Optimizing Vancomycin Soaking Protocols for Anterior Cruciate Ligament Reconstruction

<b>Background/Objectives</b>: Although current guidelines recommend soaking anterior cruciate ligament autografts in 5 mg/mL vancomycin for 20 min to reduce postoperative infections, practical constraints often limit soaking to 5 min. This study aimed to evaluate the bacterial eradication efficacy and cytotoxicity of various vancomycin concentrations and application methods within a 5 min soaking period. <b>Methods</b>: Human semitendinosus tendons were inoculated with one of four bacterial pathogens, including <i>Staphylococcus aureus</i>, <i>Staphylococcus epidermidis</i> with biofilm-producing and non-biofilm-producing strains, and <i>Enterococcus faecalis</i>. Samples were treated by direct soaking in 5 mg/mL vancomycin or gauze wrapping with 2.5, 5, or 10 mg/mL vancomycin for 5 min. Bacterial elimination was assessed using agar plating. Cytotoxicity toward human tenocytes and mesenchymal stem cells was evaluated at 6, 12, 24, and 72 h. Vancomycin release was measured using an immunofluorescence assay with the Cobas C311 Roche analyzer. <b>Results</b>: Complete bacterial eradication was achieved by direct soaking at 5 mg/mL and gauze wrapping at 10 mg/mL. All concentrations maintained cell viability above 70%, with no significant cytotoxicity. Vancomycin release was the highest in the direct soaking group, while it remained below the toxicity threshold for chondrocytes. <b>Conclusions</b>: Direct soaking at 5 mg/mL and gauze wrapping at 10 mg/mL for 5 min effectively eradicated bacterial contamination without compromising cell viability.