Odontogenesis: Methods and Protocols

562 Pages·2019·18.866 MB·English

by  Petros Papagerakis

Methods in
Molecular Biology 1922
Petros Papagerakis Editor
Odonto-
genesis
Methods and Protocols
M M B
ETHODS IN OLECULAR IO LO GY
SeriesEditor
JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
Hatfield, Hertfordshire,AL109AB,UK
Forfurther volumes:
http://www.springer.com/series/7651
Odontogenesis
Methods and Protocols
Edited by
Petros Papagerakis
School of Dentistry, University of Michigan, Ann Arbor, MI, USA
College of Dentistry, University of Saskatchewan, Saskatoon, SK, Canada
Editor
PetrosPapagerakis
SchoolofDentistry
UniversityofMichigan
AnnArbor,MI,USA
CollegeofDentistry
UniversityofSaskatchewan
Saskatoon,SK,Canada
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-4939-9011-5 ISBN978-1-4939-9012-2 (eBook)
https://doi.org/10.1007/978-1-4939-9012-2
LibraryofCongressControlNumber:2019930434
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Preface
Odontogenesis is the complex process by which embryonic cells differentiate into oral
epithelia-derived ameloblasts that secrete enamel and cranial neural crest‐derived
mesenchyme, which form odontoblasts that produce dentin and cementoblasts that make
cementum.Differentapproachesareappliedtostudygeneticandenvironmentalregulatory
controls on odontogenesis that can have dramatic influences on dental phenotypes and
genotypes observed during normaldevelopment and diseases. Thedifferent methods used
haveadvantagesandlimitations,andthisbookaimstoserveasaguidetofuturegeneration
of researchers working in the exciting field of odontogenesis. This book is divided into
6partsandcontainsatotalof41chapters.
PartIisfocusedontheestablishmentofdentalcelllinesandanimalmodelsthatserveas
tools to understand the genetic and environmental controls of tooth development. Using
these models, researchers can further enhance our understanding on how signaling mole-
culescontrolallstepsoftoothformationbycoordinatingcellproliferation,differentiation,
apoptosis, extracellular matrix synthesis, and mineral deposition. Chapters in this part
include protocols for isolation and characterization of both epithelial and mesenchymal
dental cells, establishment of stable cell lines, as well as in vivo cell lineage tracing and
classicaltissuerecombinationassaysusingthekidneycapsulemodel.
PartIIisfocusedondentalstemcellsanddentaltissueregeneration.Toothloss,caused
by dental diseases, trauma, or aging, is usually replaced by artificial materials which lack
many of the important biological characteristics of the natural tooth. Understanding the
mechanismsofstemcelldifferentiationtowarddentalphenotypesmayprovidethenecessary
foundation that will lead to novel approaches for dental tissue regeneration and stem cell
therapies in the future. Methods described in this part will help researchers to further
elucidate the complex interactions and necessary conditions driving dental cell differentia-
tion.Chaptersinthispartdetailmethodsonisolation,phenotypiccharacterization,expan-
sion,anddifferentiationprotocolsfordentalstemcellsaswellasnovelapproachesfortissue
regeneration suchas the useof multiwalled carbonnanotubes, peptides, or GelMAhydro-
gels.Italsocontainsaprotocoltostudyreparativedentinogenesisinvivo.
Part III is centered around methods to characterize gene and protein expression in
dentalcellsandtissues.Newgenesandtheirfunctionsarecontinuouslybeingdiscoveredin
experimental studies using cell lines and animal models. This part provides the necessary
knowledgeforsuccessfullymappingRNAandproteinexpressionindentaltissues.Detailed
protocolsonimmunofluorescence,insituhybridization,immunohistochemistry(including
co-localization), and the use of LNA probes for detection of low amounts of RNA are
provided.Protocolsforsilver-albumintissuestainingandisolationofsiblingproteinsfrom
boneanddentincompletethispart.
PartIVcontainsbiochemistryandimagingprotocolsthatareessentialforcharacterizing
dentalhardtissues.Thesemethods,rangingfromelectronmicroscopytomicro-CTaidedby
artificial intelligence, are critical for understanding gene function in transgenic and knock-
outmicemodelsthatmayresultinarrestedtoothdevelopmentand/orabnormalextracel-
lular matrix formation, maturation, and mineralization. Furthermore, protocols for
extraction and biochemical characterization of matrix proteins from enamel and dentin as
wellasprotocolsforexpressionandpurificationofrecombinantproteinsarealsoprovided.
v
vi Preface
Part V describes dental disease models focusing mainly on protocols to study dental
caries. Dental caries still cause a huge public health burden, and the in vitro and in vivo
modelsincludedheremayhelpindevelopingnewapproachesforprevention,diagnosis,and
treatmentofdentalcaries.Additionalchaptersincludeprotocolsofarodentdentalfluorosis
model and a method for 3D assessment of crown size and eruption space for third molars
allowingtostudytheeffectsoffluorideandthirdmolarimpaction,bothcommonlyseenin
humans.
Part VI overviews protocols on genetics, epigenetics, and clinical studies to provide
foundation for clinical research in dentistry. Whole-genome linkage analysis, association
analysis of putative candidate genes, and whole-genome association approaches now offer
exciting opportunities to discover new key genes involved in human dental development.
Thispartcontainsprotocolsfornext-generationsequencing,geneticandepigeneticstudies,
and genome-wide association studies as well as clinical protocols for measurement of early
childhood caries and saliva and supragingival fluids and biofilm collection and subsequent
analyses.
Written in the highly successful Methods in Molecular Biology™ series format, chapters
includeintroductionstotheirrespectivetopics;listsofthenecessarymaterialsandreagents;
step-by-step, readily reproducible laboratory protocols; and tips for troubleshooting and
avoiding expected pitfalls. Practical and easy-to-use Odontogenesis: Methods and Protocols
aims to guide researchers toward elucidating the secrets and mysteries of a fascinating and
uniqueorgan,thetooth!
Iamverygratefultoallparticipantsandtheircontributionstothisvolume.Weallhope
this book will serve future generations of researchers in the field of odontogenesis in their
pathwaystoexcitingdiscoveries.
AnnArbor,MI,USA PetrosPapagerakis
Contents
Preface ..................................................................... v
Contributors................................................................. xi
PART I PROTOCOLS FOR ESTABLISHMENT OF DENTAL CELLS LINES
AND ANIMAL MODELS
1 MicrodissectionandIsolationofMouseDentalEpithelialCells
ofContinuouslyGrowingMouseIncisors.................................. 3
HidemitsuHaradaandKeishiOtsu
2 EstablishmentofanImmortalizedMouseBmp2KnockoutDental
PapillaMesenchymalCellLine ........................................... 13
Wen’anXuandShuoChen
3 EstablishmentofStableCellLinesfromPrimaryHumanDentalPulp
StemCells ............................................................. 21
ElizabethGuirado,YoubinZhang,andAnneGeorge
4 IsolationofDentalStemCell-EnrichedPopulationsfromContinuously
GrowingMouseIncisors................................................. 29
AnamariaBalic
5 ApplicationofCellLineageTracingCombinedwithImmunofluorescence
intheStudyofDentinogenesis ........................................... 39
YanJing,ChaoyuanLi,andJianQ.Feng
6 TissueRecombinationandKidneyCapsuleTransplantationAssays
for theStudyofEpithelial-MesenchymalInteractions ....................... 49
LuciaJimenez-RojoandThimiosA.Mitsiadis
PART II PROTOCOLS FOR DENTAL STEM CELLS AND TISSUE REGENERATION
7 DentalMesenchymalStemCells:DentalPulpStemCells,Periodontal
LigamentStemCells,ApicalPapillaStemCells,andPrimaryTeeth
StemCells—Isolation,Characterization,andExpansionforTissue
Engineering............................................................ 59
MeyAl-HabibandGeorgeT.-J.Huang
8 PhenotypicIdentificationofDentalPulpMesenchymalStem/Stromal
CellsSubpopulationswithMultiparametricFlowCytometry ................. 77
MaximeDucret,Jean-ChristopheFarges,MariellePasdeloup,
EmelinePerrier-Groult,AndreasMueller,Fre´de´ricMallein-Gerin,
andHugoFabre
9 DentalPulpStemCells:Isolation,Characterization,Expansion,
andOdontoblastDifferentiationforTissueEngineering..................... 91
QingDong,YuanyuanWang,FatemehMohabatpour,LiZheng,
SilvanaPapagerakis,DanielChen,andPetrosPapagerakis
vii
viii Contents
10 InVitroAnalysisofIntramolecularSignalingEventsinPDLSCs
UsingConfocalandTIRFMicroscopy .................................... 103
AnnetteMerkelandAnneGeorge
11 AMouseModeltoStudyReparativeDentinogenesis........................ 111
R.C.Babb,D.Chandrasekaran,L.K.Zaugg,andP.T.Sharpe
12 MultiwalledCarbonNanotubesforDentalApplications..................... 121
PetrosKechagioglou,EleftheriosAndriotis,PetrosPapagerakis,
andSilvanaPapagerakis
13 Peptide-MediatedBiomimeticRegrowthofHumanEnamelInSitu .......... 129
KaushikMukherjee,QichaoRuan,andJanetMoradian-Oldak
14 BioengineeringToothBudConstructsUsingGelMAHydrogel.............. 139
ElizabethE.SmithandPamelaC.Yelick
15 Whole-MountInSituHybridizationofMouseEmbryos
UsingDIG-LabeledRNAProbes......................................... 151
JingyiWuandXiaofangWang
PART III PROTOCOLS FOR STUDYING GENE AND PROTEIN EXPRESSION
16 InSituHybridizationonMouseParaffinSectionsUsingDIG-Labeled
RNAProbes ........................................................... 163
JingyiWu,JianQ.Feng,andXiaofangWang
17 MethodsforInSituProteinVisualizationinDentalMineralizedTissues ...... 173
D.Hotton,A.Berdal,andA.Bolan˜os
18 InSituHybridizationinMineralizedTissues:TheAddedValue
ofLNAProbesforRNADetection ....................................... 181
G.Lignon,D.Hotton,A.Berdal,andA.Bolan˜os
19 ImmunofluorescenceProcedureforDevelopingEnamelTissues.............. 191
XuYangandEliaBeniash
20 Silver-AlbuminTissueStainingProtocoltoVisualizeOdontogenesis
inWholeEmbryos...................................................... 197
JuliaC.BoughnerandDavidM.L.Cooper
21 IsolationofSIBLINGProteinsfromBoneandDentinMatrices.............. 211
JingyiWuandXiaofangWang
22 ImmunohistochemicalCo-LocalizationofAmelogeninandAmeloblastin
inDevelopingEnamelMatrix ............................................ 219
RuchaArunBapatandJanetMoradian-Oldak
23 TheExpressionandPurificationofRecombinantMouseAmeloblastin
inE.coli............................................................... 229
JingtanSu,RuchaArunBapat,andJanetMoradian-Oldak
PART IV PROTOCOLS FOR BIOCHEMISTRYAND IMAGING
24 ProtocolsforStudyingFormationandMineralization
ofDentalTissuesInVivo:ExtractionProtocolforIsolatingDentin
MatrixProteinsfromDevelopingTeeth ................................... 239
YasuoYamakoshi,JanC.-C.Hu,MariM.Saito,andJamesP.Simmer
Contents ix
25 PurificationofDevelopingEnamelMatrixProteinsUsingPreparative
SDS-PAGE............................................................. 251
StevenJ.BrookesandClaireM.Gabe
26 UsingImageJ(FIJI)toAnalyzeandPresentX-RayCTImagesofEnamel..... 267
StevenJ.Brookes
27 ScanningElectronMicroscopy(SEM)MethodsforDentalEnamel........... 293
SteinarRisnes,MuhammadSaeed,andAmerSehic
28 MicrocomputedTomographyImaginginOdontogenesisStudies............. 309
KostasVerdelisandPhilSalmon
29 TransmissionElectronMicroscopy(TEM)andScanningElectron
Microscopy(SEM)for theExaminationofDentalHardTissues.............. 325
VictorE.Arana-ChavezandLeticiaS.Castro-Filice
PART V PROTOCOLS TO STUDY DENTAL DISEASES
30 RodentDentalFluorosisModel:ExtractionofEnamelOrgan
fromRatIncisors ....................................................... 335
M.SuzukiandJ.D.Bartlett
31 Three-DimensionalAssessmentofCrownSizeandEruptionSpace
forDevelopingThirdMolars:DataCollectionTechniquesBased
onCone-BeamComputedTomography(CBCT)........................... 341
D.F.Marchiori,G.V.Packota,andJ.C.Boughner
32 ProtocolstoStudyDentalCariesInVitro:MicrobialCariesModels .......... 357
BennettT.Amaechi,LiviaM.A.Tenuta,AntonioP.RicominiFilho,
andJaimeA.Cury
33 InVitroCariesModelsfor theAssessmentofNovelRestorativeMaterials ..... 369
BasmaSulaimanGhandourah,AnnaLefkelidou,RaedSaid,
XanthippiChatzistavrou, SusanFlannagan,CarlosGonza´les-Cabezas,
ChristopherJ.Fenno,LiZheng,SilvanaPapagerakis,
andPetrosPapagerakis
34 ProtocolstoStudyDentalCariesInVitro:pHCyclingModels............... 379
BennettT.Amaechi
35 InVivoRodentModelsforStudyingDentalCariesandPulpDisease ......... 393
JuneHsiao,YuanyuanWang,LiZheng,RuiruiLiu,RaedSaid,
LubomirHadjiyski,HeekonCha,TatianaBotero,XanthippiChatzistavrou,
QingDong,SilvanaPapagerakis,andPetrosPapagerakis
PART VI PROTOCOLS FOR GENETIC, EPIGENETIC AND CLINICAL STUDIES
36 ProtocolGenoDENT:ImplementationofaNewNGSPanel
forMolecularDiagnosisofGeneticDisorderswithOrodental
Involvement............................................................ 407
TristanRey,JulienTarabeux,Be´ne´dicteGerard,MarionDelbarre,
AntonyLeBe´chec,CorinneStoetzel,MeganaPrasad,
VirginieLaugel-Haushalter,MarzenaKawczynski,JeanMuller,
JamelChelly,He´le`neDollfus,Marie-Ce´cileManie`re,
andAgne`sBloch-Zupan
x Contents
37 ProtocolsforGeneticandEpigeneticStudiesofRareDiseases
AffectingDentalTissues................................................. 453
BrunaRabeloAmorim,PollyannaAlmeidaCostadosSantos,
CarolineLourenc¸odeLima,DeniseCarletoAndia,
JulianaForteMazzeu,andAnaCarolinaAcevedo
38 Protocols,Methods,andToolsforGenome-WideAssociation
Studies(GWAS)ofDentalTraits.......................................... 493
CaryS.Agler,DmitryShungin,AndreaG.FerreiraZandona´,
PaigeSchmadeke,PatriciaV.Basta,JasonLuo,JohnCantrell,
ThomasD.PahelJr.,BeauD.Meyer,JohnR.Shaffer,ArneS.Schaefer,
KariE.North,andKimonDivaris
39 MeasurementofEarlyChildhoodOralHealthforResearchPurposes:
DentalCariesExperienceandDevelopmentalDefectsoftheEnamel
inthePrimaryDentition................................................. 511
JeannieGinnis,AndreaG.FerreiraZandona´,GaryD.Slade,
JohnCantrell,MikafuiE.Antonio,BhavnaT.Pahel,BeauD.Meyer,
PoojanShrestha,MiguelA.Simancas-Pallares, AshwiniR.Joshi,
andKimonDivaris
40 TheSupragingivalBiofilminEarlyChildhoodCaries:Clinical
andLaboratoryProtocolsandBioinformaticsPipelinesSupporting
Metagenomics,Metatranscriptomics,andMetabolomicsStudies
oftheOralMicrobiome ................................................. 525
KimonDivaris,DmitryShungin,AdarisRodrı´guez-Corte´s,
PatriciaV.Basta,JeffRoach,HunyongCho,DiWu,
AndreaG.FerreiraZandona´,JeannieGinnis,SivapriyaRamamoorthy,
JasonM.Kinchen,JakubKwintkiewicz,NatashaButz,ApoenaA.Ribeiro,
andM.AndreaAzcarate-Peril
41 SalivaandGingivalCrevicularFluid(GCF)Collection
forBiomarkerScreening................................................. 549
PetrosPapagerakis,LiZheng,DoohakKim,RaedSaid,
AmberA.Ehlert,KevinK.M.Chung,andSilvanaPapagerakis
Index ...................................................................... 563