The treatment of chronic wounds remains a major challenge in regenerative medicine due to prolonged healing times, susceptibility to infection, and underlying conditions like diabetes. Incorporating bioactive and antibacterial nanoparticles (NPs) into wound dressings can significantly enhance their mechanical properties, structural integrity, and functionality, improving stability, biocompatibility, and healing efficacy. However, conventional methods of loading NPs in polymer matrices often lead to uneven distribution and localized toxicity. To overcome these limitations, we employ a novel in situ synthesis of copper nanoparticles (CuNPs) using an encapsulation method via the self-assembled polymerization of dopamine (DOPA) or tannic acid (TA) within collagen/carboxymethyl cellulose (Coll/CMC) 3D freeze-dried scaffolds. When CuNPs are synthesized ex situ, both DOPA and TA act as reducing and encapsulating agents. However, in situ synthesis within Coll/CMC scaffolds results in TA functioning solely as a reducing agent, while DOPA serves both as a reducing agent and, through its polymerization into polydopamine, as a stabilizing agent. The polydopamine network enhances collagen fiber adhesion to CuNPs and stabilizes them via noncovalent interactions. Notably, the DOPA-in situ/Cu sample exhibited prolonged enzymatic stability for up to 7 days. X-ray microcomputed tomography confirmed the homogeneous distribution of CuNPs throughout the scaffold. Biological assays demonstrated the enhanced antibacterial efficacy of DOPA/TA-in situ/Cu samples against Staphylococcus aureus and MRSA, along with cytocompatibility with 3T3 fibroblasts. Future research should explore the in vivo application of these scaffolds and their potential in regenerative medicine for treating infected wounds.

Central European Institute of Technology Brno University of Technology Purkynova 123 612 00 Brno Czech Republic

Department of Chemistry and Biochemistry Faculty of AgriSciences Mendel University in Brno Zemedelska 1 613 00 Brno Czech Republic

Faculty of Mechanical Engineering Brno University of Technology Technicka 2 616 69 Brno Czech Republic

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