Single-Cell Adhesion, Biofilm Formation, and Disinfectant Susceptibility of Candida albicans on CAD/CAM-Manufactured Occlusal Splint Resins

Occlusal splints used for bruxism prevention can accumulate Candida albicans biofilms, highlighting the need to evaluate disinfectant efficacy. Objectives: We aimed to evaluate the nanomechanical properties and nanoadhesion force of C. albicans cells and the efficacy of disinfectant solutions on three occlusal splint resins: 3D Bio-Splint (3D-printed), EvoBlock Monocor (milled), and Vipicril (conventional heat-polymerized resin, control). Methods: Circular specimens (10 × 1.2 mm; n = 60 per resin for the microbiological assays) were polished, immersed in artificial saliva for 56 h, and subjected to 70 brushing cycles to simulate one week of use. After mature C. albicans biofilm formation, specimens were disinfected for 10 min with 0.25% sodium hypochlorite, 2% chlorhexidine, sodium perborate (Corega Tabs), 0.1% PHMB, or PBS (control). CFU/mL counts, XTT assay, and scanning electron microscopy (SEM) analyses were performed. The surface roughness, Young’s modulus, and real-time adhesion of individual C. albicans cells onto the resins were measured by employing an atomic force microscopy (AFM) and AFM-based single-cell force spectroscopy (SCFS) with functionalized C. albicans probes. Results: The 3D-printed resin was the roughest material. Single-cell adhesion was characterized descriptively: the force and energy distributions obtained on the milled resin were shifted towards lower values in all four cell probes, whereas those obtained on the conventional and 3D-printed resins were similar. For biofilm formation, resin type, disinfection protocol, and their interaction significantly affected outcomes (p < 0.001). The printed resin showed the highest microbial load in the control group (63.4 × 106 CFU/mL), whereas the milled resin showed the lowest (3.9 × 105 CFU/mL). Chlorhexidine eliminated CFU counts in all resins, although residual metabolic activity ranged from 10.4% to 40.4%. PHMB on the printed resin showed the greatest discrepancy between residual CFU (4.9% of the control) and cell viability (93.4%), suggesting the persistence of metabolically active cells despite the reduction in cultivable microorganisms. The AFM nanocharacterization showed median Young’s modulus values of 9.92 GPa for the milled resin, 6.44 GPa for the 3D-printed resin, and 4.92 GPa for the conventional resin. Conclusions: The 3D-printed resin showed the highest surface roughness at nanoscale and was more susceptible to microbial colonization than the milled resin and more resistant to the effect of the disinfectants. Chlorhexidine was the most effective disinfectant, since it reduced the CFU to zero and significantly reduced the metabolic activity of C. albicans for all the occlusal splint resins.