Chitosan Hydrogel Enriched with Propolis from Chihuahua, Mexico: Physicochemical Characterization and Exploratory In Vivo Evaluation in a Murine Second-Degree Burn Model

Background/Objectives: Second-degree burns require strategies that address microbial contamination, oxidative stress, exudate, and tissue repair. This study aimed to develop and evaluate a marine-derived chitosan hydrogel incorporating ethanolic extract of propolis (EEP) from Chihuahua, Mexico, for second-degree burn management. Methods: EEP was characterized using parameters established in NOM-003-SAG/GAN-2017 and complementary biological assays. Chitosan hydrogels containing 1%, 3%, and 5% (w/w) EEP were evaluated for physicochemical properties, swelling, mass loss, flavonoid release, antioxidant activity, antimicrobial performance, and cell viability. The 1% EEP hydrogel was further evaluated in a murine second-degree burn model. Results: Chihuahua propolis exhibited high phenolic (27.42 ± 2.53%) and flavonoid (9.23 ± 0.314%) contents. EEP incorporation modified the chitosan matrix, providing high swelling capacity, increased structural persistence, sustained flavonoid release for 72 h, and radical-scavenging activity for 96 h. Antimicrobial activity was concentration-dependent, with the 5% EEP hydrogel reducing recoverable Escherichia coli counts below the detection limit (<102 CFU/mL). However, EEP-containing hydrogels reduced NIH-3T3 viability below the 70% ISO 10993-5 threshold under static extraction conditions. At 144 h, the 1% EEP hydrogel produced greater wound contraction than silver sulfadiazine (p = 0.021), although it did not differ significantly from the propolis-free chitosan hydrogel or untreated control. Qualitative histological assessment showed features consistent with early tissue repair, with cutaneous appendages observed in several sections from the 1% EEP group. Conclusions: Chihuahua propolis-loaded chitosan hydrogels showed promising physico-chemical, release, antioxidant, antimicrobial, and short-term in vivo findings. The in vitro reduction in metabolic activity observed under static extraction conditions warrants further evaluation using physiologically relevant exposure models, extended follow-up, and comprehensive safety assessment.