Controlled delivery of Cu2+ and Zn2+ ions from chitosan scaffolds: media-dependent release of ions and its influence on cellular responses relevant to wound healing.

Chronic wounds represent a major global healthcare burden, affecting a large patient population worldwide. A critical barrier to effective treatment is the hostile wound microenvironment, characterized by elevated reactive oxygen species and high protease activity, which lead to rapid degradation of growth factors and extracellular matrix components, thereby limiting the efficacy of conventional therapeutic strategies. Metal ions have emerged as promising alternatives in regenerative medicine due to their relative stability in such environments and their ability to regulate multiple cellular processes involved in tissue repair. In the present study, we investigate the therapeutic potential of copper (Cu2+) and zinc (Zn2+) ions incorporated into a chitosan-proline scaffold for enhancing wound healing responses. These ions exhibit concentration-dependent biological effects, where controlled levels promote collagen synthesis, cell migration, and cellular proliferation, while excessive concentrations can induce cytotoxic responses. Cytotoxicity assessment of ion solutions and extracts of ion-loaded scaffolds was performed to determine optimal concentrations that support cellular viability while maintaining therapeutic activity. Ion release behaviour in different media further provided insights into the release kinetics and helped identify concentrations suitable for biological applications. In vitro wound healing assays using fibroblast and keratinocyte cell lines demonstrated enhanced cell migration and proliferation in the presence of Cu2+ and Zn2+. Furthermore, treatment with these ions promoted collagen deposition in fibroblast cultures, indicating improved extracellular matrix formation. Gene expression analysis using RT-PCR revealed significant upregulation of wound healing markers, including collagen type I alpha 1 (Col1α1), heat shock protein 47 (HSP47), and vascular endothelial growth factor (VEGF) in L929 fibroblast cells. Collectively, these findings demonstrate that controlled delivery of therapeutic metal ions through the chitosan scaffold can effectively modulate cellular responses essential for tissue regeneration and may offer a promising innovative strategy for chronic wound management.