A novel integrated <i>in vitro</i> method for evaluating the moisturizing performance of injectable sodium hyaluronate

Abstract Injectable sodium hyaluronate (NaHA) is extensively utilized in aesthetic medicine as a dermal hydrating agent. However, there are few standardized, human-relevant preclinical methods that can reliably evaluate the moisturizing performance of those products. Existing animal and simplified cell-based models show restricted physiological relevance and insufficient sensitivity. To address this gap, we established a depot-mimicking reconstructed human full-thickness skin (RhFS) platform incorporating an inclusion-based intradermal delivery strategy. Instead of conventional mixing, this strategy mimics the clinical “depot effect” of intradermal injection, allowing for the simultaneous assessment of cellular responses and tissue-level hydration dynamics. It forms a localized NaHA depot within the dermal compartment of RhFS and preserves spatial hydration gradients, which are lost when NaHA is mixed homogenously. The platform integrates physicochemical characterization of polymer-bound water states with cellular and tissue-level functional readouts. By quantifying key biomarkers, including CD44, aquaporin-3 (AQP3), and natural moisturizing factors (NMFs), our results demonstrate that the inclusion-based delivery strategy significantly outperforms conventional mixing approaches in activating epidermal hydration pathways. Crucially, this platform effectively distinguished the moisturizing efficacy of multiple commercial NaHA formulas, thereby revealing a structure-activity relationship between water-binding states and biological outcomes. Overall, this study presents a reproducible, mechanism-informed, and human-relevant framework for preclinical performance evaluation of NaHA-based injectable biomaterials and provides a sensitive alternative to conventional animal-based approaches.