Comparative Evaluation of Chlorhexidine and Cetrimide as Irrigants in Necrotic Primary Teeth: An In vivo Study.

Context: Disinfection of the root canal system by biomechanical preparation and profuse irrigation is especially important in primary teeth due to its complex pulp canal anatomy. Appropriate selection of irrigating solution should be done in terms of tissue dissolving capacity, antibacterial efficacy, and palatability when treating pediatric cases. Aim: The aim of the study was to compare the antibacterial efficacy of 2% chlorhexidine (CHX), 0.2% cetrimide (CTR), and 0.2% CHX + 0.2% CTR against anaerobic bacteria and to test the influence of CTR added to CHX on its antibacterial action. Materials and
Methods: Seventy-five teeth that were selected for the study were divided into three groups: Group I (2% CHX), Group II (0.2% CTR), and Group III (0. 2% CHX + 0.2% CTR). Samples collected before and after irrigation were immediately processed and microbial growths were quantified. Statistical Analysis: Intergroup comparison was done using one-way analysis of variance and Newman-Keuls multiple post hoc procedures and intragroup comparison was done using paired t-test.
Results: When the mean values of Group I were compared with Group II, the data were statistically not significant (P = 0.2341), whereas Group I showed a significant difference when compared with Group III (P = 0.0107). When the mean values of Group II and Group III were compared, the data were found to be statistically not significant (P = 0.0805).
Conclusion: The efficacy of 2% CHX was found to be slightly superior when compared with that of 0.2% CTR, but the difference was not statistically significant. However, a significant difference was found between 2% CHX and combination irrigants. Copyright:

INTRODUCTION

One of the cardinal steps in pulpectomy is the disinfection of the root canals by mechanical preparation and abundant irrigation. The goal is to achieve minimal bacterial load in the root canals. Profuse irrigation is needed to get rid of the remaining infected pulp and dentin debris and to lubricate canal walls during instrumentation.[1]

Different solutions have been used for irrigating the primary root canals during biomechanical preparation. Physiological saline solution and sodium hypochlorite (NaOCl) are the most popularly used.[1] NaOCl being a cytotoxic agent poses the risk of potential injury to permanent tooth germs when it reaches the periapex.[1]

On the other hand, chlorhexidine (CHX) is a powerful antiseptic featuring both bacteriostatic and bactericidal properties.[1] A probable way to improve its antibacterial efficacy is to add different surface-active agents that could decrease its surface tension and improve the wettability.[2]

Cetrimide (CTR) (cetiltrimethylammonium bromide), a quaternary ammonium compound, can act as a cationic detergent effective against Gram-positive and negative bacteria.[3]

The aim of the present study is to compare the antibacterial efficacy of 2% CHX gluconate, 0.2% CTR, and a combination of 0.2% CHX +0.2% CTR against anaerobic bacteria.

MATERIALS AND METHODS

The study was commenced after approval from the Institutional Ethical Committee.

Seventy-five teeth of patients coming to the Department of Pedodontics and Preventive Dentistry were selected for the study.

Inclusion criteria

Age between four and 11 years[45]

Primary teeth with chronic pulpitis, chronic periapical abscess, acute exacerbation of chronic pulpitis, and chronic periapical abscess[567]

Primary molars with minimal mobility and restorable crown[5]

Minimal root resorption[5]

No sinus tract.[8]

Exclusion criteria

Teeth having furcal lesions more than 5 mm diameter or periapical lesions exceeding ten mm diameter

Children with systemic diseases and patients undergone antibiotic therapy in the past 3 months.[4]

Written consent was procured from parents before commencing the treatment.

Seventy-five teeth selected for the study were randomly assigned into three groups: Group I – 25 patients (2% CHX), Group II – 25 patients (0.2% CTR), and Group III – 25 patients (0. 2% CHX + 0.2% CTR).

Procedure

Isolation and disinfection

Tooth selected for the study was isolated under rubber dam and tooth and its surrounding clamp were disinfected with 10% povidone iodine, followed by cleaning with isopropyl alcohol after 5 min.[4]

Access preparation and sample collection

Access opening was done under aseptic conditions with sterile water-cooled high-speed diamond round bur.[4] The preirrigant samples were collected using sterile paper points having diameters analogous to that of the canals for 60 s.[4] The collected paper points were immediately transferred into test tubes containing 8 ml of thioglycollate broth, HiMedia Laboratories Pvt., Ltd., Bombay.[4]

After determining the working length radiographically, biomechanical preparation was done starting 1 mm above the apex with K-files up to three successive sizes depending on the initial canal diameter. For each instrument change, irrigation using 2 ml of respective irrigants was done using a 24 gauge needle sequentially. After drying the canal with sterile paper points, postirrigantion sample was taken using sterile paper points. This was immediately placed into another marked test tube containing 8 ml of thioglycollate broth.[4]

Processing for culture

The samples were immediately transferred to the Department of Microbiology, where processing was done within 2 h of collection of sample.[4] Both samples were streaked on to separate blood agar plates (HiMedia Laboratories Pvt., Ltd., Bombay) and incubated anaerobically using Anaerogas Pack (HiMedia Laboratories Pvt., Ltd., Bombay) in an anaerobic jar for 48 h at 37° C4. Microbial growth was quantified by counting the number of colony-forming units (CFU/ml) using a digital colony counter. Preirrigant samples are shown in Figures 1a, 2a, and 3a and postirrigant samples are shown in Figures 1b, 2b, and 3b.

Figure 1

(a) Preirrigation Group I. (b) Postirrigation Group I

Figure 2

(a) Preirrigation Group II. (b) Postirrigation Group II

Figure 3

(a) Preirrigation Group III. (b) Postirrigation Group III

RESULTS

The number of CFU/ml before and after irrigation with respective irrigants was tabulated and statistically analyzed. Intergroup comparison was done between three groups using one-way analysis of variance (ANOVA) followed by Newman–Keuls multiple post hoc procedure Table 1. Intragroup comparison was done using paired t-test [Table 2].

Table 1

Intergroup comparison using one way ANOVA

Groups	Before treatment CFU/ml×105			After treatment CFU/ml×105			Difference
	Mean	SD	SE	Mean	SD	SE	Mean	SD	SE
Group I	387.60	82.76	16.55	1.96	1.34	0.27	385.64	82.83	16.57
Group II	374.24	79.53	15.91	12.24	3.18	0.64	362.00	79.44	15.89
Group III	334.64	61.59	12.32	7.52	2.89	0.58	327.12	61.76	12.35
P	0.00001*			0.00001*			0.00001*
Pairwise comparison by Newman–Keuls multiple post hoc procedures
Group I versus Group II	P=0.5091			P=0.0001*			P=0.2341
Group 1 versus Group III	P=0.0269*			P=0.0035*			P=0.0107*
Group II versus Group III	P=0.0524			P=0.0123*			P=0.0805

CFU: Colony-forming unit, SD: Standard deviation, SE: Standard error, *P<0.05

Table 2

Intragroup comparison using paired t-test

Groups	Time points	Mean	SD	Mean difference	SD difference	Percentage of change	Paired t	P
Group I	Before	387.60	82.76	385.64	82.83	99.49	23.2798	0.00001*
	After	1.96	1.34
Group II	Before	374.24	79.53	362.00	79.44	96.73	22.7846	0.00001*
	After	12.24	3.18
Group III	Before	334.64	61.59	327.12	61.76	97.75	26.4821	0.00001*
	After	7.52	2.89

SD: Standard deviation, *P<0.05

The number of CFU/ml of anaerobic bacteria showed a mean difference of 385.64 in Group I, while Group II showed a mean difference of 362.00 and Group III showed a mean difference of 327.12 [Table 1]. When all these parameters were analyzed using one-way ANOVA, they were found to be statistically significant (P = 0.00001). Intergroup comparisons between all the three groups were done using Newman–Keuls multiple post hoc procedure [Table 1]. When the mean values of Group I were compared with Group II, the data were found to be statistically not significant (P = 0.2341), whereas Group I showed a significant difference when compared with Group III (P = 0.0107), probably because a lower concentration of CHX was used in combination. When the mean values of Group II were compared with Group III, the data were found to be statistically not significant (P = 0.0805).

When intragroup comparison was done using paired t-test, all three groups showed a statistically significant difference [P = 0.00001; Table 2].

DISCUSSION

Disinfection of the root canals before filling is a pivotal step in pulp therapy. Although mechanical preparation along with antiseptic properties of obturating materials can lower bacterial load, it is inevitable to use irrigating solutions to remove the microbiota from areas inaccessible to instrumentation. The possible organisms responsible for root canal failure are gram negative anaerobic bacteria, Enterococcus Faecalis, Staphylococcus aureus and Candida albicans.[9]

The complete removal of microbes from the root canals has become a challenging issue which led to the use of combination irrigants. Many studies have compared the properties of currently used endodontic irrigants in permanent teeth. Only a few studies were employed on primary root canals. Hence, the present study included necrotic primary teeth as the samples. Prereduced thioglycolate was used in the transportation of microbiological samples, as suggested by Carlsson and Sundqvist[10] It reduces oxygen, precluding the accumulation of superoxide radicals that can kill anaerobic bacteria; also, the presence of small amounts of agar in it can prevent the diffusion of oxygen into the medium.

CHX is a cationic agent (biguanide) which exhibits antibacterial activity. The cations of the compound associate with anions at the bacterial surface altering its integrity. Potassium ion being a small entity is the first substance to appear when the cytoplasmic membrane is damaged,[11] resulting in a bacteriostatic effect. At higher concentrations, precipitation or coagulation of the bacterial cell cytoplasm by protein cross-linking results in bactericidal action and cell death[1213] and leaving debris in the root canals,[14] which can be washed out by profuse irrigation with distilled water.

In the present study, when 2% CHX was used as the irrigant, a drastic reduction in the anaerobic bacterial count was seen post treatment which was statistically significant [P = 0.00001; Table 2]. The results obtained are in agreement with the ones obtained by Ruiz Esparza et al. (2011) wherein 2% CHX gluconate displayed a greater reduction of intracanal bacterial loading (P < 0.0001) when compared with sterile saline solution. In a similar study conducted by Delany et al.,[15] bacteriologic samples were taken before, during, immediately after, and 24 h after irrigation with CHX gluconate or with sterile saline. The CHX-treated specimens showed a highly significant reduction (P < 0.0049) in the number of microbes after instrumentation and irrigation. But unlike the present study, it was conducted on recently extracted permanent teeth.

No adverse reactions have yet been published regarding CHX when used as an irrigant or intracanal medicament. However, the adverse effects of CHX are more related to its topical application.[16] Initial symptoms include transient taste disturbances and burning sensation of the tongue. Continuous use of mouthwash can result in oral desquamation and occasional parotid gland swelling.[16] Contact with conjunctiva can result in permanent damage and contacting the tympanum can cause ototoxicity.[17] Application of CHX to intact skin can result in urticaria, Quincke's edema or dyspnea, and rare occurrence of severe anaphylactic reactions.[18] And adding to this, the burning sensation induces gagging in young children which increases their apprehension and lack of cooperative ability making it a less preferred irrigant for pediatric patients, for whom rubber dam application is not always guaranteed because of lack of cooperation and tooth anatomy. Hence, CTR (0.2%), which is a quaternary ammonium compound, was considered as an alternative irrigant to be tested. And also, one of the purposes of the study was to test the influence of surfactant CTR added to CHX on its antibacterial action. Hence, a combination of 0.2%CHX and 0.2% CTR was tested for their antibacterial action. As far as the author's knowledge, there is only one previous in vivo study, conducted by O. Oncag et al. (2003) that reports the effectiveness of CTR as irrigants in primary teeth.

CTR being a cationic surfactant has bactericidal activity along with a capacity to hamper the mechanical stability of biofilm. Both 0.2% CTR and combination (0.2%CHX + 0.2% CTR) was found highly effective against anaerobic bacteria (P 0.00001). When 0.2% CTR and combination irrigants were compared with CHX, CHX showed a better antibacterial action, probably because a higher concentration of CHX (2%) was used. However, the result was statistically significant only in comparison with the combination irrigants. The number of CHX molecules available to act with the dentin determines the strength of its antibacterial action.[17] Accordingly, a more concentrated preparation has increased antibacterial action. Similar results were obtained by Ferrer Luque et al.[19] where they reported that 2% CHX had higher residual activity than 0.2% CHX and 0.2% CTR in root canals infected with E. faecalis, even though the study was conducted on extracted permanent teeth. But unlike the present study, investigation by Isabelle Portenier et al.[20] showed that a combination of CTR (0.1%) and CHX (0.1%) was more efficient in killing bacteria when compared to 0.02% and 0.01% CHX. The above explanation holds good here also.

According to a study conducted by Shailaja et al.,[21]2% CHX + 0.2% CTR demonstrated a highly significant (P < 0.01) antimicrobial activity against E. faecalis when compared to 2% CHX alone. The results of the present study are not in agreement with this, probably because a low concentration of CHX (0.2%) was used in combination. The combination of CHX and CTR eliminated E. faecalis more rapidly than CHX alone at similar concentrations. Both CHX and CTR synergistically target the cell wall of the organism.[20] This explains the better antibacterial action of the combination irrigants when compared to CTR alone in the present study, even though the results are nonsignificant (P 0.0805).

An interesting finding was reported by Maria Teresa (2014)[19] that alternating the use of CHX and CTR depicted a significantly higher percentage of reduction than when used in combination. CTR was always applied first which facilitated the destructuring of the Extracellular Polymeric Substance (EPS) matrix followed by CHX so that it was able to act directly on E. faecalis, possibly exerting its bactericidal potential to a higher degree. However, a greater efficacy of CTR (commercial product-smear clear) as opposed to CHX was previously reported by Dunavant et al.[22] Unlike the results of the present study, Oncag et al.(2003)[4] reported that Cetrexidin (0.2% CHX + 0.2% CTR) was significantly more effective than 0.9% saline in killing anaerobic bacteria with a statistically significant difference and with 2% CHX showing a statistically not significant difference.

CONCLUSION

From the present study following conclusions can be drawn:

2% CHX, 0.2% CTR, and combination of 0.2% CHX and 0.2% CTR are effective irrigants against anaerobic bacteria in necrotic primary teeth

The efficacy of 2% CHX was slightly superior when compared to 0.2% CTR and 0.2% CHX + 0.2% CTR

0.2% CTR is proved to have antibacterial action against anaerobic bacteria and can be used as an alternative to 2% CHX

Addition of CTR did not adversely affect the action of CHX.

Limitations of the study

The samples were taken immediately after irrigation and microbiological processing done. Further studies have to be done to document the overall efficacy of all four irrigants over a longer period

The property of substantivity of chlorhexidine was not tested.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.