the vocal folds are located at the narrowest point of the airway, adverse hostÀbiomaterial responses can impair upper airway patency and create life-threatening complications. For instance, 2À5% of patients were reported to have postoperative inflammatory reactions including edema, dysphonia, and dyspnea to injectable biomaterials derived from hyaluronic acid (HA). [4][5][6] Although the incidence is rare, severe airway reactions could lead to an emergency admission for intensive care and require intubation procedures for breathing support. [5,7] Inevitably, this immunemediated rejection response is detrimental to the longevity, performance, and integration of the biomaterial in the vocal fold mucosa.Upon biomaterial introduction into a host, the immune system quickly responds to the iatrogenic trauma and foreign