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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 2  |  Issue : 2  |  Page : 23-29

The effects of human amniotic fluid on periodontal ligament fibroblast cell viability, proliferation, and cytokine/growth factor expression


1 Department of Periodontology, Indiana University School of Dentistry, Indianapolis, IN, USA
2 Biomedical Sciences and Comprehensive Care, Indiana University School of Dentistry, Indianapolis, IN, USA

Date of Web Publication19-Aug-2019

Correspondence Address:
Prof. L Jack Windsor
Department of Biomedical Sciences and Comprehensive Care, Indiana University School of Dentistry, 1121 West Michigan Street, DS 271, Indianapolis, IN 46202
USA
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/GFSC.GFSC_10_19

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  Abstract 


Background: The importance of the amniotic fluid (AF) to the fetus is clear. However, very few studies have been published to examine the potential uses of this fluid in various areas such as tissue regeneration. AF contains epidermal growth factor, transforming growth factor-alpha, transforming growth factor beta-1, insulin-like growth factor-I, erythropoietin and granulocyte colony-stimulating factor, as well as hyaluronic acid and hyaluronic acid-stimulating factor. Previous studies suggest that AF can increase fibroblast proliferation and chemotaxis, and decrease apoptosis as well as promote wound healing. Furthermore, evidence showed that human AF inhibits hyaluronidase, elastase, and cathepsin. The current study examined the effects of human AF on periodontal ligament fibroblasts (PDLF) in terms of cell toxicity, cell proliferation, and cytokine/growth factor expression. Materials and Methods: Cytotoxicity of AF on PDLF was determined using lactate dehydrogenase assays. PDLF proliferation was determined using water-soluble tetrazolium-1 assays. Cytokine/growth factor expression was determined on AF-treated PDLF, AF alone, and PDLF alone utilizing protein arrays. Results: Human AF at 10% and below did not affect cell growth and was not toxic. AF-treated PDLF cells showed a decrease in cytokine/growth factor levels compared to the sum of cytokine/growth factor levels in AF only and cells only for 39 of the 80 proteins examined (48.8%). Of the 39 examined cytokines, 20 inflammatory cytokines, 11 cell cycle cytokines, 1 anti-inflammatory cytokine, and 7 other cytokines were decreased. Conclusion: Human AF at the examined concentrations was not toxic to PDLF cells and did not influence their proliferation. In addition, AF (10%) caused a decrease in the total protein levels of cytokines/growth factors expressed in 39 of the 80 proteins examined (48.8%). Of the 39 examined cytokines, 20 inflammatory cytokines, 11 cell cycle cytokines, 1 anti-inflammatory cytokine, and 7 other cytokines were decreased.

Keywords: Amniotic fluid, cellular proliferation, cytokines, cytotoxicity, fibroblasts, periodontal ligament


How to cite this article:
Ibraheem AG, Blanchard SB, Al-Hijji SM, Al-Nasr-Allah K, Windsor L J. The effects of human amniotic fluid on periodontal ligament fibroblast cell viability, proliferation, and cytokine/growth factor expression. Int J Growth Factors Stem Cells Dent 2019;2:23-9

How to cite this URL:
Ibraheem AG, Blanchard SB, Al-Hijji SM, Al-Nasr-Allah K, Windsor L J. The effects of human amniotic fluid on periodontal ligament fibroblast cell viability, proliferation, and cytokine/growth factor expression. Int J Growth Factors Stem Cells Dent [serial online] 2019 [cited 2019 Dec 16];2:23-9. Available from: http://www.cellsindentistry.org/text.asp?2019/2/2/23/264708




  Introduction Top


Amniotic fluid (AF) is essential for fetal development, nutrition, and protection. Human AF is complex in nature, and very few studies have been published to identify other potential uses outside the mother's womb. Shimberg[1] in 1938 reported using AF in the treatment of joint disease in 68 patients with various orthopedic conditions and reported that AF accelerates a defense-repair mechanism within the joints.

Maternal plasma contributes the water and solutes to the AF by first passing across the amnion through transmembranous flow, across the fetal vessels on the placental surface through intramembranous flow, and across fetal skin. Fetal kidneys develop after 8 weeks of gestation, which allows the fetus to contribute to the contents and volume of the AF. However, this contribution through fetal urination and oral, nasal, tracheal, and pulmonary fluids secretion becomes significant only during the second half of the pregnancy as the fetus' skin keratinizes and acts as a barrier that blocks the early transfer of fluids.[2],[3] The AF volume peaks by 28 weeks of gestation reaching approximately 800 mL where it plateaus to then decline by week 42 to approximately 400 mL.[4] Underwood examined the contents of the AF for cytokines and growth factors.[5] These included epidermal growth factor, transforming growth factor-alpha (TGF-α), TGF-β1, and insulin-like growth factor-I (IGF-1). AF also contains erythropoietin and granulocyte colony-stimulating factor, as well as hyaluronic acid and hyaluronic acid-stimulating factor.

Periodontal ligament fibroblasts (PDLF) predominate in the periodontal ligament connective tissues. Given the major role that they play in the development and function of the periodontal ligaments, they have been described by Ten Cate as the “architect, builder, and caretaker of connective tissue.”[6] Therefore, the focus of this study was to examine the effects of AF on the viability and proliferation of human PDLFs. In addition, the cytokine/growth factor expression from AF-treated PDLF cells was examined to determine the potential of AF on periodontal regeneration.


  Materials and Methods Top


Cell culture

Human PDLFs were purchased from ScienCell Research Laboratories (Carlsbad, San Diego, CA, USA). PDLFs were grown at 37°C in 5% CO2 in low glucose (1 g/L) Dulbecco's Modified Eagle's Media (DMEM) (Hyclone Logan, Utah) supplemented with 10% fetal bovine serum, 200 mM L-glutamine, 100 U/mL penicillin, 50 /mL gentamycin, and 250 /mL fungizone.

Human AF collected from a single donor was a gift from MiMedx (MiMedx© Marietta, GA, USA). It was collected from a single consenting mother having cesarian surgery and then lyophilized. DMEM without serum was used to reconstitute the freeze-dried AF to its original volume. A previous study[7] showed that the effects of human AF on human skin fibroblast proliferation were similar when pooled AF donors samples or individual AF donors samples were used. Based on the concentration range published in Chrissouli's paper,[7] concentrations of 1.25%, 2.5%, 5%, and 10% human AF were utilized in the water-soluble tetrazolium (WST) and lactate dehydrogenase (LDH) assays to determine cytotoxicity and cell proliferation.

Cell toxicity

Cellular membrane integrity was monitored by the permeability assay based on the determination of the release of LDH into the media (Cytotoxicity Detection KitPLUS Roche Applied Science, Mannheim, Germany).

Human PDLFs were seeded in 6-well plates (100,000 cells/well) in DMEM plus serum and incubated overnight to allow the cells to attach. The media was then removed and concentrations of AF (1.25, 2.5, 5 and 10%) in serum-free DMEM were added. After 72 h, the assays were performed per manufacturer's instructions (Roche Applied Science, Mannheim, Germany). Absorbance was recorded at 490 nm in a microplate reader (Titertek, Multiskan MCC, Flow Laboratories, McLean, VA, USA). The high controls (total cell death) were generated by the addition of lysis mix to the control wells per the manufacturer.

The experiments were repeated three times, and the mean values were calculated. The percentage release of LDH was calculated from the treated cells by comparing it with the maximum release of LDH (total cell death). To determine the cytotoxicity, the absorbance value of the background was subtracted from the experimented samples. The cytotoxicity was calculated as follows:

Cytotoxicity (%) = (experiment value-low control)/(high control-low control) × 100%.

Proliferation

Human PDLFs were seeded in 6-well plates (100,000 cells/well) in DMEM plus serum and incubated overnight to allow the cells to attach. The media was then removed, and the human PDLFs were exposed to concentrations of AF (1.25%, 2.5%, 5%, and 10%) diluted in serum-free DMEM After 72 h, assays were performed per manufacturer's instructions (WST, Roche Applied Science, Penzberg, Germany). Briefly, the media in the six-well plates was removed, and the cells washed 3 times with 2 mL of serum-free DMEM. The cell proliferation reagent WST-1 was added, and the plate was incubated per manufacturer's instructions.

A 100 μL sample from each well of the six-well plates was placed in a 96-well plate, and the absorbance of the samples against the negative control as the blank was measured using a microplate reader (Titertek) at 450 nm. The experiment was repeated three times, and the mean values calculated.

The absorbance values of each sample were compared with the untreated cell control, by percentage, in the following equation:

Cell proliferation (%) = Absorbance value of AF condensate treatment/absorbance value of no AF treatment × 100%.

Cytokine and growth factor arrays

Protein arrays [Table 1] (RayBio Human Cytokine Antibody Array 5, RayBiotech, Norcross, GA, USA) were used to detect cytokine/growth factor expression from the human PDLFs after exposure to human AF (MiMedx) as described by the manufacturer (RayBio). The highest concentration of AF (10%) that was not toxic and did not affect cell growth was used to treat the cells for 3 days in serum-free DMEM.
Table 1: Cytokine/growth factor array

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Media (1 mL) from three repeated experiments were analyzed by protein arrays. The membranes were blocked for 30 min, incubated for 3 h with the collected samples, washed, incubated for 2 h with biotin-conjugated antibodies, washed, and incubated with horseradish peroxidase-conjugated streptavidin for 2 h as per the manufacturer. Detection agents supplied in the array kits were mixed and applied to each membrane for 2 min. The cytokine/growth factors on the membranes were then visualized by autoradiography on X-ray film. Signal intensities were quantified with a Bio-Rad Gel Doc XR imaging system and analyzed with Quantity One software (Bio-Rad, Laboratories, Hercules, CA, USA). The experiments were repeated three times and the mean values calculated.

Statistical methods

The WST and LDH data were subjected to one-way analysis of variance (ANOVA) followed by the Tukey's honestly significant difference test for pair-wise comparisons among the groups (P < 0.05). To determine the level of cytokine/growth factor expression, the optical densities of the visible dots on the membrane were measured. For each membrane, the densities were adjusted for the background by subtracting the average value of the negative controls and then normalized by dividing by the average of the positive controls. The data were then converted back to the original scale by multiplying by the average of the positive controls for the first membrane. Three membranes were used for each of the three groups (AF + cells, AF only, and cells only). Group comparisons were made using one-way ANOVA, followed by pair-wise tests using Fisher's protected least significant differences to control the overall significance level at 5%. In addition to the direct comparisons among the three groups, the AF + cells group was compared against the sum of the AF only and cells only groups to evaluate nonadditive effects of the AF and cells; a nonsignificant test for this effect indicates the effect could be additive, whereas a significant test could indicate the effect is either synergistic (AF + cells is significantly greater than the sum of AF only and cells only) or inhibitory (AF + cells is significantly less than the sum of AF only and cells only). Tests of normality were performed before performing the ANOVAs. Statistical analyses were performed using SPSS 23.0 for Windows software system (SPSS Inc., Chicago, IL, USA) and SAS version 9.4 (SAS Institute Inc., Cary, NC, USA).


  Results Top


Cytotoxicity and cell proliferation

Cell proliferation results [Figure 1]a showed that AF at concentrations of 1.25% (86.1 ± 1.31, 0.073), 2.5% (95.5 ± 4.25, 0.858), 5% (95.8 ± 2.04, 0.886), and 10% (90.4 ± 4.02, 0.286) (mean ± standard error [SE], P value) did not affect PDLF cell proliferation. Cytotoxicity [Figures 1]b showed that AF at concentrations of 1.25% (21 ± 0.53, 0.636), 2.5% (21.5 ± 1.31, 0.467), 5% (19.2 ± 0.73, 0.998), and 10% (17.2 ± 1.85, 0.892) (mean ± SE, P value) were not toxic to PDLF cells. Concentrations of more than 10% AF were not tested.
Figure 1: (a) Cell proliferation versus amniotic fluid concentration showing no significant difference (P < 0.05) in cell proliferation compared to 0% amniotic fluid, (b) cell viability versus amniotic fluid concentration showing no significant difference (P < 0.05) in viability compared to 0% amniotic fluid

Click here to view


Cytokine array

AF-treated PDLF cells (AFC) showed a significant decrease (P < 0.05) in cytokine/growth factor levels compared to the sum of cytokine/growth factor levels in AF only and cells only for 39 of the 80 cytokine/growth factor (48.8%) examined [Table 2]. Of the 39 examined cytokines, 20 inflammatory cytokines [Table 2], 11-cell cycle cytokines [Table 3], 1 anti-inflammatory cytokine [Table 4], and 7 other cytokines [Table 4] were decreased. AFC showed inhibitory effects compared to the sum of AF only and cells only for BDNF, BLC, fibroblast growth factor (FGF)-6, FGF-7, FGF-9, FGF-4, FLT-3 Ligand, Fractalkine, GCP-2, GRO a/b/g, IFN-gamma, IGFBP-2, IGFBP-4, IL-1 beta, IL-15, IL-16, IL-3, IL-4, IL-12, IP-10, LIF, LIGHT, Leptin, M-CSF, MCP-4, NT-4, osteoprotegerin (OPG), OPN, OSM, PARC, PDGF-BB, POS, RANTES, SCF, SDF-1 alpha, TARC, TIMP-2, TNF alpha, TNF beta, and VEGF-A.
Table 2: Inflammatory cytokines/growth factors that showed significant decrease P<0.05

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Table 3: Cell cycle cytokines/growth factors that showed significant decrease P<0.05

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Table 4: Anti-Inflammatory and other cytokines/growth factors that showed significant decrease P<0.05

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AFC demonstrated a significant increase in GRO a/b/g (P = 0.038), GRO alpha (P = 0.025), IGFBP-1 (P = 0.026), and IL-7 (P = 0.039) compared to their levels in cells only. IL-6 showed significantly higher levels (P = 0.004) in AFC compared to its levels in AF only. OPG levels were significantly lower (P = 0.015) in AF only versus in cells only and showed a significant increase (P = 0.01) in AFC compared to its levels in AF only. OPG levels in AFC were, however, significantly lower (P = 0.009) than the sum of its values in AF only and in cells only. The AF only levels of IGFBP-1 were significantly higher (0.019) than in cells only while the AF only levels of SDF-1 alpha and TARC were significantly less (P = 0.045 and P = 0.049) than in cells only. The AFC levels of SDF-1 alpha and TARC, however, were significantly less (P = 0.016 and P = 0.017) than the sum of their values in AF only and cells only. The cytokines that were increased in AFC versus C or AFC versus AF was increased due to an additive effect of their values in AF only and C only. No significant increase (P < 0.05) was reported in cytokines levels in AFC versus AF + C.

Images taken of X-rays of the cytokine/growth factor arrays for different groups are shown in [Figure 2]. Forty-one cytokines were not altered [Supplemental Table 1][Additional file 1].
Figure 2: (a) X-ray of cytokine/growth factor array of amniotic fluid treated periodontal ligament fibroblasts cells, (b) X-ray of cytokine/growth factor array of amniotic fluid only, (c) X-ray of cytokine/growth factor array of periodontal ligament fibroblasts cells only

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  Discussion Top


The current study is the first to explore the effects of AF on PDLF cells. This study examined cytotoxicity, cell proliferation, and cytokine/growth factor expression from PDLF cells exposed to AF. The results showed that AF at concentrations of 1.25%, 2.5%, 5%, and 10% had no effects on cytotoxicity and cell proliferation of the PDLF cells. Concentrations of more than 10% AF were not tested.

A study by Chrissouli showed significant cell proliferation of human skin fibroblasts treated with amniotic fluid at concentrations 1%–50% (v/v) and 0.1% v/v fetal bovine serum.[7] Their study used a different fibroblast type, and fetal bovine serum (0.1% v/v) was included in the media with the AF. These differences could account for the differences seen between their study and the current study that showed no PLDF cell proliferation. Also, in a recent study,[8] the effects of AF on neonatal foreskin keratinocytes and fibroblasts were examined in terms of cytotoxicity and cell proliferation. The study showed that cell viability did not differ for the keratinocytes and fibroblasts. AF caused a decrease in keratinocyte proliferation and showed no difference in neonatal foreskin fibroblast proliferation.[8] The lack of cellular proliferation in their study is consistent with results from the current study.

The results of the current study show a downregulation in 20 inflammatory cytokines, including pro-inflammatory cytokines IL-1 beta, TNF-alpha, and IFN-gamma. These cytokines were found to be elevated in newborns with evidence of perinatal brain damage when compared to controls.[9] Infants who developed white matter brain damage had elevated postnatal levels of circulating IFN-gamma in umbilical cord blood and neonatal blood compared to those who did not develop brain damage.[10] A study undertaken to explore the association between the mean neonatal concentration of inflammatory mediators and chemokines of normal children compared to children with congenital cerebral palsy found that the concentrations of IL-1, IL-8, IL-9, TNF-α, and RANTES were higher.[11] IL-8 levels were not increased in the current study. However, IL-1, TNF-α, and RANTES levels were decreased. IL-9 levels were not examined in the present study.

The current study shows significant inhibition of TNF-α (P = 0.026) levels in AFC compared to the sum of the growth factor levels in AF only and cell only. TNF-α is a well-known mediator of bone resorption.[12] TNF-α may act independently or along with IL-1 to synergistically initiate the resorptive process.[12],[13] Therefore, the ability of AF to downregulate IL-1 beta and TNF-α may suggest a protective function of AF. OPG was also downregulated in the current study. Its levels in AFC were significantly less (P = 0.009) than the sum of its values in AF only and in cells only. OPG is a decoy receptor that binds to receptor activator of nuclear factor-kB receptor and downregulates osteoclast formation and therefore, inhibits bone resorption.[14] Given the developmental stage of the fetus, OPG might not be needed since osteoclastic activity would not be expected.

In the current study, IL-6 expression showed a trend toward increasing (P = 0.082) on treating PDLF cells with AF. Certain cytokines are known to overlap in function and are sometimes present in antagonistic groups. IL-6 is one of those overlapping cytokines in that it can function as an anti-inflammatory or pro-inflammatory cytokine.[15] In its pro-inflammatory role, it causes B-cell activation, which leads to the production of IL-1, a pro-inflammatory cytokine.[16] IL-1 enhances bone resorption, stimulation of matrix metalloproteinase production, and prostaglandin synthesis.[16] Interestingly, in the current study, IL-1 beta was significantly downregulated (P = 0.012) in AFC compared to the sum of the growth factor levels in AF only and cells only. In its anti-inflammatory role, IL-6 inhibits TNF, and IL-1 production from macrophages.[15] In an animal study, the anti-inflammatory function exhibited by IL-6 could not be compensated for by IL-10, which is another anti-inflammatory cytokine.[17] IL-10 levels were unchanged in the current study. IL-6 in its anti-inflammatory role is considered crucial in regulating the levels of pro-inflammatory cytokines thereby sometimes playing a crucial anti-inflammatory role.[17]

In the current study, numerous cytokine/growth factors related to cell cycle regulation were downregulated. FGFs control a broad range of biological cell functions such as proliferation, survival, migration, and differentiation. Multiple FGF isoforms were downregulated in AFC, which may explain why PDL fibroblast cell proliferation was not observed when treated with AF in the current study. Other cell cycle cytokines/growth factors that were downregulated included IGFBP 2 and 4, PDGF-BB, VEGF-A, LIF, SCF, and SDF-1 alpha. IL-4, an anti-inflammatory cytokine, was downregulated in the current study. It is unclear why it might be downregulated.

The current data suggest that AF promotes an anti-inflammatory state that provides a nonreactive state that is crucial for the developing fetus. This study suggests that AF may be useful in downregulating inflammation but may not induce cell proliferation. Therefore, AF may be valuable in treating inflammation but appears unlikely to be of any benefit for the regeneration of periodontal tissues involving PDLFs.

Regardless of these interesting findings, some limitations of the present study need to be addressed. For example, the AF used was from one individual. AF from different individuals should be examined to observe the variations between individuals. In addition, it would be valuable to do ELISA on some of the major cytokines in the different groups such as IL-1, GRO a/b/g, VEGF, PDGF, IL-4, TIMP 2, and OPG.


  Conclusion Top


The study found that AF had no effects on cell viability and was not toxic at the concentrations examined. AF (10%) downregulated numerous inflammatory cytokines/growth factors.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Shimberg M. The use of amniotic-fluid concentrate in orthopaedic conditions. J Bone Joint Surg Am 1938;20:167-77.  Back to cited text no. 1
    
2.
Cunningham FG, Leveno KJ, Bloom SL, Spong CY, Dashe JS, Hoffman BL, et al. “Williams obstetrics” (24th edition). New York, NY: McGraw-Hill Education; 2014. p. 1376.  Back to cited text no. 2
    
3.
Gilbert WM, Brace RA. Amniotic fluid volume and normal flows to and from the amniotic cavity. Semin Perinatol 1993;17:150-7.  Back to cited text no. 3
    
4.
Brace RA, Wolf EJ. Normal amniotic fluid volume changes throughout pregnancy. Am J Obstet Gynecol 1989;161:382-8.  Back to cited text no. 4
    
5.
Underwood MA, Gilbert WM, Sherman MP. Amniotic fluid: Not just fetal urine anymore. J Perinatol 2005;25:341-8.  Back to cited text no. 5
    
6.
Ten Cate AR, Deporter DA, Freeman E. The role of fibroblasts in the remodeling of periodontal ligament during physiologic tooth movement. Am J Orthod 1976;69:155-68.  Back to cited text no. 6
    
7.
Chrissouli S, Pratsinis H, Velissariou V, Anastasiou A, Kletsas D. Human amniotic fluid stimulates the proliferation of human fetal and adult skin fibroblasts: The roles of bFGF and PDGF and of the ERK and Akt signaling pathways. Wound Repair Regen 2010;18:643-54.  Back to cited text no. 7
    
8.
Papanna R, Won JH, Mann LK, Loose D, Fletcher S. 64: Amniotic fluid selectively inhibits proliferation of keratinocytes: A potential mechanism for delayed fetal wound healing. Am J Obstet Gynecol 2019;220:S51-2.  Back to cited text no. 8
    
9.
Dammann O, O'Shea TM. Cytokines and perinatal brain damage. Clin Perinatol 2008;35:643-63, v.  Back to cited text no. 9
    
10.
Hansen-Pupp I, Harling S, Berg AC, Cilio C, Hellström-Westas L, Ley D, et al. Circulating interferon-gamma and white matter brain damage in preterm infants. Pediatr Res 2005;58:946-52.  Back to cited text no. 10
    
11.
Nelson KB, Dambrosia JM, Grether JK, Phillips TM. Neonatal cytokines and coagulation factors in children with cerebral palsy. Ann Neurol 1998;44:665-75.  Back to cited text no. 11
    
12.
McCauley LK, Nohutcu RM. Mediators of periodontal osseous destruction and remodeling: Principles and implications for diagnosis and therapy. J Periodontol 2002;73:1377-91.  Back to cited text no. 12
    
13.
Pfeilschifter J, Chenu C, Bird A, Mundy GR, Roodman GD. Interleukin-1 and tumor necrosis factor stimulate the formation of human osteoclastlike cells in vitro. J Bone Miner Res 1989;4:113-8.  Back to cited text no. 13
    
14.
Sodek J, McKee MD. Molecular and cellular biology of alveolar bone. Periodontol 2000 2000;24:99-126.  Back to cited text no. 14
    
15.
Opal SM, DePalo VA. Anti-inflammatory cytokines. Chest 2000;117:1162-72.  Back to cited text no. 15
    
16.
Okada H, Murakami S. Cytokine expression in periodontal health and disease. Crit Rev Oral Biol Med 1998;9:248-66.  Back to cited text no. 16
    
17.
Xing Z, Gauldie J, Cox G, Baumann H, Jordana M, Lei XF, et al. IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses. J Clin Invest 1998;101:311-20.  Back to cited text no. 17
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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