The Invasion of Bacterial Biofilms into the Dentinal Tubules of Extracted Teeth Retrofilled with Fluorescently Labeled Retrograde Filling Materials

Abstract

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 Enterococcus faecalis 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 (p = 0.004). Deeper filling penetration was associated with shallower penetration of both dead and live, or live alone, bacteria (p = 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.

Comparative Analysis of Bacterial Colonization in Simulated Vertical Root Fractures of Root Canals Filled with Various Obturation Materials using Confocal Laser Scanning Microscopy

“…Our results reveal a pattern of bacterial invasion and lling penetration in which the buccolingual direction was preferred to the mesiodistal direction, regardless of the lling material. This nding is in accordance with the results of previous investigations [10,14,48]. The deeper penetration of both the lling material and bacteria in the buccolingual direction may be associated with an anatomicalphysiological phenomenon termed "the butter y effect," which refers to the butter y-like appearance observed on the cross-sections of roots as a result of higher sclerosis in the tubuli at the distal and mesial sides of the root canal [32,49,50].…”

“…CLSM is a valuable modality of choice alongside more traditional SEM and PCR-based techniques to identify viable bacteria in dentinal tubules [10,[12][13][14][15]. Importantly, using CLSM with the live/dead staining method allows us to assess the extent of contamination and vitality of bacteria in the dentinal tubules [10,[12][13][14][15]. No study has used CLSM to evaluate the bacterial colonization in VRF of extracted human teeth lled with different materials.…”

“…Confocal laser scanning microscopy (CLSM) has been used in previous studies to examine the nature of bacterial colonization in VRFs [10,[12][13][14][15]. However, there is still a need to investigate the relationship between the type of intracanal lling material and the bacterial colonization of VRF.…”

Degradation of Fusobacterium nucleatum biofilm and quantity of reactive oxygen species due to a combination of photodynamic therapy and 2.5% sodium hypochlorite

“…The minimal and maximal bacterial penetration depths into the dentinal tubules were 1 µm and 1480 µm, respectively, with a mean of 167 µm. 39 Thus, a complementary/supportive technique to increase the effectiveness of root canal treatment is needed. 40 The average biofilm degradation in the PDT-only group (0.78) was lower than the control group (1.06).…”

Clinical applications of antimicrobial photodynamic therapy in dentistry

“…aPDT is not an exception as the light propagation and PS penetration inside dentinal tubules are restricted. Moreover, reports have shown that bacteria can migrate into dentinal tubules up to a depth of 1,000 μm, where oxygen as a vital component of aPDT is absent ( Balhaddad et al, 2020 ; Rosen et al, 2020 ). To overcome these issues, some studies used hydrogen peroxide solution that provided pre-treatment of the biofilm, resulting in better PS penetration and increased available oxygen in the environment ( Garcez et al, 2011 ; Garcez and Hamblin, 2017 ).…”