| Abstract|| |
Background: Long-term use of intracanal medicaments such as calcium hydroxide (CH) reduces the fracture resistance of dentin. The present study was undertaken to evaluate the fracture resistance of radicular dentin on long-term use of CH, chlorhexidine (CHX) with lycopene (LP).
Aim: To compare the fracture resistance of radicular dentin when intracanal medicaments such as CH, CHX with LP were used for 1-week and 1-month time interval.
Settings and Design: Sixty single-rooted extracted human permanent premolars were collected, and complete instrumentation was done. Samples were divided into three groups based on intracanal medicament used.
Materials and Methods: Group 1 - no medicament was placed (CON), group 2 - mixture of 1.5 g of CH and 1 ml of 2% CHX (CHCHX), group 3 - mixture of 1.5 g of CH, 1 ml of CHX and 1 ml of 5% LP solution (CHCHXLP). After storage period of each group for 1-week and 1-month, middle 8 mm root cylinder was sectioned and tested for fracture resistance.
Statistical Analysis: Results were analyzed using paired t-test.
Results: At 1-month time interval, there was a statistically significant difference in fracture resistance between CHCHX and CHCHXLP groups.
Conclusion: Addition of LP has not decreased the fracture resistance of radicular dentin after 1-month.
Keywords: Calcium hydroxide; fracture resistance; intracanal medicament; lycopene
|How to cite this article:|
Madhusudhana K, Archanagupta K, Suneelkumar C, Lavanya A, Deepthi M. Effect of addition of lycopene to calcium hydroxide and chlorhexidine as intracanal medicament on fracture resistance of radicular dentin at two different time intervals: An in vitro study. J Conserv Dent 2015;18:205-9
|How to cite this URL:|
Madhusudhana K, Archanagupta K, Suneelkumar C, Lavanya A, Deepthi M. Effect of addition of lycopene to calcium hydroxide and chlorhexidine as intracanal medicament on fracture resistance of radicular dentin at two different time intervals: An in vitro study. J Conserv Dent [serial online] 2015 [cited 2019 Jul 20];18:205-9. Available from: http://www.jcd.org.in/text.asp?2015/18/3/205/157248
| Introduction|| |
Calcium hydroxide (CH) is widely used in dentistry for various procedures such as pulp capping, apexification and as intracanal medicament. CH is used as intracanal medicament to decrease the microbial load during inter appointment treatment regimen.  CH has various properties such as antibacterial activity,  antiexudative action,  biocompatibility,  ability to induce mineralized tissue,  ability to dissolve necrotic tissue  and ability to inactivate bacterial endotoxin. , Antimicrobial activity of CH is attributed due to its high alkaline pH. The hydroxyl ions at high pH disrupt the nutritional relationship causing lethal effects on microorganisms.
Chlorhexidine (CHX) is another intracanal medicament that is routinely used in endodontics for reducing microbial growth.  It has properties such as substantivity and broad antibacterial spectrum. Studies have proven that when CH is combined with CHX have synergistic effect on reducing microbial growth. , CHX exerts its action by increasing the membrane permeability of low molecular weight ions like potassium and phosphorous at low concentration and precipitation of cytoplasmic contents at a higher concentrations.
Calcium hydroxide is used as an intracanal medicament for apexification procedures and nonsurgical endodontic treatment of large periapical lesions for longer periods.  Although CH is proven to be good intracanal medicament in reducing the microbial growth,  literature (Doyon et al.,  Yassen and Platt  Zarei et al.  ) have shown that there is decrease in mechanical properties of radicular dentin when CH is used as intracanal medicament for more than 5 weeks. Study by Prabhakar et al.  has shown that when CH is mixed with 0.2% CHX, there is no alteration in fracture resistance of radicular dentin after 30 days.
Recently, there has been growing interest in evaluating the effect of natural antioxidants in dentistry. Studies by Arumugam et al.,  Abraham et al.  have shown that addition of antioxidants like lycopene (LP), grape seed extract on bleached enamel have increased bond strength of enamel to composite resin restorations. No previous study has been undertaken to use antioxidants with intracanal medicaments to improve fracture resistance of radicular dentin.
The aim of the present study is to evaluate change in fracture resistance of radicular dentin when CH, CHX combination is mixed with natural antioxidant like LP for 30 days.
The tested null hypothesis was that there would be no difference in the fracture resistance of radicular dentin when intracanal medicaments like CH with CHX and CH, CHX with LP, were used for 1-week and 1-month time interval.
In the PICO analysis format:
- Problem - Decrease in fracture resistance of radicular dentin when intracanal medicaments were used for longer periods to reduce microbial growth.
- Intervention - CH and CHX combination when used as intracanal medicament, change in fracture resistance of radicular dentin.
- Comparison - CH, CHX and LP combination when used as intracanal medicament, change in fracture resistance of radicular dentin.
- Outcome - Change in fracture resistance.
| Materials and Methods|| |
Extracted human mandibular single-rooted premolars (n = 60) were selected. Teeth with root cracks, caries, restorations and previous endodontic treatments were excluded. After extraction, the soft tissues, dental calculus, and stains were immediately removed from teeth and stored in normal saline at 4°C for further use.
An endodontic access cavity was prepared in each tooth and the working length was determined by visualizing the tip of a size 15 k-file (Dentsply Maillefer, India) extending beyond the apical foramen and subtracting 1 mm from the length of the file. Later, cleaning and shaping of root canals were done using ProTaper rotary instruments (Dentsply Maillefer, India) till F2. Along with instrumentation, 1 ml of 3% NaOCl (Prime Dental Product, India) was used as an intracanal irrigant between uses of each succeeding file. Further, the canals were finally rinsed with sterile saline to remove any dentin debris remained in canal after instrumentation.
Teeth were randomly assigned into three groups, each containing 20 samples. Group 1, the control group (CON, n = 20), no medicament was applied in the canal. Group 2 (CHCX, n = 20) contains 1.5 g of CH powder (Prime Dental Product, India) and 1 ml of 2% CHX (Calypso, Septodont, India) as intra canal medicament. Group 3 (CHCXLP, n = 20) contain paste of intracanal medicament formed by mixing 1.5 g of CH powder, 1 ml of 2% CHX and 1 ml of 5% LP solution. About 5% LP solution is prepared by mixing 5 g of LP (Mynutramart, India), which is in the form of powder to 100 ml of distilled water.
In groups 2 and 3, the intracanal medicament paste was applied to the root canal spaces with a sterile lentulospiral (Dentsply Maillefer, India) in a slow speed hand piece (NSK, India) and tamped on the canal space up to the cementoenamel junction level. Later, the access openings of all teeth were sealed with glass ionomer cement (GC Company, India) and sealed apically with flowable composite (Tetricflow, Ivoclar Vivadent, India). Then, each group was subdivided into 10 teeth in each, depending on storage period for 1-week and 1-month respectively. After each storage period, the middle 8 mm root cylinder was sectioned with a low-speed diamond disc and straight hand piece for each tooth. The root cylinder was irrigated with distilled water to remove the medicaments.
Fracture resistance of each 8 mm root cylinder was tested using Instron Universal Testing Machine (UTM) (autograph). The root cylinders were positioned vertically on lower fixed platform of UTM using a double-sided adhesive tape. A loading fixture was lowered until the tip rested on root cylinder. Then, a vertical loading force was applied at a crosshead speed of 1 mm/min until the root cylinder was fractured. The load at fracture was measured and expressed in Newton units and analyzed using paired t-test.
| Results|| |
[Table 1] represents that the mean fracture resistance of the samples at 1-week period was numerically >1-month period in all the groups, but a significant statistical difference was observed in CHCHX group. [Table 2] shows the intragroup analysis of fracture resistance of all the three groups at 1-week and 1-month period. There was no statistical difference between all the groups at 1-week. At 1-week period, fracture resistance was high in control group numerically than the other two groups. At 1-month period, fracture resistance was high in control group, and there is a statistical significance difference between control and CHCHX group and between CHCHX and CHCHXLP group. There was no statistical difference between CON - CHCHXLP groups.
|Table 2: Intragroup analysis of groups at 1-week and 1-month time interval|
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Graph 1 represents the mean fracture resistance of all the groups at 1-week and 1-month time interval. At 1-week, there was no statistical difference in the values whereas there was numerical difference between the control - CHCHX and CHCHX - CHCHXLP groups. Between 1-week and 1-month time interval, there was a significant statistical difference in CHCHX group.
| Discussion|| |
The aim of this study is to investigate whether the addition of natural antioxidant LP to CH-CHX mixture as intracanal medicament has any influence on fracture resistance of radicular dentin. The tested null hypothesis was rejected because there was a statistical significance difference in fracture resistance of CHCHXLP group.
Human mature permanent teeth were chosen in the study as an increase in frequency of fracture with immature teeth has been reported because of incomplete root development and subsequent thinner dentinal walls. To simulate the routine clinical conditions and to include its effect also 3% sodium hypochlorite has been used a common irrigant between the succeeding files during cleaning and shaping procedure of all the samples.
In the present study, the mean compressive strength of samples in control group shows higher values than the groups with intracanal medicament, at 1-week and 1-month period [Table 1]. This shows the process of dissolution, denaturation and hydrolysis of the organic structure of radicular dentin due to medicaments. Doyon et al.  stated that there may be disruption in collagen fibers and hydroxyapatite crystals interaction at alkaline pH due to CH, which may negatively influence the mechanical properties of radicular dentin.
Calcium hydroxide has been used in endodontic treatment of human teeth, often overextended periods of time. Literature supports that there was decrease in fracture resistance of radicular dentin when CH was used as intracanal medicament. , In the systematic review by Yassen and Platt many authors (Doyon et al. (2005), Andreasen et al. (2006), Rosenberg et al. (2007), Twati et al. (2009))  quoted that strength of radicular dentin was significantly decreased at a period of >1-month. So, time would be required for CH to penetrate into dentin and denature the collagen fibrils making dentin susceptible to fracture.
In the present study in CHCHX group, there was a significant decrease in fracture resistance of radicular dentin after 1-month time interval [Table 1]. Although this combination has proven to be synergistic in decreasing the microbial growth, it has negative effect on fracture resistance of radicular dentin. This may be mainly because of the release of reactive oxygen species (ROS) by CHX in an alkaline environment. ROS are small, short-lived, and highly reactive radicals formed by incomplete one-electron reduction of oxygen. 
There was no statistical difference between all the groups at 1-week. At 1-month period, fracture resistance was high in control group, and there is a statistical significance difference between control and CHCHX group and between CHCHX and CHCHXLP group [Table 2].
In the present study when antioxidants like LP was added to the mixture of CH and CHX, there was no significant decrease in fracture resistance of radicular dentin at 1-week and 1-month interval. Though there is decrease in numerical value, this is not a statistically significant difference.
Antioxidants are substances that considerably delay or inhibit oxidation of the oxidizable substrate at lower concentrations.  LP, the red pigment of ripe tomatoes, watermelons, red chillies and guavas, is a tetraterpene with eight isoprene units composed entirely of carbon and hydrogen. This highly unsaturated hydrocarbon contains 11 conjugated and two nonconjugated carbon-carbon double bonds. It is a strong antioxidant and free radical-scavenger because it possesses the property of quenching singlet oxygen.  It neutralizes the hydroxyl radicals and stimulates many antioxidative enzymes such as superoxide dismutase, glutathione peroxidase, glutathione reductase and protects cell membranes from lipid peroxidation. LP has the potential to quench singlet oxygen, scavenge various free radicals like nitrogen dioxide (NO 2 ), Thiyl (RS), and sulfonyl (RSO 2 ). ,
Mageshwaran et al.  conducted a study on ROS counteraction by antioxidants against Enterococcus faecalis in agar diffusion test by mass spectrometer. They concluded that CH and CHX combination group produced excessive amount of ROS, which are detrimental to the host tissues and reduced ROS formation in CH, CHX and LP combination group. The results of present study showed the decreased fracture resistance of radicular dentin in CH-CHX group which could be due to the release of ROS. In the present study CHCHXLP group, the LP might have neutralized the ROS produced in CHCHX combinations and resulted in no statistical significance difference in compressive strength of radicular dentin at 1-week and 1-month time interval.
The quenching of ROS by LP may be due to its structure containing high number of conjugated double bonds, which has the maximum oxygen-quenching ability. This is a physical quenching process that is, the carotenoid remains intact and can undergo further cycles of single oxygen quenching. Further, LP effectively deactivates the electronically excited sensitizer molecules that are involved in the generation of radicals and singlet oxygen. ,
The null hypothesis of this study is that there would be no difference in the fracture resistance of radicular dentin after addition of LP to CH-CHX mixture at 1-week and 1-month time interval was rejected. This is because there is a significant difference in increased compressive strength of radicular dentin in CHCHXLP when compared to CHCHX group.
| Conclusion|| |
Within the limitations of this study, it can be concluded that addition of LP to CH and CHX mixture has not decreased the fracture resistance of radicular dentin after 1-month. However, this result is more encouraging and need to be validated with a larger sample size, long-term evaluation with in vitro models and randomized control trials before extrapolating these results into the clinical scenario.
| References|| |
Doyon GE, Dumsha T, von Fraunhofer JA. Fracture resistance of human root dentin exposed to intracanal calcium hydroxide. J Endod 2005;31:895-7.
Manzur A, González AM, Pozos A, Silva-Herzog D, Friedman S. Bacterial quantification in teeth with apical periodontitis related to instrumentation and different intracanal medications: A randomized clinical trial. J Endod 2007;33:114-8.
Allard U, Stromberg U, Stromberg T. Endodontic treatment of experimentally induced apical periodontitis in dogs. Endod Dent Traumatol 1987;3:240-4.
Lu Y, Liu T, Li X, Li H, Pi G. Histologic evaluation of direct pulp capping with a self-etching adhesive and calcium hydroxide in beagles. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:e78-84.
Leonardo MR, Hernandez ME, Silva LA, Tanomaru-Filho M. Effect of a calcium hydroxide-based root canal dressing on periapical repair in dogs: A histological study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:680-5.
Hasselgren G, Olsson B, Cvek M. Effects of calcium hydroxide and sodium hypochlorite on the dissolution of necrotic porcine muscle tissue. J Endod 1988;14:125-7.
Buck RA, Cai J, Eleazer PD, Staat RH, Hurst HE. Detoxification of endotoxin by endodontic irrigants and calcium hydroxide. J Endod 2001;27:325-7.
Tanomaru JM, Leonardo MR, Tanomaru Filho M, Bonetti Filho I, Silva LA. Effect of different irrigation solutions and calcium hydroxide on bacterial LPS. Int Endod J 2003;36:733-9.
Vaghela DJ, Kandaswamy D, Venkateshbabu N, Jamini N, Ganesh A. Disinfection of dentinal tubules with two different formulations of calcium hydroxide as compared to 2% chlorhexidine: As intracanal medicaments against Enterococcus faecalis
and Candida albicans
: An in vitro
study. J Conserv Dent 2011;14:182-6.
Sinha N, Patil S, Dodwad PK, Patil AC, Singh B. Evaluation of antimicrobial efficacy of calcium hydroxide paste, chlorhexidine gel, and a combination of both as intracanal medicament: An in vivo
comparative study. J Conserv Dent 2013;16:65-70.
Bhardwaj A, Ballal S, Velmurugan N. Comparative evaluation of the antimicrobial activity of natural extracts of Morinda citrifolia
, papain and aloe vera (all in gel formulation), 2% chlorhexidine gel and calcium hydroxide, against Enterococcus faecalis
: An in vitro
study. J Conserv Dent 2012;15:293-7.
Aggarwal V, Miglani S, Singla M. Conventional apexification and revascularization induced maturogenesis of two non-vital, immature teeth in same patient: 24 months follow up of a case. J Conserv Dent 2012;15:68-72.
Yassen GH, Platt JA. The effect of nonsetting calcium hydroxide on root fracture and mechanical properties of radicular dentine: A systematic review. Int Endod J 2013;46:112-8.
Zarei M, Afkhami F, Malek Poor Z. Fracture resistance of human root dentin exposed to calcium hydroxide intervisit medication at various time periods: An in vitro
study. Dent Traumatol 2013;29:156-60.
Prabhakar AR, Hadakar SG, Raju OS. Comparative evaluation of pH and antibacterial effect of various calcium hydroxide combinations on E. faecalis
and its effect on root strength: An in vitro
study. Contemp Clin Dent 2012;3:42-7.
Arumugam MT, Nesamani R, Kittappa K, Sanjeev K, Sekar M. Effect of various antioxidants on the shear bond strength of composite resin to bleached enamel: An in vitro
study. J Conserv Dent 2014;17:22-6.
Abraham S, Ghonmode WN, Saujanya KP, Jaju N, Tambe VH, Yawalikar PP. Effect of grape seed extracts on bond strength of bleached enamel using fifth and seventh generation bonding agents. J Int Oral Health 2013;5:101-7.
Yeung SY, Huang CS, Chan CP, Lin CP, Lin HN, Lee PH, et al.
Antioxidant and pro-oxidant properties of chlorhexidine and its interaction with calcium hydroxide solutions. Int Endod J 2007;40:837-44.
Chapple IL. Reactive oxygen species and antioxidants in inflammatory diseases. J Clin Periodontol 1997;24:287-96.
Waris G, Ahsan H. Reactive oxygen species: Role in the development of cancer and various chronic conditions. J Carcinog 2006;5:14.
Subhash K, Bose C, Agrawal BK. Effect of short term supplementation of tomatoes on antioxidant enzymes and lipid peroxidation in type-II diabetes. Indian J Clin Biochem 2007;22: 95-8.
Stahl W, Sies H. Bioactivity and protective effects of natural carotenoids. Biochim Biophys Acta 2005;1740:101-7.
Mageshwaran T, Ebenezar AR, Madhanamadhubala M, Kavitha S, Mahalaxmi S. Counteraction of reactive oxygen species and determination of antibacterial efficacy of proanthocyanidin and lycopene when mixed with calcium hydroxide and chlorhexidine mixture: An in vitro
comparative study. J Conserv Dent 2012;15:337-41.
Dr. Koppolu Madhusudhana
Department of Conservative Dentistry and Endodontics, Narayana Dental College, Chinthareddypalem, Nellore - 524 002, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]