INTRODUCTION: Fracture fixation devices (FFD) have infection rates ranging from 5 to 10% for closed fractures and even higher rates up to 30% for open fractures1 causing a devastating complication. Clinically, implant infections are prevented by systemic antibiotic prophylaxis, the placement of antibiotic laden bone cements, and the use of minimally invasive surgical procedures2. However, conventional biomaterials such as bone cement are compatible with a limited number of antibiotics, and they show poor antibiotic release profiles which can lead to antimicrobial resistant development.
Cationic antimicrobial peptides (AMPs) have shown to successfully kill antimicrobial resistant bacteria. However, the application of AMPs in FFD is quite limited. Therefore, we studied the incorporation of the AMP SAAP-1483 in a polymeric coating printed by drop on demand (DOD) on titanium fixation plates which were 3D printed using the selective laser melting (SLM) technology. These devices were tested in vitro and in vivo in a biomaterial-associated infection mouse model against Staphylococcus aureus.
METHODOLOGY: SAAP-148 was loaded into a polymer-based composite ink and printed by DOD printer on SLM 3D printed titanium coupons and implants. The release of the peptide was carried out in phosphate buffer saline solution at 37ºC and 120 rpm. The concentration of the peptide was measured by the µBCA assay. The antimicrobial experiments were performed with S. aureus JAR 06.01.31, an orthopaedic clinical strain. A JIS assay was used to evaluate the surface microbicidal activity of the coating in vitro. The antimicrobial efficacy of the coatings was evaluated in an in vivo subcutaneous biomaterial-associated infection mouse model inoculated with 25 µL containing 106 colony forming units (CFU) of S. aureus, and evaluated by quantitative culture at 1 and 4 days post-surgical infection.
RESULTS: The release assay showed a burst release of SAAP-148 during the first 1 and 24 hours, with 300 µg/mL and 200 µg/mL, respectively, followed by a sustained released of 40 to 60 µg/mL for the next 14 days. Moreover, the coating releasing SAAP-148, exhibited a complete killing in vitro and a significant reduction in numbers of CFU at 1 day post-surgery in the biomaterial and surrounding tissue in the in vivo biomaterial-associated mouse model compared to uncoated titanium.
CONCLUSIONS: The peptide was successfully incorporated in the polymer-based composite showing proper release kinetics and successful antimicrobial activity in vivo. The next step is to evaluate the antibacterial activity of the coating in a bone fixation plate infection mouse model.
ACKNOWLEDGEMENTS: This research was funded by the research project PRINTAID, the EU Framework Programme for Research and Innovation within Horizon 2020 - Marie Skłodowska-Curie Innovative Training Networks under grant agreement No. 722467.
1 Depypere M., et al., Pathogenesis and management of fracture-related infection. Clin. Microbiol. Infect. 26: 572–578 (2020)
2 Kavanagh, N., et al. Staphylococcal osteomyelitis: disease progression, treatment challenges, and future directions. Clinical microbiology reviews, 31(2), e00084-17. (2018)
3 de Breij, A., & Riool, M., et al., The antimicrobial peptide SAAP-148 combats drug-resistant bacteria and biofilms. Science Translational Medicine, 10(423) (2018).