Apical Negative Pressure irrigation presents tissue compatibility in immature teeth

Abstract Aim: To compare the apical negative pressure irrigation (ANP) with conventional irrigation in the teeth of immature dogs with apical periodontitis. Methods: Fifty-two immature pre-molar root canals were randomly assigned into 4 groups: ANP (n=15); conventional irrigation (n=17); healthy teeth (control) (n = 10); and teeth with untreated apical periodontitis (control) (n=10). After induction of apical periodontitis, teeth were instrumented using EndoVac® (apical negative pressure irrigation) or conventional irrigation. The animals were euthanized after 90 days. The sections were stained by HE and analyzed under conventional and fluorescence microscopy. TRAP histoenzymology was also performed. Statistical analyses were performed with the significance level set at 5%. Results: There was difference in the histopathological parameters between ANP and conventional groups (p<0.05). The ANP group showed a predominance of low magnitude inflammatory infiltrate, a smaller periodontal ligament, and lower mineralized tissue resorption. There were no differences in the periapical lesion extensions between the ANP and conventional groups (p>0.05). However, a lower number of osteoclasts was observed in the ANP group (p<0.05). Conclusion: The EndoVac® irrigation system presented better biological results and more advanced repair process in immature teeth with apical periodontitis than the conventional irrigation system, confirming the hypothesis.


Introduction
Infection control in endodontic therapy is extremely important to achieve a successful outcome in teeth with apical periodontitis 7,16,25,27 . This can be obtained through the steps involved in the endodontic treatment 15 , including irrigation 8 . The association between irrigation and biomechanical preparation optimizes the cleaning of root canals 6,22 .
Although the conventional irrigation system is widely used 12 , in 2007 a novel system called EndoVac ® (Discus Dental, Culver City, CA, USA) was launched to the dental market. Instead of positive pressure, the EndoVac ® system uses an apical negative pressure irrigation (ANP) and has been considered a promising disinfection protocol in the endodontic literature 8,23 .
Previous studies demonstrated that the ANP decreases the risk of irrigant solution extrusion through the apical foramen 9,14,26 . This system enables the circulation of irrigant solution to all working lengths (WL) 2 and facilitates microbiological control 13,23 . The ANP is also 10,18 and debris, mostly at the apical third of a root canal 1,4,10,11 .
The anatomy of the apical third in the root canals of immature teeth increases the risk of accidental injection of irrigant solution into the periapical tissues 3,26 . However, an in vitro study demonstrated that open apex teeth had similar extrusion to closed apex teeth when the ANP was used 20 .
To date, no in vivo studies comparing the EndoVac ® system with the conventional irrigation system separately in immature teeth have been published. Therefore, the aim of this in vivo study was to perform the histopathological and histoenzymological evaluation to compare the EndoVac ® system with the conventional irrigation in immature dog teeth with experimentally induced apical periodontitis. The hypothesis is that ANP presented better biological results in immature teeth with apical periodontitis than the conventional irrigation system.

Material and methods
This research project was approved by the Institutional Animal Ethics Committee (#006/2012).
All the experimental procedures were performed as in our previous studies 8,24 . After this period, a rubber dam was used to isolate the teeth and the temporary restoration was removed. Root canal disinfection was performed 3 mm shorter than the radiographic apex, followed by WL determination, established 1 mm shorter than the radiographic apex. The complementation of root canal disinfection was performed at the WL. ANP and conventional groups were instrumented with K-type were irrigated with 10 mL of NaOCl 2.5% for both the EndoVac ® system and conventional system. The recommended protocol for the EndoVac ® system includes 2 main steps: macro-irrigation and micro-irrigation. In this particular study with immature teeth, canals were irrigated using the macro-cannula only after determining the apical size of the canal due to the large apical size, as previously published 23 .

HE-staining
After 90 days, the animals were euthanized. The maxillas and mandibles with teeth were dissected and sectioned to obtain individual roots. The histotechnical procedures were performed as previously published 5,8 .

Apical Negative Pressure irrigation presents tissue compatibility in immature teeth
The HE-stained sections were analyzed in an Axio Imager.M1 microscope (Zeiss, Göttingen, Germany), using scores based on the following histopathological mild (score 1), moderate or severe (score 2); (b) thickness of periodontal ligament: normal (score 1), slightly increased (score 2), moderately increased (score 3), or severely increased (score 4); and (c) process of resorption of mineralized tissues: absent (score 1) or present (score 2). In addition, descriptions of the apical and periapical regions were conducted for each group.

Fluorescence microscope morphometry
In the ANP, conventional, and apical periodontitis groups, the area of the periapical lesion was measured in square millimeters in the HE-stained sections using described 5,19,24 .
In the healthy teeth group, the thickness of the healthy periodontal ligament area was measured by drawing a line perpendicular to the root apex, located 0.5 mm above the opening of each specimen, to delimit the maximum height of the measured area.

Ta r t r a t e -r e s i s t a n t a c i d p h o s p h a t a s e histoenzymology (TRAP)
The TRAP activity was performed to mark the osteoclasts 8,19 . The sections were deparaffinized, hydrated, and placed in a solution of 50% ethanol/ acetone for 1 minute and dried at room temperature. There was an invagination of partially mineralized connective tissue into the root canal in the ANP specimens. In the healthy specimens, the osteoclast count region was established as described for the were expressed in cell numbers.

Statistical Analysis
The GraphPad Prism 5.a (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analyses.
Chi-square or Fisher's exact test were used to evaluate the scores. One-way ANOVA with Tukey's post hoc test was used to evaluate mean difference. The level

Microscopic analysis of apical and periapical regions
The ANP group presented mixed and diffused to moderate (26.7%). The periapical region showed rich neovascularization and better repair process with connective tissue in the region of the large apical foramen to the middle third of the root canal. This tissue originated from the periodontal tissue that invaginated into the root canal in 53.6% of the cases, In 80% of the specimens there was no dentin, bone, or cementum resorption (Figure 1).
In the conventional group, there were mixed and The majority of the cases (58.8%) were moderately inflamed. The periodontal ligament was severely increased in 82.4% of the cases, with areas of edema was severe bone, cementum, and dentin resorption.
In some cases, there were no cementoblasts on the cement surface and unrepaired root resorption was also frequent (Figure 2A

Conclusion
The results from this in vivo study allowed us to conclude that the negative pressure irrigation (EndoVac ® ) demonstrated better biological results than the conventional irrigation in immature teeth with apical periodontitis and presented a more advanced studies should be performed in order to provide additional information for dental practice.