Synchrotron cryo-soft X-ray microscopy reveals bacterial interactions with nanostructured calcium phosphate substrates.

Nanostructured synthetic bone grafts offer a promising strategy to prevent bacterial colonisation while supporting bone regeneration. Here, calcium-deficient hydroxyapatite (CDHA) nanopillars synthesized from α-tricalcium phosphate exhibit contact-killing activity against Gram-positive Bacillus subtilis. Unlike inert bactericidal surfaces, CDHA exchanges ions with the surrounding environment, introducing chemical interactions alongside mechanical damage. Using synchrotron cryo-soft X-ray tomography and spectromicroscopy, we visualise bacterial ultrastructure and intracellular ionic changes in fully hydrated cells at subcellular resolution. Some bacteria exposed to the nanotopographies display membrane rupture, cytosolic leakage, and multivesicular body formation, indicating severe stress responses. XANES cryo-spectromicroscopy and linear absorption coefficient analysis identify a bacterial subpopulation with increased intracellular calcium, correlating with calcium release from the substrate and confirmed by confocal microscopy. However, this calcium accumulation does not affect viability, consistent with the halotolerant nature of B. subtilis. These findings provide insights into the combined effects of nanotopography and surface chemistry on bacterial responses.