Evaluation of antimicrobial action and push-out bond strength and compressive strength using mineral trioxide aggregate and triple antibiotic medicament combination as root-end filling material: A novel in vitro study

Abstract

INTRODUCTION Endodontic therapy aims to obtain a fluid-tight seal between the root canal system and the surrounding periodontium. Usually, the routine endodontic procedures are done in orthograde approach, and when this is not feasible, a retrograde approach using root-end preparation and filling through surgical intervention is recommended. Microorganisms play an important role in the outcome of endodontic success. As the root canal has a complex anatomy, it is not possible to eliminate the microbes and to seal all the portal of entries by orthograde approach; hence, root-end filling in retrograde preparations is advocated.[1] The aim of root-end filling material is to obtain an excellent seal apically in the surgically treated root apex. A root-end filling material should have improved sealing ability, and also possess antimicrobial properties to avoid the entry of bacteria and their products from the root canal system to the periapical area or vice versa. Enterococcus faecalis is a common microbial organism found in oral flora. It is most frequently found in endodontically treated teeth with persistent lesions and therefore carries a huge attention during endodontic treatment. The incidence of E. faecalis in periapical lesion ranges from 22% to 74%, as it can invade dentinal tubules and sustain for a long period of time under unfavorable conditions such as lack of canal nutrients, alkaline conditions of calcium hydroxide medication, and its adhesion to the root dentin and cementum.[2] Mineral trioxide aggregate (MTA) is considered an ideal retrograde filling material as it promotes the formation of cementum, periodontal ligament, and bone, therefore induces regeneration rather than repair. It has an excellent sealing ability, biocompatible, and less cytotoxicity.[3] However, certain drawbacks of MTA include difficulty in manipulation and limited antimicrobial activity. It is not much effective against various microbes such as E faecalis, Streptococcus mutans, and Actinomyces viscosus from the root canal microbiota.[1] Owing to the varied species of the microorganisms in the root canal system, it necessitates the use of an effective retrograde filling material during retro preparations that possess effective antimicrobial action. Triple antibiotic paste (TAP) contains ciprofloxacin, metronidazole, and minocycline. It is widely used as an intracanal medicament during regeneration and revascularization procedures. It is found to be effective against several intracanal microbes.[4] MTA has been used in combination with chlorhexidine (CHX) and tetracycline for enhancing its antimicrobial effect.[5] Since TAP has wide endodontic applications as an effective antimicrobial agent; however, to the best of our knowledge, literature is scarce indicating the combination of MTA with TAP as root-end filling material; therefore, the aims of the present study are threefold (i) to evaluate antimicrobial activity of MTA in addition to TAP against E faecalis, (ii) to determine the effect of this combination on push out, and (iii) compressive strength when used as retrograde filling materials in in vitro models. MATERIALS AND METHODS The present study was conducted after obtaining Institutional Ethical clearance (Ref No: IEC/2020-21/22). Bacterial strain and media The E. Faecalis (ATCC 29212) were subcultured from the stock cultures on sheep blood agar media (HiMedia Laboratories, Mumbai, India) maintained at 37°C for about 24 h. Inoculum was prepared by transferring the microbial colonies with a sterilized inoculation loop to sheep blood agar plate and the cultural strains were confirmed by Gram staining. Preparation of the test samples Preparation of TAP was done using metronidazole 400 mg (Abbott Pvt Ltd., Mumbai, India), ciprofloxacin 200 mg (Cipla Pvt Ltd, Mumbai, India), and minocycline 100 mg (Research-Lab Fine Chem Industries, Mumbai, India) in ratio 1:1:1 were grounded in a mortar and pestle. Fine sieve was used to remove heavy filler particles and to obtain a fine powder. The carrier was prepared by mixing macrogol and propylene glycol (MP) (SMPLY, Mount Pleasant, Tennesse) in a 1:1 ratio. The combined powder was then mixed with the MP carrier and in the ratio of 5:1 on a glass slab with a stainless steel spatula (Hoshino et al.). A final concentration of 1 mg/ml of TAP was used in this study. Preparation of ProRoot MTA (Dentsply Maillefer, Ballaigues, Switzerland) was done according to the manufacturer's instructions by adding the powder and liquid ratio in 3:1 ratio to obtain putty-like consistency. Mixing was done on a glass slab using a metal spatula. Experimental samples in each group were prepared by mixing MTA and TAP on a sterile mixing pad with a stainless steel spatula vigorously until a uniform mix is obtained for about 10 s as follows: Group 1: MTA pellets Group 2: MTA and triple antibiotic mixed in 2:1 ratio by volume Group 3: MTA and triple antibiotic mixed in 1:1 ratio by volume. Antimicrobial activity The antimicrobial activity was determined by agar diffusion test and the medium used was blood Agar which contains tryptone protein base, protein digest of soyabean, agar, sodium chloride, and 5% sheep blood (HiMedia Laboratories, Mumbai, India). To evaluate the antimicrobial activity against E. faecalis, the test materials of above mentioned three groups were used. Three wells measuring about 6 mm × 4 mm dimensions were prepared in each agar plate using bore drills followed by immediate placement of test samples. For pre diffusion of the test materials, the plates were set aside at room temperature for 2 h and were maintained for 24 h at 37°C. The diameters of zones of inhibition were analyzed using a precision ruler. The study was performed in triplets, and the average of three reading was taken as the final reading. Push-out bond strength To evaluate the push-out bond strength, 30 extracted human maxillary central incisors were collected and decoronated to a standard length of 13 mm. The roots were instrumented with a rotary ProTaper Universal file system (Dentsply Maillefer, Ballaigues, Switzerland) till size F5 and irrigation was carried out with 3 ml of 3% sodium hypochlorite (Chemdent, India) and normal saline (Vishal Private limited, India) after using each instrument, followed by obturation with guttapercha (Dentsply Maillefer, Ballaigues, Switzerland) and AH Plus root canal sealer (Dentsply Maillefer, Ballaigues, Switzerland). The roots were sectioned horizontally, 3 mm above the apical foramen perpendicular to the long axis of the tooth with a diamond bur (Mani, Tochigi, Japan)). Root-end preparation to a depth of 3 mm was performed using ultrasonics stainless steel retro tip size 2 (Dentsply Maillefer, Ballaigues, Switzerland). The specimens were randomly divided into three groups (n = 10 each) and retro filling was done using test materials as mentioned earlier in the above tests. In all the specimens, a second horizontal section was made 4 mm above the previous section using a diamond disk (Mani, Tochigi, Japan) and low-speed handpiece (NSK, Tochigi Japan). Acrylic resins rings of 4 mm × 16 mm dimensions were prepared and the specimens were embedded into it. For push-out testing, each specimen was loaded under a universal testing machine (UNITEK 9450, India) at a speed of about 0.5 mm/min. Compressive strength To evaluate the compressive strength, cylindrical specimens of the test materials of each group were fabricated using molds measuring about 6 mm in height and 3 mm in diameter and were kept under 100% humidity at 37°C until use and divided into three groups (n = 10), same as mentioned in the above tests. For compressive strength analysis, each specimen was loaded with a 5 kN load at a speed of 0.5 mm/min under a universal testing machine (UNITEK 9450, India) and the values were noted. Statistical analysis Data analysis was done using Statistical Package for the Social Sciences (SPSS) software version 23 (IBM Statistics, Chicago, USA). For continuous variables, mean ± standard deviation were used. The difference of the means of analysis variables between more than two independent groups was analyzed by Kruskal–Wallis Test at a (P < 0.05) for statistical significance. RESULTS The zones of inhibition for antimicrobial activity are shown in [Table 1]. MTA + TAP (1:1 ratio) had greater zone of inhibition (34.3 mm) followed by MTA + TAP (2:1 ratio) (34.0 mm), and MTA alone had least zone of inhibition (30.7 mm) with statistical difference between them [Table 2].Table 1: Krussal-Wallis test for antimicrobial activity and push-out bond strength and compressive strengthTable 2: Intragroup comparison of P value for different parameters using Kruskal-Wallis testThe push-out bond strength was found to be higher in MTA alone (5.3 MPa), followed by MTA + TAP (2:1 ratio) (5.1 MPa) and least in MTA + TAP (1:1 ratio) (5.0 MPa), with no statistical difference between them. Compressive strength was higher in MTA alone (39 MPa), followed by MTA + TAP (1:1 ratio) (38 MPa) and MTA + TAP (2:1 ratio) (37 MPa) with no statistical difference between them (P < 0.05) [Tables 1 and 2]. DISCUSSION The primary intention of the materials used for root-end filling is to deliver an adequate seal apically and to prevent the ingress of microbes into the apical periodontium. Sometimes, in spite of obtaining a perfect seal, root-end filling materials leave microscopic spaces between material and the cavity of root-end preparation and cause microleakage leading to reinfection. Therefore, retrograde filling materials should be biocompatible, prevent microleakage, and also possess antimicrobial effect to prevent the microbial ingress.[6] E. faecalis is a facultative anaerobic, Gram-positive microbe. It has the ability to thrive in unfavorable conditions such as environment having low oxygen, high pH, at a wide range of temperatures, and deprived state of nutrients. The occurrence of E. faecalis in endodontically treated teeth is nine times more than the untreated infected teeth. Hence, its prevention and elimination are paramount for endodontic success.[7] MTA contains tricalcium aluminate, tricalcium silicate, bismuth oxide, and has hydrophilic fine particles which comes in contact with moisture or blood and tends to harden. MTA fulfills ideal requirements of root-end filling material such as it is nonresorbable, biocompatible, nontoxic, and exhibits minimal leakage around the margins. Hydroxyapatite crystals are formed over MTA when it comes in contact with tissue synthetic fluid. This induces the formation of calcified structures in endodontic treatments. Therefore, it is considered to be ideal material for root-end filling.[8] However, MTA has some short comings such as high cost, long setting time, difficulty in handling, and possesses limited antimicrobial activity. It is shown that MTA is less effective against certain microbes such as E faecalis.[9] TAP is a combination of ciprofloxacin, metronidazole, and minocycline. Metronidazole, as a nitroimidazole compound, has lethal action on anaerobes and is known to be antimicrobial agent against protozoa and anaerobic bacteria. Minocycline is bacteriostatic against Gram-positive and Gram-negative bacteria. It also increases the amount of interleukin-10, which is an inflammatory cytokine. Ciprofloxacin is bactericidal in action and exhibits greater antimicrobial activity against Gram-negative bacteria but limited activity against Gram-positive ones. Many anaerobic bacteria are resistant to ciprofloxacin. Hence, it is often used with metronidazole in treating mixed infections. Therefore, TAP can affect Gram-negative, Gram-positive, and anaerobic bacteria, and this combination is effective against most of the odontogenic microorganisms.[5] Kim and Kim stated that TAP had greater inhibition zones against E. faecalis than calcium hydroxide.[10] In another study, TAP and CHX were added to MTA, it was found that the combination of MTA with TAP exhibited higher antimicrobial activity.[11] The results of present study are in accordance with the above studies, with Group 3 (MTA and TAP 1:1) showing maximum antimicrobial activity followed by Group 2 (MTA and TAP 2:1) and least with Group 1 (MTA alone). Therefore, TAP enhances the antimicrobial activity of MTA when added in equal volume. The criteria to select a material for root-end filling should not only depend on the sealing ability and dimensional stability but also on its effective antimicrobial activity. The success of root-end filling depends on adaptation between the material and dentin, as it is important to maintain the integrity of the dentin-cement interface.[12] Push-out bond strength test is an efficient method to evaluate the bond strength of a material to dentin as there is uniform stress distribution at the dentin-cement interface. The bond strength between retrograde filling material and dentin should provide sufficient adaptation and greater interface between the dentin and material.[13] Compressive strength, on the other hand, is an indirect measure of material setting and also an important property affecting the clinical performance of any material.[14] Hence, push-out bond strength and compressive strengths of the test materials were evaluated in this study. Studies show that 1 mg/mL of TAP caused significantly less reduction in dentinal microhardness when compared to 1 g/mL TAP; hence, it is recommended to use 1 mg/mL.[1516] Hence, we used 1 mg/mL concentration of TAP in our study. It is shown that TAP induces loss of dentine and an increase in roughness considerably.[1718] As dentin roughness increases, it causes lower wettability of dentine and also lowers inorganic component of root dentin; in addition, the high pH (2.9) of TAP can demineralize the root dentine.[19] The results of the present study showed that push-out bond strength and compressive strength of Group 1 (MTA alone) were higher compared to other two groups containing MTA in combination with TAP; however, there was no statistical difference between the three groups which is in accordance to the above-mentioned studies.[171819] The application of TAP as an antimicrobial agent is not restricted to the field of endodontics alone, it is also having its significant use in field of operative dentistry. Glass ionomer cement (GIC) containing TAP was found to be active against Lactobacillus casei and Streptococcus mutans. Yesilyurt et al.[20] reported that if 1.5% TAP is incorporated to GIC, the compressive strength, the physical properties, and the bonding strength to dentine are not modified. In another study, it was stated that there was no difference in compressive strength on the addition of different concentrations of CHX to MTA.[21] Hence, the addition of antimicrobial agents in small quantities does not inhibit the physical properties of MTA rather enhances its antimicrobial properties. Therefore, the overall results of present study show that TAP enhances antimicrobial activity of MTA against E. faecalis without affecting other properties such as compressive strength and push-out bond strength to a greater extent. Therefore, this combination proves to be an effective root-end filling material. This study aims to address the combination of MTA and TAP as a retro filling material; however, certain limitations of the study include (i) the use of agar diffusion method, inspite of its common application, Several factors affect the accuracy of this method such as the contact between the agar gel and test material, thickness and viscosity of the gel, the test material may dissolve and diffuse through the medium, the storage conditions and incubation period of the agar plates might affect the accuracy and reproducibility of the results. (ii) As this is an in vitro study, it does not mimic the clinical conditions since retro preparations are usually done in periapical areas where it comes in contact it various factors such as tissue fluids and blood, hence remains a matter of concern. (iii) Studies using other microbes should be undertaken evaluating the efficacy of MTA + TAP combination. (iv) Tooth discoloration remains a matter of concern with TAP due to the presence of minocycline. However, this study is a basic approach; therefore, further studies have to be undertaken in future addressing the above concerns using other methods of evaluating the antimicrobial activity, larger sample size, and other in vivo and in vitro studies for the effective use of this combination. CONCLUSION The combination of TAP with MTA as a root-end filling material increases the antimicrobial activity against resistant microorganism E. Faecalis without much compromising the compressive and push-out bond strength hence, this combination can be advocated as an alternative material for root-end filling. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.