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Objectives and questions: The increasing prevalence of antibiotic resistance progressively compromises the efficacy of conventional antibiotic therapies and contributes to persistent, difficult-to-treat infections. Consequently, alternative antimicrobial strategies are urgently needed. In this context, photosensitizer (PS)-loaded biomaterials represent a promising approach, particularly when combined with conventional antibiotic-based therapies, as they may improve localized drug biodistribution and enhance control of infection sites. This is especially relevant in the management of orthopaedic infections. This study aims to develop multifunctional hybrid hydrogel-fibrous dressings for the management of difficult-to-treat orthopaedic wound infections. Methylene blue (MB)-loaded hydrogels were synthetized to evaluate their photoantimicrobial activity against Staphylococcus aureus strains, as well as their biocompatibility with Saos-2 cells, in order to identify optimal hydrogel compositions for wound treatment.

Material and methods: Synthetic hydrophilic polymer aqueous solutions, with or without oil addition, were supplemented (or not) with MB and crosslinked in order to formulate stable photosensitizer-loaded hydrogels. For in vitro experiments, the different hydrogel formulations were eluted in PBS for 24 h at 37°C under agitation (100 rpm). Eluates were collected and mixed with bacterial suspensions of orthopaedic S. aureus strains to assess antimicrobial activity. In parallel, Saos-2 cells were exposed to the eluates to evaluate biocompatibility. In both cases, photodynamic treatment (PDT) was performed using light cycles at 650 nm. Antimicrobial efficacy was assessed by comparing bacterial growth onset times, correlated with CFU counts, between experimental groups. Cytocompatibility was determined using multiple cell viability assays (resazurin, neutral red uptake, and LIVE/DEAD staining). The MB release from the hydrogels was quantified using UV-Vis spectroscopy.

Results: Unloaded hydrogel formulations showed no antimicrobial activity, irrespective of light exposure. In contrast, all MB-loaded hydrogel formulations demonstrated antimicrobial effects against the clinical S. aureus strains following light irradiation. Oil supplementation within the hydrogel formulations significantly enhanced MB release during elution compared to the hydrogels without oil. All formulations were well tolerated by Saos-2 cells, with no detectable PDT-related cytotoxicity.

Discussion and conclusions: Overall, these results highlight the potential of MB-loaded hydrogels as a photoantimicrobial dressing to reduce S. aureus burden under controlled light activation. Ongoing studies aim to further optimize the system and evaluate its efficacy in infected Galleria mellonella larvae models.