Freezing Nitrogen-Ethanol Composite Effectively Eradicates Staphylococcus aureus Biofilm on Prosthetic Surfaces

Periprosthetic joint infection (PJI) remains one of the most challenging complications after arthroplasty, largely due to biofilm formation on prosthetic surfaces, which protects bacteria from antibiotics and host defenses. This study investigated whether a freezing nitrogen-ethanol composite (FNEC), a semisolid cryogenic material combining liquid nitrogen and ethanol, can effectively eradicate Staphylococcus aureus biofilms on prosthetic components.

Biofilms of S. aureus were established on plastic spacers and metallic knee prostheses. Specimens were treated for 15 min with 75% ethanol, liquid nitrogen (LN), or FNEC. Bacterial viability was assessed using LIVE/DEAD fluorescence staining, while eradication efficacy was confirmed through broth inoculation and optical-density (OD₆₀₀) measurements. Additional indirect-exposure experiments evaluated the contribution of FNEC's freezing effect, and time-dependent testing determined the minimal effective exposure duration.

FNEC achieved the most complete biofilm eradication across both plastic and metallic surfaces. LIVE/DEAD staining demonstrated widespread cell death with minimal residual viability. Broth cultures from FNEC-treated samples remained clear, with OD₆₀₀ values indistinguishable from sterile controls, whereas LN and untreated samples showed significant bacterial growth. Indirect experiments confirmed that FNEC's cryogenic effect alone substantially contributed to bacterial death. Time-course analysis revealed that a 5-min FNEC exposure was sufficient for complete sterilization.

FNEC exhibited potent bactericidal and biofilm-removal capability, outperforming ethanol and LN alone. Its combined chemical and cryogenic effects enabled rapid and complete eradication of S. aureus biofilms within 5 min. These findings support FNEC as a promising adjunctive strategy for intraoperative biofilm management and may facilitate single-stage revision surgery for PJI.