Angmar-Mansson

Bayram

Brookes

Hubbard

Murray

Scientific Advisory Board

Skobe

Smith

Tarasevich

Jennifer Kirkham

Steven J. Brookes

Thomas G. H. Diekwisch

Henry C. Margolis

Ariane Berdal

Michael J. Hubbard

Crossref

Frontiers in Physiology

Enamel Research: Priorities and Future Directions

Kirkham, J

Brookes, SJ

Diekwisch, TGH

Margolis, HC

Berdal, A

Hubbard, MJ

University of Melbourne Institutional Repository

Enamel research: Priorities and future directions

White Rose Research Online

OPINIONpublished: 20 July 2017doi: 10.3389/fphys.2017.00513Frontiers in Physiology | www.frontiersin.org 1 July 2017 | Volume 8 | Article 513Edited by:Agnes Bloch-Zupan,University of Strasbourg, FranceReviewed by:Michel Goldberg,Institut National de la Santé et de laRecherche Médicale, FranceJung-Wook Kim,Seoul National University, South KoreaJan Hu,University of Michigan, United StatesBernhard Ganss,University of Toronto, CanadaColin Robinson,University of Leeds, United Kingdom*Correspondence:Jennifer Kirkhamj.kirkham@leeds.ac.ukSpecialty section:This article was submitted toCraniofacial Biology and DentalResearch,a section of the journalFrontiers in PhysiologyReceived: 28 April 2017Accepted: 05 July 2017Published: 20 July 2017Citation:Kirkham J, Brookes SJ,Diekwisch TGH, Margolis HC,Berdal A and Hubbard MJ (2017)Enamel Research: Priorities andFuture Directions.Front. Physiol. 8:513.doi: 10.3389/fphys.2017.00513Enamel Research: Priorities andFuture DirectionsJennifer Kirkham 1*, Steven J. Brookes 1, Thomas G. H. Diekwisch 2, Henry C. Margolis 3,Ariane Berdal 4 and Michael J. Hubbard 51Division of Oral Biology, School of Dentistry, St James’s University Hospital, University of Leeds, Leeds, United Kingdom,2 Texas A&M University College of Dentistry, Dallas, TX, United States, 3Center for Biomineralization, The Forsyth Institute,Cambridge, MA, United States, 4Centre de Recherche des Cordeliers, INSERM UMRS 1138, University Paris-Diderot, Paris,France, 5Department of Paediatrics, University of Melbourne, Melbourne, VIC, AustraliaKeywords: enamel, research priorities, amelogenesis, odontogenesis, future directionsAt the close of the 2016 Enamel 9 International Symposium, the collective wisdom and views ofenamel researchers were sought and discussions held in order to provide a consensus view of thepriorities and future directions in enamel research. Researchers considered progress in the fieldsince the Enamel VIII conference held in 2011, together with the research strengths and strategicgaps in our current knowledge portfolio, synthesizing this information toward recommendationsfor future advancement. We aim to present these recommendations and identified priorities here,drawing upon the Closing Session discussions and those held throughout the Symposium aspresented in the Supplemental data.It is clear that significant advancements have been made in enamel research over the last 5 yearssince Enamel VIII. Despite these advances, it is equally clear that many issues highlighted thereas future priorities (Scientific Advisory Board, 2011) are still pressing. The greater our technicalcapabilities and knowledge base, the more questions and unknowns we uncover and the morewe realize what we do not know. More than 100 researchers from 28 countries attended Enamel9 and their reported findings provide indisputable evidence of the quality, breadth and vigor ofenamel research across the globe. Attendance at the Symposium by >30 early career researchersgives us confidence of the continuing vibrance and sustainability of the enamel field. In addition,it was particularly gratifying to see so many examples of interdisciplinary effort used to addressincreasing numbers of research questions in an integrated way, as was prioritized at Enamel VIII.Our first priority reflects the need to encourage and hasten the development of this valuable trend.PRIORITY 1—STRONGER OUTCOMES THROUGHINTERDISCIPLINARY, INTEGRATIVE AND TRANSLATIONALAPPROACHESFuture advancements will be made at the interfaces of disciplinary boundaries (Nature News, 2015)and we encourage such collaborations as the preferred models for future working.We propose positioning enamel within the context of the whole organism and not viewingthis tissue in isolation. Enamel is unique but the cells that are central to its formation respondto equivalent signals and insults and share common pathways with cells throughout the body.For example, we need to improve our understanding of the regulatory/signaling pathwaysthat contribute toward enamel development and how these align with, or differ from, thosein other systems. These are important issues in framing our future research questions as wemove toward translating findings from bench to chairside to population. We note the greatlyincreased participation by clinicians at Enamel 9 and recommend clinical involvement inframing translational research questions through identification of the clinical challenges and byKirkham et al. Enamel Research: Priorities and Future Directionscontextualization of existing research. This is particularlyimportant if we wish to remain responsive to advances and shiftsin the direction of clinical treatment (e.g., minimally interventivedentistry, biomimetics) and open up new frontiers such asregenerative therapies and tissue engineering.PRIORITY 2—BETTER INTERFACING OFBIOLOGY WITH PATHOLOGYResearch into the dysregulation of amelogenesis andbiomineralization defects has significantly expanded overthe past 5 years, adding vastly to classical understandingobtained from studies of normal tissue. Enamel VIII predictedthat the extraordinary pace of technical advancement in geneticswith concomitant decreases in costs for whole exome sequencingwould see all genes underlying amelogenesis imperfecta (AI)identified by Enamel 9. We now know that this is far fromreality, despite the fact that >18 genes have been identifiedwhere mutations underlie AI, with new variants—and newgenes—being reported regularly (Smith et al., 2017) Each findingbrings new pointers to direct our lines of enquiry in to thenormal mechanisms of enamel formation, the basis of its highlycomplex architecture and the pathogenesis of disease. There areclearly many more AI-associated genes to be discovered butwe face a significant lag in our understanding of pathologicalmechanisms and phenotyping of affected tissues.Increased awareness of the high prevalence of MolarHypomineralization (MH)1, (Hubbard et al., in press) highlightsboth a significant public health challenge and the needto better understand the pathology (and ultimately theprevention) of enamel opacities. It is likely that MH hasmulti-factorial causation, with environmental/lifestyle factorsplaying an important role but potentially with an underlyinggenetic component increasing individuals’ susceptibility. Givensuggestions that MH prevalence has increased in recent times,there may be parallels with other “lifestyle” related diseasesthat have seen greatly increased prevalence over relatively shorttimescales, such as cardiovascular disease and diabetes, whereprevalence patterns reflect both patients’ lifestyle and theirgenetic background. Individuals’ susceptibility to enamel carieshas already been linked to specific genetic polymorphisms(Bayram et al., 2015) and it seems likely that this paradigmcould also account for variable susceptibility to fluorosis.Improved understanding of the relationship between genes andenvironmental factors in the patterns of normal and abnormalenamel development should be a major priority.PRIORITY 3—BETTER STANDARDIZATIONOF EXPERIMENTAL VARIABLESAttendees identified the urgent need for standardization ofenamel phenotyping, both in AI and MH and also in animalmodels more generally. This includes the need to considereffects in different tooth types (molars as well as incisors) and1http://www.thed3group.org/prevalence.htmlin different tissues and compartments (including non-dental).The need to standardize phenotyping at each level, includingdetailed histological, ultrastructural, and molecular phenotypingin animal models and, wherever possible, of affected human teethis paramount.PRIORITY 4—BETTER USE OF ANIMALMODELSAnimal models have added greatly to our understanding ofthe fundamental biology, chemistry and physical properties ofenamel and will continue to do so but it is important that weunderstand the disadvantages and limitations of any models (andthis applies equally in vitro and in vivo) when using them to betterunderstand pathobiology and draw conclusions in comparisonswith human teeth. We recommend that a standardized (andupdatable) “check list” for phenotyping would greatly facilitateour combined understanding of the effects of gene mutationsand knock-outs and enable cross-comparisons to be made withwild-type animals and “equivalent” human teeth. It would alsomean that we could understand the limitations of our modelsand increase the rigor of our findings. For example, the recentreports of ER stress as a pathomechanism in AI and the viewof AI as a proteopathy (Brookes et al., 2014) signals that weshould be aware that confounded observations could arise frominadvertent stressing of the ameloblasts in in vivo models. Majordifferences in fluoride metabolism between rodents and humans(Angmar-Mansson and Whitford, 1984) is another importantexample.PRIORITY 5—BETTER COLLABORATIONFOR STRONGER COLLECTIVE VOICE ANDOUTPUTSCompared with many others areas of scientific endeavor, enamelresearchers are generally few and far between, making it difficultto be competitive at both topic and individual-performancelevels. For example, it is likely that the “low hanging fruit” ofgene discovery in AI has been picked by small groups in multiplecountries but we know that for many AI patients, no causativegene has yet been identified. At Enamel 9, the proposal wasmade to establish an enamel genetics research network to supporteffective international collaboration by sharing findings in acommunity of trust (“GEnamel”: “Genetics for understandingEnamel”). We hope that GEnamel will provide a paradigm forfurther inter-group collaborations and data sharing. In turn, itwas agreed that this network might usefully interface with atranslational research and education network recently establishedto promote issues associated with developmental dental defects(The D3 Group)2. Together with the potential for more frequentEnamel Symposia (see below), such combined initiatives wouldmove toward an identifiable international community witha strong collective voice without inhibiting the independent2http://www.thed3group.orgFrontiers in Physiology | www.frontiersin.org 2 July 2017 | Volume 8 | Article 513Kirkham et al. Enamel Research: Priorities and Future Directionsapproaches to enamel research that often generate new growthpoints.PRIORITY 6—HARNESSING NEWTECHNOLOGIESFurthering our understanding of pathology and the fundamentalprocesses of normal enamel development can be acceleratedby adopting new ways of looking at old problems. Recentadvances in computational evolutionary genetics and molecularpaleobiology bring new perspectives on the role of specificmolecules in enamel formation (Qu et al., 2015). Additionalmaterials science perspectives on enamel and its bio-inspiredanalogs would enormously benefit translational researchseeking to develop new biomimetic materials and regenerativeapproaches to dental diseases. The need for greater inclusionof materials scientists in enamel research was recognized atEnamel VIII but this remains a strategic gap that has yet to beclosed. Computer modeling of the complex data sets now beinggenerated across multiple physiological compartments is alsopoorly represented in the enamel field. There is an urgent needto pro-actively recruit scientists from these disciplines to join usin both framing and answering questions.New techniques in imaging and high resolution sampleinterrogation, such as focussed ion beam microscopy (Smithet al., 2016) and neutron reflectometry (Tarasevich et al.,2013) are yielding previously unobtainable insights, for examplein elucidating the earliest stages of amelogenesis, the elusive“mineralization front” and the physico-chemical nature ofthe earliest enamel minerals. We predict that improvedimaging techniques will help provide answers to outstandingquestions in respect of our understanding of the mineralizationfront and the complex four dimensional movements ofameloblasts in dictating enamel structure. However, we stilllack technical capability in tissue culture, notably threedimensional (3D) in vitro models and long term organoculturefor investigating the relationship between ameloblasts, theenamel matrix and the other constituents of the enamelorgan. We have long recognized that ameloblasts and thestratum intermedium form a functional syncytium (Skobe et al.,1995) and that studying ameloblasts in isolation seriouslycurtails both scope and significance of findings but a 3Dculture system eludes us still and remains a much neededpriority.PRIORITY 7—INCREASEDATTRACTIVENESS TO FUNDING BODIESFunding for enamel research remains a problem world-widedespite the fact that dental disease is a major public healthburden. Affecting about 3 billion people, untreated cariesin adults was the number one disease reported in the lastGlobal Burden of Disease study (Murray et al., 2012). Theinternational dearth of “programme grant” funding schemesthat support multi-disciplinary teams and enable the integratedscience approach we favor here (Priority 1) is a seriousproblem. We urge all colleagues to impress upon fundersthe advantages of such an approach. Increased attractivenessto funders should follow from improvements to our researchquality, quantity, utility and visibility (Priorities 1–6). Weacknowledge the continued support for the Enamel Symposiafrom NIH and the participation at Enamel 9 by Dr JasonWan (NICDR), illustrating a working relationship that weshould aspire to emulate with research funders across theglobe. The classical funding envelope might also be expandedthrough translational initiatives involving industry, professionalbodies and government. Enamel is the most highly mineralizedmammalian tissue and its exquisitely ordered structure confersuniquemechanical properties of significance to a broad audience,inside and outside of the health sciences. There is muchwe and others can learn from this tissue in the designof new materials and therapeutics. Opportunities thereforearise for generating research funding from our nationalresearch councils (e.g., for fundamental discovery, appliedand translational research), industry (e.g., oral healthcare,orthobiologics, pharmaceutical) and charitable foundations (e.g.,Wellcome Trust, Gates Foundation).PRIORITY 8—INCREASED AND MOREEFFECTIVE COMMUNICATIONGiven how rapidly recent findings have impacted our thinkingplus the ongoing emergence of new technologies, our finalrecommendation is that due consideration be given to holdingthe Enamel Symposia more frequently and/or that additionalshort, sharply focussed meetings be held alongside larger events(e.g., the IADR global congress). We also need to ask how wemight help researchers, especially those new to our field, accessrecommendations from previous Enamel Symposia and findkey work published between meetings. These challenges mightbe met through an easily accessible website, increasing ourcurrency and ensuring that past knowledge and thinking are notlost.In conclusion, the Enamel Symposia uniquely bring togetherresearchers from across the world, engendering a sense ofcommunity that fosters collaboration and exchange of ideas toaccelerate research. We look forward to the next Symposium,Enamel 10, where progress against these identified priorities—and advances currently unforeseen—will be considered.AUTHOR CONTRIBUTIONSJK drafted the paper; all co-authors added intellectual content.All authors agreed to the final manuscript.ACKNOWLEDGMENTSWe gratefully acknowledge the excellent work of Greg Baugh andJulie McDermott in transcribing the Q&A and Panel Discussionsand Claire Smith for her help in proof reading. We are indebtedto the Session Moderators at the Enamel 9 Symposium for theircontributions toward editing the transcripts, including OphirFrontiers in Physiology | www.frontiersin.org 3 July 2017 | Volume 8 | Article 513Kirkham et al. Enamel Research: Priorities and Future DirectionsKlein (Session1); Ariane Berdal (Session 2); Janet Moradian-Oldak (Session 3); Jim Simmer (Session 4); Michael Hubbard(Session 5); Agnès Bloch-Zupan (Session 6); John Bartlett(Session 7); Elia Beniash (Session 8); Henry Margolis (Session 9),and Steven Brookes (Session 10).SUPPLEMENTARY MATERIALThe Supplementary Material for this article can be foundonline at: http://journal.frontiersin.org/article/10.3389/fphys.2017.00513/full#supplementary-materialREFERENCESAngmar-Mansson, B., and Whitford, G. M. (1984). Enamel fluorosis relatedto plasma F levels in the rat. Caries Res. 18, 25–32. doi: 10.1159/000260743Bayram, M., Deeley, K., Reis, M. F., Trombetta, V. M., Ruff, T. D., Sencak, R. C.,et al. (2015). Genetic influences on dental enamel that impact caries differbetween the primary and permanent dentitions. Eur. Oral Sci. 123, 327–334.doi: 10.1111/eos.12204Brookes, S. J., Barron, M. J., Boot-Handford, R., Kirkham, J., and Dixon,M. J. (2014). Endoplasmic reticulum stress in amelogenesis imperfecta andphenotypic rescue using 4-phenylbutyrate. Hum. Mol. Genet. 23, 2468–2480.doi: 10.1093/hmg/ddt642Hubbard, M. J., Mangum, J. E., Perez, V. A., Nervo, G. J., and Hall, R. K. (in press).Molar hypomineralisation: a call to arms for enamel researchers. Front. Physiol.doi: 10.3389/fphys.2017.00546Murray, C. J., Vos, T., Lozano, R., Naghavi, M., Flaxman, A. D., Michaud,C., et al. (2012). Disability-adjusted life years (DALYs) for 291 diseases andinjuries in 21 regions, 1990–2010: a systematic analysis for the Global Burdenof Disease Study 2010. Lancet 380, 2197–2223. doi: 10.1016/S0140-6736(12)61689-4Nature News (2015). Why interdisciplinary research matters. Nature 525:305.doi: 10.1038/525305aQu, Q., Haitina, T., Zhu, M., and Ahlberg, P. E. (2015). New genomicand fossil data illuminate the origin of enamel. Nature 526, 108–111.doi: 10.1038/nature15259Scientific Advisory Board, M. (2011). Enamel biology and future researchdirections. Eur. J. Oral Sci. 119, 377–379. doi: 10.1111/j.1600-0722.2011.00934.x.Skobe, Z., Stern, D. N., and Prostak, K. S. (1995). “The cell biology of amelogenesis,”in Dental Enamel: Formation to Destruction, eds C. Robinson, J. Kirkham, andR. Shore (Boca Raton, FL: CRC Press), 23–57.Smith, C. E., Hu, Y., Hu, J. C., and Simmer, J. P. (2016). Ultrastructure of earlyamelogenesis in wild-type, Amelx−/−, and Enam−/− mice: enamel ribboninitiation on dentin mineral and ribbon orientation by ameloblasts.Mol. Genet.Genomic Med. 4, 662–683. doi: 10.1002/mgg3.253Smith, C. E. L., Poulter, J. A., Antanaviciute, A., Kirkham, J., Brookes, S. J.,Inglehearn, C. F., et al. (2017). Amelogenesis imperfecta; genes, proteins, andpathways. Front. Physiol. 8:435. doi: 10.3389/fphys.2017.00435Tarasevich, B. J., Perez-Salas, U., Masica, D. L., Philo, J., Kienzle, P., Krueger, S.,et al. (2013). Neutron reflectometry studies of the adsorbed structure of theamelogenin, LRAP. J. Phys. Chem. B 117, 3098–3109. doi: 10.1021/jp311936jConflict of Interest Statement: The authors declare that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.The reviewer CR declared a shared affiliation, though no other collaboration,with the authors JK and SB to the handling Editor, who ensured that the processmet the standards of a fair and objective review.Copyright © 2017 Kirkham, Brookes, Diekwisch, Margolis, Berdal and Hubbard.This is an open-access article distributed under the terms of the Creative CommonsAttribution License (CC BY). The use, distribution or reproduction in other forumsis permitted, provided the original author(s) or licensor are credited and that theoriginal publication in this journal is cited, in accordance with accepted academicpractice. No use, distribution or reproduction is permitted which does not complywith these terms.Frontiers in Physiology | www.frontiersin.org 4 July 2017 | Volume 8 | Article 513

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