|Year : 2018 | Volume
| Issue : 2 | Page : 49-55
A comparative evaluation of inflammatory response of various root canal irrigants in charles foster rat models
Vishal Singh1, Anshu Sharma2
1 Department of Conservative Dentistry and Endodontics, DJ College of Dental Sciences and Research, Modinagar, Uttar Pradesh, India
2 Department of Pedodontics and Preventive Dentistry, Genesis Institute of Dental Sciences and Research, Ferozpur, Punjab, India
|Date of Web Publication||30-May-2018|
Department of Pedodontics and Preventive Dentistry, Genesis Institute of Dental Sciences and Research, Ferozpur, Punjab
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: The aim is to evaluate the inflammatory response of root canal irrigants such as sodium hypochlorite, mixture of sodium hypochlorite (NaOCl) with ethylenediaminetetraacetic acid (EDTA) versus a new root canal irrigant mixture of tetracycline isomer, an acid, and a detergent (MTAD). Materials and Methods: Eighteen Charles foster rats were injected with 0.1 ml of the irrigant solution; normal saline served as control. Biopsies of the skin were obtained at 2 h, 2 days, and 2 weeks. The chronic and acute inflammatory responses were studied in specimen. Results: Three percent NaOCl-induced maximum acute inflammatory response, followed by mixture of 3% NaOCl with 17% EDTA. MTAD did not evoke any significant acute inflammatory response. All the irrigants evoked almost equal chronic inflammatory reaction. During 2 h study 3% NaOCl, being the most inflammatory, mixture of 3% NaOCl with 17% EDTA being the least inflammatory. Chronic inflammatory reactions further aggravated in each treatment group at 2 days except in MTAD-treated rats skin which sloughed before 2 weeks with no sign of resolution of chronic inflammation. Conclusion: This study suggests that NaOCl evokes rapid acute inflammatory response compared to other irrigants under study, but subsided in 2 days. Its combination with EDTA also had similar inflammatory response as NaOCl alone, but of lower magnitude, suggesting NaOCl with EDTA as better root canal irrigant. MTAD caused necrosis of the skin tissue at 2 days and sloughing; thereafter, this study does not suggest its use as a root canal irrigant.
Keywords: 3% sodium hypochlorite and 17% ethylenediaminetetraacetic acid, 3% sodium hypochlorite, albino rats, inflammatory response, mixture of tetracycline isomer, an acid, and a detergent
|How to cite this article:|
Singh V, Sharma A. A comparative evaluation of inflammatory response of various root canal irrigants in charles foster rat models. J Interdiscip Dentistry 2018;8:49-55
|How to cite this URL:|
Singh V, Sharma A. A comparative evaluation of inflammatory response of various root canal irrigants in charles foster rat models. J Interdiscip Dentistry [serial online] 2018 [cited 2018 Aug 21];8:49-55. Available from: http://www.jidonline.com/text.asp?2018/8/2/49/233618
| Clinical Relevance to Interdisciplinary Dentistry|| |
Tissue inflammatory reaction of a root canal irrigant is a prudent factor in determining choice of preferred endodontic irrigant for clinician.Various endo perio lesions can be treated successfully with proper choice of irrgants and clinical manuevre.
| Introduction|| |
Central dogma of root canal infection is due to infection of root canal space as a sequela to a profound carious lesion, periodontal infections, and cracks in the crown due to trauma. The main purpose of endodontic treatment is the debridement of the root canal system and to have an access for delivery of antimicrobial substances. Historically, numerous compounds have been suggested as endodontic irrigants, ranging from inert substances (sodium chloride saline) to highly toxic and allergenic biocides such as formaldehyde. Root canal irrigants may act as a double-edged sword. On the one hand, they may eliminate the causative agent and necrotic debris, and on the other hand, they may cause an inflammatory reaction to healthy tissues. Inflammatory response elicited by various irrigants forms a prudent parameter which dictates the use and success of these root canal irrigants. Since comparative evaluation of these irrigants on the basis of inflammatory response may dictate there clinical feasibility. Currently, the mainstay of disinfection of root canal disinfectants is sodium hypochlorite (NaOCl), which has the capability of dissolving necrotic tissue and inactivating bacterial endotoxins. NaOCl dissolves the organic component of the smear layer due to its proteolytic effect on the collagen present in the smear layer. Although NaOCl appears to be the most desirable single endodontic irrigant, it cannot dissolve inorganic dentin particles, and thus prevent the formation of a smear layer during instrumentation. Demineralizing agents such as ethylenediaminetetraacetic acid (EDTA) have therefore been recommended as adjuvants in the root canal therapy. EDTA, however, when used alone has little if any antibacterial activity. It works in conjugation with NaOCl., Therefore, by facilitating cleaning and removal of infected tissue, EDTA contributes to the elimination of bacteria in the root canal.
Relatively new irrigant mixture of tetracycline isomer, an acid, and a detergent (MTAD) which is a mixture of 3% doxycycline hyclate, 4.25% citric acid, and 0.5% polysorbate 80 (Tween-80) detergent which has the desirable property of disinfecting, removing smear layer, and eradicating Enterococcus faecalis bacterium that is resistant to several medications, from the root canal system., The present study aimed to rule out the pathophysiologic responses of conventional irrigants such as NaOCl, NaOCl with EDTA to that with nascent materials such as BioPure MTAD.
| Materials and Methods|| |
The inflammatory effects of the root canal irrigants on the subcutaneous tissue of rats were examined. Eighteen Charles–foster rats weighing 100–150 g were used for in vivo experiments. The animals were housed in a temperature-controlled environment (23°C–25°C) with water and food ad libitum. Subcutaneous rat tissue reaction to the following irrigating solutions was evaluated: 0.9% sterile saline (control); 3% NaOCl (Cmident Delhi, India limited); 3% NaOCl and 17% EDTA (PrevestDenpro limited Jammu, India) in 1:1 ratio volume by volume; Biopure MTAD (mixture of 3% doxycycline hyclate, 4.25% citric acid, and 0.5% polysorbate-80 [Tween 80] detergent) solution (Dentsply, Tulsa, Oklahoma, USA). Under general anesthesia with placing cotton soaked in anesthetic ether in a jar and placing the animal in the jar, the dorsal skin of the animals was shaved and cleaned with 5% iodine solution. Using a permanent marker pen, 4 circles were demarcated on the dermis of each rat leaving at least 2 cm between each circle. Using a syringe, 0.1 ml of each root canal irrigant was injected subcutaneously into 4 circles. Left scapula was injected with control, right scapula was injected with 3% NaOCl, left thigh was injected with a mixture of 3% NaOCl with 17% EDTA, and right thigh was injected with MTAD. Evaluations were made 2 h, 2 days, and 2 weeks after injection, in each examination period, using general anesthetic as described above. The dorsal skin was shaved and tissue specimens of 4-mm diameter were excised with a scalpel from six animals from the injection sites, stored in 10% formal alcohol (90 ml ethyl alcohol and 10 ml formaldehyde) solution for 48 h, washed in running water to remove traces of the fixating solution, and thereafter, embedded in paraffin blocks using the standard procedures. Slides prepared were examined under compound light microscope with on objective of ×4, ×10, ×40, and ×100 (for use under oil immersion) the eyepiece was binocular with a magnification of ×5 with a prism having a magnification of ×2.5 (Nikon. Japan). The area showing maximum infiltration of inflammatory cells in the dermis and subcutaneous tissue were identified under ×125 magnification. Counting of inflammatory cells was done under ×250 magnification per field in terms of neutrophils, lymphocytes, plasma cells, and macrophages. Based on the count per high-power field (×250) per slide, the inflammatory response was studied, and the data obtained were subjected to statistical analysis. SPSS Statistics version 17.0 (SPSS inc. Chicago, IL, USA) was used for statistical analysis and all data were expressed as mean and standard deviation. Student's t-test was used to detect the level of significance within the groups. Level of significance was set at the level of P < 0.05.
| Results|| |
Acute inflammatory response was assessed by the neutrophil count during the entire period of the study (2 h–2 weeks), whereas the chronic inflammatory response was studied by counting chronic inflammatory cells (lymphocytes, plasma cells, and macrophages) 3% NaOCl elicited maximum acute inflammatory response (10.16 ± 2.78) [Table 1] which was significantly higher than control (P< 0.001). Acute inflammatory response induced by 3% NaOCl within 2 h subsided in 2 days and 2 weeks sample which was not different to control group [Table 1], [Graph 1] and [Figure 1] and [Figure 2].
|Figure 1: Control group (normal saline) (a) at 2 h (H and E, ×125), (b) at 2 h (H and E, ×250), (c) at 2 days showing hair follicles and histiocytes (H and E, ×125), (d) at 2 days showing lymphocytes and histiocytes in the dermis (H and E, ×250), (e) at 2 weeks showing hypertrophic mucosa and acanthosis at the site of injection (H and E, ×125), (f) at 2 weeks showing hypertrophic mucosa and acanthosis at the site of injection (H and E, ×250)|
Click here to view
|Figure 2: Three percent sodium hypochlorite (a) at 2 h showing infiltration of inflammatory cells (H and E, ×125), (b) at 2 h showing mild lymphocytes and neutrophils infiltration (H and E, ×250), (c) at 2 days showing necrosis, ulceration, and congested blood vessels (H and E, ×125), (d) at 2 days showing inflammation around muscle fibers (H and E, ×250), (e) at 2 weeks showing flattening of epithelium (H and E, ×125), (f) at 2 weeks showing few lymphocytes (H and E, ×250)|
Click here to view
Mixing of 3% NaOCl with 17% EDTA caused acute inflammatory reactions not statistically different from control (P > 0.05). Similar trend was seen during the period of study though there was substantial decrease in number of cells per high-power field at 2-day interval (9.33 ± 5.31–2.66 ± 1.21) and at 2 weeks (0.50 ± 0.54) [Figure 3]. MTAD evoked minimal acute inflammatory response at 2 h interval which was not significant as compared to control (P > 0.05). At 2-day interval of histopathological studies, no acute inflammatory cell was seen. However, at this point of time, the histological sample showed gross necrosis of skin sample on microscopic examination. At 2-week interval, few neutrophils were seen (1.16 ± 1.16) implying that MTAD did not evoke any acute inflammatory response. All the irrigants used in this study evoked significant chronic inflammatory response as compared to the control except combination of 3% NaOCl and 17% EDTA P > 0.05 [Table 2] and [Graph 2]. Three percent NaOCl evoked brisk chronic inflammatory response (12.66 ± 2.06, P < 0.001) when compared to control at 2 h interval. At 2-day interval, chronic inflammatory response was further enhanced (22.50 ± 8.43, P < 0.05) which showed a subsiding trend though it was still significantly higher than the control (P< 0.01). Mixture of 3% NaOCl with 17% EDTA though showed a chronic inflammatory response, but it was not significant as compared to control. However, this combination of two agents showed significantly higher chronic inflammatory response at 2-day interval (14.66 ± 5.00, P < 0.01) and at 2-week interval (7.16 ± 1.72, P = 0.001). MTAD also evoked significant chronic inflammatory response at 2 h (9.5 ± 5.54, P < 0.05), at 2 days (13.00 ± 3.68, P = 0.001), and at 2 weeks (36.5 ± 4.5, P < 0.001). An increasing chronic inflammatory reaction was observed during the entire period of the study in contrast to other test agents which showed increasing chronic inflammatory trend up to 2 days and thereafter resolving trend was seen. At 2-day interval, 3% NaOCl and MTAD differed significantly (P = 0.04) while other comparisons did not (mixture of 3% NaOCl and 17% EDTA: MTAD, P = 0.883; 3% NaOCl: mixture of 3% NaOCl and 17% EDTA, P = 0.096). At 2-week interval, the statistical significance of 3% NaOCl alone or in combination with 17% EDTA in comparison to MTAD was significant (P = 0.000 for both groups), whereas there was no significant difference when comparison between 3% NaOCl and mixture of 3% NaOCl with 17% EDTA was done (P = 0.909).
|Figure 3: Three percent sodium hypochlorite + 17% ethylenediaminetetraacetic acid (a) at 2 h showing large areas of hemorrhage, necrosis (H and E, ×125), (b) at 2 h showing hemorrhage, necrosis, and neutrophills (H and E, ×250), (c) at 2 days showing intact epithelium and mild lymphocytic infiltration and congested blood vessels (H and E, ×125), (d) at 2 days showing hemorrage and dilated blood vessels (H and E, ×250), (e) at 2 weeks showing fibrosis an hemosidrin laden macrophages (H and E, ×125), (f) at 2 weeks showing hemosiderin laden macrophages (H and E, ×250)|
Click here to view
|Table 2: Chronic inflammatory response (lymphocytes, plasma cells, and macrophages)|
Click here to view
| Discussion|| |
Root canal irrigants form an integral part of endodontic therapy since it facilitates not only biomechanical preparation but also assists in disinfecting the canal. Various irrigants have been used for the purpose solely or in combination, namely, NaOCL, EDTA, chlorhexidine, and relatively newer irrigants such as MTAD (mixture of tetracycline isomer, citric acid, and detergent [Tween 80]) during clinical application of root canal irrigants. Although the main aim is to facilitate instrumentation and disinfection, biologic response of living tissues has been a matter of debate since time immemorial. NaOCl has been the most commonly used endodontic irrigant because of its antimicrobial and tissue-dissolving activity ,, which primarily acts by inhibition of glucose oxidation and depletion of adenine nucleotides; it is the medicament of choice due to its efficacy against pathogenic organisms and pulp digestion. Its concentration for use in endodontics today varies from 0.5% to 5.25%. At low concentrations, it will dissolve mainly necrotic tissue, whereas at higher concentrations tissue dissolution is better but it also dissolves vital tissue, a generally undesirable effect. It has been shown that clinical effectiveness does not increase conclusively for concentrations higher than 1% EDTA (17% disodium salt, pH 7.0) (Zehnder 2006) has little if any antibacterial activity. It works in conjugation with NaOCl., Therefore, by facilitating cleaning and removal of infected tissue [Figure 4], EDTA contributes to the elimination of bacteria in the root canal. MTAD is a relatively newer root canal irrigant which removes smear layer as well as encompasses antibacterial activity against E. faecalis, a notorious microorganism seen in the root canals of human teeth. E. faecalis – formerly classified as part of the Group D Streptococcus system – is a Gram-positive commensal bacterium inhabiting the gastrointestinal tracts of humans and other mammals. Although basically, E. faecalis is an inhabitant of the gastrointestinal tract. It has been frequently found in root canal-treated teeth in prevalence values ranging from 30% to 90% of the cases. Root canal-treated teeth are about nine times more likely to harbor E. faecalis than cases of primary infections. All the available root canal irrigants induce certain amount of inflammatory response from host tissue, and it is expected that this inflammatory response should be as minimum as possible, and this is one of the basic parameters which dictate the success of irrigant in endodontics. Several studies have been conducted, in which irrigants were compared to know the inflammatory reactions. However, the present study stands apart since acute as well as chronic response is judged separately. In the present study, the aim was to justify judicious clinical use of some commonly used irrigants. For this purpose, inflammatory response was tested in rat models by injecting 3% NaOCl, 3% NaOCl + 17% EDTA, and MTAD at intervals of 2 h, 48 h, and 2 weeks. Acute inflammatory response was assessed by the neutrophil counts, whereas the chronic inflammatory response was studied by counting chronic inflammatory cells (mononuclear cells). Three percent NaOCl elicited maximum acute inflammatory response (10.16 ± 2.78) which subsided in 2 days (1.83 ± 0.75) and 2 weeks sample (0.50 ± 0.83). This is in accordance with study conducted by. NaOCl has shown maximum acute inflammatory response in initial periods as compared with control (P< 0.001), thereafter it resolves. Three percent NaOCl evoked brisk chronic inflammatory response (12.66 ± 2.06, P < 0.001) when compared to control at 2 h interval. At 2 days interval, chronic inflammatory response was further enhanced (22.50 ± 8.43, P < 0.01) which showed a subsiding trend. Mixing of 3% NaOCl with 17% EDTA caused acute inflammatory reactions not statistically different from control (P > 0.05). This can be attributed to the fact that EDTA neutralizes the proteolytic effect of NaOCl by chelation of Cl - ion. Similar trend as observed with NaOCl, that is, decline in number of cell was seen during the 2 days study period (9.33 ± 5.31–2.66 ± 1.21) and at 2 weeks (0.50 ± 0.54). This means that whether alone or in combination with NaOCl and EDTA shows a decrease in inflammatory response. This finding is in accordance to the finding suggested by. However, this combination of two agents showed significantly higher chronic inflammatory response at 2 days interval (14.66 ± 5.00, P < 0.01) and at 2 weeks interval (7.16 ± 1.72, P = 0.001). These findings are attributed to the fact that EDTA is potent anticoagulant, due its property of chelating calcium which is Factor IV in coagulation cascade (Ganong Physiology). Although initially acute inflammation was not seen with this combination, histopathological examination revealed massive hemorrhage. This may account for persisting chronic inflammation during the entire period of the study. A significant breakthrough came with MTAD; studies have shown that MTAD as a final rinse is capable of removing the smear layer with minimal erosive changes on the surface of dentin. Another study  investigated the amount of tissue loss after exposing bovine pulp and dentin to various concentrations of NaOCl, EDTA, or MTAD. Their results showed that various concentrations of NaOCl removed organic components of pulp and dentin effectively. The major difference between the actions of these solutions is a high binding affinity of doxycycline present in MTAD for the dentin. In this in vivo study, MTAD evoked minimal acute inflammatory response at 2 h interval. At 2-day interval, no acute inflammatory cell was seen, and at 2-week interval, few neutrophils were seen (1.16 ± 1.16) implying that MTAD did not evoke any acute inflammatory response. Our observations support the previous studies. Basic constituent in MTAD is tetracycline which is active against rickettsia microorganisms primarily, but its activity against E. faecalis has also been shown to be promising along with minimal inflammatory response which supports its use. The only fact which questions use of MTAD is significant chronic inflammatory response as observed in our study at all study intervals. MTAD evoked significant chronic inflammatory response at 2 h (9.5 ± 5.54, P < 0.05), at 2 days (13.00 ± 3.68, P = 0.001), and at 2 weeks (36.5 ± 4.5, P < 0.001). An increasing chronic inflammatory reaction was observed during the entire period of the study in contrast to other test agents which showed increasing chronic inflammatory trend up to 2 days, and thereafter, resolving trend was seen. This supports the fact the hypothesis that MTAD is a better irrigant in terms of tooth wear and antibacterial efficacy, but high chronic inflammation poses a major drawback in its further success. Studies have concluded that NaOCl has a higher inflammatory response at the end of 14 and 30 days  when used in a concentration of 5.25%. Similar variations have been observed with MTAD. MTAD showed consistent disinfection of infected root canals with 5.25% NaOCl/15% EDTA. All these observations converge at a point that certain parameters are key to guide success of irrigants in root canal therapy mainly concentration of the irrigant, and evaluation of cellular infiltrate for inflammation. In this study, the attempt has been made to evaluate the inflammatory responses of commonly used irrigants in vivo which suggests that NaOCl evokes rapid acute inflammatory response as compared to other irrigants under study, but at the same time inflammation, subside in a short time of 2 days. Its combination with EDTA also behaved in similar pattern as NaOCl used alone, but the inflammatory reaction evoked were of lower magnitude, implying that as far as inflammatory are concerned combination of NaOCl with EDTA is a better option as a root canal irrigant. However, MTAD as it caused necrosis of the skin tissue at 2 days and sloughing; thereafter, this study does not suggest its use as a root canal irrigant. To resolve these complexities, further investigations are required with larger sample size to prove the clinical efficacy of available irrigants, for which this study may serve as a template.
|Figure 4: Mixture of tetracycline isomer (a) at 2 h showing normal skin and hair follicles (H and E, ×125), (b) at 2 h showing normal squamous epithelium and few lymphocytes (H and E, ×250), (c) at 2 days showing sloughed off squamous epithelium and fibrosis (H and E, ×125), (d) at 2 days showing necrosis of epithelium and fibrosis (H and E, ×250), (e) at 2 weeks showing ulceration and chronic granulation tissue (H and E, ×125), (f) at 2 weeks showing dense mononuclear cell infiltration chronic granulation tissue (H and E, ×250)|
Click here to view
| Conclusion|| |
The present study was conducted to evaluate the chronic and acute inflammatory dermal response of various irrigants used in endodontics using rat model for the study. Three percent NaOCl-induced maximum acute inflammatory response, followed by mixture of 3% NaOCl with 17% EDTA. MTAD, however, did not evoke any significant acute inflammatory response but induced degenerative histopathological changes in the rat skin tissue at 2-day interval. All the irrigants used in this study evoked almost equal chronic inflammatory reaction. After 2 hr follow up 3% NaOCl was observed to be the most inflammatory and mixture of 3% NaOCl with 17% EDTA being the least inflammatory irrigant in the present study. The chronic inflammatory reactions further aggravated in each treatment group at 2-day interval except for MTAD-treated rat skin which sloughed before 2 weeks, and the peripheral tissue showed no sign of resolution of chronic inflammation. Therefore, it can be concluded mixture of 3% NaOCl with 17% EDTA is the safest irrigant as it evoked least acute and chronic inflammatory response than all the other irrigants used in our study. Whereas, MTAD appears to be most injurious, as it showed no signs of resolution of the chronic as well as acute inflammatory response.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Zehnder M. Root canal irrigants. J Endod 2006;32:389-98.
Gomes-Filho JE, Aurélio KG, Costa MM, Bernabé PF. Comparison of the biocompatibility of different root canal irrigants. J Appl Oral Sci 2008;16:137-44.
Serper A, Calt S. The demineralizing effects of EDTA at different concentrations and pH. J Endod 2002;28:501-2.
Giardino L, Ambu E, Savoldi E, Rimondini R, Cassanelli C, Debbia EA, et al.
Comparative evaluation of antimicrobial efficacy of sodium hypochlorite, MTAD, and tetraclean against Enterococcus faecalis
biofilm. J Endod 2007;33:852-5.
Torabinejad M, Johnson WB. Irrigation Solution and Methods for Use. US Patent and Trademark Office; 25 December, 2003.
Grawehr M, Sener B, Waltimo T, Zehnder M. Interactions of ethylenediamine tetraacetic acid with sodium hypochlorite in aqueous solutions. Int Endod J 2003;36:411-7.
Torabinejad M, Khademi AA, Babagoli J, Cho Y, Johnson WB, Bozhilov K, et al.
Anew solution for the removal of the smear layer. J Endod 2003;29:170-5.
Yesilsoy C, Whitaker E, Cleveland D, Phillips E, Trope M. Antimicrobial and toxic effects of established and potential root canal irrigants. J Endod 1995;21:513-5.
Jiménez-Rubio A, Segura JJ, Llamas R, Jiménez-Planas A, Guerrero JM, Calvo JR, et al. In vitro
study of the effect of sodium hypochlorite and glutaraldehyde on substrate adherence capacity of macrophages. J Endod 1997;23:562-4.
Serper A, Ozbek M, Calt S. Accidental sodium hypochlorite-induced skin injury during endodontic treatment. J Endod 2004;30:180-1.
Barrette WC Jr., Hannum DM, Wheeler WD, Hurst JK. General mechanism for the bacterial toxicity of hypochlorous acid: Abolition of ATP production. Biochemistry 1989;28:9172-8.
Molander A, Reit C, Dahlén G, Kvist T. Microbiological status of root-filled teeth with apical periodontitis. Int Endod J 1998;31:1-7.
Onçaǧ O, Hoşgör M, Hilmioǧlu S, Zekioǧlu O, Eronat C, Burhanoǧlu D, et al.
Comparison of antibacterial and toxic effects of various root canal irrigants. Int Endod J 2003;36:423-32.
Segura JJ, Calvo JR, Guerrero JM, Jimenez-Planas A, Sampedro C, Llamas R, et al.
EDTA inhibits in vitro
substrate adherence capacity of macrophages: Endodontic implications. J Endod 1997;23:205-8.
Ganong WF. Review of Medical Physiology. 22nd
ed. New Delhi: McGraw Hill Companies Publication; 2008. p. 544-6.
Beltz RE, Torabinejad M, Pouresmail M. Quantitative analysis of the solubilizing action of MTAD, sodium hypochlorite, and EDTA on bovine pulp and dentin. J Endod 2003;29:334-7.
Zhang W, Torabinejad M, Li Y. Evaluation of cytotoxicity of MTAD using the MTT-tetrazolium method. J Endod 2003;29:654-7.
Johal S, Baumgartner JC, Marshall JG. Comparison of the antimicrobial efficacy of 1.3% NaOCl/BioPure MTAD to 5.25% NaOCl/15% EDTA for root canal irrigation. J Endod 2007;33:48-51.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]