UNDERSTANDING THE BACTERICIDAL POTENTIAL OF NANOSTRUCTURED CALCIUM PHOSPHATES (continuation)

One of the main challenges we face when using synthetic bone grafts to restore bone function is the risk of infection. One strategy to fight infection is to use synthetic bone grafts with bactericidal topographies consisting of nanopillars capable of deforming and breaking down the bacterial wall. We have recently developed synthetic calcium phosphates with potentially bactericidal nanotopographies. However, the precise mechanisms governing this antimicrobial process remain elusive. Moreover, being CaP a bioactive material, the ionic exchanges with the environment can influence the microbial response. To elucidate these mechanisms, we propose cryo-soft X-ray tomography and spectromicroscopy at the Ca L2,3-absorption edge. In this study, bacteria (Bacillus subtilis, BS) were incubated on CaP materials with different nanotopographies (small and large pillars) and control surfaces (flat CaP and glass). Tomography at the Ca L2-edge allowed to detect higher levels of Ca concentration in the l cytosol of some bacteria exposed to the CaP surfaces, revealing a gradient: flat CaP > large pillars > small pillars > glass coverslip. Ca maps at the Ca L2-edge visualized the bacteria's shape on the CaP materials but not on the glass coverslip. XANES spectra confirmed the presence of Ca within some bacteria. Moreover, tomographies at 520 eV (maximum carbon absorption contrast) revealed notable differences in the cellular ultrastructure between BS incubated on small CaP pillars and the glass control. These differences included the formation/absence of multivesicles, changes in membrane integrity, and varying degrees of DNA condensation. However, due to the limited time, we were not able to analyse the samples of bacteria incubated on large nanopillars and a flat CaP surface. We request 1 day (3 shifts) to acquire tomographies at 520 eV of bacteria incubated on these two surfaces to complete our study.