Periprosthetic infections are leading causes of revision surgery resulting in significant increased patient comorbidities and costs. Considerable research has targeted development of biomaterials that may eliminate implant-related infections.1 This in vitro study was developed to compare biofilm formation on three materials used in spinal fusion surgery – silicon nitride, PEEK, and titanium – using one gram-positive and one gram-negative bacterial species.
Materials and Methods
Several surface treated silicon nitride (Si3N4, MC2®, Amedica Corporation, Salt Lake City, UT), poly-ether-ether-ketone (PEEK, ASTM D6262), and medical grade titanium (Ti6Al4V, ASTM F136) discs Ø12.7 × 1mm were prepared or acquired for use in this well-plate study. Each group of discs (n=3) were ultrasonically cleaned, UV-sterilized, inoculated with 105Staphylococcus epidermidis (ATCC® 25922™) or Escherichia coli (ATCC® 14990™) and placed in a culture medium of phosphate buffered saline (PBS) containing 7% glucose and 10% human plasma on a shaking incubator at 37°C and 120 rpm for 24 or 48 hrs. Coupons were retrieved, rinsed in PBS to remove planktonic bacteria, placed in a centrifuge with fresh PBS, and vortexed. The bacterial solutions were serially diluted, plated, and incubated at 37°C for 24 to 48 hrs. Colony forming units (CFU/mm2) were counted using applicable dilution factors and surface areas. A two-tailed, heteroscedastic Student's t-test (95% confidence) was used to determine statistical significance.
Biofilm adhesion results are provided in Figures 1 and 2 for S. epi. and E. coli, respectively. For S. epi. at 24 hrs, biofilm growth on PEEK was about three orders of magnitude greater than on Ti6Al4V or any Si3N4 material (all p<0.005). Ti6Al4V also had more bacteria than the Si3N4 samples, but was only significant for as-fired and nitrogen-annealed treatments. Similar trends and significance for S. epi. were observed at 48 hrs. For E. coli, biofilm formation on PEEK was significantly greater than all other materials at both 24 and 48 hrs. Bacterial growth on Ti6Al4V was also statistically greater than all Si3N4conditions, with the possible exception of nitrogen-annealed Si3N4. By 48 hrs, PEEK remained 2 orders of magnitude above Ti6Al4V, and 2.5–3 orders of magnitude greater than the Si3N4 conditions. Ti6Al4V was also significantly greater than all of the Si3N4 treatments at 48 hrs.
Si3N4, PEEK, and Ti6Al4V surfaces demonstrated significant differences in bacterial adhesion and proliferation for both gram-positive S. epi. and gram-negative E. coli, particularly at 48 hrs post-inoculation. The various Si3N4 samples showed the most favorable bacterial resistance for both species tested. The exact mechanism of the bacteriostatic behavior of Si3N4 is still under investigation; but it may result from chemical interaction with the material's surface to form peroxynitrite (a powerful oxidative agent).2
Identifying biomaterial surfaces that resist biofilm adhesion is an important emerging strategy in addressing implant-related infections. Si3N4 is a new biomaterial with the apparent potential to suppress biofilm formation.