Functionalization of titanium implants with phase-transited lysozyme for gentle immobilization of antimicrobial lysozyme

This work deals with the implementation of a gentle method for the coating of titanium surfaces with lysozyme in order to prevent microorganisms adhesion and proliferation and, consequently, implant failure. Titanium medical grade 5 (Ti6Al4V) discs having either polished surface or biomimetic surface (BAS) were pretreated with oxygen plasma to clean the surface and, at the same time, increase its hydrophilicity. Functionalization consisted in a first layer of phase-transited lysozyme (PTL) for durable anchorage. This layer was treated with citric acid which was used as a spacer for, in a final step, ionically bind bioactive lysozyme without altering its enzymatic activity. Functionalized discs were characterized using a variety of techniques (profilometry, SEM-EDX, FTIR-ATR, water contact angle, mechanical friction) and their enzymatic activity was demonstrated against M. lysodeikticus. Lysozyme functionalization prevented Staphylococcus aureus adhesion while maintaining the high cytocompatibility of the discs with relevant cell lines. Relevantly, stability of the coating was demonstrated after prolonged storage in PBS, freeze-drying cycles and conventional γ-ray sterilization protocols; the disc maintaining the enzymatic activity of lysozyme. Even after friction against bone-mimicking material resembling implantation screwing, the functionalized discs still retained more than 70% enzyme activity. Overall, this affordable approach could be useful to endorse titanium implants with antimicrobial properties.