|Year : 2016 | Volume
| Issue : 1 | Page : 23-30
Comparison of the effect of ethylenediamine tetra-acetic acid, chlorhexidine, etidronic acid and propolis as an irrigant on the microhardness of root dentin: An in vitro study
Sumita A Bhagwat, Tamara Ann Lopez, Lalitagauri P Mandke
Department of Conservative Dentistry and Endodontics, DYPU College of Dentistry, Navi Mumbai, Maharashtra, India
|Date of Web Publication||12-Apr-2016|
Sumita A Bhagwat
Department of Conservative Dentistry and Endodontics, DYPU College of Dentistry, Navi Mumbai, Maharashtra
Source of Support: None, Conflict of Interest: None
Aim: This in vitro study was carried out to compare of the effect of 17% ethylenediamine tetra-acetic acid (EDTA), 2% chlorhexidine (CHX), 18% etidronic acid (HEBP), and 4% propolis as an irrigant on the microhardness of root dentin. Materials and Methods: The sample size for the study was 100. Each specimen consisted of a longitudinally sectioned half of a root of a single-rooted tooth which was embedded in acrylic resin. The prepared specimens were divided randomly into five groups of twenty specimens each. Each group was treated with the irrigants to be tested. Group I was the control - the specimens were treated with distilled water. The specimens in Group II were treated with sodium hypochlorite (NaOCl) followed by EDTA. Specimens in Group III were treated with NaOCl followed by CHX. Specimens in Group IV were treated with NaOCl followed by HEBP, and specimens in Group V were treated with NaOCl followed by propolis. Following this, all the specimens were placed on the Vickers hardness tester and three readings were taken for each specimen. An average reading was obtained for each group. The results were tabulated and statistically analyzed to determine which of the irrigant solutions had the least effect on the microhardness of root dentin. Results: Eighteen percent HEBP had the least effect on the root dentin microhardness, followed by 4% propolis and 2% CHX. Seventeen percent EDTA showed maximum effect on the microhardness of the dentin. Conclusion: Under the limitations of this study, 18% HEBP and 4% propolis show promise for use as irrigants because of less detrimental effect on the hardness of root dentin. More studies are needed on demineralization depth and the sealability of resin sealers in the radicular dentin after the use of propolis and HEBP.
Keywords: Chlorhexidine, ethylenediamine tetra-acetic acid, etidronic acid, microhardness, propolis, root dentin
|How to cite this article:|
Bhagwat SA, Lopez TA, Mandke LP. Comparison of the effect of ethylenediamine tetra-acetic acid, chlorhexidine, etidronic acid and propolis as an irrigant on the microhardness of root dentin: An in vitro study. J Dent Res Rev 2016;3:23-30
|How to cite this URL:|
Bhagwat SA, Lopez TA, Mandke LP. Comparison of the effect of ethylenediamine tetra-acetic acid, chlorhexidine, etidronic acid and propolis as an irrigant on the microhardness of root dentin: An in vitro study. J Dent Res Rev [serial online] 2016 [cited 2023 Jan 31];3:23-30. Available from: https://www.jdrr.org/text.asp?2016/3/1/23/180112
| Introduction|| |
Irrigation of the root canal system provides gross debridement, lubrication, destruction of microbes, dissolution of tissues and helps in cleaning areas that are inaccessible for mechanical cleansing. Apart from beneficial effects, irrigants may exhibit detrimental effects on the dentin or root canal filling materials. Reports have indicated that the use of popular irrigants ethylenediamine tetra-acetic acid (EDTA) and sodium hypochlorite (NaOCl) can demonstrate erosion of canal walls.
An irrigation protocol with etidronic acid (1-hydroxyethylidene-1, 1-biphosphonate, called HEBP for short) or also etidronate, has been suggested as an alternative to EDTA because of its chelating ability. However, there has been only one study to evaluate the effect of HEBP on root dentine microhardness in the literature. Propolis, another new irrigant, has also not been evaluated for its effect on microhardness of root dentine.
This study was an attempt to evaluate and compare the effect of four irrigants on the microhardness of root dentine using a Vicker's microhardness test. Two of these are the commonly used 17% EDTA and 2% chlorhexidine (CHX) and the other two are relatively newer irrigants 18% HEBP and a 4% solution of propolis.
| Materials and Methods|| |
- Fifty single rooted, freshly extracted human teeth
- DPI cold cure acrylic resin (powder and liquid)
- Aluminum molds (2 cm in diameter, 2 cm in height)
- Wax spatula
- Enamel bowl
- Distilled water
- 3% NaOCl solution (Prime Dental Products Ltd., Mumbai, India)
- 17% EDTA solution
- Dent wash by Prime Dental Products Ltd., Mumbai, India
- 2% CHX solution (Dentochlor) (Ammdent, Mohali, India)
- 50% HEBP solution (Sequacel HD) (Excel Industries Ltd., Mumbai, India)
- 500 mg propolis tablets (Forever Bee Propolis, Aloe Vera of America Inc., Dallas, Texas, USA)
- Large glass trays
- Vickers diamond tester/indenter.
Preparation of the solutions
- Preparation of 1.3% NaOCl solution using 3% NaOCl solution and distilled water:
142 ml of distilled water + 108 ml of 3% NaOCl solution = 250 ml of 1.3% NaOCl
- Preparation of 18% HEBP solution using 50% HEBP solution and distilled water:
160 ml of distilled water + 90 ml of 50% HEBP solution = 250 ml of 18% HEBP solution
- Preparation of 4% propolis solution using 500 mg propolis and distilled water:
One 500 mg tablet of propolis dissolved in 120 ml of warm distilled water = 4% propolis solution
Hundred recently extracted human single rooted teeth were collected. Decoronation at the cementoenamel junction and longitudinal sectioning starting from the cervical end with a low speed diamond disc was carried out. The segments were all smoothened on a grinding stone using distilled water as lubricant. The separated lingual and buccal segments were mounted horizontally in autopolymerizing resin so that the root dentine was exposed. To make the specimens, the autopolymerizing resin was poured into aluminum cylinders 2 cm in height and 2 cm diameter. The dentine surfaces of the mounted specimens were smoothened with a series of fine emery papers under distilled water to remove surface scratches and polished with alumina suspension.
The specimens were divided into five groups randomly, each group having twenty specimens. All groups were treated with four different irrigants and one group with distilled water as per the following protocol:
- Group I: Specimens were treated with distilled water for 20 min. This group served as control group
- Group II: Specimens were treated with 1.3% NaOCl for 20 min followed by 17% EDTA for 1 min
- Group III: Specimens were treated with 1.3% NaOCl for 20 min followed by 2% CHX digluconate for 5 min
- Group IV: Specimens were treated with 1.3% NaOCl for 20 min followed by 18% HEBP for 5 min
- Group V: Specimens treatment with 1.3% NaOCl up to 20 min followed by 4% propolis up to 5 min.
A Vickers microhardness tester (Reichert Austria Make, Sr. No. 363798) was used to measure the surface hardness of the dentin of each specimen. The indentations were made with a Vickers diamond indenter at a minimum of three separated locations on each specimen. The locations were selected in three regions of the canal wall: Apical, middle, and cervical. Indentations were made on the sectioned surface of each specimen with a 100 g load for a dwell time of 30 s. [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5] and [Figure 6] respectively show the indentation of one sample in each of the groups. Each reading at the cervical, middle, and apical third of the root was made by taking an average of four readings at the four corners of each indentation. Then an average of the three readings was taken to make one average reading per sample and the values were added and an average obtained to produce one hardness value for every specimen. These measurements were then converted into Vickers numbers.
|Figure 1: Root dentin following treatment with distilled water (Group I) as taken with an optical microscope (×600)|
Click here to view
|Figure 2: Root dentin following treatment with sodium hypochlorite followed by 17% ethylenediamine tetra-acetic acid (Group II) as taken with an optical microscope (×600)|
Click here to view
|Figure 3: Root dentin following treatment with sodium hypochlorite followed by 2% chlorhexidine (Group III) as taken with an optical microscope (×600)|
Click here to view
|Figure 4: Root dentin following treatment with sodium hypochlorite followed by 18% etidronic acid (Group IV) as taken with an optical microscope (×600)|
Click here to view
|Figure 5: Root dentin following treatment with sodium hypochlorite followed by 4% Propolis (Group V) as taken with an optical microscope (×600)|
Click here to view
|Figure 6: Graph showing the average microhardness of dentin when treated with the various irrigants in Vicker Hardness numbers on Y-axis|
Click here to view
| Results|| |
[Appendix 1 [Additional file 1]] gives the microhardness readings of the tested specimens of each group.
[Table 1] gives the mean values of microhardness for each group and the standard deviation. The microhardness data was statistically analyzed by one-way ANOVA [Table 2] and intergroup comparison of means was examined employing post-hoc Tukey's multiple comparison test [Table 3]. Statistical significance was established at P < 0.05 level.
|Table 1: The mean values of microhardness for each group and the standard deviation|
Click here to view
| Discussion|| |
It is important to know the mechanical properties of dentin to understand how occlusal forces are distributed along the tooth and for prediction of absorption of stresses, especially how they change with age, pathology, and clinical treatments. 
Irrigating solutions used in endodontic treatment cause changes in the properties of dentin. Alteration in the calcium/phosphate ratio changes the proportion of organic and inorganic components. This affects the microhardness, permeability, and solubility of dentin. Many studies show that various concentrations of chelating solutions and also citric acid bring about a reduction in dentin hardness. Increased exposure time also increases this effect. NaOCl is not a chelating agent but it can change the ratio of calcium to phosphate in the dentin of the canal. NaOCl is routinely used in endodontics. Its primary action is to remove organic residue found within smear layer and in the small and slender canals that are inaccessible by mechanical instrumentation. 
Many authors recommend the combined use of EDTA and NaOCl in the irrigation process but more knowledge about related changes in dentin biomechanical properties is needed. 
It is believed that enlargement of canals must be accompanied by excellent irrigation so that the prepared canal becomes as bacteria-free as possible.  One problem with the commonly used irrigants is that most of them deplete the root dentin of Ca ions thus decreasing the microhardness and increasing the potential for weakening of the dentinal walls.  The most commonly used irrigants are NaOCl, EDTA, hydrogen peroxide, CHX, and saline. Other than saline, studies have shown that all the others produce a decrease in dentine microhardness and an increase in the brittleness of the dentin and consequently the root canal-treated teeth more prone to fracture.  Hence there has been extensive research in the exploration of newer irrigants to study and test their efficacy for use as irrigants in endodontics.
The minimum inhibitory concentration for NaOCl is 5.25% for 40 min and for CHX is 2% for a minute.  Since our study assessed only the chemical action as final rinse, none of the irrigant activation protocols was used in this study. A standard time of 20 min for all groups was the irrigation time with 1.3% NaOCl, which represents the routine in clinical scenario. This was followed by a rinse of 17% EDTA for 1 min in Group II,  2% CHX for 5 min in Group III,  18% HEBP for 5 min in Group IV  and 4% propolis for 5 min in Group V.  It was found that there was insignificant alteration in apatite/collagen ratio and nearly no change in flexural strength with 1.3% NaOCl for 20 min of irrigation. 
HEBP belongs to a class of chemicals known as phosphonates. It is used systemically in patients suffering from osteoporosis or Paget's disease  as it prevents bone resorption. It has been suggested as an option to EDTA and citric acid because of its chelating ability. It is a weak chelator showing minimum effect on the microhardness or root dentine. In addition, it does not react with NaOCl, unlike EDTA, which strongly reacts with hypochlorite rendering the NaOCl ineffective. 
Patil and Uppin evaluated the effect of widely used endodontic irrigants on root dentin microhardness and surface roughness and found that 5% and 2.5% NaOCl solutions, 3% H 2 O 2 , 17% EDTA solution, all decreased the dentin microhardness. 
Saleh and Ettman reported decreased dentin microhardness after irrigation with 3% H 2 O 2 and 5% NaOCl or 17% EDTA for 60 s. 
Slutzky-Goldberg et al. measured microhardness at depths of 500, 1000, and 1500 μm from the lumen of the root canal. A decrease in microhardness was found at 500 μm between the control and samples irrigated with 6% NaOCl and 2.5% NaOCl at all irrigation periods. 
Thangaraj et al. conducted a study, the results of which indicated that irrigation of root canals with 17% EDTA solution reduced the microhardness of root canal dentin as the irrigation time was increased, and this observation is similar to the study done by Ari et al., De-Deus et al., Eldeniz et al., and Sayin et al. 
Oliveira et al. concluded that CHX and NaOCl solutions significantly caused a reduction to the microhardness of root canal dentin at 500 and 1000 μm from the pulp-dentin interface. 
There has been a good deal of research work carried out in the domain of irrigants used in endodontics. HEBP is a new potential alternative to EDTA because of its chelating ability.  However, there has been only one study to evaluate the effect of HEBP on root dentine microhardness in the literature.
Literature reveals a study was done to compare the antimicrobial efficacy of propolis (a new addition to the ever-expanding list of innovative irrigants), NaOCl and saline as intracanal irrigants. The results of this study indicated that propolis has antimicrobial activity equal to that of NaOCl.  However, the effect of propolis on the microhardness of dentin in the root canal has yet to be estimated.
Microhardness determination provides indirect evidence of change in mineral content in the dental hard tissues. Chemical irrigants cause alterations in the chemical structure of dentin and change the ratio of calcium to phosphorus in the surface. Changes in the Ca:P ratio may change permeability and solubility of dentin. This can alter the nature of its adhesive property to sealers. This may further lead to compromised sealer penetration and a degree of apical microleakage. It is known that EDTA strongly reacts with NaOCl rendering it ineffective. CHX also reacts with NaOCl to form a precipitate. Hence it is imperative to device a contemporary, safe, and efficient irrigation protocol during root canal preparation. 
Eighteen percent HEBP and 4% propolis solution have been proposed as irrigants and their use as an intracanal irrigant is being extensively studied. However, the effect of HEBP and propolis on the dentin microhardness had not been appraised so far.
Hence, our study evaluated the difference in effect of 17% EDTA, 2% CHX, 18% HEBP, and 4% propolis solutions when used as irrigants, on the microhardness of human root canal dentine using the Vicker's microhardness tester.
After treatment of root dentin with the various irrigants for the stipulated period of time, the surface hardness was ascertained in all specimens using a Vickers microhardness tester (Reichert Austria Make, Sr. No. 363798). The indentations were formed with a Vicker's diamond indenter at three separated locations. Three readings were averaged for each specimen to a produce a single value, following which a final average reading obtained for each group.
The microhardness data were tabulated and statistically analyzed by one-way ANOVA and the intergroup comparison was done by employing Tukey's multiple comparison test post hoc. Statistical significance was fixed at P < 0.05 level.
[Table 1] gives the mean and standard deviation values of the root dentine microhardness readings for all the groups. The microhardness values of control (Group I), EDTA treated (Group II), CHX treated (Group III), HEBP treated (Group IV), and propolis treated (Group V) were 55.99 ± 2.99, 41.49 ± 2.92, 42.87 ± 1.98, 55.64 ± 6.94, and 49.76 ± 4.07, respectively. [Table 2] and [Table 3] show the results of the statistical analysis using ANOVA one-way test and Tukey multiple comparisons, respectively.
There was statistical significance in differences in microhardness for all the groups (P < 0.05). Intergroup comparison demonstrated that the decrease in microhardness was of statistical significance among the experimental groups and also between the experimental and the control groups (P < 0.05).
In our study, it was concluded that 18% HEBP, a chelating agent, when used as an intracanal irrigant had the least effect on dentin microhardness. Use of 4% propolis for the same period of time caused a greater reduction in the dentin microhardness. The greatest reduction in dentin microhardness was caused by 17% EDTA. The use of 2% CHX brought about a reduction in dentin microhardness greater than that caused by propolis and HEBP, but less than that caused by the use of EDTA. Thus our study proved that 18% HEBP had the least effect on dentin microhardness.
Nowadays, there is a trend to use natural materials as a cure for a variety of diseases. Propolis is one such natural substance. The Egyptian and Greek civilizations recognized the many healing qualities of propolis. It was also used for healing sores and ulcers by Hippocrates, the founder of Modern Medicine. 
Propolis is a resinous material that honeybees collect from plants and mix with wax and other substances. Propolis is used as a sealant for unwanted open spaces in the hive. Propolis is composed of resin and balsams, pollen, and other constituents which are amino acids, minerals, Vitamins A, B complex and the active biochemical substance called bioflavenoid (Vitamin P), phenols and aromatic compounds. It is commonly brown in color. Flavenoids are well-known plant compounds, which have antibacterial, antifungal, antiviral, antioxidant besides having anti-inflammatory properties. They are the most common group of polyphenolic compounds in human diet and are found in plants. 
Propolis has been found to be very effective against Gram-positive and Gram-negative bacteria. Research has shown its antioxidant, antibacterial, antifungal, and antiviral, anti-inflammatory, antitumor properties. In the dental field, current research involving propolis highlights its antimicrobial and anti-inflammatory properties particularly in caries progression, oral surgery, oral pathology, periodontitis, and endodontics. 
In 2003, Al-Qathami and Al-Madi compared the antimicrobial efficacy of propolis, NaOCl, and saline as intracanal irrigants. The results of this study indicated that propolis has an antimicrobial activity equal to that of NaOCl. 
In 2006, Oncag et al. conducted a study that revealed that propolis had good in vitro antibacterial activity against Enterococcus faecalis in the root canals and suggested that it is used as an intracanal medicament. 
In 2012, Zare Jahromi et al. conducted a study comparing the antimicrobial efficacy of propolis with that of calcium hydroxide and found that propolis was more successful at inhibiting bacterial growth. 
In our study, the effect of 4% propolis irrigant on the microhardness of root dentin was tested, and it was found that it affected the microhardness <17% EDTA irrigant and 2% CHX irrigant did.
HEBP belongs to a class of chemicals known as phosphonates. It is obtained by the reaction of glacial acetic acid and phosphorous trichloride. It is a water white to pale yellow liquid, its density is 28-1.38 g/ml at 20°C, it is water miscible and stable under ordinary conditions. HEBP is noted for excellent CaCO 3 scale inhibition and stabilization of chlorine. It is widely used in industry as a sequestering agent and calcium carbonate scale inhibitor in cooling water treatment, for boiler water treatment, industrial and institutional cleaners, preventing stains in swimming pools, and in personal care products. 
In 2000, Kόηόk et al. stated that Re-186 HEBP (Re-186 HEBP) is suitable for palliative treatment of metastatic bone pain. They concluded that Re-186 HEDP is a highly effective agent in the control of bone pain in patients with prostate, breast, and rectum cancer. 
In 2005, Zehnder et al. evaluated citric acid-NaOCl, EDTA-NaOCl, and HEBP-NaOCl mixtures for their antimicrobial capacity. EDTA and citric acid negatively interfered with NaOCl, while HEBP did not. 
In 2012, Neelakantan et al. tested the impact of chelation by NaOCl and HEBP while instrumenting the canal, and employing a final rinse of either EDTA or NaOCl and HEBP on the bond strength of dentin using an epoxy resin sealer (AH Plus). It was found that groups that used NaOCl and HEBP irrigation protocol while instrumenting root canal had significantly greater bond strength than those groups which followed the NaOCl + EDTA irrigation. 
If anti-microbial activity and effect on the microhardness of root dentin were the only requirements of an endodontic irrigant, the results of our study and previous studies would indicate that propolis is as good or even better than NaOCl. However, NaOCl possesses other significant attributes that propolis is not known to possess. NaOCl can dissolve pulp tissues as well as necrotic tissue. Further investigation is to be carried out to investigate the effectiveness of propolis in pulp and necrotic tissue debridement. 
Etidronic acid has been proposed as an alternative to EDTA because of its ability to bring about chelation. It is a weak chelator which shows minimal influence on the microhardness of root dentine. In addition, it has no chemical action with NaOCl, unlike EDTA which shows a strong reaction with hypochlorite thus making the NaOCl ineffectual. 
Thence, both HEBP and propolis show encouraging results as intracanal irrigants. Exhaustive studies need to be initiated to study other properties of propolis and HEBP which will prove the utility of both as feasible choices as compared to irrigants used and recommended presently.
| Conclusion|| |
- Seventeen percent EDTA exhibited the greatest reduction in microhardness of the root dentine
- Two percent CHX exhibited a reduction in dentine microhardness, greater than that of HEBP and propolis
- Four percent propolis exhibited a decrease in root dentine microhardness, less in comparison with EDTA and CHX, but >18% HEBP
- Eighteen percent HEBP exhibited minimal effect on root dentine microhardness.
Within the practical limits of this study, 18% HEBP and 4% propolis show promise as irrigants with lesser ramifications on the dentine mineral content. Further investigation into the depth of demineralization and the sealability of resin sealers is imperative in order to provide greater and predictable data on the clinical performance of HEBP and propolis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Barón M, Llena C, Forner L, Palomares M, González-García C, Salmerón-Sánchez M. Nanostructural changes in dentine caused by endodontic irrigants. Med Oral Patol Oral Cir Bucal 2013;18:e733-6.
Jaju S, Jaju PP. Newer root canal irrigants in horizon: A review. Int J Dent 2011;2011:851359.
Zehnder M. Root canal irrigants. J Endod 2006;32:389-98.
Kandaswamy D, Venkateshbabu N. Root canal irrigants. J Conserv Dent 2010;13:256-64.
Goldman LB, Bogis J, Cavlieri R, Lin PS. Scanning electron microscopic study of the effects of various irrigants and combinations on the walls of prepared root canals. J Endod 1982;11:487-92.
De-Deus G, Namen F, Galan J Jr., Zehnder M. Soft chelating irrigation protocol optimizes bonding quality of Resilon/Epiphany root fillings. J Endod 2008;34:703-5.
Al-Qathami H, Al-Madi EM. Comparison of sodium hypochlorite, propolis and saline as root canal irrigants: A pilot study. Saudi Dent J 2003;15:100-3.
Zhang K, Kim YK, Cadenaro M, Bryan TE, Sidow SJ, Loushine RJ, et al.
Effects of different exposure times and concentrations of sodium hypochlorite/ethylenediaminetetraacetic acid on the structural integrity of mineralized dentin. J Endod 2010;36:105-9.
Russell RG, Rogers MJ. Bisphosphonates: From the laboratory to the clinic and back again. Bone 1999;25:97-106.
Dineshkumar MK, Vinothkumar TS, Arathi G, Shanthisree P, Kandaswamy D. Effect of ethylene diamine tetra-acetic acid, MTAD™, and HEBP as a final rinse on the microhardness of root dentin. J Conserv Dent 2012;15:170-3.
Patil CR, Uppin V. Effect of endodontic irrigating solutions on the microhardness and roughness of root canal dentin: An in vitro
study. Indian J Dent Res 2011;22:22-7.
Saleh AA, Ettman WM. Effect of endodontic irrigation solutions on microhardness of root canal dentin. J Dent 1999;27:43-6.
Thangaraj D, Ballal V, Acharya S. Determination of calcium loss and its effect on microhardness of root canal dentin following treatment with 17% ethylenediaminetetraacetic acid solution at different time intervals - An in vitro
study. Endodontology 2009;21:9-15.
Oliveira LD, Carvalho CA, Nunes W, Valera MC, Camargo CH, Jorge AO. Effects of chlorhexidine and sodium hypochlorite on the microhardness of root canal dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;104:e125-8.
Rathod S, Brahmankar R, Kolte A. Propolis - A natural remedy. Indian J Dent Res Rev 2011:50-2.
Oncag O, Cogulu D, Uzel A, Sorkun K. Efficacy of propolis as an intracanal medicament against Enterococcus faecalis
. Gen Dent 2006;54:319-22.
Zare Jahromi M, Toubayani H, Rezaei M. Propolis: A new alternative for root canal disinfection. Iran Endod J 2012;7:127-33.
Slutzky-Goldberg I, Maree M, Liberman R, Heling I. Effect of sodium hypochlorite on dentin microhardness. J Endod 2004;30:880-2.
Küçük NO, Ibis E, Aras G, Baltaci S, Ozalp G, Bedük Y, et al.
Palliative analgesic effect of Re-186 HEDP in various cancer patients with bone metastases. Ann Nucl Med 2000;14:239-45.
Zehnder M, Schmidlin P, Sener B, Waltimo T. Chelation in root canal therapy reconsidered. J Endod 2005;31:817-20.
Neelakantan P, Varughese AA, Sharma S, Subbarao CV, Zehnder M, De-Deus G. Continuous chelation irrigation improves the adhesion of epoxy resin-based root canal sealer to root dentine. Int Endod J 2012;45:1097-102.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]