|Year : 2018 | Volume
| Issue : 1 | Page : 25-33
Calcium chloride dihydrate affects the biological properties of white mineral trioxide aggregate on dental pulp stem cells: An in vitro study
Hany Mohamed Aly Ahmed1, Norhayati Luddin2, Thirumulu Ponnuraj Kannan3, Khairani Idah Mokhtar4, Azlina Ahmad3
1 Department of Restorative Dentistry, Faculty of Dentistry, University of Malaya, 50603, Kuala Lumpur, Malaysia
2 School of Dental Sciences, Universiti Sains Malaysia, Kubang Kerian, 16150 Kelantan, Malaysia
3 School of Dental Sciences, Universiti Sains Malaysia; Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, 16150 Kelantan, Malaysia
4 Kulliyyah of Dentistry, International Islamic University Malaysia, Bandar Indera Mahkota, 25200 Kuantan, Pahang, Malaysia
|Date of Web Publication||10-Jan-2018|
Assoc. Prof. Norhayati Luddin
School of Dental Sciences, Universiti Sains Malaysia, Kubang Kerian, 16150, Kelantan
Source of Support: None, Conflict of Interest: None
Introduction: Biological testing of biomaterials on dental pulp stem cells (DPSCs) is one recent advance in endodontic research. The aim of this study was to compare the cytotoxicity, cell attachment properties, and dentinogenic differentiation potential of extracts of white mineral trioxide aggregate (WMTA)/calcium chloride dihydrate CaCl2.2H2O combination (fast-set WMTA [FS WMTA]) to that of WMTA on DPSCs.
Materials and Methods: The cytotoxicity and cell attachment properties were evaluated on DPSCs using methyl-thiazol-diphenyltetrazolium assay and under scanning electron microscope, respectively. After 1, 3, and 7 days of incubation, the expression of four dentinogenic gene markers (BGLAP, DSPP, RUNX2, and SPP1) was examined using the real-time polymerase chain reaction. Mann-Whitney test and one-way analysis of variance were used for statistical analysis (P = 0.05).
Results: While WMTA showed favorable cytotoxicity and cell attachment properties, FS WMTA demonstrated severe/moderate cytotoxicity at three successive concentrations (P < 0.05), and the cell attachment properties were less favorable. However, DPSCs treated with FS WMTA extracts showed higher expressions of dentinogenic gene markers than WMTA (P < 0.05). BGLAP and SPP1 were down- and up-regulated in both groups at all-time intervals, respectively. DSPP was upregulated only in WMTA at day 3 compared to days 1 and 7 in FS WMTA. RUNX2 was upregulated at all-time intervals only in FS WMTA.
Conclusions: The addition of CaCl2.2H2O increases the cytotoxicity but enhances the dentinogenic differentiation potential of WMTA on DPSCs.
Keywords: Calcium chloride dihydrate, cell attachment, cytotoxicity, dentinogenic differentiation, white mineral trioxide aggregate
|How to cite this article:|
Ahmed HM, Luddin N, Kannan TP, Mokhtar KI, Ahmad A. Calcium chloride dihydrate affects the biological properties of white mineral trioxide aggregate on dental pulp stem cells: An in vitro study. Saudi Endod J 2018;8:25-33
|How to cite this URL:|
Ahmed HM, Luddin N, Kannan TP, Mokhtar KI, Ahmad A. Calcium chloride dihydrate affects the biological properties of white mineral trioxide aggregate on dental pulp stem cells: An in vitro study. Saudi Endod J [serial online] 2018 [cited 2021 Jan 23];8:25-33. Available from: https://www.saudiendodj.com/text.asp?2018/8/1/25/222760
| Introduction|| |
Mineral trioxide aggregate (MTA) was introduced in the field of endodontics as a root-end filling material. Studies demonstrated the favorable biological profile of white MTA (WMTA),, but difficult handling properties and prolonged setting time continue to be its main drawbacks., As an attempt to overcome these drawbacks, a number of setting accelerators at different concentrations have been examined such as calcium chloride (CaCl2) and others.,,,
Studies have proved the ability of MTA/CaCl2 combination to maintain good sealing ability and high pH values., However, the biological profile of this combination is contradictory. Two studies reported favorable biological responses of WMTA/CaCl2 combination when applied in an animal model and on L929 mouse cells., Conversely, one report has demonstrated undesirable cellular responses when this combination is challenged on MG-63 cells.
Literature shows considerable progress with regard to the molecular mechanisms of osteogenic/dentinogenic differentiation of cells after exposure to MTA formulations;,,,,,, however, the exact mechanism of hard tissue formation is yet not well understood. Studies have examined the dentinogenic differentiation potential of WMTA on different cell populations. Tani-Ishii et al. confirmed the ability of WMTA to induce upregulation of osteogenic genes (such as osteocalcin) in osteoblasts. Other investigations examined this osteogenic/dentinogenic differentiation property on different cell types such as cementoblasts, osteosarcoma cell line, human dental pulp cells,, and stem cells from apical papilla. In addition, WMTA was reported to support cell attachment and osteogenic gene marker (runt-related transcription factor 2) expression in osteoblasts.
Despite appreciable efforts presented in previous studies, information on the biological properties of WMTA mixed with CaCl2 on human cells related to the tooth is scarce. Therefore, this study aimed to compare the cytotoxicity, cell attachment properties, and dentinogenic differentiation potential of extracts of WMTA/CaCl2.2H2O combination (fast-set WMTA [FS WMTA]) to that of WMTA on dental pulp stem cells (DPSCs). The research hypotheses were that both WMTA and FS WMTA show comparable cytotoxicity, cell attachment properties, and dentinogenic differentiation potential on DPSCs.
| Materials and Methods|| |
Preparation of materials
First group (WMTA) – 1 g of WMTA (Dentsply, USA) was mixed with 250 μl of liquid provided by the manufacturer. CaCl2 in the dihydrate form (CaCl2.2H2O) (Merck, Darmstadt, Germany) was selected to be the setting accelerator. One gram of WMTA mixed with 10% CaCl2.2H2O and 200 μl of liquid provided from the manufacturer served as the second group.
All experimental procedures were carried out in a purifier Class II biological safety cabinet (Labconco, MO, USA) under aseptic condition. All surfaces were disinfected with 70% prepared ethanol spray, before and after use. Human DPSC lines isolated from third molars (adult) and characterized were purchased from AllCells (CA, USA). The complete growth medium was prepared by supplementing 500 ml of basal cell growth medium (AllCells), with 50 ml of human mesenchymal stem cell stimulatory supplements (AllCells). DPSCs were then thawed and cultured.
Optimization of the experiment
Pilot studies were carried out to determine the maximum concentration of the extracts where the serial dilution would be prepared. Nearly 200 mg/ml was selected as the maximum concentration. The study samples were prepared, sterilized using ultraviolet light for 30 min, introduced into sterile 15 ml centrifugation tubes, and then, the prepared culture media were added into each tube and incubated for 7 days at 37°C.
5 × 103 cells in 100 μl of prepared media were added into each well of sterile 96-well plate (Thermo Scientific Nunc, Roskilde, Denmark). Five replicates were prepared for each concentration (each group has five concentrations) then incubated at 37°C and 5% CO2 for 24 and 72 h. Each experiment was performed three times on three successive passages (DPSCs – P7-9).
Application of the extracts
On day 7, the extracts were passed through a sterile 0.2 μm filter (Pall, Ann Arbor, MI, USA). The extracts were then prepared at five serial dilutions (200, 100, 50, 25, and 12.5 mg/ml). The media in the seeded 96-well plate was then replaced by the materials extracts, and the last row served as the control. The plate was then incubated at 37°C and 5% CO2 for 24 and 72 h.
Application of the methyl-thiazol-diphenyltetrazolium assay
Thirty microliter of methyl-thiazol-diphenyltetrazolium (MTT) solution was added into each well at each interval of time. After 3–4 h, the content of each well was replaced by 100 μl of dimethyl sulfoxide. The optical density (OD) was immediately measured using an ELISA reader (Sunrise, Tecan, Austria) at a test wavelength of 570 nm (reference wavelength was 600 nm). The cell viability values were then calculated using the following formula:
Cell viability (%) = ([A – B]/[C − B]) × 100
Where A is the OD of test group, B is the OD of blank wells, and C is the OD of control group. The cytotoxicity profile was classified according to Zhang et al. (>90%: noncytotoxic, 60%–90% slight cytotoxicity; 30%–60%: moderate cytotoxicity; 0%–30%: severe cytotoxicity).
Cell attachment properties
The cell attachment properties were examined as described in a previous study. Acrylic molds were fabricated, sterilized, and the cements were added after mixing. After setting, each assembly was sterilized, and 250 μl of prepared medium having 105 cells (P5, 6) was added onto the top of every sample and left for 30 min. Subsequently, 5 ml of prepared medium was added, and the plate was incubated at 37°C and 5% CO2 for 24 and 72 h. After each time interval, the samples were washed using a prepared phosphate-buffered solution and prefixed in buffered 2.5% glutaraldehyde solution (Merck, Germany), and the samples were then dehydrated in ethanol at five concentrations (30%, 50%, 10 min each; 70%, 90%, 100%, 100%, 5 min each). The samples were then fitted onto aluminum stubs, coated with gold using a sputter coating machine (Leica EM SCD005, Czech Republic), and then viewed under scanning electron microscope (SEM) (Quanta 450 FEG, FEI Netherlands, Eindhoven, The Netherlands). Empty acrylic molds and acrylic molds filled with phenolic-based cement (Pulpotec, Produits Dentaires SA, Switzerland) served as negative and positive control groups, respectively.
Dentinogenic differentiation potential using real-time polymerase chain reaction
Based on results of MTT assay (results section), extracts at 12.5 mg/ml concentration presented the best noncytotoxic cell viability values for both groups. Therefore, this concentration was selected for examining the dentinogenic differentiation potential on DPSCs. [Table 1] shows the genes used in this study.
|Table 1: Gene names and symbols used for evaluation of the dentinogenic differentiation potential of the test materials|
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After application of the extracts and at each time interval, the RNA was extracted from the DPSCs. The protocol for RNA extraction designed by Ambion kit (Life technologies, CA, USA) was followed. The concentration of RNA was measured using a spectrophotometer (Eppendorf, Hamburg, Germany). RNA electrophoresis on denaturing agarose gel was performed for checking the quality of RNA, and the RNA was converted to cDNA using a thermocycler (Eppendorf, Germany).
The polymerase chain reaction (PCR) reaction mix was prepared for each group according to the manufacturer's instructions (10 μl of TaqMan Fast MM + 1 μl of the probe + 9 μl [2 μl of cDNA + 7 μl of nuclease-free water]). The plate was then covered by a clear transparent film (Applied Biosystems, CA, USA), centrifuged at 3000 rpm for 2 min, and then introduced into a real-time PCR machine (7500 software, Applied Biosystems, USA). The cycle threshold (cT) values were then determined, and the level of gene expression was classified. The delta cT (mean cT of test gene – mean cT of beta actin) and delta delta cT (mean delta cT untreated – mean delta cT treated) values were then calculated.
Results obtained from MTT assay were analyzed using Mann–Whitney test. One-way analysis of variance followed by post hoc (Tukey's honest significant difference) was performed to analyze the data collected for the dentinogenic differentiation potential (Statistical Package for the Social Sciences (SPSS) version 20, Chicago, IL, USA). The level of significance was set at 0.05 (P = 0.05).
| Results|| |
Results showed that the addition of CaCl2.2H2O to WMTA reduced the cell viability values, and the difference was significant in 6 out of 10 concentrations at the two time intervals (P < 0.05) [Figure 1]. Slight and noncytotoxic activities were observed in WMTA at concentrations <100 mg/ml at both time intervals. However, FS WMTA demonstrated severe cytotoxic activity at two successive concentrations (200 and 100 mg/ml) after 24 and 72 h. At the concentration of 50 mg/ml, FS WMTA showed moderate cytotoxic activity. Noncytotoxic activities were only observed in FS WMTA at the concentration of 12.5 mg/ml [Figure 1].
|Figure 1: Intergroup comparisons between the cell viability values of white mineral trioxide aggregate (WMTA) and fast-set white mineral trioxide aggregate (FS WMTA) on dental pulp stem cells, (a) after 24 h, (b) after 72 h, using Mann–Whitney test. The level of significance was set at 0.05 (* = P < 0.05)|
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Cell attachment properties
DPSCs adhered over the surface of WMTA and at the interface (between the mold and materials) after 24 h of incubation [Figure 2]a and [Figure 2]b. After 72 h, there was increased numbers of DPSCs with prominent cytoplasmic processes interacting with the surface of test materials and with neighboring cells [Figure 2]c and [Figure 2]d. In general, it was noted that DPSCs were more confluent at the interface than the surface of test materials. On the other hand, DPSCs attached onto FS WMTA and at the interface after 24 and 72 h, but the cells were fewer and more difficult to identify as compared to WMTA, especially at the interface [Figure 2]e, [Figure 2]f, [Figure 2]g, [Figure 2]h. A matrix-like layer of DPSCs was observed at some areas on the top of FS WMTA [Figure 2]h. [Figure 2]i, [Figure 2]j, [Figure 2]n shows the cell attachment properties of the control groups after 24 and 72 h. DPSCs attached and spread with prominent cytoplasmic processes over the empty acrylic molds; however, cells were round with few cytoplasmic processes on the top and at the interface of Pulpotec samples [Figure 2]i, [Figure 2]j, [Figure 2]n.
|Figure 2: Cell attachment properties of white mineral trioxide aggregate and fast-set white mineral trioxide aggregate (white arrows) on dental pulp stem cells. (a) White mineral trioxide aggregate at the interface (24 h), (b) white mineral trioxide aggregate on the top (24 h), (c) white mineral trioxide aggregate at the interface (72 h), (d) white mineral trioxide aggregate on the top (72 h), (e) fast-set white mineral trioxide aggregate at the interface (24 h), (f) fast-set white mineral trioxide aggregate on the top (24 h), (g) fast-set white mineral trioxide aggregate at the interface (72 h), (h) fast-set white mineral trioxide aggregate on the top (72 h), (i) acrylic mold (control) 24 h, (j) acrylic mold (72 h), (k) Pulpotec (control) at the interface (24 h), (l) Pulpotec on the top (24 h), (m) Pulpotec at the interface (72 h), (n) Pulpotec on the top (72 h)|
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Multicomponent plot and classification of gene expression using the cycle threshold values
Results showed that the expression of RUNX2 gene was the highest in all groups [Table 2]. The expression of SPP1 gene was higher than BGLAP and DSPP genes. The latter two genes basically showed low and very low expressions [Table 2]. The expression of DSPP gene was undetermined in FS WMTA (day 3) and WMTA (day 7) [Table 2].
|Table 2: Expression levels of genes in dental pulp stem cells treated with extracts of normal set formulations|
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Relative quantification using delta cycle threshold values (comparison of the expression of genes)
[Table 3] shows the mean values, standard deviation, and statistical analysis after 1, 3, and 7 days. Intergroup comparisons are listed in [Table 4].
|Table 3: Mean, standard deviation, and one-way analysis of variance statistical analysis for the delta cycle threshold values for all genes after 1, 3, and 7 days of incubation|
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|Table 4: Post hoc intergroup comparisons for all genes after 1, 3, and 7 days of incubation|
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- BGLAP – The difference in delta cT values of WMTA/FS WMTA comparisons was statistically significant at days 1 and 7 (P < 0.05) [Table 3]. Intergroup comparisons show significant differences between WMTA and control [Table 4]
- DSPP – The difference in delta cT values of WMTA/FS WMTA comparisons was statistically significant at day 1 (P < 0.05) [Table 3]. DSPP was not expressed in WMTA and FS WMTA groups at day 7 and 3, respectively
- RUNX2 – At all-time intervals, the difference between groups was statistically significant (P < 0.05) [Table 3]. With the exception of WMTA/FS WMTA (days 3), the difference in delta cT values was statistically significant (P < 0.05) [Table 4]
- SPP1 – At all-time intervals, the difference between groups was statistically significant (P < 0.05) [Table 3]. The intergroup comparisons showed significant differences between all groups [Table 4].
Relative quantification using delta delta cycle threshold values
The expression of each gene was normalized with the control group to determine the up- and down-regulation of each gene. A comparison of the up/downregulations between WMTA and FS WMTA showed that the addition of CaCl2.2H2O to WMTA resulted in an upregulation of BGLAP gene (day 7), DSPP gene (day 1, 7), and RUNX2 gene (day 1, 7). FS WMTA increased the upregulation of SPP1 gene at all-day intervals though WMTA induced an upregulation to DSPP (day 3), and a more upregulation of RUNX2 gene (day 3) compared to FS WMTA [Figure 3].
|Figure 3: Relative quantification of the genes expressions (the gene expression of the control group was set at 0). (a) After 1 day, (b) after 3 days, (c) after 7 days|
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| Discussion|| |
In vitro cytotoxicity tests are simple, relevant, and essential stages of the biocompatibility screening process. Since MTA is a hydrophilic material likely to release ionic components, it is apt to interfere with intracellular enzyme activities. The MTT assay was selected in this study to assess the activity of mitochondrial dehydrogenase in DPSCs. Results of the present study demonstrated considerable variations in the cellular responses of DPSCs toward WMTA and FS WMTA, thus rejecting the research hypothesis. In contrast to the favorable responses of WMTA, extracts from FS WMTA showed severe/moderate toxicity to DPSCs at three successive concentrations. This is contradicted with results of a recent study  which demonstrated favorable cytotoxic activity of WMTA/CaCl2 combination on isolated DPSCs. Differences in results might be attributed to different materials preparation and/or methodological procedures in which the direct method was used for cytotoxicity evaluation. However, in this study, the extraction dilution method was selected to examine the cytotoxic effects of leachable elements from study samples on cells that are distant to and in close contact with the cement.
The quality and quantity of cell attachment onto retrograde filling materials is generally agreed to be a valid criterion for evaluations of the biocompatibility of dental biomaterials., The favorable attachment properties of WMTA to DPSCs observed in this work are consistent with others., Studies examined the cell attachment properties of WMTA/CaCl2 combination show contradicting results. One investigation compared three combinations of FS WMTA including CaCl2, and SEM observations of MG-63 osteosarcoma cells demonstrated unfavorable attachment to the surfaces of WMTA mixed with CaCl2, which is consistent to this study. Another study showed favorable cell attachment properties of WMTA/CaCl2 combination. Differences in results could be explained by the different cell line used, materials preparation, or methodological procedures.
The less favorable attachment properties of FS WMTA on DPSCs at the interface indicate that elutes of the test material may affect the attachment and spreading of cells at areas around the material; this finding is consistent with the results demonstrated by the MTT colorimetric assay when the effects of extracts of FS WMTA on DPSCs were examined.
The technological innovation of real-time PCR has become increasingly important in research laboratories due to its capacity for generating quantitative results. BGLAP, DSPP, RUNX2, and SPP1 genes were selected as target genes and were examined at different time intervals (1, 3, and 7 days) to evaluate the ability of extracts to induce early and late expression of these target genes in DPSCs. There were two attempts to extract the RNA from DPSCs after 14 days of incubation. However, it was noted that the RNAs extracted from DPSCs incubated in extracts of FS WMTA were not enough because at day 14, the cells were very few in number compared to WMTA. A similar observation has been reported in another study when cementoblasts were examined with intermediate restorative material (IRM) and MTA, and the cells did not yield enough RNA with IRM due to its cytotoxic effect.
BGLAP is an intermediate/late osteogenic marker gene.,, Results showed that the expression of BGLAP in DPSCs was low and downregulated at all-time intervals. Results obtained from this study are inconsistent with two studies which reported upregulation of BGLAP as early as 1 day after treatment on MC3T3-E1 osteoblast cells and human dental pulp cells, respectively., On the other hand, Hakki et al. found that BGLAP was downregulated with OCCM cementoblasts even after 5 days of incubation. These results prove that different cell types could express BGLAP differently.
DSPP is a late marker gene for odontoblast differentiation – an important prerequisite for tooth development and mineralization. DPSCs have the capacity to differentiate into odontoblast-like cells. Results showed that the expression of DSPP was very low. With the exception of WMTA (day 3) and FS WMTA (days 1 and 7), the expression of DSPP was downregulated when compared with the control. Our findings are consistent to one study performed on the ability of WMTA to induce odontoblast-like differentiation of pulp cells. In contrast, Schneider  did not observe an increase in the expression of DSPP when stem cells from apical papillae were treated with WMTA for 7, 14, and 21 days. Notably, the authors reported the presence of a low percentage of undifferentiated cells assuming that such a mixed cell population would affect the expression of DSPP.
RUNX2 is the earliest and the most specific marker for bone formation. Results showed that the expression of RUNX2 is high in both groups but the upregulations at all-time intervals were only observed with FS WMTA compared to upregulation at day 3 for WMTA. Data presented in this investigation are consistent to one study which found that the expression of RUNX2 in dental pulp stromal cells cultured on WMTA increased at day 2 and then gradually decreased until day 7. Results are contradicting to another investigation  which found that the expression of RUNX2 in osteoblasts was low when osteoblasts were treated with WMTA for 24 h, but the expression was relatively higher during 1 week of growth and differentiation on WMTA. Inconsistency in results might be attributed to the different cell line used, and differences in methodological procedures in which the cells were harvested from the surfaces of WMTA while this study examined DPSCs in material extracts.
SPP1 is one of the intermediate osteogenic gene markers that plays an important role in osteogenesis. Results showed that FS WMTA extracts enhanced the upregulation of SPP1 significantly. The expression levels demonstrated in this study are consistent to one study that showed a continuous upregulation of SPP1 of WMTA-treated MG63 osteosarcoma cell line along the days 1, 3, 7, and 15. Nevertheless, findings presented in this study are inconsistent with one investigation examined the effects of WMTA on the expression of SPP1 in cementoblasts. The authors observed decreased SPP1 transcripts in the groups treated with WMTA except for those at 0.2 and 0.002 mg/ml concentrations of WMTA when compared with control group at day 5. Differences in results might be attributed to the different cell line used, and the powder form used for examining the mineralization potential of WMTA.
In general, it can be concluded that the addition of CaCl2.2H2O to WMTA resulted in an upregulation of downregulated genes, namely, DSPP (WMTA - day 1 and WMTA - day 7) and RUNX2 (WMTA - day 1, WMTA - day 7), more upregulation of SPP1 (at all-time intervals). This observation indicates that FS WMTA can enhance the expression of osteogenic/dentinogenic gene markers in DPSCs compared to WMTA, thus rejecting the research hypothesis. This might be attributed to the higher pH values obtained by this combination compared to pure cements.
Combining the cytotoxic and osteogenic/dentinogenic differentiation potential profiles could explain the findings presented in animal models demonstrated by Parirokh et al. and Bortoluzzi et al. Parirokh et al. found more inflammation/necrosis and less calcified bridge formation in GMTA/CaCl2-treated dental pulp compared to GMTA alone (GMTA - Grey MTA), while Bortoluzzi et al. found that the pulp tissue response was similar for WMTA with and without CaCl2. Both studies together with the in vitro observations presented in this study indicate that the more cytotoxic effects exerted by MTA/CaCl2 combination are compensated with a higher induction for osteogenic/dentinogenic genes expression. The more dentinogenic differentiation potential of FS WMTA on DPSCs compared to WMTA supports its potential application for direct pulp capping to enhance the induction of hard tissue formation. However, the undesirable cytotoxic effect of this combination is of particular concern. Perhaps, its indication in teeth with immature roots is advantageous because of the high healing potential and vasculature.
The cytotoxicity and dentinogenic differentiation potential of WMTA and FS WMTA were evaluated using in vitro models, which is one limitation of this study. Experimental models may not typically simulate the clinical situation where the material is applied on vital tissues having different types of cells, blood, and interstitial fluids. The application of test materials in a biological biosystem may affect the response of related cell populations to the material. The dentinogenic differentiation potential on other cell populations related to the clinical situation such as osteoblasts, odontoblasts, and cementoblasts was not examined.
| Conclusions|| |
The addition of CaCl2.2H2O increases the cytotoxic activity but enhances the dentinogenic differentiation potential of WMTA on DPSCs. Future studies are warranted to validate findings presented in this report.
The authors would like to express their gratitude to Mrs. Ee Bee Choo, Mr. Hazhar Hassan and Mr. Yushamdan Yusof (Nor Laboratory, School of Physics), Ms. Jamilah Afandi, Mr. Johary Othman (Microscopy Unit, School of Biological Sciences), Mr. Nik Fakuruddin (SEM unit, School of Health Sciences), and members of the craniofacial laboratory (School of Dental Sciences), Universiti Sains Malaysia (USM), for their technical support.
Financial support and sponsorship
This study was supported by the USM Research University grant number 1001/PPSG/813056.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]
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