Authors: Antoci Valentin Jr. BS PhD, Ketonis Constantinos BS, Davidson Helen M. BS, Parvizi Javad MD FRCS, Freeman THerese A. PhD, Adams Christopher S. PhD, Shapiro Irvine M. PhD, Hickok Noreen J. PhD; Rothman Institute, Philadelphia, PA
Title: Novel Antibiotic-modified Implant Inhibits Bacterial Attachment After Multiple Challenges
Purpose: In this study, we assess whether an implant that is covalently modified with antibiotics retards bacterial biofilm formation after multiple bacterial challenges and when covered with serum proteins. We also test its effect on osteoblast adhesion.
Methods: To modify the surface, 1 mm diameter Ti6Al4V wires were passivated and coupled sequentially with two linkers and vancomycin. This vancomycin-derivatized surface, in parallel with underivatized surfaces, was challenged with S. epidermidis in tryptic soy broth (TSB) under static conditions, at 37?XC for 24 h. Bacterial colonization was evaluated by immunofluorescent microscopy using the Cyto9 vital stain as well as by direct bacterial counts. Scanning electron microscopy and retention of crystal violet were used to measure biofilm formation. The specificity of the immobilized antibiotic was tested using the gram negative organism E. coli as vancomycin has no activity against this organism. To simulate clinical scenarios, in this case, delayed repeat infection, the implants were challenged with S. epidermidis, stripped by detergent lysis, and rechallenged up to 7 times. To simulate the complex environment of implantation, rods were precoated with FBS and coverage with serum proteins was verified by detection of fibronectin and vancomycin using immunohistochemistry. The serum-coated surface was also challenged with bacteria and evaluated for bacterial adhesion using the Cyto9 staint. Finally, to assess the interaction of bone cells with the derivatized surface, osteoblastic MC3T3 cells were cultured on vancomycin-modified rods and morphology evaluated by CellTracker Green.
Results: The vancomycin-Ti rod inhibited S. epidermidis biofilm formation (Ci L104 cfu) over the 30 h of incubation. Specifically, vancomycin-modified surfaces showed minimal biofilm formation as detected by scanning electron microscopy, cyto9 staining, and crystal violet staining. In contrast, control titanium was encased in an abundant bacterial slime at the same time points, with extensive surface colonization, and biofilm formation. Furthermore, the vancomycin-modified surface inhibited bacterial coverage even in the presence of fibronectin and similar serum proteins, suggesting that it would retain its activity in a complex physiological environment. Importantly, when chronically exposed to bacteria by repeated strippings and re-challenge, the vancomcyin-modified surface continued to inhibit bacterial colonization even after up to seven 24 h challenges. Vancomycin-modified titanium showed significantly less bacteria and biofilm formation under all conditions. Despite its antibacterial effects, the vancomycin-modified surface allowed osteoblast colonization and proliferation, suggesting that normal biocompatibility and bone growth is possible after clinical implantation.
Discussion and Conclusion: Eradication of periprosthetic infection is challenging mostly due to formation of biofilm. We have demonstrated that vancomycin covalently bound to Ti implants not only inhibits immediate bacterial colonization, but can inhibit biofilm formation after multiple challanges. Further, the surface maintains activity even after coverage by serum proteins. The observed effects were specifically due to the antibiotic and not the chemistry or other physical changes to the surface, as the modified surface had no effect on E. coli colonization. Therefore, vancomycin-modified surfaces are successful antibacterial tools against PPI while still maintaining a compatible environment for bone growth.
