Evaluation of the Effect of Long-term Use of Three Intracanal Medicaments on the Radicular Dentin Microhardness and Fracture Resistance: An in vitro study.

OBJECTIVES: The aim of this research was to evaluate the effect of long-term use of three intracanal medicaments on the radicular dentin microhardness and fracture resistance.
MATERIAL AND METHODS: A chemomechanical preparation was done using the Protaper rotary instruments up to F3. The teeth were stored in an incubator at 37°C at 100% humidity and were categorized in three groups by random allocation, namely: Triple Antibiotic Paste (TAP), Calcium hydroxide paste (Apexcal) and Ledermix. Following medicament application, the access openings of all teeth were sealed with 4 mm thickness of cavit. The samples were stored for periods of 1 week, 1 month and 3months. Two dentin cylinders measuring 5mm and 3mm were obtained from each sample. The cervical third was used for fracture resistance and the middle third was used for micro hardness evaluation. The microhardness testing was done using a Knoop microhardness tester, and the fracture resistance testing was done using the universal testing machine.
RESULTS: Calcium hydroxide showed maximally negative effect on the physical properties of radicular dentin compared to TAP (p= 0.0100 at one month and Ledermix (p=0.0001 at one month). With an increase in the application time, there was an increased deterioration in the physical properties of radicular dentin.
CONCLUSION: Long-term placement of calcium hydroxide, Triple Antibiotic Paste, and Ledermix (p= 0.0001at 3 months) significantly affects the microhardness and fracture resistance of radicular dentin.

Introduction

The root canal system is comprised of the main canal and various complexities such as lateral canals, ramifications, loops, isthmuses, deltas (). In infected root canals, bacteria, which persist deep within the dentin, may be difficult to eradicate and trigger periapical pathosis (). During a root canal preparation almost half of the root canal surface and ramifications may remain untouched ().

An adequate chemo-mechanical preparation to obtain a three-dimensional seal is important to attain a successful endodontic therapy (). Failure of endodontic treatment is most commonly related to the perseverance of pathogenic microbes that thrive very well even after a thorough cleaning and shaping is done ().

Combined use of adequate isolation techniques, biomechanical preparation, effective irrigating solutions and techniques, along with a proven disinfecting medicament play a major role in determining the success of a root canal therapy ().

Medicaments play a major role in preparing the root canals for long term treatment protocols, especially in situations of regenerative endodontics and apexification. Calcium hydroxide (CH), Triple Antibiotic Paste (TAP), Double Antibiotic Paste (DAP) and Ledermix are the most commonly used intracanal medicaments ().

Calcium hydroxide (CH) is widely used to achieve disinfection within the root canal. It is also used for the induction of apexogenesis and apexification, in management of internal eradicular resorptive defects, various perforations and in treating periradicular pathoses; nevertheless, it has been used as a dental-pulp capping agent (, ).

Bystrom et al. confirmed the fact that E. faecalis is the most resilient strain against CH (, ).

Various studies have proved that long-standing use of CH may have an adversative effect on the physical properties of radicular dentin (). Almost 50% of reduction in microtensile fracture strength (MTFS) of teeth was seen by Rosenberg et al. while they were studying long-term application of CH (7-84 days) (). Even the mean elastic modulus of bovine dentine increased drastically, as seen by Kawamoto et al. (2008), thus making it more prone to fracture ().

Lately, a newer formulation called ‘triple antibiotic paste’ (TAP) has been given by Hoshino et al. Ciprofloxacin, metronidazole and minocycline together form TAP (). The abovementioned antibiotics exhibit effective antimicrobial and antibacterial properties ().

Trauma and periapical pathosis in immature permanent teeth impede the deposition of minerals by the disruption in blood flow thereby causing cessation of complete root development (). The apexification technique promotes apical closure by either placement of a MTA apical plug or with periodic changes of intracanal medicaments (, ). Another treatment option is the Regenerative endodontic procedure which is designed to replace the damaged structures to reestablish the pulp-dentin complex thereby maintaining the vitality of pulp (). According to Fouad, a complete disinfection is required for a higher level of efficacy than is needed in clinical endodontics (). Thus, the establishment of a sustained sterile microenvironment within the root canal which is achieved through adequate decontamination procedures will give the newly formed tissue adequate time to establish itself within the endodontic environment ().

Disinfection of the root canal is considered to be a crucial step as infection prevents regeneration, repair and stem cell activity (, ). The American Association of Endodontics (AAE) protocol advocates the use of TAP in regenerative endodontic procedures (). Its use is also advocated in the treatment of radicular resorptive defects, root fracture, and also in the non-surgical management of periapical pathosis ().

The use of TAP is associated with a few limitations; Tooth discoloration is the biggest drawback of this medicament, which arises due to the presence of minocycline which has a negative impact on the esthetics of the patient, especially when used in the anterior teeth (, ). TAP, when used for long term, has shown to cause an increased radicular dentin demineralization and an associated reduction in microhardness (, ).

Ledermix™ paste is a commercially available intracanal medicament paste that was introduced by Schroeder and Triadan in 1960. It is a polyethylene glycol-based paste containing tetracycline antibiotic, demeclocycline HCl (conc. of 3.2%), and a corticosteroid, triamcinolone acetonide, conc. 1% (, ). It is efficient in alleviating pain, in the inhibition of inflammatory root resorption and as an antimicrobial agent (). In cases of root resorption and large periapical lesions, Ledermix is kept for a prolonged period of time (, ). Long term exposure of enamel to Ledermix causes a drastic reduction in the microhardness which is due to the aqueous tetracycline component. Due to lack of literature in this field this study was designed to evaluate the long-term impact of these medicaments on properties such as microhardness and fracture resistance of radicular dentin.

The aim of this work is to compare and evaluate the effect of long-term use of three intracanal medicaments on the radicular dentin microhardness and fracture resistance.

Null hypothesis

There is no difference in the effect of long term placement of calcium hydroxide, Triple Antibiotic Paste and Ledermix on the radicular dentin micro hardness and fracture resistance.

Material and methods

Source of data

The study was conducted in Department of Conservative Dentistry and Endodontics, KAHER’ s KLE VK Institute of Dental Sciences’, KLE Academy of Higher Education and Research (KLE University) Belagavi.

The microhardness testing was conducted at M.I.T (Manipal Institute of Technology). The fracture resistance testing was performed at K.L.E Engineering College Belagavi. A total of 180 extracted, human permanent single rooted premolar teeth were selected.

Selection criteria

Extracted human permanent single rooted premolar teeth with patent canals and the teeth with apical width corresponding to #20 K-file or less were included. Carious teeth, teeth with apical width more than #20 K-file size, teeth with calcified canals, teeth with fracture/crack or a restoration, teeth with internal/external resorption were excluded.

Methodology

180 extracted, human single rooted premolar teeth were selected. Cleaning of visible blood and gross debris was done using an ultrasonic scaler. The extracted teeth were handled according to OSHA (Occupational Safety and Health Administration) guidelines. 0.1% thymol solution was used for the storage of the samples until use.

A round bur with a high-speed handpiece was used to make an access cavity in each tooth specimen. Apical patency was attained using a size 10 K file ((Mani. Inc., Tochigi, Japan. A 15 K-file (Mani. Inc., Tochigi, Japan) was extended 1 mm beyond the apical foramen by visualizing its tip, following which 1 mm was removed from the predetermined working length. Shaping and cleaning was carried out using the Protaper (Dentsply, India) rotary instruments till F3 with endo motar(X -Smart, Dentsply, India). In between each instrumentation change, the canals were irrigated with 2mL of 3% NaOCl (Vishal Dentocare Pvt. Ltd., Ahmedabad India), followed by 2mL of 17% EDTA (DEOR Deo Smear-Off, India) as the final irrigant. The canals were subsequently rinsed with 5-mL of sterile saline using a 27-gauge needle. Following irrigation of teeth, sterile paper points (Dentsply, India) were used to dry the root canals.

The teeth were stored in an incubator at 37°C at 100% humidity. The teeth were stored for periods of 1 week, 1 month and 3months.

Medicament application

180 specimens were allocated to the following three groups:

Group I: Triple Antibiotic Paste (TAP); Group II: Calcium hydroxide paste (Apexcal); Group III: Ledermix

Group I: Triple Antibiotic Paste (TAP)

1st experimental group (n = 60), For the preparation of TAP, 1mg / ml of each antibiotic powders (USP-grade) in equal quantities of metronidazole, ciprofloxacin and minocycline in a ratio of 3:1 was combined with polyethylene glycol. A sterile lentulo spiral was used for the introduction of the prepared medicament into the root canals with a slow-speed handpiece. Following this, the medicament was compacted to the level of the CEJ using sterile pluggers (Sybron endo).

Group II: Calcium hydroxide paste (Apexcal)

For the second experimental group (n = 60), commercially available Ca (OH) 2 paste (Apexcal) was used. The medicament application to the root canal space was done in a similar manner as described previously.

Group III: Ledermix

For the third experimental group (n = 60), commercially available Ledermix paste was used. The paste was introduced into the root canals as elaborated previously. Following medicament application, 4mm of cavit (3M ESPE) was used to seal all the prepared specimens. After the coronal seal was achieved, flowable composite (Ivoclar Vivadent) was used to obtain an apical seal.

Preparation of root specimens

A low-speed diamond saw (Agar Scientific) under constant water cooling was used for decoronation of the prepared samples, 0.5mm radicular to the CEJ. The decoronated segment was then sectioned horizontally in order to obtain 2 radicular dentin discs. Fracture resistance assessment was performed on the 5-mm section cervical to the CEJ and microhardness evaluation was made on the middle third 3-mm root section. 5 mL of sodium hypochlorite in combination with gentle, manual agitation with a ProTaper F3 instrument followed by final irrigation with 5 mL of EDTA was used for the elimination of the medicament from each prepared section.

Microhardness testing

A Vickers Microhardness Tester (MCS Mechatronic) was used to measure the microhardness of each tooth specimen. Three indentations were created on the polished surface of each specimen with the help of a 50-g load held straight to the polished side for 15 s. The indentations were made 500 lm from the pulp dentin interface. The optical microscope was used to observe indentations created. The mean of the values obtained for the three indentations was representative for each specimen at each depth.

Fracture resistance testing

The Universal testing machine (MCS Mechatronic) was used for assessment on each 5-mm root section. The lower platform was used to place the specimen vertically with the coronal side facing upwards. A cylindrical loading fixture with a radius = 1.9 mm was fixed to the upper crosshead until the spherical tip touched the root specimen. A loading force at a cross head speed of 0.5 mm per minute was applied until the root cylinder fractured and was expressed in newtons.

Results

Comparisons of three groups with micro-hardness and fracture resistance at different time points are respectively shown in Figure 1 and Figure 2. Data analysis with Tukey’s multiple posthoc for hardness and fracture resistance is shown in Table 1 and Table 2.

Figure 1

Comparison of three groups with micro hardness at different time points

Figure 2

Comparison of three groups with fracture resistance at different time points

Table 1

Pair wise comparison of three groups with micro hardness at different time points by Tukey’s multiple posthoc procedures

Times	Groups vs	Groups	Mean Difference	Std. Error	p-value
1 week	Calcium hydroxide vs	Tap	-22.99	5.44	0.0001**
	Calcium hydroxide vs	Ledermix	-60.83	5.44	0.0001**
	Tap vs	Ledermix	-37.84	5.44	0.0001**
1month	Calcium hydroxide vs	Tap	-21.97	2.31	0.0001**
	Calcium hydroxide vs	Ledermix	-20.90	2.31	0.0001**
	Tap vs	Ledermix	1.08	2.31	0.8880
3 months	Calcium hydroxide vs	Tap	-27.80	2.32	0.0001**
	Calcium hydroxide vs	Ledermix	-25.99	2.32	0.0001**
	Tap vs	Ledermix	1.81	2.32	0.7180

**p<0.01

Table 2

Pair wise comparison of three groups with fracture resistance at different time points by Tukey’s multiple posthoc procedures

Times	Groups vs	Groups	Mean Difference	Std. Error	p-value
1 week	Calcium hydroxide vs	Tap	-15.80	5.78	0.0220*
	Calcium hydroxide vs	Ledermix	-22.70	5.78	0.0010**
	Tap vs	Ledermix	-6.90	5.78	0.4610
1month	Calcium hydroxide vs	Tap	-17.60	5.81	0.0100*
	Calcium hydroxide vs	Ledermix	-26.65	5.81	0.0001**
	Tap vs	Ledermix	-9.05	5.81	0.2720
3 months	Calcium hydroxide vs	Tap	-13.20	5.85	0.0700
	Calcium hydroxide vs	Ledermix	-27.70	5.85	0.0001**
	Tap vs	Ledermix	-14.50	5.85	0.0420*

*p<0.05’, **p<0.01

Statistical analysis

The ANOVA test revealed a significant difference in three groups with microhardness and fracture resistance at different time points by one-way ANOVA between all the three groups i.e., Group I (Calcium hydroxide), Group II (Triple Antibiotic Paste) and Group III (Ledermix). The lowest scores for microhardness were shown by Group I (Calcium hydroxide) (49.73±5.25%), (39.10±3.37%), (31.09±1.18%) at 1 week, 1 month and 3 months, respectively. The lowest scores for fracture resistance were shown by Group I (Calcium hydroxide) (737.05±14.92%), (725.40±15.74%), (711.35±15.84%) at 1 week, 1 month and 3 months, respectively. This was significantly different from the other two groups. Group II (Triple Antibiotic Paste) showed the scores for microhardness of (72.72 ± 7.93%), (61.07 ± 5.42%), (58.89 ± 6.85%), and the fracture resistance scores were (752.85 ± 16.76%), (743.00 ± 16.54%), (724.55 ± 16.28%) at 1 week, 1month and 3 months respectively. Group III (Ledermix) had scores for microhardness of (110.56 ± 28.25%), (60.00 ± 10.95%), (57.08 ± 10.64%), and the fracture resistance scores were (759.75 ± 22.30%), (752.05 ± 22.18%), (739.05 ± 22.59%) at 1 week, 1month and 3 months respectively. The same values are shown in Figures 1 and 2.

The pairwise comparison of the three experimental groups is described in detail in Table 1 and 2, and a significant difference was found between the Calcium hydroxide (Group I),Triple Antibiotic Paste (Group II), Ledermix (Group III)on the radicular dentin microhardness and fracture resistance.

Discussion

Due to the complexity of the root canal system, biomechanical preparation by itself is incompetent in completely eradicating microorganisms. Recent research has revealed that the bacteria present in instrumented canals prior to completion of treatment can increase in number and reach their pretreatment count in about 2-4 days (). Amongst the persistent bacteria within the root canal E. faecalis is the most resilient microorganism that causes persistent periapical lesions. In these cases, the use of intracanal medicaments is encouraged (). The application time of intracanal medicaments varies depending upon its clinical use which may range from 1 to 4 weeks for decontamination of the root canal and even extend up to 11 weeks in cases of endodontic regeneration.

It has been affirmed that microhardness assessment gives an indirect substantiation of the mineral loss or gain in mineralized dental tissues as it is dependent upon the amount of calcified matrix per square millimeter (). Arends and Bosch stated that microhardness testing is the most practical method of indirect quantitative analysis by accessing the amount of demineralization of dental hard tissues. Microhardness test provides an insight about the dentinal interaction with different medicaments ().

Dentin may differ significantly between teeth and is associated with considerable number of variations. The tubular density of dentin has found to increase from cervical to apical areas of radicular dentin, which results in an inverse correlation amongst radicular dentinal microhardness and radicular tubular density. This may cause alterations in the results due to the variances in adjacent areas of the dentinal tissue (). Hence, in the present study, the microhardness measurement was performed in the middle-third of the root structure for each sample.

Vickers microhardness testing was chosen since it is less affected by measurement errors. Despite small sized samples and surface conditions the specimens can be evaluated with good accuracy (). There have been cumulative reports of demineralization, associated surface weakening and deterioration in the mechanical properties of radicular dentin which eventually led to microcracks and development of vertical root fracture following long term placement of intracanal medicaments ().The assessment of the mechanical properties of radicular dentin can be done by fracture resistance studies as it relates to the amount of demineralization. Hence, the fracture resistance evaluation was chosen as the second parameter to be tested in this study. In this study, the force was generated at an angle of 0 o, which results in the development of a splitting stress over the tooth specimen. The stresses induced would be minimal because of less bending moments (). In numerous clinical studies the immature permanent teeth treated by endodontic regeneration have shown an increase in the radicular dentinal wall thickness limited mainly to the middle and the apical third rather than the cervical region, thus weakening this region with an increased incidence of fracture; therefore, the cervical third of the root was chosen for fracture resistance test (, ).

As NiTi Protaper instruments are intended to cover the entire range of treatment with fewer files which integrate greater flexibility, unparalleled efficacy and enhanced safety. They have been used over stainless steel instruments in order to prepare the root canals. For the standardization of the root canal, each root was prepared up to Protaper size F3, which corresponds to ISO #no. 30.

Manual agitation was done carefully in an attempt not to abrade the radicular dentin since it has been advocated that the persistence of endodontic intracanal medicament residues interact with the smear layer during instrumentation of the root canal (, ).

Hoshino et al. () recommended the usage of TAP (3Mix) – in a ratio 1:1:1. 
- Ciprofloxacin 200mg, Metronidazole 500mg, Minocycline 100mg. Ciprofloxacin and Minocycline have found to have minimal inhibitory concentration of 5 and 20 μg against E. feacalis and Metronidazole was stated to have no inhibitory effects (57). However, as a combination (100μg each /ml) they have proven to inhibit entirely the growth of every strain (58). In regenerative endodontics, increased concentrations of TAP cause cytotoxic effects on the stem cells of dental pulp and apical papilla. Therefore, decreased concentration in a range of 0.1 mg/mL to 2 mg/mL has been recommended.

The lowest scores for microhardness and fracture resistance were shown by Group I Calcium hydroxide at 1 week, 1 month and 3 months, respectively. The present result could be explained by the alkaline pH of calcium hydroxide and its low molecular weight which denatures the collagenous matrix (). The results of the present study are consistent with a recently performed study by Yassen et al. which showed that Ca (OH)2 produced substantial collagen degradation of superficially present root canal dentine after a short duration of 1-week (). It is well understood that the collagen constituent is accountable for the toughness of the mineralized hard tissues, hence any defect produced compromises the mechanical properties making it more susceptible to fracture, which has been seen in this study after 3 months. Long term application of Ca (OH) 2 also enhances crack propagation (). Increased susceptibility to fracture was seen in this study’s 3-month fracture resistance data. Group II (Triple Antibiotic Paste) showed the scores for microhardness of (72.72±7.93%), (61.07±5.42%), (58.89±6.85%), and the fracture resistance scores were (752.85±16.76%), (743.00±16.54%), (724.55±16.28%) at 1 week, 1month and 3 months respectively. This may be credited to various acids commonly mixed with antibiotics to preserve their chemical steadiness and to regulate their tonicity. The results of the present study totally reject the null hypothesis.

Minocycline causes chelation of calcium, which in turn contributes to demineralization (, ). Studies show that increased concentration of TAP is directly proportional to the surface roughness (). This also causes reduction in the inorganic content and loss of dentin, which decreases the wettability of dentin surfaces (). The results obtained in the present research are in line with studies conducted by Yassen et el (2013) and Kinney et al. (2003) that demonstrated that 1 g/mL TAP caused severe reduction in microhardness and fracture resistance of the radicular dentin at time intervals of 1 week and 1 month. TAP increases the organic content (, ). The reason attributed to the change in the chemical properties of radicular dentin following the use of TAP is due to its acidic ability (pH = 2.9) and associated surface demineralization (). TAP has a less negative effect on the chemical structure of radicular dentin compared to CH which was also found in the present study. Time period being an important factor, the increased exposure time is directly proportional to demineralization (). The results obtained by Madhusudhana K et al. are consistent with the results of the present study ().

The samples exposed to Ledermix intracanal medicament showed less reduction in the radicular dentin microhardness and fracture resistance. This could be attributed to its lower pH (8.76) compared to the Ca (OH)2 intracanal medicament (65). It has been proposed that a higher alkaline pH may cause denaturation of the organic matrix or result in the collapse of the dentine inorganic matrix. Studies have found that the denaturing of organic matrix is promoted by high pH (). There is encapsulation of the organic matrix by the inorganic hydroxyapatite, and the penetration of the intracanal medicament requires greater time (e.g., 4 weeks in some studies), thus making the dentinal structure more brittle with an increased susceptibility to fracture.

Further in vivo studies are needed in order to apply the results obtained from the present study in clinical setting. There is insufficient evidence to current protocols followed in regenerative endodontics. This has been proposed based upon research and previous case reports. The area of regenerative endodontics is still in its budding stage where we need evidence-based data to determine the best treatment time, the best medicament(s), and the best concentration without compromising the physical properties of the tooth.

Conclusions

Within the parameters of this in vitro study, the null hypothesis has been rejected and the following conclusions can be drawn: The results of the present study suggest avoiding long term use of intracanal medicaments which are generally used in regenerative endodontics to obtain a sterile environment to promote growth. The three-month application of Ca (OH)2, TAP and Ledermix intracanal medicaments significantly decreased the fracture resistance and microhardness of radicular dentin compared to the 1-week application. A prolonged exposure of intracanal medicaments has adverse effects on chemical and mechanical properties of root canal dentin.