| Summary: | In this study, we evaluated the invasion of bacteria into the dentinal tubules of retrofilled extracted human teeth, and the influence of different fluorescently labeled retrograde filling materials on the bacterial invasion and viability, by means of confocal laser scanning microscopy (CLSM). The root apices of extracted teeth were cut, prepared, and filled retrogradely using either intermediate restorative material (IRM), mineral trioxide aggregate (MTA), or Biodentine. The roots were filled with <i>Enterococcus faecalis</i> bacteria from their coronal part for 21 days. Then, 3-mm-long apical segments were cut to get root axial slices, and the bacteria were fluorescently stained and evaluated by CLSM. Bacterial penetration into the dentinal tubules favored the bucco-lingual directions. The filling materials penetrated up to 957 µm into the tubuli, and the bacteria, up to 1480 µm (means: 130 and 167 μm, respectively). Biodentine fillings penetrated less and the associated bacteria penetrated deeper into the tubuli compared to MTA or IRM (<i>p</i> = 0.004). Deeper filling penetration was associated with shallower penetration of both dead and live, or live alone, bacteria (<i>p</i> = 0.015). In conclusion, the current study enables better understanding of the microbiological–pathological course after endodontic surgical procedures. It was found that even with retrograde fillings, bacteria invade deep into the dental tubules, where deeper filling penetration prevents deeper penetration of the bacteria and adversely affects the viability of the bacteria.
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