In Situ Mineralization of Alginate Hydrogels with Nisin: A Bioactive Material for Bone Applications

The rise of antibiotic‑resistant pathogens in osteoarticular infections underscores the need for alternative antimicrobial strategies acting locally within bone tissue. Antimicrobial peptides (AMPs) such as nisin offer broad‑spectrum activity and low resistance potential, yet their clinical use is limited by rapid degradation and systemic clearance in vivo. To address this limitation, we developed alginate hydrogels capable of encapsulating and protecting nisin while enabling localized antimicrobial action. These hydrogels exhibited clear antibacterial activity against Staphylococcus aureus and S. epidermidis in a 3D agar diffusion assay, generating inhibition halos of 1.84 ± 0.34 mm in width. They also achieved complete eradication of planktonic cultures, corresponding to a ≥5-log reduction in CFU.

To enhance the bone related properties of the system, the hydrogels were mineralized using complementary strategies: (i) encapsulation of preformed calcium phosphate (CaP) powders (hydroxyapatite and β-TCP), and (ii) a novel in situ alginate mineralization method designed to improve the stability and homogeneity of crosslinking and CaP distribution throughout the hydrogel. Notably, hydrogels prepared with our novel method showed enhanced storage stability, remaining active for at least two additional weeks compared to standard preparations. Varying the duration of in situ mineralization enabled the formation of distinct CaP phases, such as hydroxyapatite and brushite, as confirmed by XRD. Importantly, CaP functionalization did not compromise antimicrobial performance, with average halo widths of 1.71 ± 0.30 mm. Preliminary analyses indicate that mineralization increases the mechanical robustness of the hydrogels, with further mechanical, rheological, and stability studies underway. These characterizations will guide the selection of the final application format (injectable, mouldable, or scaffold-like). Ongoing work also focuses on assessing bioactivity through ion release profiling and apatite formation in SBF.

Overall, this work presents a promising antimicrobial and bioactive hydrogel platform that integrates AMP delivery with CaP mineralization, offering potential for localized infection control and bone regeneration in osteoarticular applications.