The Ability of Quantitative, Specific, and Sensitive Point-of-Care/Chair-Side Oral Fluid Immunotests for aMMP-8 to Detect Periodontal and Peri-Implant Diseases

Alassiri, Saeed; Parnanen, Pirjo; Rathnayake, Nilminie; Johannsen, Gunnar; Heikkinen, Anna-Maria; Lazzara, Richard; van der Schoor, Peter; van der Schoor, Jan Gerrit; Tervahartiala, Taina; Gieselmann, Dirk; Sorsa, Timo

doi:https://doi.org/10.1155/2018/1306396

Disease Markers

On this page

Abstract Introduction Discussion Conflicts of Interest Acknowledgments References Copyright Related Articles

Special Issue

The Use of Saliva in the Diagnosis of Oral and Systemic Diseases

View this Special Issue

Review Article | Open Access

Volume 2018 | Article ID 1306396 | https://doi.org/10.1155/2018/1306396

The Ability of Quantitative, Specific, and Sensitive Point-of-Care/Chair-Side Oral Fluid Immunotests for aMMP-8 to Detect Periodontal and Peri-Implant Diseases

Saeed Alassiri,¹Pirjo Parnanen,¹Nilminie Rathnayake,²Gunnar Johannsen,²Anna-Maria Heikkinen,¹Richard Lazzara,³Peter van der Schoor,^2,3Jan Gerrit van der Schoor,³Taina Tervahartiala,¹Dirk Gieselmann,³and Timo Sorsa^1,2

Academic Editor: Waszkiewicz Napoleon

Received12 Mar 2018

Accepted21 Jun 2018

Published05 Aug 2018

Abstract

The analysis of the disease-specific oral and systemic biomarkers in saliva and oral fluids (i.e., mouth rinse, gingival crevicular fluid (GCF), and peri-implantitis fluid (PISF)) is demanding. Several hosts and microbial factors may influence their expression, release, and levels. The type of saliva/oral fluids utilized for the diagnostics affects the analysis. High sensitivity and specificities together with sophisticated methods and techniques are essential for valuable outcome. We describe here recently developed practical, convenient, inexpensive, noninvasive, and quantitative mouth rinse and PISF/GCF/chair-side/point-of-care (PoC) lateral-flow aMMP-8 immunoassays (PerioSafe and ImplantSafe/ORALyser) to detect, predict, and monitor successfully the course, treatment, and prevention of periodontitis and peri-implantitis, respectively. The tests have been independently and successfully validated to differentiate periodontal and peri-implant health and disease in Finland, Germany, Netherland, Sweden, Turkey, Nigeria, Malawi, and USA. The clinical use of salivary/oral fluid biomarkers to identify oral and systemic conditions requires additional studies utilizing these noninvasive screening, diagnostic, and preventive aMMP-8 PoC/chair-side technologies.

1. Introduction

Diagnosis of periodontal and peri-implant diseases are mostly based on an array of clinical measurements and indices including pocket probing depths, bleeding on probing, and assessment of clinical attachment level together with radiographic findings. Additional information such as medical, hereditary, and specific features and the amount of dental plaque have also been recorded [1, 2]. These laborous diagnostic procedures require not only multiple manual recordings but also professional examiners with trained expertise. So far, these clinical and radiographic diagnostic procedures are the best currently available ones for diagnosing and monitoring the disease course, treatment, and maintenance [1]. However, they can only assess the past experience, current extent, and severity of the periodontal and peri-implant diseases. Since these clinical and radiographic analyses of periodontitis and peri-implantitis have low sensitivity and low positive predictive value [1], no reliable information can be obtained regarding the diseases’ current activities and their future courses [1]. Also, the episodic progression of periodontitis and peri-implantitis makes the accurate assessment of disease activity and progression difficult and complicated [1]. Neutrophil collagenase, also called matrix metalloproteinase (MMP)-8, polymorphonuclear (PMN) leukocyte collagenase, or collagenase-2, has been identified and characterized as a major collagenolytic enzyme that causes active periodontal and peri-implant degeneration (APD) in periodontitis and peri-implantitis [3–7]. MMP-8 can resolve and regulate inflammatory and immunological cascades by processing nonmatrix bioactive substrates such as chemokines, cytokines, serpins, and complement components. Physiological levels of MMP-8 can exert protective and defensive anti-inflammatory characteristics [5]. Increased levels of especially active MMP-8 (aMMP-8), but not latent, inactive proform, have been found in periodontitis- and peri-implantitis-affected oral fluids (saliva, mouth rinse, gingival crevicular fluid (GCF), and peri-implant sulcular fluid (PISF)) [8–10]. A key characteristic of active periodontal and peri-implant diseases is the sustained pathological elevation and activation of MMP-8 in periodontal and peri-implant tissues, which are reflected in oral fluids [4]. Consequently, aMMP-8 is a promising biomarker candidate for diagnosing and assessing the progression and course of these episodic oral inflammatory tissue destructive and degenerative diseases [3, 4]. More importantly, aMMP-8 in oral fluids can also serve as a predictive and preventive adjunctive biotechnological tool to indicate [4, 7, 8, 11] and time the preventive interventions (secondary prevention or supportive periodontal/peri-implant therapy [12, 13]) and to inhibit or reduce the conversion of preperiodontitis (formerly gingivitis) and preperi-implantitis (formerly mucositis) to periodontitis and implantitis, respectively.

With this background, we describe the documentation of currently commercially available quantitative reader-based aMMP-8 oral fluid specific point-of-care/chair-side lateral-flow reader-equipped immunotests, that is, PerioSafe and ImplantSafe/ORALyser, for periodontal and peri-implant diseases, respectively.

2. PerioSafe and ImplantSafe, aMMP-8 Oral Fluid PoC/Chair-Side Tests, in Chronic Periodontitis and Peri-Implantitis: Diagnostic Utilizations and Monitoring the Effects of Treatments

Recently, lateral-flow point-of-care (PoC)/chair-side tests (PerioSafe and ImplantSafe), discovered in Finland and further developed in Germany [4, 14], have been developed based on earlier described technologies and monoclonal antibodies [4, 5, 14, 15]. The tests, PerioSafe and ImplantSafe, and reader (ORALyser) have been developed and manufactured by Medix Biochemica Ltd (Espoo, Finland) and dentognostics GmbH (Jena, Germany) and are commercially available from Dentognostics GmbH (Jena, Germany). In fact, the PoC/chair-side aMMP-8 lateral-flow immunotests resemble the classical pregnancy and/or recently described HIV-PoC tests [16, 17]. The aMMP-8 oral fluid tests can be used according to the manufacturer’s instructions [5]. PerioSafe measures and analyses the levels of aMMP-8 in mouth rinse and ImplantSafe in PISF and GCF; thus, PerioSafe is patient-specific and ImplantSafe is site-specific [5, 14]. PerioSafe and ImplantSafe test-sticks can be quantitated by the ORALyser reader in 5 min PoC/chair-side [4]. PerioSafe and ImplantSafe with ORALyser quantitation are reliable, quantitative, noninvasive, safe, and inexpensive adjunctive point-of-care diagnostic tools for diagnosis, screening, monitoring, and prevention of periodontal and peri-implant diseases [4, 5].

Chronic periodontitis (), peri-implantitis (), and their healthy controls ( and 30, resp.) were characterized clinically and from X-rays as described earlier [11, 15, 18, 19]. Ten clinically and X-ray-diagnosed adult chronic periodontitis patients [11, 15] were all (100%) diagnosed to be aMMP-8 positive by PerioSafe visual test, and their aMMP-8 lateral-flow test-sticks were quantitated by ORALyser before (all >20 ng/ml, visually (+)) and after periodontal treatment, scaling and root planing (SRP) or anti-infective treatment (Figures 1(a) and (b)). SRP or anti-infective treatment was found to reduce the pocket depths and the bleeding of probing [11, 15]. SRP also affected the aMMP-8 levels in mouth rinse from positive (+) to negative (−) by visual estimation of the test results and from >20 ng/ml (positive (+)) to <20 ng/ml (negative (−)) by ORALyser reader quantitation (Figure 1(a)). Systemically and periodontally healthy dental students (age 22–24, ) served as healthy controls (Figure 1(b)).

We further demonstrated here that the ImplantSafe aMMP-8 PoC/chair-side sulcular fluid test site specifically in 5 min detects the peri-implantitis sites () differentiating them from clinically healthy peri-implant sites () and can be utilized for monitoring treatment (Figures 2(a) and 2(b)). Peri-implantitis and healthy sites were diagnosed clinically and by X-rays as described [18]. Peri-implantitis sites were surgically treated according to the Swedish national guidelines [19]. We also analysed quantitatively aMMP-8 by immunofluorometric assay (IFMA) [15] and all forms of MMP-9 densitometrically by quantitated gelatin-zymography [20]. Similar to ImplantSafe PoC/chair-side findings, elevated aMMP-8 levels could be detected by IFMA in all peri-implantitis sites (29 = 100% ImplantSafe-positive >20 ng/ml (124,60 ± 22.50 ng/ml)) differing from clinically healthy sites all having low aMMP-8 levels (32 sites/<20 ng/ml (18.60 ± 3.46 ng/ml)) all being ImplantSafe PoC-negative (Figure 2(b)). This difference was statistically significant (, Wilcoxon test) (Figure 2(a)). Surgical treatment of peri-implantitis sites according to the Swedish national guidelines [19] caused the positive ImplantSafe tests to be negative (Figure 2(b)). This pilot case-control peri-implantitis study shows both 100% sensitivity and specificity for ImplantSafe test. Currently, this test is available also as a reader-equipped/quantitated-PoC tool, that is, ORALyser reader [4]. MMP-9 or gelatinase-B, analysis by gelatin zymography from these same PISF samples, revealed that any form of MMP-9 or total MMP-9 was not able to differentiate peri-implant health and disease (Figure 2(a)). Our pilot case-control studies have received approval from the local ethical committee of Stockholm Community, Sweden (2016-08-24/2016/1:8 and 2016-1-24) and the Helsinki University Central Hospital, Finland (nro260/13/03/00/13).

Figure 2

Peri-implant sulcular fluid (PISF) collected from 29 peri-implantitis and 32 periodontally healthy sites were tested for elevated aMMP-8 by ImplantSafe PoC/chair-side test (+/−), analysed for aMMP-8 (ng/ml) by immunofluorometric assay (IFMA), and by quantitated gelatin zymography for all molecular weight forms of gelatinase-B (MMP-9, zymographic densitometric units) (a). Implantitis sites were tested before and after surgical treatment by ImplantSafe (+/−); treatment caused positive sites to be negative (b). Elevated aMMP-8 levels in PISF detected by ImplantSafe positivity and IFMA associated significantly with peri-implantitis (, Wilcoxon test ()) and could thus be conveniently PoC detected in 5 min by ImplantSafe. Any forms or total MMP-9 did –not differentiate peri-implantitis and healthy sites (a).

3. Discussion

Currently, the practical and quantitative PoC/chair-side lateral-flow oral fluid aMMP-8 immunoassays have been successfully developed and are commercially available. The tests have been independently and successfully validated in Finland, Nigeria, Germany, Holland, Malawi, Turkey, Sweden, and USA [4, 5, 14, 21–24]. The tests have diagnostic sensitivity and specificity 76–90% and 96%, respectively, corresponding to odds ratio of >72 [4, 5, 14, 21]. The test results are quantitatively available by the reader in 5 min PoC/chair-side [4]. The tests have been shown to be useful to screen susceptible sites and patients, differentiate active and inactive periodontitis and peri-implantitis sites, predict the future disease progression, and monitor the treatment—with or without different adjunctive medications—response, and maintenance therapy [4, 5]. As demonstrated in the present study, the tests excellently differentiated periodontal and peri-implant health and diseases. Additionally, the test can identify initial and alarm or early periodontitis (preperiodontitis) in genetically predisposed adolescents [21]. Thus, the test is effective in both adult and adolescent populations and can act in “a gene”-test manner [14, 21]. Pathologically elevated aMMP-8 associates, reflects, and precedes the active phase of periodontal and peri-implant diseases, that is, APD [4, 7, 8]. This forms the basis of predictive value for aMMP-8 oral fluid tests in periodontal and peri-implant diseases; thus, the test is positive ahead or before clinical manifestations and/or radiographic outcomes of APD [4, 5, 8]. The tests can detect enough early subclinical and silent developing preperiodontitis and preperi-implantitis [4, 21]. The tests are very suitable for monitoring the disease’s development and progression as well as timing and targeting the preventive and therapeutic interventions (i.e., secondary prevention and/or supportive periodontal/peri-implant therapy) [12, 13]. In comparison to bleeding on probing, aMMP-8 PoC/chair-side test is much more sensitive to distinguish periodontal health and disease [14]. Noteworthy, bleeding on probing always causes bacteraemia. Noninvasive PerioSafe and ImplantSafe testings never cause bacteraemia.

Regarding other PoC technologies for periodontitis and peri-implantitis, Ritzer et al. [25] described peri-implantitis/periodontitis online diagnosis utilizing elegantly the tongue as a 24/7 detector allowing diagnosis by “anyone, anywhere, and anytime.” Their clever technology utilizes chewing gum-containing peptide sensors as protease cleavable linkers between a bitter taste substance and a microparticle. MMPs upregulated in oral cavity and fluids (i.e., saliva and GCF/PISF) are presented to be responsible for cleaving the sensor in chewing gum generating the bitter taste consequently to be detected by the tongue as a 24/7 in vivo and online sensor [25]. Nonetheless, their elegant 24/7 tongue sensor assay is not MMP-specific for the main collagenolytic MMP, MMP-8, upregulated and activated in periodontitis- and peri-implantitis-affected periodontal and peri-implant tissue, gingiva and oral fluids, including saliva, GCF, PISF, and mouth rinse. The sensor is sensitive also to cleavages by MMP-1, MMP-13, and MMP-9 present and upregulated in the diseased periodontitis and peri-implantitis tissue and oral fluids [25]. Furthermore, no information was provided regarding specificity, sensitivity, and predictability of the 24/7 tongue technology regarding periodontitis and peri-implantitis. PerioSafe and ImplantSafe exert 76–90% specificity and >96% sensitivity [4, 5]. Also, the susceptibilities of the protease cleavable linker 24/7 sensor to catalytically competent and effective microbial proteases expressed and released by dysbiotic oral periodontopathogens and candidial species were not addressed [26, 27]. Recently, a PoC/chair-side device, comparable to PCR-detection, for Porphyromonas gingivalis related to chronic periodontitis detection has been developed [28]. A chair-side assay for GCF calprotectin has been described [29]. Calprotectin does not degrade anything [29].

PerioSafe and ImplantSafe are specific and sensitive for aMMP-8, a major destructive and collagenolytic factor for APD [4, 5]. Overall, PerioSafe and ImplantSafe, aMMP-8 quantitative oral fluid PoC/chair-side lateral-flow immunotests, are the first clinically validated commercially available diagnostic, prognostic, quantitative, predictive, noninvasive, and preventive PoC/chair-side technologies for periodontal and peri-implant diseases [4, 5]; the tests can be used by dental and medical professionals linking these disciplines [4]. Potentially, such aMMP-8 PoC tests can eventually be adapted for other medical disciplines [30, 31].

Conflicts of Interest

Professor Timo Sorsa is an inventor of US patents 5652223, 5736341, 5866432, and 6143476. All other authors declare no conflicts of interests.

Acknowledgments

This study has been supported by grants from the Helsinki University Hospital Research Foundation (TYH 2016251, TYH 2017251, TYH2018229, Y1014SLO17, and Y1014SLO18), Helsinki, Finland, and Karolinska Institutet, Stockholm, Sweden.

References

D. F. Kinane, P. G. Stathopoulou, and P. N. Papapanou, “Periodontal diseases,” Nature Reviews Disease Primers, vol. 3, article 17038, 2017.
View at: Publisher Site | Google Scholar
N. P. Lang, A. Joss, T. Orsanic, F. A. Gusberti, and B. E. Siegrist, “Bleeding on probing. A predictor for the progression of periodontal disease?” Journal of Clinical Periodontology, vol. 13, no. 6, pp. 590–596, 1986.
View at: Publisher Site | Google Scholar
A. E. Herr, A. V. Hatch, D. J. Throckmorton et al., “Microfluidic immunoassays as rapid saliva-based clinical diagnostics,” Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 13, pp. 5268–5273, 2007.
View at: Publisher Site | Google Scholar
T. Sorsa, D. Gieselmann, N. B. Arweiler, and M. Hernández, “A quantitative point-of-care test for periodontal and dental peri-implant diseases,” Nature Reviews Disease Primers, vol. 3, article 17069, 2017.
View at: Publisher Site | Google Scholar
T. Sorsa, A. M. Heikkinen, J. Leppilahti et al., “Active matrix metalloproteinase-8: contributor to periodontitis and a missing link between genetics, dentistry, and medicine,” in Pathogenesis of Periodontal Diseases, N. Bostanci and G. N. Belibasakis, Eds., pp. 51–57, Springer International Publishing, Cham, 2018.
View at: Publisher Site | Google Scholar
E. F. de Morais, J. C. Pinheiro, R. B. Leite, P. P. A. Santos, C. A. G. Barboza, and R. A. Freitas, “Matrix metalloproteinase-8 levels in periodontal disease patients: a systematic review,” Journal of Periodontal Research, vol. 53, no. 2, pp. 156–163, 2018.
View at: Publisher Site | Google Scholar
E. F. de de Morais, A. N. Dantas, J. C. Pinheiro et al., “Matrix metalloproteinase-8 analysis in patients with periodontal disease with prediabetes or type 2 diabetes mellitus: a systematic review,” Archives of Oral Biology, vol. 87, pp. 43–51, 2018.
View at: Publisher Site | Google Scholar
W. Lee, S. Aitken, J. Sodek, and C. A. G. McCulloch, “Evidence of a direct relationship between neutrophil collagenase activity and periodontal tissue destruction in vivo: role of active enzyme in human periodontitis,” Journal of Periodontal Research, vol. 30, no. 1, pp. 23–33, 1995.
View at: Publisher Site | Google Scholar
S. Mancini, R. Romanelli, C. A. Laschinger, C. M. Overall, J. Sodek, and C. A. G. McCulloch, “Assessment of a novel screening test for neutrophil collagenase activity in the diagnosis of periodontal diseases,” Journal of Periodontology, vol. 70, no. 11, pp. 1292–1302, 1999.
View at: Publisher Site | Google Scholar
R. Romanelli, S. Mancini, C. Laschinger, C. M. Overall, J. Sodek, and C. McCulloch, “Activation of neutrophil collagenase in periodontitis,” Infection and Immunity, vol. 67, no. 5, pp. 2319–2326, 1999.
View at: Google Scholar
J. M. Leppilahti, M. A. Kallio, T. Tervahartiala, T. Sorsa, and P. Mäntylä, “Gingival crevicular fluid matrix metalloproteinase-8 levels predict treatment outcome among smokers with chronic periodontitis,” Journal of Periodontology, vol. 85, no. 2, pp. 250–260, 2014.
View at: Publisher Site | Google Scholar
P. Axelsson, B. Nyström, and J. Lindhe, “The long-term effect of a plaque control program on tooth mortality, caries and periodontal disease in adults. Results after 30 years of maintenance,” Journal of Clinical Periodontology, vol. 31, no. 9, pp. 749–757, 2004.
View at: Publisher Site | Google Scholar
D. Ziebolz, G. Schmalz, D. Gollasch, P. Eickholz, and S. Rinke, “Microbiological and aMMP-8 findings depending on peri-implant disease in patients undergoing supportive implant therapy,” Diagnostic Microbiology and Infectious Disease, vol. 88, no. 1, pp. 47–52, 2017.
View at: Publisher Site | Google Scholar
S. O. Nwhator, P. O. Ayanbadejo, K. A. Umeizudike et al., “Clinical correlates of a lateral-flow immunoassay oral risk indicator,” Journal of Periodontology, vol. 85, no. 1, pp. 188–194, 2014.
View at: Publisher Site | Google Scholar
P. Mäntylä, M. Stenman, D. F. Kinane et al., “Gingival crevicular fluid collagenase-2 (MMP-8) test stick for chair-side monitoring of periodontitis,” Journal of Periodontal Research, vol. 38, no. 4, pp. 436–439, 2003.
View at: Publisher Site | Google Scholar
T. Shochet, I. A. Comstock, N. T. N. Ngoc et al., “Results of a pilot study in the U.S. and Vietnam to assess the utility and acceptability of a multi-level pregnancy test (MLPT) for home monitoring of hCG trends after assisted reproduction,” BMC Women's Health, vol. 17, no. 1, p. 67, 2017.
View at: Publisher Site | Google Scholar
T. Prazuck, S. Karon, C. Gubavu et al., “A finger-stick whole-blood HIV self-test as an HIV screening tool adapted to the general public,” PLoS ONE, vol. 11, no. 2, article e0146755, 2016.
View at: Publisher Site | Google Scholar
A. Mombelli and N. P. Lang, “The diagnosis and treatment of peri-implantitis,” Periodontology 2000, vol. 17, no. 1, pp. 63–76, 1998.
View at: Publisher Site | Google Scholar
http://www.socialstyrelsen.se/english.
T. Sorsa, T. Salo, E. Koivunen et al., “Activation of type IV procollagenases by human tumor-associated trypsin-2,” Journal of Biological Chemistry, vol. 272, no. 34, pp. 21067–21074, 1997.
View at: Publisher Site | Google Scholar
A. M. Heikkinen, T. Raivisto, K. Kettunen et al., “Pilot study on the genetic background of an active matrix metalloproteinase-8 test in Finnish adolescents,” Journal of Periodontology, vol. 88, no. 5, pp. 464–472, 2017.
View at: Publisher Site | Google Scholar
S. Izadi Borujeni, M. Mayer, and P. Eickholz, “Activated matrix metalloproteinase-8 in saliva as diagnostic test for periodontal disease? A case-control study,” Medical Microbiology and Immunology, vol. 204, no. 6, pp. 665–672, 2015.
View at: Publisher Site | Google Scholar
K. Lorenz, T. Keller, B. Noack, A. Freitag, L. Netuschil, and T. Hoffmann, “Evaluation of a novel point-of-care test for active matrix metalloproteinase-8: agreement between qualitative and quantitative measurements and relation to periodontal inflammation,” Journal of Periodontal Research, vol. 52, no. 2, pp. 277–284, 2017.
View at: Publisher Site | Google Scholar
N. Johnson, J. L. Ebersole, R. J. Kryscio et al., “Rapid assessment of salivary MMP-8 and periodontal disease using lateral flow immunoassay,” Oral Diseases, vol. 22, no. 7, pp. 681–687, 2016.
View at: Publisher Site | Google Scholar
J. Ritzer, T. Lühmann, C. Rode et al., “Diagnosing peri-implant disease using the tongue as a 24/7 detector,” Nature Communications, vol. 8, no. 1, pp. 264–272, 2017.
View at: Publisher Site | Google Scholar
M. T. Nieminen, D. Listyarifah, J. Hagström et al., “Treponema denticola chymotrypsin-like proteinase may contribute to orodigestive carcinogenesis through immunomodulation,” British Journal of Cancer, vol. 118, no. 3, pp. 428–434, 2017.
View at: Publisher Site | Google Scholar
P. Pärnänen, J. H. Meurman, and T. Sorsa, “The effects of Candida proteinases on human proMMP-9, TIMP-1 and TIMP-2,” Mycoses, vol. 54, no. 4, pp. 325–330, 2011.
View at: Publisher Site | Google Scholar
N. M. O’Brien-Simpson, K. Burgess, J. C. Lenzo, G. C. Brammar, I. B. Darby, and E. C. Reynolds, “Rapid chair-side test for detection of Porphyromonas gingivalis,” Journal of Dental Research, vol. 96, no. 6, pp. 618–625, 2017.
View at: Publisher Site | Google Scholar
J.-I. Kido, S. Murakami, M. Kitamura et al., “Useful immunochromatographic assay of calprotectin in gingival crevicular fluid for diagnosis of diseased sites in patients with periodontal diseases,” Journal of Periodontology, vol. 89, no. 1, pp. 67–75, 2017.
View at: Publisher Site | Google Scholar
T. Myntti, L. Rahkonen, I. Nupponen et al., “Amniotic fluid infection in preterm pregnancies with intact membranes,” Disease Markers, vol. 2017, Article ID 8167276, 9 pages, 2017.
View at: Publisher Site | Google Scholar
M. T. Nieminen, P. Vesterinen, T. Tervahartiala et al., “Practical implications of novel serum ELISA-assay for matrix metalloproteinase-8 in acute cardiac diagnostics,” Acute Cardiac Care, vol. 17, no. 3, pp. 46-47, 2015.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2018 Saeed Alassiri et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

2687

Downloads

1573

Citations