Table Of ContentErmannoBonucci
Biological Calcification
Ermanno Bonucci
Biological Calcification
Normal and Pathological Processes
in the Early Stages
With48Figures
123
ProfessorDr.ErmannoBonucci
DipartimentodiMedicinaSperimentaleePatologia
UniversitàLaSapienza–PoliclinicoUmbertoI
VialeReginaElena,324
00161Roma
Italia
e-mail:[email protected]
LibraryofCongressControlNumber:2006928438
ISBN-103-540-36012-3Springer-VerlagBerlinHeidelbergNewYork
ISBN-13978-3-540-36012-4Springer-VerlagBerlinHeidelbergNewYork
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(GottholdEphraimLessing,EineDuplik,VIII,26-27,1778)
IfGodclaspedalltruthsinhisrighthand,andinhisleftheldonlytheever-
livingquestfortruth–thoughIaddthatIwillalways,eternally,err–andif
hesaidtome:“Chose”,thenIwouldhumblygrasphislefthandandanswer:
“Father,givemethis!Thepuretruthisreservedforyoualone”.
(translatedbyAnthonyJohnson,
DepartmentofEnglishStudies,UniversityofPisa)
Preface
This book follows a precursor volume devoted to biological calcification, is-
sued by the CRC Press, Boca Raton (Florida) in 1992. Several basic aspects
of the calcification process were analyzed in it by outstanding authors who
hadunquestionedcompetenceintheirrespectiveresearchareas.Itsmainaim
was that of giving readers access to a series of papers which, even though
they discussed divergent aspects of biological calcifications drawn from the
study of systems as different as vertebrate skeletons and mollusks, in vitro
cultures and unicellular organisms, ectopic calcification and urinary stones,
provided elements permitting a coherent approach to a comprehensive view
ofthecalcificationprocessinbiologicaltissues.
Now, almost 15 years after the publication of that book, a great variety
of new data from a wide spectrum of biological organisms and systems has
enriched our knowledge of the normal and pathological mechanisms which
canleadtocalcification.Evenso,thiswholeprocessisstillproblematic:thenew
knowledge,conceptsandideashaveoftensuggestedthatadefinitivesolution
was close at hand, but the local mechanism through which the inorganic
substanceislaiddowninorganicmatricescontinuestobeanelusive,largely
enigmatictopic.
Thistantalizingsituationiscertainlygroundedintheintrinsicdifficulties
of the problem, but persisting doubts appear to derive from at least two ex-
trinsic issues: first, the topics falling under the general heading ‘Biological
Calcifications’areexceedinglynumerousandvarious;second,theyareneces-
sarilyfacedbyinvestigatorswhopossessdistinctkindsofspecializationand
competence, and apply different, often highly sophisticatedmethodsto very
dissimilarcalcifiedtissues.Thispotentiallyproductivesituationispartlyun-
derminedbythefactthattheresultsproducedbysomanystudiesarescattered
throughadauntingnumberofpublicationswhicharequiteoftenrepetitive,or
expressconflictingstandpoints,mayonlybeavailabletoarestrictednumber
of those who are interested, are often written in technical language that is
extremelyspecialized,andlessthantransparenttoallreaders,andtendtoset
uppalisadesroundspecificscientificdomains.
Thisvolumeaimstoofferacriticalrevisionoftheoldandrecentliterature
onthevariousaspectsofthecalcificationprocess,withspecialreferencetoits
earliest phases as seen in different tissues and organisms. Its scope extends
beyondanupdatingofpreviouslyavailabledata;itschaptershavetheoverall
purpose of collectingresults from as many different sources as possible and
VIII Preface
attempting, on the basis of personal experience, to achieve integration in
definitiveconclusionsthatinallcasesaimtomakethewholerangeofresults
cohereintoacomprehensivetheoryofcalcificationmechanismsinbiological
systems.Agreatdealofefforthasbeenputintothetaskofselecting–while
avoiding arbitrary or subjective exclusions – the results that have withstood
criticalattitudesandthetestofexperience,thatarecommontodifferenttissues
and/or organisms, and/or that appear to be typical of one or many of them,
andtoreportthemassimplyaspossiblesothattheybecomefullyintelligible
toallreaders.
Toensureaunifiedtreatmentofthevarioustopicsasfarasthisisattainable,
Ihavechosentobetheonlyauthorofthebook,inspiteoftheriskofappearing
presumptuousandthelikelihoodofincurringblameforinadvertentomissions
andmistakes.Thatchoicehasthejustification,broughtbypersonaleditorial
experience,thatanundulylargenumberofauthorswouldotherwisehavebeen
needed to cover all the topics to be included, and that the final result would
probablyhavebeenanoversizedvolume(ifnotseveralvolumes)comprising
amultiplicityofcomplete,exhaustivechaptersbutinevitablylackingasingle
focusorconclusion,whichare,conversely,theoverridingaimofthisbook.
Asthiswriterhasbeeninvolvedforover40yearsinstudiesonthebiological
calcificationprocess,mostlycarriedoutusingmorphologicalmethods,acer-
tainprevalenceofmorphologicaldatacouldhavefounditswayintodiscussions
on the various aspects of the problem. Moreover, such a long application to
thesametopichasmeantthatsomeideashavealreadytakenshape,especially
thoseaboutthenatureoftheearliestinorganicparticles,theorganic-inorganic
relationshipsthatunderliecrystaldevelopment,andthepossibilitythatbiolog-
icalcalcificationsoccurthroughthesamebasicmechanism(s)inallorganisms.
Thesepersonalideashave,however,notbeenallowedundueprominence;the
theories,proposals,opinions,suggestionsandconclusionscurrentlyavailable
intheliteraturehavebeenviewedasimpersonallyaspossible.Forthisreason,
alargenumberofreferencesappearattheendofeachchapter.Tosomeread-
erstheymayappearredundant,buttheydoofferthoseinterested–especially
younginvestigators–thechancetopersonallyverifythepointsmadeinthis
bookandbuildupanindependentview.Inthiscontext,oldreferenceshavenot
beenomitted.Besidestheirhistoricalvalue,theyallowthehard-wonprogress
ofscientificknowledgetobesurveyed,andforestalltherisk–lessrare than
isusuallythought–ofreplicatingresearchthathasalreadybeencarriedout.
In line with these concepts, the concluding remarks at the end of chapters
havethefunctionoffocusingthereader’sattentiononasetofmaintopicsand
problems,notofadvancingpreconstitutedsolutionsorpersonalopinions.
Inspiteofhiswishtobeinclusive,theauthoriswellawarethatomissions
and mistakes are a constant hazard; in this sense, I apologize in advance.
Thisbookhasbeenwrittenintheconvictionthatallresults–eventhosethat
currentlyappeartobeobsolete,incongruousorinconsistent–mayplayapart
inthesearchfortruth,providedtheyarerootedinseriousstudiesandopen
Preface IX
minds. Perfection is hard, if not impossible, to attain and – at least in the
biologicalfield–whatappearstodaytobeanincontrovertibletruthmayturn
outtomorrowtobeamistake.Readerswhosharethisconceptwillperhapsbe
kindenoughtoallowforoccasionalimperfections.
This book could not have been produced without the help of a number
of people, too many to be listed one by one. Yet the author wishes to recall
with gratitude the memory of Antonio Ascenzi and Vittorio Marinozzi, the
two scientists who introduced him to the study of normal and pathological
calcifiedtissues.Inaddition,heissoindebtedtohisdirectco-workersfortheir
continuous scientific advice and support and for their technical assistance
that he cannot avoid mentioning them and expressing his warmest thanks
to Paola Ballanti, Silvia Berni, Carlo Della Rocca, Renato Di Grezia, Martina
Leopizi,PatriziaMocetti,GiulianaSilvestriniandLucioVirgilii.Theinvaluable
assistanceofAnthonyJohnson,DepartmentofEnglishStudies,Universityof
Pisa,inimprovinglanguagestyleandcorrectinggrammarmistakes,andthe
friendlysuggestionsofDrMarianninaFailla,aregratefullyacknowledged.The
electronicfacilitiesofthelibraryofthe“LaSapienza”University,Rome,have
greatly facilitated the writing of this book. The more personal studies have
beensupportedbygrantsfromthe“LaSapienza”University,Rome,theItalian
NationalResearchCouncilandtheItalianMinistryofEducation.Theconstant
supportoftheauthor’swife,AnnadeMatteis,andtheencouragementofhis
children Francesca and Alessandro, have been indispensable in overcoming
severaldifficultmomentsalongtheroad.
Rome,Spring2006 ErmannoBonucci
Contents
1 Introduction 1
References.................................................................................. 2
2 HistoricalNotes 5
2.1 Introduction....................................................................... 5
2.2 Before1960........................................................................ 7
2.3 After1960.......................................................................... 11
2.4 CalcificationorMineralization?............................................. 13
References.................................................................................. 13
3 Methodology 23
3.1 Introduction....................................................................... 23
3.2 BiophysicalMethods............................................................ 23
3.2.1 X-rayDiffraction...................................................... 24
3.2.2 NeutronDiffraction.................................................. 27
3.2.3 Selected-areaElectronDiffraction(SAED)................... 27
3.2.4 EnergyDispersiveX-rayElementalAnalysis(EDX)....... 28
3.2.5 NuclearMagneticResonance(NMR)Spectroscopy....... 28
3.2.6 ProtonNuclearMagneticResonanceMicroscopy.......... 29
3.2.7 ElectronSpinResonance(ESR).................................. 30
3.2.8 EnergyFilteringElectronMicroscopy(EFEM).............. 30
3.2.9 InfraredSpectroscopy(IRS)....................................... 31
3.2.10 RamanMicrospectroscopy........................................ 31
3.2.11 AtomicForceMicroscopy(AFM)................................ 32
3.2.12 OtherBiophysicalTechniques.................................... 33
3.3 BiochemicalMethods .......................................................... 33
3.3.1 Electrophoresis........................................................ 33
3.3.2 Chromatography...................................................... 34
3.3.3 Enzyme-linkedAssay................................................ 36
3.3.4 DensityGradientFractionation.................................. 36
3.3.5 SequentialDissociativeExtraction.............................. 38
3.3.6 InVitroSystems....................................................... 38
3.4 MorphologicalMethods....................................................... 39
3.4.1 PreparativeProcedures............................................. 40
3.4.2 Fixation.................................................................. 40
XII Contents
3.4.3 Embedding ............................................................. 42
3.4.4 Decalcification......................................................... 43
3.4.5 HistochemistryandImmunohistochemistry................ 49
3.5 ConcludingRemarks........................................................... 50
References.................................................................................. 51
4 TheNatureandCompositionoftheInorganicPhase 67
4.1 Introduction....................................................................... 67
4.2 Vertebrates......................................................................... 68
4.2.1 Bone ...................................................................... 68
4.2.2 Cartilage,Dentin,Cementum,Tendon ........................ 73
4.2.3 Enamel................................................................... 74
4.3 LowerVertebrates ............................................................... 77
4.3.1 Enameloid .............................................................. 78
4.3.2 Isopedine................................................................ 79
4.4 Invertebrates...................................................................... 79
4.5 UnicellularOrganisms......................................................... 81
4.6 PathologicalCalcifications.................................................... 83
4.6.1 Mitochondria.......................................................... 84
4.6.2 VascularCalcification............................................... 85
4.7 CongenitalorAcquiredDiseases;
GeneticallyEngineeredAnimals............................................ 86
4.8 ConcludingRemarks........................................................... 88
References.................................................................................. 89
5 TheShapeofInorganicParticles 105
5.1 Introduction.......................................................................105
5.2 Vertebrates.........................................................................105
5.2.1 Bone ......................................................................105
5.2.2 Tendons..................................................................116
5.2.3 Dentin....................................................................118
5.2.4 Cementum..............................................................118
5.2.5 Cartilage.................................................................119
5.2.6 Enamel...................................................................120
5.3 LowerVertebrates ...............................................................121
5.3.1 Enameloid ..............................................................121
5.3.2 Isopedine................................................................122
5.4 Invertebrates......................................................................122
5.5 UnicellularOrganisms.........................................................125
5.6 PathologicalCalcifications....................................................126
5.6.1 CalcifiedMitochondria.............................................127
5.6.2 VascularCalcification...............................................127
5.7 CongenitalorAcquiredDiseases;
GeneticallyEngineeredAnimals............................................128
Contents XIII
5.8 ConcludingRemarks...........................................................129
References..................................................................................130
6 TheSizeofInorganicParticles 145
6.1 Introduction.......................................................................145
6.2 Vertebrates.........................................................................146
6.2.1 Bone ......................................................................146
6.2.2 Tendon...................................................................149
6.2.3 Dentin....................................................................150
6.2.4 Cementum..............................................................151
6.2.5 EpiphysealCartilage.................................................151
6.2.6 Enamel...................................................................152
6.3 LowerVertebrates ...............................................................155
6.4 Invertebrates......................................................................155
6.5 UnicellularOrganisms.........................................................157
6.6 PathologicalCalcification.....................................................157
6.7 CongenitalorAcquiredDiseases;GeneticallyModifiedAnimals 158
6.8 ConcludingRemarks...........................................................159
References..................................................................................160
7 CalcifyingMatrices:BoneandTendons 167
7.1 Introduction.......................................................................167
7.2 TheOrganicMatrixofBone:Collagen....................................168
7.3 TheOrganicMatrixofBone:Non-collagenousComponents......175
7.3.1 Proteoglycans..........................................................176
7.3.2 Gla-proteins............................................................180
7.3.3 Glycoproteins(Phosphoproteins)...............................184
7.3.4 Lipids.....................................................................197
7.3.5 MatrixVesicles ........................................................199
7.4 TheOrganicMatrixofTendons.............................................200
7.5 ConcludingRemarks...........................................................202
References..................................................................................204
8 CalcifyingMatrices:DentinandCementum 231
8.1 Introduction.......................................................................231
8.2 CollagenofDentin..............................................................231
8.3 DentinNon-collagenousProteins:Proteoglycans.....................233
8.4 DentinNon-collagenousProteins:Gla-proteins.......................236
8.4.1 Osteocalcin.............................................................236
8.4.2 DentinMatrixGla-protein.........................................237
8.5 DentinNon-collagenousProteins:Phosphoproteins.................237
8.5.1 Phosphophoryn.......................................................238
8.5.2 Osteonectin.............................................................240
8.5.3 Osteopontin............................................................240
