Biofilm formation, its removal, and consequent effect on the osteoblast response to titanium surfaces: A model for re-osseointegration.

Abstract

The aim of the study was to characterize pathogenic biofilm formation on titanium surfaces, the ability to remove the biofilm and the osteoblast response to infected and cleaned titanium surfaces as a model for re-osseointegration. Multispecies biofilm composed of Pseudomonas. gingivalis, F. nucleatum, S. sanguis, and A. naeslundii were grown on smooth, acid-etched, and acid-etched-aluminum-sprayed titanium surfaces. Bacterial viability was determined with live/dead staining. The biofilm was removed mechanically or together with adjunctive antibiotics. The osteoblast (Saos2) response to previously infected, treated and non-infected titanium surfaces were measured according to 4'-6-diamidino-2-phenylindole staining. Alkaline phosphatase levels and receptor activator of nuclear factor kappa-Β ligand/osteoprotegerin expression were measured with enzyme-linked immunosorbent assay and immunofluorescence staining, respectively The inflammatory environment was established by using differentiated HL-60 cells (neutrophils) pre-inoculated onto the biofilm clusters that were more prominent and less scattered on infected titanium surfaces before osteoblast attachment. Biofilm formed on all the tested surfaces, with an increased thickness on rough surfaces and no differences in bacterial viability. All the treatments reduced the amount of biofilm, but none led to bacteria-free surfaces. The treated surfaces showed reduced osteoblast attachment and reduced alkaline phosphatase activity compared with non-infected surfaces. Additionally, treated surfaces showed an osteoblast shift to a pro-osteoclastic-induction phenotype, compared with non-infected surfaces. The presence of experimental inflammation before osteoblast attachment reduced the levels of osteoblast attachment compared with that of the non-inflamed control. Biofilm removal from titanium surfaces is incomplete when hand instruments are used alone or in combination with antibiotics. The treated surfaces showed impaired osteoblast attachment and function, particularly in the presence of inflammation, which may prevent or decrease the ability for re-osseointegration.