Topographic characterization of multispecies biofilms growing on dental implant surfaces: An in vitro model

Objectives To describe the early biofilm formation over whole dental implants with its micro- and macrostructure, using an in vitro multispecies biofilm model. Material and methods Six bacterial strains (Streptococcus oralis, Actinomyces naeslundii, Veillonella parvula, Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans) were used to develop in vitro biofilms over whole titanium implants (growth times 12, 24, 48, 72, 96, and 120 hr). The morphology of biofilms was studied by confocal laser scanning microscopy and scanning electron microscopy, and the bacterial dynamics through quantitative polymerase chain reaction. As primary outcome variable, numbers of each species [colony-forming units per milliliter (CFU/ml)] at different incubation times were compared using the one-way analysis of variance and post hoc testing with Bonferroni's correction. Furthermore, implants were fixed in methacrylate stents to reproduce the clinical situation and biofilms were developed and analyzed by scanning electron microscopy. Results Bacteria colonized implants in a short period of time. Biofilms reached a mature state at 96 hr, exhibiting different ratios of live/dead cells depending on their location, being the peaks of the threads the areas harboring more live bacteria. The densities of each bacteria fluctuated in time, reaching its maximum at 96 hr. Even though the coefficients of variation were high, percentages were similar to those published previously using implant surface specimens, rather than whole implants. Conclusion Dental implants can be colonized by different bacterial species, developing into a mature and well-structured biofilm, which depending on the location may exhibit different degree of bacterial vitality.