Azithromycin-loaded chitosan films: a multifunctional platform for oral tissue regeneration.

This study aimed to create and benchmark azithromycin-loaded chitosan composite films optimized for oral regenerative surgery using a systematic 32 design-of-experiments (DoE) model. Nine formulations (CS LMW/MMW/HMW; 1-3% gelatin; 1-3% glycerol) were evaluated for solid-state interactions, mechanical response, swelling, drug-release kinetics, bioactivity, antimicrobial efficacy, and post-hydration surgical handling. FTIR and SEM confirmed the presence of a physically integrated polymer network. Mechanical testing revealed formulation-dependent flexibility, with the optimal membrane (CS HMW, 2% gelatin, 1% glycerol) exhibiting a Young's modulus of 82.43 kPa and a yield stress of 2569.6-2620.1 kPa, thereby balancing stiffness and plastic deformation. All films ensured zero-order azithromycin release (R2 = 0.96-0.98) with anomalous diffusion contribution (Peppas n = 0.41-0.46). Swelling index and dissolution were strongly correlated (r = 0.84), while sustained-release profiles aligned with enhanced anti-inflammatory effects. At clinically relevant extract levels, films preserved viability of fibroblasts and osteoblasts (MTT) for at least 72 h, whereas pure AZC at 600 µM induced cytostatic effects. Scratch assays confirmed that chitosan supports cell migration, while azithromycin provides antibacterial and local immunomodulatory activity without stimulating proliferation. Films produced clear growth inhibition zones against K. pneumoniae and S. aureus, with no antifungal effect on C. albicans. After controlled hydration in human blood and iPRF, membranes remained non-permeating barriers, gained temporary adhesiveness, and retained adapted shape. Ex vivo porcine mandible and maxilla models demonstrated strong adhesion, elasticity after hydration, and stability during suturing and bone-augmentation coverage without the need for fixation. This platform offers predictable, sustained drug release, tunable mechanics, and hydration-triggered conformability, along with quantifiable antibacterial and anti-inflammatory bioactivity, thereby supporting future preclinical in vivo oral regeneration studies.