Journal of Endodontics Research - http://endodonticsjournal.com
Inflammatory response to different endodontic irrigating solutions
http://endodonticsjournal.com/articles/91/1/Inflammatory-response-to-different-endodontic-irrigating-solutions/Page1.html
By JofER editor
Published on 09/6/2008
 
M. Tanomaru Filho, M. R. Leonardo, L. A. B. Silva, F. F. Anibal & L. H. Faccioli.
Department of Endodontics, School of Dentistry of Araraquara, University of the State of Sao Paulo, Araraquara, SP, Brazil.
Department of Clinical Pediatrics, School of Dentistry of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil.
School of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil.
School of Pharmaceutical Science, University of Sao Paulo, Ribeirao Preto, SP, Brazil.


Aim.
The aim of this study was to evaluate the inflammatory response to irrigating solutions injected into the peritoneal cavity of mice.

Conclusions.
In this study, a 0.5% sodium hypochlorite solution caused irritating tissue reactions and showed higher inflammatory responses. Two percent chlorhexidine digluconate was biocompatible, suggesting that it can be an alternative or a complement to sodium hypochlorite during irrigation.

Introduction - Materials and methods.
M. Tanomaru Filho, M. R. Leonardo, L. A. B. Silva, F. F. Anibal & L. H. Faccioli.
Department of Endodontics, School of Dentistry of Araraquara, University of the State of Sao Paulo, Araraquara, SP, Brazil.
Department of Clinical Pediatrics, School of Dentistry of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil.
School of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil.
School of Pharmaceutical Science, University of Sao Paulo, Ribeirao Preto, SP, Brazil.


Introduction.
During the biomechanical preparation of infected teeth with no pulp vitality, special attention must be given to the elimination of bacteria and their subproducts from the root-canal system. These factors play an important role in the development and maintenance of periapical lesions and thus, the success of root-canal treatment (Leonardo et al.1994, Sjogren et al. 1997). Therefore, biomechanical preparation must be supplemented by irrigating solutions that have an antibacterial effect (Bystrom & Sundqvist 1983, Jeansonne &White1994, Leonardo et al.1995, Leonardo 1998).
The most popular irrigating solution is sodium hypochlorite because of its physico-chemical and antibacterial properties. It is recommended for treatment of teeth without pulp vitality, particularly at high concentrations, because these have greater antibacterial effect than diluted solutions (Yesilsoy et al. 1995, Ayhan et al. 1999). However, more concentrated solutions can irritate periapical and apical tissues (Spangberg 1973). Since irrigating solutions can contact periapical tissue during biomechanical preparation, biocompatibility with these tissues is important. Thus, irrigating solutions with antibacterial activity and biocompatibility, such as chlorhexidine, have been proposed as an alternative for treatment of teeth with infected root canals. The antibacterial effect and long-acting nature of 2% chlorhexidine digluconate (Delany et al.1982, Ohara et al. 1993, Jeansonne &White1994, White et al.1997, Komorowski et al. 2000) has led researchers to recommend its use in root-canal treatment (Jeansonne & White 1994, White et al.1997, Leonardo et al.1999).The biocompatibility of chlorhexidine has been reported (Southard et al. 1989); however, its biological response when associated with sodium hypochlorite has not been studied.
The objective of this study was to analyze the inflammatory response of 0.5% sodium hypochlorite and 2% chlorhexidine injected into the peritoneal cavity of mice.

Materials and methods.
All animals were housed in facilities approved by the UNESP, Brazil. The use of animals for this research was approved by the local Ethical Committee for Animal Research and NIH guidelines stated in the ‘Principles of Laboratory Animal Care’ (NIH guidelines 1985) were followed.

Cellular migration to the peritoneal cavity.
Sixty male Swiss mice (6-8 weeks old, 15-20 g body weight) were randomly divided into three groups of 20 mice each. Each group received a 0.3-mL intra-peritoneal injection:
  1. phosphate buffered saline (control, PBS:7.2g sodium chloride,1.38 g of monobasic sodium phosphate and 6.96 g of dibasic potassium phosphate);
  2. 0.5% sodium hypochlorite;
  3. 2% chlorhexidine digluconate.
The solutions were prepared at the Chemistry Institute of Araraquara, UNESP, Brazil.
Five animals from each group were sacrificed at 4, 24, 48 h or 7 days (168 h) after intra-peritoneal injection with an anaesthetic overdose. Four milliliters of PBS/ 0.05% sodium citrate was injected for intra-peritoneal washing. An aliquot was separated and centrifuged, and the supernatant was stored at _20 8C for cell counting. Total and differential cell count Twenty-microlitre aliquots of peritoneal liquid were diluted and homogenized in 400 mL of Turk solution (15 mL glacial acetic acid, 0.2m L genetian violet, 500 mL distilled and deionized water) and then placed in Neubauer chamber (Improved Neubauer, Levy Hemacytometer, Hausser Scientific, Gaithersburg, MD, USA). Total inflammatory cell count was performed with an optic microscope (Zeiss, Jena, Germany, 120x).
Twenty microlitres of intra-peritoneal washing were centrifuged (Cytospin 3, Shandon Inc., Pittsburgh, PA, USA), at 900 _ g, for 3 min. Smear slides were dried at room temperature and were stained with Rosenfeld stain (Faccioli et al. 1990) for differential inflammatory cell, polymorphonuclear neutrophil and mononuclear cell counts (macrophage and lymphocyte). Neutrophil and mononuclear cell counts were performed with an optic microscope (Jenamed 2, Zeiss) with an immersion lens (1000x).

Protein leakage.
The supernatant of the peritoneal cavity stored at _20 8C was used for evaluation of protein leakage, as a parameter of oedema. Protein concentration was determined using Comassie stain (Pierce, Rockford, IL, USA), according the instructions of the manufacturer.

Statistical analysis.
The inflammatory cell migration was analyzed by the Kruskal-Wallis test. The groups were also compared using the Miller test.

Results.
Cellular migration to peritoneal cavity.
The response to the injection of the solutions into the peritoneal cavity was evaluated by the number of neutrophils (Fig.1) and mononuclear cells (Fig. 2) at 4, 24, 48 and 168 h after the injection. At 4 and 24 h post-injection, the number of neutrophils was similar in all groups (P > 0.05). The chlorhexidine group had a similar number of neutrophils at all time periods as the control group. There was a significant increase in the number of neutrophils in the 0.5% sodium hypochlorite group from 48 to168 h (P < 0.05). The migration of mononuclear cells into the peritoneal cavity was similar in all groups at 4 and 24 h post-injection (P > 0.05), and the chlorhexidine group was similar to the control group at all time periods. There was an increase in neutrophils from 48 to168 h post-injection (P < 0.05) for the0.5% sodium hypochlorite solution.

Protein leakage to periodontal cavity.
There was a significant increase in protein leakage in the groups injected with 0.5% sodium hypochlorite compared to the PBS group at 4-48 h (P < 0.05). Protein leakage was similar for 2% chlorhexidine and PBS at all time periods (P > 0.05) (Fig. 3).

Figure 1. Migration of neutrophils into the peritoneal cavity of mice at different time periods induced by the injection of 0.5% sodium hypochlorite, 2% digluconate chlorhexidine and phosphate buffered saline (PBS). Results are reported as mean +SEM.

Migration of neutrophils into the peritoneal cavity of mice at different time periods induced by the injection
*Statistically different (P < 0.05).
**Statistically different (P < 0.01).


Figure 2. Migration of mononuclear cells into the peritoneal cavity of mice at different time periods induced by the injection of 0.5% sodium hypochlorite, 2% digluconate chlorhexidine and phosphate buffered saline (PBS). Results are reported as mean +SEM.

Migration of mononuclear cells into the peritoneal cavity of mice at different time periods induced by the injection
*Statistically different (P < 0.05).
**Statistically different (P < 0.01).


Figure 3. Protein leakage into the peritoneal cavity of mice at different time periods induced by the injection of 0.5% sodium hypochlorite, 2% digluconate chlorhexidine and phosphate buffered saline (PBS). Results are reported as mean +SEM.

Protein leakage into the peritoneal cavity of mice at different time periods induced by the injection of 0.5%sodium hypochlorite, 2% digluconate chlorhexidine and phosphate buffered saline
*Statistically different (P < 0.05).
**Statistically different (P < 0.01).


Discussion - References.
Discussion.
The migration of inflammatory cells into the peritoneal cavity of mice has been used to evaluate inflammatory response caused by endodontic materials (Silva et al. 1997) and calcium hydroxide dressings (Nelson Filho et al.1999). In this study, the solutions did not cause a significant increase in neutrophils or mononuclear cells in the peritoneal cavity at 4 and 24 h (P > 0.05). However, at 48-168 h, the 0.5% sodium hypochlorite solution caused a greater increase in the number of neutrophils in the peritoneal cavity (P < 0.05). This response is probably due to tissue irritation caused by the capacity of sodium hypochlorite to dissolve organic tissue (Grossman & Meiman 1941, Gordon et al. 1981), and is increased at higher concentrations. Yesilsoy et al. (1995) and Spangberg (1973) reported the irritating effect of sodium hypochlorite, particularly at high concentrations. Leonardo et al. (1984) observed periapical and apical tissue irritation after biomechanical preparation of dog’s teeth using 4% sodium hypochlorite solution.
Two percent chlorhexidine solution has a wide spectrum of antibacterial effect as well as prolonged residual effect (Jeansonne & White 1994,White et al. 1997, Leonardo et al.1999), suggesting its use as an irrigating solution in infected root canals. This solution has also shown to be non-irritating to tissue. The use of 2% chlorhexidine as a periodontal irrigant did not cause obvious toxic effect son gingival tissue (Loe&Schiott1970, Southard et al.1989).
At all experimental periods, chlorhexidine was statistically similar to the control group (P > 0.05). The absence of oedema observed by protein leakage suggested no significant tissue damage, indicating biocompatibility of chlorhexidine (Yesilsoy et al.1995).
Jimenes-Rubio et al. (1997) reported that5.25%sodium hypochlorite and 1% glutaraldehyde significantly decreased macrophage adhesion capacity, an important factor during inflammation. Segura et al. (1999) observed that0.12%chlorhexidine solution inhibited macrophage adherence, however, at lower intensity than 5.25% sodium hypochlorite.
Although chlorhexidine had better biological results compared to sodium hypochlorite, it does not dissolve tissue or inactivate bacterial LPS. Therefore its use has been recommended as an alternative to sodium hypochlorite in patients who are allergic to hypochlorite or in teeth with incomplete roots (Jeansonne & White 1994). Fuss & Trope (1996) have recommended its use in crestal perforations because a biocompatible medicament is essential to prevent inflammatory response in proximity to the epithelial attachment. It can also be used for final irrigation due to its broad spectrum of antimicrobial activity (Delany et al. 1982, Ohara et al. 1993, Jeansonne &White1994).

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