The Effects of Double Antibiotic Paste and Amoxicillin-clavulanate Paste Used in Endodontic Regeneration on Microhardness of Radicular Dentine: An In vitro Study.

OBJECTIVE: Double antibiotic paste is a root canal medicament used in regenerative endodontics and its long-term exposure to dental hard tissues was shown to reduce its mechanical properties. The amoxicillin-clavulanate paste is a medicament effective against endodontic pathogens and is effective in endodontic regeneration. Its effect on radicular dentine is yet to be investigated. The purpose of this study is to investigate and compare the effects of double antibiotic paste and amoxicillin-clavulanate paste on the microhardness of radicular dentine.
MATERIALS AND METHODS: The root canal of 45 premolars was mechanically prepared and divided into three groups for the placement of intracanal medicament: Group 1 (n = 15) double antibiotic paste, Group 2 (n = 15) amoxicillin-clavulanate paste, and Group 3 control group (n = 15). All the specimens were subject to microhardness testing at 500 and 1000 microns depth for 1 week, 1 month, and 3 months and analyzed.
RESULTS: There is no significant reduction in microhardness of radicular dentin at 1 week on all the three groups. At 1 month and 3 month period, the amoxicillin-clavulanate paste is found to have no significant effect on dentin microhardness when compared to double antibiotic paste.
CONCLUSION: The use of amoxicillin-clavulanate paste as an intracanal medicament does not cause significant weakening of dentin even after its long-term use. Copyright:

INTRODUCTION

Endodontic regeneration is a procedure aimed to restore the vitality of damaged tissue in the canal space to promote root maturation of immature permanent tooth with necrotic pulp. Further root development and reinforcement of the dentinal walls by deposition of hard tissues, thus strengthening the root in comparison to the traditional apexification procedures are the advantages of this procedure.[1] In necrotic and infected teeth, bacterial colonization and biofilm formation occur causing contamination of the root canal due to bacterial toxin. Root canal infection negatively influences regeneration by damaging stem cells in the periapical tissues.[2] Disinfection in endodontic regeneration sets the stage for a stem cell–conducive environment promoting deposition of hard tissues.[3]

Mechanical preparation, irrigation with disinfectant solutions, and intracanal medicaments are used to achieve root canal disinfection in endodontic procedures. Aggressive shaping and cleaning could damage the fragile and thin root canal walls of the incompletely developed permanent tooth and in which intracanal medicaments have a vital role in obtaining disinfection of the root canal system. One of the widely accepted intracanal medicament is calcium hydroxide because of its antimicrobial properties. But calcium hydroxide dressing for a prolonged period could expose the tooth to root fracture.[4] Local application of antibiotics has been investigated as intracanal medicaments.[5] The triple antibiotic paste is a combination of metronidazole, ciprofloxacin, and minocycline found to be effective in reducing viable bacteria in regenerative protocols.[6] In double antibiotic paste, minocycline is not used now to overcome the discoloration caused by triple antibiotic paste and both triple and double antibiotic have antibacterial effect on human dentine.[7]

Amoxicillin-clavulanate (augmentin) is a combination of semisynthetic antibiotic amoxicillin and the lactamase inhibitor, clavulanate potassium. Amoxicillin-clavulanate possesses the properties of broad-spectrum antibacterial activity and in particular bactericidal activity against most Gram-positive and Gram-negative microorganisms.[8] It was used initially as a component of triple antibiotic paste to substitute for minocycline.[910] Amoxicillin-clavulanate is an antibiotic found to be 100% effective against common endodontic pathogens.[1112] The use of the amoxicillin-clavulanate paste is found to be effective in resolving periapical lesions and promoting apical closure of an immature tooth with necrotic pulp as an intracanal medicament.[13] In regenerative therapy, prolonged application of antibiotics as an intracanal medicament is essential. The use of single antibiotic paste is desirable compared to antibiotic combinations as in triple and double antibiotic paste which can increase the risk of bacterial resistance even for use on short duration.[1415]

In regenerative endodontic therapy, antibiotic paste has to be placed in the root canal for several weeks. This antibiotic paste is bound to produce changes on the dentinal surface when used for longer periods. Acids are added to some antibiotics to maintain chemical stability, to control tonicity, or to ensure physiological compatibility.[16] The longer exposure times of dentine to these acidic antibiotic pastes result in significantly greater demineralization compared with shorter time periods.[16] Studies have shown that long-term exposure of dental hard tissues to double antibiotic paste might cause its demineralization and could negatively affect the mechanical properties.[17] The use of amoxicillin-clavulanate paste in endodontic regeneration extends for a longer duration and thus, it is imperative to find its effect on mechanical properties of dentin. This study was done to find the effect of amoxicillin-clavulanate paste on microhardness of radicular dentin and to compare it with that of double antibiotic paste at 1 week, 1 month, and 3 months' time period.

MATERIALS AND METHODS

The study was done on intact mandibular premolar teeth with single root and root canal which was indicated for orthodontic extraction at the age group of 12–15 years. The forty five teeth were stored at 0.1% thymol solution at 4°C until use. Digital radiographs were exposed from the facial and proximal views to ensure the presence of single root canal.

Endodontic access cavity preparation was done. The working length was determined and the root canals were mechanically prepared up to F3 using ProTaper universal Ni-Ti rotary file (Dentsply Maillefer, Switzerland). Canals were irrigated between files with 1 ml of 1.5% sodium hypochlorite (NaOCl). A final rinse was performed using 5 ml of saline. Root canals were then dried with sterile paper points and were randomly assigned to three treatment groups of 15 teeth each as per the use of intracanal medications. In Group 1, the double antibiotic paste was used; in Group 2 amoxicillin-clavulanate paste was used; and in Group 3, no medicament was used for intracanal dressing, which forms the control group.

Sample preparation

The double antibiotic paste was prepared freshly by mixing USP grade antibiotic powders compounded of equal portions of metronidazole and ciprofloxacin (Kerala State Drugs and Pharmaceuticals, Alappuzha District, Kerala) with reverse osmosis water in the P/L ratio of 2.5:1 in a concentration of 0.1 mg/ml. The amoxicillin-clavulanate paste was prepared freshly by mixing amoxicillin-clavulanate powder (Kerala State Drug and Pharmaceuticals, Alappuzha District, Kerala) with polymer solution of carbopol. The powder liquid ratio of 1:1 in a concentration of 0.1 mg/ml (Department of Pharmacology, Government Dental College, Trivandrum) was used for the preparation of amoxicillin-clavulanate paste. In both Groups 1 and 2, the paste was applied in the canal with a sterile lentulo spiral and tamped in the root canal space to the level of cementoenamel junction. The tooth was sealed apically with flowable composite and the access opening of all the teeth was sealed with cavit with a thickness of 4 mms. The 15 samples of each group were further subdivided into five teeth each and were kept in normal saline at 37°C for 1 week, 1 month, and 3 months, respectively, before testing. Decoronation of the teeth was done at the level of 0.5 mm radicular to the cementoenamel junction using a low-speed diamond saw under water cooling. A middle 3 mm root cylinder was horizontally sectioned from each root using a water-cooled diamond saw for microhardness testing. The root cylinders from the experimental groups were irrigated with distilled water to remove the medicament.

Microhardness testing

The specimens were mounted on special mold and the coronal sides of these were polished using a Buehler polishing unit with metasery 250 grinder powder and with 220,280, 400,800, and 1000 grit papers and finally using a 1 μm diamond polishing suspension. As a final cleaning step, the polished specimen was sonicated in de-ionized water for 3 min. Microhardness measurement was performed using a Knoop Microhardness tester (CLEMEX) on the polished side of each root cylinder at 500 μm and 1000 μm from the pulp–dentin interface. At each depth, 3 indentations were made using 50 g load oriented perpendicular to the indentation surface for 15 s. The indentation was carefully observed in an optical microscope with a digital camera and image analyzing software, allowing the precise measurement of the diagonals. The representative hardness value of each specimen at each depth was obtained as the mean of the result for the three indentations.

RESULTS

The data were analyzed and compared using independent sample t-test and ANOVA.

The effect of intracanal medicaments on microhardness of radicular dentine at a depth of 500 and 1000 μm at different time periods is shown in Table 1. In Group 1 (double antibiotic paste), there is a significant decrease in microhardness from 1 week to 1 month and to 3 months. In Group 2 (amoxicillin-clavulanate paste), and in Group 3 (control group), there is decrease in microhardness from 1 week to 1 month and to 3 months but not statistically significant.

Table 1

Comparison of Group 1, Group 2, and Group 3 at 1 week, 1 month, and 3 months’ time period at 500 μm and 1000 μm using one-way ANOVA

	Groups	1 week	1 month	3 months	ANOVA (P)
500 μm	Group 1	53±1.52	47±1.14	44±1.48	0.000
	Group 2	54±1.22	53±1.00	52±1.34	0.205
	Group 3	53±1.41	52±1.52	51±0.84	0.307
1000 μm	Group 1	61±1.00	56±1.22	52±1.00	0.000
	Group 2	63±1.00	62±1.22	62±2.34	0.552
	Group 3	62±3.00	61±0.71	61±1.58	0.668

The intergroup comparison at each time period on microhardness at a depth of 500 μm and 1000 μm is shown in Table 2. In 1 week time period, a comparison of microhardness values between the three groups is not significant. At 1 month and 3 months, the time period comparison of microhardness between three groups is significantly different.

Table 2

Intergroup comparison at 1 week, 1 month, and 3 months at 500 μm and 1000 μm using one-way ANOVA

Depth	Month	Groups	n	Mean	SD	SEM	P
500 μm	1 week	Group 1	5	53.60	1.51658	1.48324	0.537
		Group 2	5	54.00	1.22474	1.34164
		Group 3	5	53.00	1.41421	1.14018
	1 month	Group 1	5	47.60	1.14018	0.50990	0.000
		Group 2	5	53.00	1.00000	0.44721
		Group 3	5	52.40	1.51658	0.67823
	3 months	Group 1	5	44.20	1.48324	0.66332	0.000
		Group 2	5	52.60	1.34164	0.60000
		Group 3	5	51.60	1.14018	0.50990
1000 μm	1 week	Group 1	5	61.00	1.000	0.44721	0.293
		Group 2	5	63.00	1.000	0.44721
		Group 3	5	62.00	3.000	1.34164
	1 month	Group 1	5	56.00	1.22474	0.54772	0.000
		Group 2	5	62.00	1.22474	0.54772
		Group 3	5	61.00	0.70711	031623
	3 months	Group 1	5	52.00	1.00000	0.44721	0.000
		Group 2	5	62.00	2.34521	1.04881
		Group 3	5	61.00	1.58114	0.70711

SD: Standard deviation, SEM: Standard error of mean

The results of intergroup comparison at 1 month and 3 months at 500 μm and 1000 μm depth are shown in Table 3. At 1 month, Group 1 showed significantly lower microhardness than Group 2 and Group 3. Comparison between Group 2 and Group 3 is found to be not significant. At 3 months, Group 1 showed significantly lower microhardness than Group 2 and Group 3. On intergroup comparison, the microhardness value of Group 2 and Group 3 is found to be not significant.

Table 3

Intergroup comparison done in 1 month and 3 months at 500 μm and 1000 μm

Time period	Groups	Mean	SD	P	Groups	Mean	SD	P
	500 μm				1000 μm
1 month	Group 1	53.60	1.51658	0.000	Group 1	56.00	1.22474	0.000
	Group 2	54.00	1.22474		Group 2	62.00	1.22474
	Group 2	53.00	1.00000	0.481	Group 2	62.00	1.22474	0.153
	Group 3	52.40	1.51658		Group 3	61.00	0.70711
	Group 1	53.60	1.51658	0.001	Group 1	56.00	1.22474	0.000
	Group 3	52.40	1.51658		Group 3	61.00	0.70711
3 months	Group 1	44.20	1.48324	0.000	Group 1	52.00	1.00000	0.000
	Group 2	52.60	1.34164		Group 2	62.00	2.34521
	Group 2	52.60	1.34164	0.240	Group 2	62.00	2.34521	0.452
	Group 3	51.60	1.14018		Group 3	61.00	1.58114
	Group 1	44.20	1.48324	0.000	Group 1	52.00	1.00000	0.000
	Group 3	51.60	1.14018		Group 3	61.00	1.58114

SD: Standard deviation

DISCUSSION

Infected pulp does not provide an environment conducive to host tissue regeneration. Hence, effective disinfection is a prerequisite to pulpal revascularization. Chemical disinfection plays a crucial role before endodontic regeneration in reducing the microbial load within the necrotic canal space. High concentrations of NaOCl and antibiotic intracanal medicaments were found to have a negative effect on endodontic regeneration,[718] whereas studies by Ruparel et al. proved that low concentration of antibiotic paste has no harmful effect on stem cells of apical papillae survival and also found to be effective against endodontic pathogens.[19] Therefore, the recent recommendations suggest the use of 1.5% NaOCl root canal irrigant, followed by low concentration of antibiotic mixtures ranging from 0.1 to 1 mg/mL as an interappointment medicament for endodontic regeneration.[3] Based on these concepts, 1.5% NaOCl was used as root canal irrigant during root canal preparation and antibiotic paste of concentration 0.1 mg/ml was used as intracanal medicament in the present study.

The double antibiotic paste is effectively used in endodontic regeneration.[7] Amoxicillin-clavulanate is an effective antibiotic against common endodontic pathogens.[12] It acts by inhibition of bacterial cell wall synthesis. Nosrat et al. used amoxicillin-clavulanate paste as an intracanal medicament for an immature tooth with acute abscess for 5 weeks which showed complete periapical healing and formation of the root apex.[13] In a recent study, it has been found that amoxicillin-clavulanate paste significantly reduced intracanal bacterial biofilm which is comparable to that of triple antibiotic paste.[20] The effect of amoxicillin-clavulanate paste on stem cells of apical papilla has been investigated and found that it is similar to triple antibiotic paste and at lower concentrations, there is better survival of stem cells with minimal detrimental effect.[19] The effect of amoxicillin-clavulanate paste on the mechanical properties of radicular dentine has not investigated extensively. Hence, in this present study, the amoxicillin-clavulanate paste was investigated for its effect on microhardness of radicular dentine.

Regenerative endodontic treatment involves several appointments and may take several months for completion. Usually, 2–4 week application of intracanal medicaments provides therapeutic effects of antibiotics on the tooth.[2122] It is found that treatment periods from 1 week to 15 days were used in some cases and is found to extent even up to 11 weeks.[23] Long-term exposure of dental hard tissue to medicaments may negatively affect the chemical, physical, and mechanical properties of radicular dentine.[24] Hence, in the present study, to evaluate both short-term and long-term effects of double antibiotic paste and amoxicillin-clavulanate paste on microhardness of radicular dentine, the test was conducted in three time intervals that are 1 week, 1 month, and 3 months.

Triple antibiotic paste and double antibiotic paste have a negative effect on microhardness of radicular dentine when used as intracanal medicament in endodontic regeneration.[1525] This is substantiated by the study in which the chemical integrity of dentin was evaluated after various endodontic regenerative protocols.[17] In the above study, a significant reduction in phosphate/amide ratio was found in dentin treated with triple antibiotic paste compared to untreated control dentin, which indicates a net superficial demineralization effect of medicaments. This was explained by the strong acidic nature of triple antibiotic paste (pH = 2.9). Similarly, the lower microhardness of double antibiotic paste also might be due to this type of demineralization related to ph of the antibiotic used. At 1 week, 1 month, and 3 months evaluation, amoxicillin-clavulanate paste showed only negligible effect on microhardness than double antibiotic paste at the same period. This might be due to nearly neutral ph of amoxicillin-clavulanate antibiotic paste without any demineralization effect of radicular dentine which needs further studies.

Microhardness tests is a means to measure the hardness of teeth which requires only a tiny area of specimen surface for testing and is easy and quick. Compared to Vickers microhardness test, load impression in the Knoop microhardness test can be applied to brittle materials without cracking, and also the longer diagonal obtained in this test is easier to read than the short diagonal of the Vickers microhardness method.[26] Based on this concept, the Knoop microhardness test was preferred in this study. This study on the effect of microhardness of dentin using double antibiotic paste and amoxicillin-clavulanate paste to the control group was done at 500,000 μm from the pulp dentin interface. There was no significant reduction in dentinal microhardness between the three groups at 1 week time. The double antibiotic paste had a significant effect on microhardness of dentine up to 1000 μm than the control group at 1 month and 3 months and dentinal microhardness reduces significantly from 1 week to 1 month and 3 months. Compared with amoxicillin-clavulanate paste at 500,000 μm, at 1 month and 3 months period, the double antibiotic paste showed a significant reduction in microhardness of dentin. There was no significant change in microhardness between amoxicillin-clavulanate paste and control group at 1 week, 1 month, and 3 months at 500,000 μm. The effect of double antibiotic paste on microhardness of radicular dentin was analyzed by Yassen et al. at 1 week, 1 month, and 3 months against a control group. They concluded that at 500,000 μm, the intracanal application of double antibiotic paste caused a significant decrease in root dentine microhardness after 1 and 3 months.[16] In another study, the application of double antibiotic paste for 4 weeks significantly reduced the microhardness value compared to baseline.[25]

Antibiotics reduce the microbial load in the root canal to provide a conductive environment for regeneration. The use of a single antibiotic reduces the possibility of bacterial resistance compared to antibiotic combinations. The amoxicillin-clavulanate paste is effective against common endodontic pathogens and its influence on the reduction of dentinal microhardness at 500,000 μm is found to be not significant compared to double antibiotic paste at all time periods. This effect is similar to the control group where no intracanal medicament was used. At a concentration of 0.1 mg/mL, amoxicillin-clavulanate paste has no detectable effect on the survival of stem cells of the apical papilla.[19] Within the limitations of this study, the result suggests that amoxicillin-clavulanate paste was used to cause only minimal demineralization and weakening of dentin at all time periods observed. It is an acceptable choice as an intracanal medicament in regenerative endodontics, especially when the treatment period is of longer duration.

The empirical results reported herein should be considered in the light of some limitations. Regenerative treatment is usually performed in an immature tooth with open apex, but in this in vitro study, the procedure was done in the mature tooth as we are investigating only the effect of various antibiotic paste on microhardness of radicular dentin. For the same above reason, instrumentation was done up to ProTaper universal F3 for the standardization of the specimen. Because of the relatively small number of samples in this study, we should consider the present findings as a preliminary one and further study with large number of sample size should confirm these results. It is an in vitro study and uniformity of the procedure was maintained in all the tooth samples.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.