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Aluminum and bone: Review of new clinical circumstances associated with Al(3+) deposition in the calcified matrix of bone

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GENERAL REVIEW

Aluminum and bone: Review of new clinical circumstances associated with Al 3+

deposition in the calcified matrix of bone

Aluminium et os : revue de nouvelles circonstances cliniques Al

3+

peut être localisé dans la matrice osseuse

D. Chappard

a,b,∗

, P. Bizot

c

, G. Mabilleau

a,b

, L. Hubert

c

aGroupeétudesremodelageosseuxetbiomatériaux(GEROM),LHEA,IRIS-IBSinstitutdebiologieensanté, CHUd’Angers,LUNAMuniversitéNantesAngersLeMans,49933Angerscedex,France

bServicecommund’imagerieetanalysesmicroscopiques(SCIAM),IRIS-IBSinstitutdebiologieensanté, CHUd’Angers,LUNAMuniversité,49933Angerscedex,France

cDépartementdechirurgieosseuse,CHUd’Angers,49933Angerscedex,France

KEYWORDS Aluminum;

Bone;

Mineralization;

Biomaterial;

Titanium;

TA6V;

Metaloxidation;

Exostosis;

Metaltoxicity

Summary Severaldecadesago,aluminumencephalopathyassociatedwithosteomalaciahas beenrecognizedasthemajorcomplicationofchronicrenalfailureindialyzedpatients.Removal ofaluminumfromthedialysatehasledtoadisappearanceofthedisease.However,aluminum depositoccursinthehydroxyapatiteofthebonematrixinsomeclinicalcircumstancesthat are presented inthis review. We have encounteredaluminum in bone inpatients with an increasedintestinalpermeability(coeliacdisease),orinthecaseofprolongedadministration ofaluminumanti-aciddrugs.Acolocalisationofaluminumwithironwasalsonotedincasesof hemochromatosisandsicklecellanemia.Aluminiumwasalsoidentifiedinaseriesofpatients withexostosis,afrequent benignbonetumor.Corrosionofprostheticimplantscomposedof gradeVtitanium(TA6Visanalloycontaining6%aluminumand4%vanadium)wasalsoobserved inaseriesofhiporkneerevisions.Aluminumcanbeidentifiedinundecalcifiedbonematrix stainedbysolochromeazurine,ahighlyspecificstainallowingthedetectionof0.03atomic%.

Colocalizationofaluminumandirondoesnotseemtobethefruitofchancebutthecellularand molecularmechanismsarestillpoorlyunderstood.Histochemistryissuperiortospectroscopic analyses(EDSandWDSinscanningelectronmicroscopy).

©2015ElsevierMassonSAS.Allrightsreserved.

Correspondingauthor.Groupeétudesremodelageosseuxetbiomatériaux(GEROM),LHEA,IRIS-IBSinstitutdebiologieensanté,CHU d’Angers,LUNAMuniversitéNantesAngersLeMans,49933Angerscedex,France.

E-mailaddress:daniel.chappard@univ-angers.fr(D.Chappard).

http://dx.doi.org/10.1016/j.morpho.2015.12.001 1286-0115/©2015ElsevierMassonSAS.Allrightsreserved.

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MOTSCLÉS Aluminium; Tissuosseux; Minéralisation; Biomatériaux; Titane; TA6V; Oxydation métallique; Exostose;

Toxicitédesmétaux

Résumé L’encéphalopathie aluminiqueassociée àuneostéomalacie a étéreconnueil y a plusieursdécenniescommeunecomplicationmajeuredel’insuffisancerénalechroniquechez lespatientsdialysés.L’éliminationdel’aluminiumdesbainsdedialyseaconduitàladisparition decettemaladie.Cependant,desdépôtsd’aluminiumpeuventsurvenirdansl’hydroxyapatite de la matrice osseuseau cours denouvelles circonstancescliniques qui sontdécrites dans cette revue.Nous avonsobservéde l’aluminiumdansl’os depatients quiprésentaientune perméabilitéintestinaleaccrue(maladiecœliaque)ouencasd’administration prolongéede médicamentsanti-acidescontenantdel’aluminium.Unecolocalisationdel’aluminiumetdu ferestaussirencontréedanslescasd’hémochromatoseetdedrépanocytose.Lacorrosiondes implantsprothétiquesorthopédiquescomposésdetitanedegradeV(TA6V—unalliageconte- nant6%d’aluminiumet4%devanadium),aaussiétéobservéedansunesériedeprothèsesde hancheoudegenouaprèsrévisionprothétique.L’aluminiumaaussiétéidentifiédansunesérie depatientsjeunesavecexostose,unetumeurosseusebénigne.L’aluminiumpeutêtreidentifié danslamatriceosseusenondécalcifiéeparlacolorationausolochromeazurine,uncolorant hautementspécifiquequipermetladétectionde0,03 %d’aluminiumenfractionatomique.La colocalisationdel’aluminiumetdufernesemblepasêtrelefruitduhasardmaislesmécanismes cellulairesetmoléculairesd’entréedel’aluminiumetduferdanslacellulesontencoremal connus.L’histochimieapparaîtcommeuneméthodesupérieureauxanalysesspectroscopiques (EDSetWDS)coupléesàlamicroscopieélectroniqueàbalayage.

©2015ElsevierMassonSAS.Tousdroitsréservés.

Introduction

Bonematrixiscomposedoftwophases:

• an organic phase is elaborated by osteoblasts and is mainlycomposedoftypeIcollagenmicrofibrilsandnon- collagenousproteins;

Figure1 Transmissionelectronmicroscopyofthebonematrix.A) Undecalcifiedultrathinsectionshowingthe mineralphase composedofhydroxyapatite crystals intheform oftablets. B) After decalcification,the organicphase composedofcollagen microfibrilsisclearlyunmasked.

Microscopieélectroniqueàtransmissiondelamatrice osseuse.A)coupeultrafinenondécalcifiéemontrantlaphaseminérale constituéedecristauxd’hydroxyapatiteprésentssousformedetablettes.B)Aprèsdécalcification,laphaseorganiquecomposée demicrofibrillesdecollagèneestclairementdémasquée.

• amineralphasewhichiscomposedofnumeroushydroxy- apatite crystals (HA) deposited between the collagen microfibrils(Fig.1).

The formula for biological HA is Ca10(PO4)6(OH)2. In mature bone, nucleation of the HA crystals seems to be due to non-collagenous proteins such as the bone sialoprotein (BSP) deposited at the mineralization front

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c a b

PO43- OH- Ca2+

O2-

large channel Ø 0.3 -0.45nm small

channel Ø 0.25nm

Figure2 Projectionontheplane(001)ofthecrystallographic structureofhydroxyapatitewiththelocalizationofthesmall and largechannels. The large channel, centered on anOH groupandoutlinedbyPO43,isthesiteforionicsubstitutions.

Projectionsurleplan(001)delastructurecristallographique de l’hydroxyapatite montrant la localisation des petits et grands tunnels.Le grand tunnel,centrésurun groupe-OH—

estentouré pardesPO43,ils’agitdu sitepossiblepour les substitutionsanioniques.

[1]. Alkaline phosphatase, elaborated and secreted by osteoblasts in the osteoid tissue (seams of recently syn- thetized organic bone matrix) allows the development of the mature HA crystals from the crystal nuclei. The enzymesubstratesarepyridoxalphosphate,ATP,ADP,AMP, glucose-1-AMPc, glucose-6-phosphate, pyrophosphate and phosphor-ethanolaminpresentintheextracellularfluids[2].

Alkaline phosphatasealsoacts asa phosphatetransporter tothemineralizationfront,allowingtheaccretionofphos- phatesonthecalciumatomsoftheHAcrystal.

Fromacrystallographicpointofview,thearrangement ofCa2+,PO43andOHgroupsinthecrystalcanbemodeled asinFig.2[3].Inthisatomiclattice,twochannelscanbe described:asmallone(0.25nmindiameter)andalargeone (0.3—0.45nm in diameter). In this large channel, a num- ber of atom substitutionsare possible. Hydroxidegroups, forexample,canbesubstitutedbyanionssuchasF;phos- phatecanbesubstitutedbyother anions(e.g.carbonate) andcalciumcanbereplacedbymetalliccationssuchasFe3+, Sr2+,Pb2+...The non-exhaustivelistof thesesubstitutions appearsonTable1[3].Someofthesesubstitutions(Fand Sr2+)havebeenproposedintherapeuticforthetreatment ofosteoporosis(thesedrugsarenon-longerusedinclinical practice).Itshouldbenotedthatanysubstitutionchanges thecrystal properties(e.g.F-increasesacidresistanceof HAtohydrolysis)withaconsequenceatthetissuelevelon thebonequality.

Histochemistryisapowerfultooltocharacterizeminer- alization ofthe bone matrix andto identifythepresence ofcertainmetalionsabnormallypresentinHAwherethey can alter bone quality.The Perls’staining is a worldwide admitted histochemical stain for iron.The method works onsoft and hard tissues. In the presence of ferrocyanide ions,Fe3+ (butnotFe2+)isprecipitated asa highlywater- insolublebluecomplexalsotermedPrussianblue.Inbone,it

Table 1 Non-exhaustive list of cationic (for Ca2+) and anionic substitutions (for PO43 andOH). The carbonate substitutionofaphosphateiscalled␣substitution,andfor ahydroxide,a␤substitution.␤substitutionincreasesinthe humanbonematrixduringageing.

Listenonexhaustivedessubstitutionscationiques(pourle Ca2+)etanioniques(pourPO43etOH).Lasubstitutiondes phosphatesparlescarbonatesestappeléesubstitution,et unhydroxylparuncarbonatesubstitution.Lasubstitution

augmenteaucoursduvieillissement.

Ca2+ PO43 OH

Pb2+ Na+ Fe3+ VO43 CO32 SiO44 F CO32

Cd2+ Li+ La3+ AsO43 SO42 GeO44 CI O2 Zn2+ K+ Al3+ MnO43 HPO42 I S2

Ni2+ Ag+ Br

Mn2+

Ba2+

Sr2+

Mg2+ Lacuna Lacuna

ispreferabletoavoidtheuseacounterstaindyeifonewants toclearlyidentifytheironbandsinthebonematrix[4—7].

Fe3+ isdepositedinthecementorarrestlineswhenaBSU isformed,eitherincorticaland/ortrabecularbone.These lines are known to contain specific proteins (osteopontin andosteocalcin)thatcanbindmetalions.Decalcificationof bonesectionsabolishesthestainingbecauseFe3+isboundto theHAcrystals.Ithasbeenshownbyaverysensitivemethod (microX-rayfluorescenceanalysiswithasynchrotron)that ZnandPbionswhicharealsopresentintheinterstitialfluid can accumulatein the cement lines by uptake in the HA crystalandattachmenttotheseproteins[8].

Histochemical identification of aluminum in bone was extensivelystudiedinpatientswithrenalfailurewhodevel- oped encephalopathy and osteomalacia (see below). The differentmethodsonlyworkonundecalcifiedbonesections [9]. Aluminum can be stained by the aluminon technique using aurine tricarboxylic stain [4,10]. The method was foundmoresensitive thanatomicabsorptionspectrometry [11]. Several authors have reported that the regressive staining withsolochrome azurine B(also termed Mordant blueBorchromeazurolB-CI43830)[12]givesbetterresults andidentifiesmorebandsinthebonematrix[13—16].We have found that spectroscopic methods such as scanning electron microscopy (SEM) coupled with X-ray energy dispersivespectroscopy(EDS)cannotidentifyaluminumand irononbonesamplesduetothelowconcentrationofthese metalsintheHAcrystals.Thelimitof detectionofEDSis 0.05%(atomic%)[17,18].Wehaveidentifiedthesemetals bywavelengthdispersivespectroscopy(WDS),coupledwith SEM,andthelocalconcentrationsofthesemetalsisinthe range of 0.03—0.035% at a maximum [19]. However, this methoddoesnotallowmappingimagesofthetissues.

In vitro models of calcification

A number of models have been described to mimic in vitro the calcification of the bone matrix. We have developed a synthetic polymer (poly (2-hydroxyethyl)

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Figure3 Scanningelectronmicroscopyofcalcospheritesdevelopedonapolymer. A)Control calcospheritescomposedofHA tabletsdevelopedinastandardbodyfluid.B)Calcospheritesdevelopedinthepresenceofironinthebodyfluid;notethereduced sizeindicatinganinteractionwithmineralization.C)SurfaceofthepolymerincubatedinthepresenceofAl3+;thereisanalmost completeinhibitionofthecalcospheritesgrowth.D)EDSanalysisofthecalcospheritesinBindicatingthepresenceoftracesof ironinHAcomposedofcalcium/phosphatestabilizedwithmagnesium.E)EDSanalysisofthemineraldevelopedinCindicatingthe presenceoftraceofaluminumtogetherwithcalcium,phosphateandmagnesium.

Microscopieélectroniqueàbalayagedecalcosphéritesdéveloppéssurunpolymèrefonctionnaliséavecdesgroupements—COOH.

A)Lescalcosphéritessontprésentsàlasurfacedumatériauetsedéveloppentparnucléationsuccessivedansleliquidebiologique.

B)Croissancedescalcosphéritesenprésencedeferàl’intérieurdumilieudeculture.Laprésenceduferréduitconsidérablement leurtaille.C)Absencededéveloppementdecalcosphériteslorsquedesionsaluminiumsontajoutésdanslemilieudeculture.La surfacedupolymère,avecquelquestracescalcifiées,estbienvisible.D)AnalyseEDSdescalcosphéritesprésentssurlaFig.B : notezlaprésenced’unpetitpicdeferdansl’hydroxyapatitecomposéedecalciumetphosphatestabilisépardumagnésium.E) AnalyseEDSdescalcosphéritesprésentssurlaFig.C:laprésenced’aluminiumestaussimiseenévidencelorsquelaconcentration estsupérieureà0,05 %desatomes.

methacrylate—pHEMA)whichcanbechemicallymodifiedby carboxymethylation.CarboxymethylatedpHEMAreproduces the structure of acid proteins of the bone matrix known toinducemineralization(e.g.BSP)[20].Whenplacedina syntheticbodyfluidhavingthesameioniccompositionthan theextracellularfluids,pelletsmadeofthispolymerinduce mineralization. Calcified globules made of HA develop on the polymer surface within 15days (Fig. 3A). These mineralized globules are similar in size and composition tothecalcospheritesdescribedinthecartilaginousgrowth plateoflongbones.Calcospheritesarealsoobservedinthe wovenboneofrapidlyformingbones(callus,metaplasia...).

The carboxymethylated pHEMA model has been used to evaluate the effects of drugs known to interfere with

calcification[21]andalsometalionswhichcanbedissolved in thebodyfluid.Fe3+ ionsat theconcentrationof 20,40 or 60␮M/L reduce thesizeof thecalcospherites(Fig.3B) [22].SEM-EDS identifiedironinthe calcospheritesat very lowamount(>0.05atomic%)(Fig.3D).Cobalt,nickeland chromium(thesemetalspresentinstainless-steelorthope- dic prostheses)canalso modify thesize ofcalcospherites [23].Strontiumhasalsoaneffectonthecalcospheritesina dosedependentmanner.However,Sr2+canbeprogressively elutedfromthecrystals[24].

Wehaverecentlyinvestigatedtheinfluenceofaluminum in this model [25]. Aluminum chloride was added in the bodyfluidattheconcentrationof20,40,and60␮g/LAl3+. Aluminum strongly reduced the growth of calcospherites

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Figure 4 A) Osteomalaciain apatient with dialyzed renal insufficiency. Goldner’strichromeidentifiesmineralized bone matrix ingreen andosteoid tissue inred;undecalcified sec- tion.Notetheconsiderableamountofosteoidtissueandthe thicknessofosteoidseamsindicatingamineralizationdefect.

B)Histochemicalidentificationofaluminumbythesolochrome azurinemethod.Al3+isinblue,boneisfaintlystainedinorange.

A) Ostéomalacie chez un patient dialysé pour une insuffi- sance rénalechronique. Trichrome de Goldner : la matrice osseuse minéralisée est en vert, le tissu ostéoïde en rouge surdes coupes non décalcifiées.B) Identification histochim- iquedel’aluminiumparlacolorationausolochromeazurine.

Al3+apparaîtsousformedebandesbleues,l’osesttrèslégère- mentcoloréenorange.Notezlaquantitéconsidérabledetissu ostéoïde et l’épaisseur des bordures ostéoïdes indiquant un troubledeminéralisation.

(and the highest concentration has completely inhibited their formation) (Fig. 3D). SEM-EDS identified Al3+ in the HA calcospherites (Fig. 3E). Furthermore, other pellets were incubated with pieces of aluminum foil and, here again, a strong inhibition of the calcospherite formation wasobserved.ThisindicatesthatAl3+canbereleasedfrom aluminumfoilsbyoxidationinthebodyfluid.

Bone, aluminum and kidney

During the 1970s, a series of patients with renal insuf- ficiency, who were dialyzed, developed the dialysis

Figure5 A)Athreadedhipprosthesiscoveredbyathickcoat- ingofaluminadeposedbyaplasmatorch(arrows).Thickand polishedsection afterundecalcified embedding; themetalis inblack.B) Histochemical identificationof aluminumby the solochrome azurine method in another patient who had the sametypeofprosthesis.Themetallicparthasbeenremoved beforeembeddingandsectioningata7␮mthickness.Al3+isin blue,thecalcifiedbonematrixinorangeandnon-mineralized boneisunstained (arrow)and formsathick layerfacingthe prosthesis,indicatingalocalizedmineralizationdefect.

A)Prothèsedehanche filetéerecouverteparunépaisdépôt d’alumine déposé par torche à plasma (flèches) ; tranche épaisseetpoliesansdécalcification.Lemétalapparaîtennoir.

B)Identificationhistochimiquedel’aluminiumparlatechnique au solochrome azurine. La partie métallique a été enlevée avantinclusionpourréaliserdescoupesde7md’épaisseur.

Al3+ est en bleu,la matrice calcifiée en orange et l’osnon minéraliséestincolore(flèche) indiquantundéfaut localde minéralisation.

encephalopathyassociated withosteomalacia (a mineral- izationdefectcharacterizedbyanaccumulationofosteoid tissue)(Fig.4A)[26,27].Encephalopathywasrelatedtothe aluminum contentin the dialysate because water, at the time,waspurifiedbyaluminagelsinthewatertreatment plants.Dialysispatientshadanadditionalcauseofaluminum intoxication: they were treated by aluminum hydroxide asa countermeasure for hyperphosphatemia. In the bone frompatients withdialysisosteomalacia, the presence of aluminumwas identifiedaslinear bandsby histochemical methods;thisreflects thedepositionofAl3+ alongthecal- cificationfrontandcementlineswithinthecalcifiedbone matrix(Fig.4B)[9,11].Indialyzedpatients,ironandfluoride werealsoidentified by severalauthors but the consensus wasthataluminumhadthemostdeleteriouseffectsonbone mineralization[28].Aluminumwasalsofound tointerfere

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Figure6 A)BoneharvestedinthevicinityofaTA6Vhipprosthesisreplacedduringahipprosthesisrevision.Arrowsidentifyblack metallicdebrisinthemarrowcontainedintheHaversiancanals.Goldner’strichrome,undecalcifiedbone.B)Sectionfromthesame patient,metallicweardebriscomposedofTA6Vareevidenced(arrow).Bluebandsofaluminumarepresentinthecalcifiedbone matrixoftheHaversiancanalsurroundingthemetalparticles.Solochromeazurinestaining.C)Boneharvestedfromthefemoral headofapatienttreatedbyathreadedscrewforahipfractureseveralyearsago.Fibrosishasdevelopedinsidethethreads(the metalhasbeenremoved).Goldner’strichrome,undecalcifiedsection.D)Sectionfromthesamepatientstainedwithsolochrome azurine.Aluminumdepositsarevisibleasbluebandsinthebonefacingthescrew.Thesofttissues(whichdonotcontaincalcium phosphate)areunstained.

A)Tissuosseuxprélevéauvoisinaged’uneprothèseenTA6Vutiliséepourletraitementd’unefracturedehanche.Lavisaété laisséeenplacependantdenombreusesannées.LesflèchesidentifientdesdébrismétalliquesdanslescanauxdeHavers.Trichrome deGoldner,coupenondécalcifiée.B)Coupevoisineobtenuechezlemêmepatient ;lesdébrismétalliquessontconstituésde particulesd’usureduTA6V(flèche).Desbandesbleues(aluminium)sontprésentesdanslamatriceosseusecalcifiée,descanaux deHaversautourdesparticulesmétalliques;colorationausolochromeazurine.C)Fragmentdetêtefémoraled’unpatienttraité parunevisfiletéepourunefractureducolfémoralsurvenuedenombreusesannéesauparavant.Unefibroses’estdéveloppéeà l’intérieurdufiletage(lemétalaétéenlevéavantl’inclusion).ColorationdeGoldner,coupenondécalcifiée.D)Coupeadjacente provenantdumêmepatientetcolorée parlesolochromeazurine.Desdépôtsd’aluminiumsontvisibles sousformedebandes bleuesdansletissuosseuxenfacedelavis.Lestissusmous(necontenantpasdephosphatedecalcium)sontincolores.

directlywithboneremodelingbyreducingboneformation byosteoblasts [29,30].An indirect effectwasalsoidenti- fiedasaluminumcanaccumulateintheparathyroidglands andalters theparathyroid hormonelevelsby reducing its synthesis[31].

Bone, aluminum and biomaterials

Severalreports havestressedtheimportanceofaluminum released by biomaterials as a toxic for bone. A ceramic coating of prosthetic stems has been proposed to favor osseointegration.HAandaluminacoatingswerewidelyused in the 1990s. However, alumina-coated prostheses were associated with a juxta prosthetic mineralization defect (similartothedialysisosteomalacia)duetoanaccumulation ofAl3+ intheanchoringbone.AdirectreleaseofAl3+ from thecoatingwasfoundafterhistochemicalstaining(Fig.5) [32,33].Abonecementcontainingaluminumfluorosilicate

wasusedinoto-surgeryandcausedseverallethalcasesof aluminumencephalopathy[34].

Biomaterials are now widely used in biomedicine and their applications are numerous: heart valves, artificial heart, dentaland orthopedic implants, tracheal and vas- cularprostheses...Morethanhalfofbiomaterialsproduced byindustryareusedinanintra-osseoussite(cement,syn- theticbonefillingmaterialorprosthesis).Biomaterialsare inertobjectsplacedindirectcontactwiththebiologicalflu- ids.Theycanattackthesurfaceofmetallicbiomaterialsas theycontainnumerousanions(Cl,PO43,HCO3,SO42...), cations (Mg2+, Na+, Ca2+, Fe3+...), dissolved oxygen and free radicaloxygen species. This liquid microenvironment appears to have an oxidizing power equal to 1/3 this of the ocean water and ¼ this of the air [35]. In addition, thebodytemperatureincreasestheoxidizingcapacityand local pH variationsarefrequent [36]. Corrosionof metal- licbiomaterialscanoccurindifferenttypesofconditions:

atthemetaljointgrains(asmetalsarepolycristalline),by

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Figure7 A)Genetichemochromatosiswithosteoporosis.Perl’sstainingidentifiesnumerousbandsofiondepositedinthebone matrix.B)Histochemicaldetectionofaluminumbysolochromeazurineinthesamepatient.C)Histochemicaldetectionofaluminum inacaseofsicklecellanemia.D)Histochemicaldetectionofiron(Perl’sstaining)inthesamepatient.Notethebluelines(arrows) indicatingthepresenceofironinthebonematrix(unstained)andthepresenceofnumeroussiderophagescontainingironinthe bonemarrow(bluespots).

A)Hémochromatosegénétiqueavecostéoporose.ColorationdePerl’sidentifiantdenombreusesbandesdeferdanslamatrice osseuse.B)Détectionhistochimiquedel’aluminiumparlesolochromeazurinechezlemêmepatient.C)Détectionhistochimique del’aluminiumdansuncasdedrépanocytose.D)Détectionhistochimiquedufer(colorationdePerls)chezlemêmepatient.Notez laprésencedelignesbleues(flèches)indiquantlaprésencedeferdanslamatriceosseusenoncoloréeetlaprésencedenombreux sidérophagesintramédullairescontenantdufer(tâchesbleues).

galvaniccurrents(ifdifferentmetalsarepresent,byalocal electrolytic phenomenon) and by the generation of wear debrisfromarticulatingbiomaterials(especiallyinthecase oforthopedicprosthesis).

We haverecently reporteda seriesof32 patientswith a revision for a total reconstructionafter aseptic loosen- ingofanalumina-on-aluminahipprosthesis[37].Thistype of prosthesis is known to have the best friction torque [38,39].In thesepatients,the periprosthetictissues were analyzed in the search of alumina debris.In 14 patients, asmallfragmentofperiprostheticbonewasalsoanalyzed afterundecalcifiedembedding.Sectionswereobtainedona heavydutymicrotomeandstainedbyaGoldner’strichrome (for the identification of osteoid tissue and mineralized phase), solochrome azurine staining (for identification of Al3+)andPerls’staining.Aluminagrainswereneverencoun- tered in the soft tissues. Metal particles were present in 3patientsandSEM-EDSrevealedthattheywerecomposed oftheTA6Vtitaniumalloycontaining6%aluminumand4%

vanadium.TA6V(orgradeVtitaniumASTM)isthemetalused inorthopedicsforprostheses,screws,cups...).Histochem- icalanalysisrevealedthattheFe3+ wasnotpresent inthe bone matrix but aluminum bands wereobserved for each patient. In a control series of 8 patients having received aprosthesis withoutalumina(e.g.,metal-on-polyethylene ormetal-on-metal),thesamemethodologyofanalysiswas

used.Al3+wasalsoidentifiedinthebonematrixfromthese patients as linear bands in the vicinity of the prosthesis orthemetalweardebris(Fig.6A—B).It islikelythatAl3+

comes from oxidation of the metallic parts of the pros- thesis(stem, cup, screw)and an origin from thealumina cannotbeexcluded. However,the longterm tolerance of thealumina-on-alumina prostheses,andthe fact thatthe amount of aluminum bandsin the bone matrix were sim- ilar in the other group, is a plea for an oxidation of the TA6Vratherthanaionicdecompositionofthisverystable alumina[40].TheAl3+ionreleasefromalumina-on-alumina andmetal-on-metaltotalhipprostheseswasdosed inthe serumfromaseriesofpatientsandtheAl3+contentwasnot differentbetweenthetwogroupsandwithacontrolgroup withoutprosthesis[41].

Presenceofaluminuminthebonematrixisalsocommon inorthopedic samplesobtained afterablation of titanium material.Fig.6C—Dillustratesthereleaseofaluminumfrom athreadedscrewinafemoralheadplacedmorethanten yearsbeforefor treatingahipfracture.The materialwas changedduringrevisionforatotalhiparthroplasty.Insuch cases,thecontinuousreleaseofaluminumfromthemetallic biomaterialisprovenbythedepositionofnumerousrepet- itivebandsmimickingthegrowthlinesonatreetrunk.Ina compilationstudy,thereleaseofmetalionswasconfirmed in the body fluids of patients with metallic biomaterials

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Figure8 A) X-rayimageofanisolatedexocytosis ofthelower femoralextremityina13years-old-child.B)A whole exocy- tosisstudiedundecalcified.Thedystrophicproliferativecartilageisindicatedbyarrows.Athinshell ofcorticalbonesurrounds atrabecularnetwork.Goldner’sstaining.C)Histochemicalidentificationofaluminumbythesolochromeazurinemethodinthis exostosis.

A)Radiographied’uneexostoseisoléedel’extrémitéfémorale inférieurechezunenfantde13ans(flècheverte).B)Exostose complèteétudiéesansdécalcification.Lacouchedecartilagedystrophiqueproliférativeestindiquéepardesflèches.Unefine coquedetissuosseuxcorticalentoureunréseautrabéculaire.TrichromedeGoldner.C)Identificationhistochimiquedel’aluminium parlesolochromeazurinedansl’ostrabéculairedel’exostose.

byvarioustechniquesincludingInductivelyCoupledPlasma Mass Spectrometry (ICP-MS) and graphite furnace atomic absorptionspectrometry (GF-AAS) [42].Although titanium is protected bya film of titanium oxides,the TA6V-based devicescanreleasealuminumafterprolongedimplantation [43].

Thiswillcertainlybeobservedinthenextfutureinden- talimplantology.Classically,dentalimplantswereprepared with c.p. grade II titanium. It is more ductile than the TA6V alloy(gradeV), whichcontains aluminum andvana- dium. Some industrial companieshave recently proposed TA6Vimplantsonthe market,especiallyshortimplantsto beusedinareaswithalowbonemass.Titaniumcanbeeas- ilyoxidizedandalteredinmouthbytheactionofnumerous oxidants(fluoride containing toothpastes,reactive oxygen species from polymorphonucleated cells and bacteria, or organicacids ofthesaliva)[44].The releaseofaluminum fromTA6Vdentalimplantscouldrepresentasanitaryprob- leminthenextdecade.

Bone, aluminum and diseases associated with iron metabolism

Genetic hemochromatosis (GH) is an autosomal recessive diseaseresponsibleforanironoverload.Themostfrequent

form is due to a mutation of the HFE gene. In these patients,osteopeniaandosteoporosisarefrequent(28—50%

of patients), together with the liver diseasewhich is the moreseverefactor[45].Apositiveironstainingisreported in thebone matrix ofthese patientsand appearstoform linearbands. Surprisingly,aluminum canalsobe detected aslinearbandsinthesameareasinsomepatients(Fig.7).

Fe3+ is also identified by histochemistry in the bone matrixofpatientswithotheriron-relateddiseasessuchas␤- thalassemiaandsicklecellanemia[46,47].Themechanisms oftheFe3+ localizationinbonearenotclearlyunderstood but the roleof metaltransporter proteins (such asferro- portin)hasbeenrecentlyreviewed[48].Intwopatientswith sicklecellanemia,acolocalizationofAl3+withFe3+inbone wasobservedinourlaboratory.

Bone, aluminum and digestive diseases

Aluminumisabsorbedbythegutasevidencedbyanumber ofpapers intherat[49,50].Itisalsoabsorbedinhumans asshowninpatientsreceivingphosphatebindersindialysis patients [51].Aluminum absorptionfrom thegut leads to abonedeposition,eveninthepresenceofanormalkidney function. Al3+ absorption was increased in a patient with apeptic ulceranda long-termhistoryof gastroprotection

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[52].Wereportedapatientwithaceliacdiseasewhowas usingoralmedicationscontainingaluminum.Shedeveloped osteoporosis with very low bone mineral density values (T score=−6.18 at the lumbar spine). Al3+ was identified bysolochrome azurinestaining ina transiliacbonebiopsy [53].Wealsoobservedsimilarfindingsinapatientwithlow bonemineraldensity(Tscore=−3.56atthelumbarspine) andfracturesoftheosilium. Thepatienthadalsoalong history of dyspepsia and was using aluminum-containing gastroprotectorsfordecades;hereagain,Al3+wasidentified in thebone biopsy. Parenteralnutritionis also associated with an increased aluminum absorption (released from nutrition solutions containing contaminated casein, from glass vials...) [54]. Aluminum has also been identified as a possible environmental causalfactor in Crohn’s disease [55].Aluminum enhancescolicinflammation inmice [56].

Lessons fromthe past (dialysispatients) and fromanimal studiesindicatethatchronicaccumulationofaluminumin the bone is likely tooccur in patients withinflammatory diseasesofthedigestivetract.Althoughtheseinflammatory boweldiseasesareknowntoinduceosteoporosisbythem- selves[57],itismostprobablethataluminumdepositionin thebonematrixcontributestotheboneloss.However,this remainstobeprovenbybonebiopsystudies.

Calcified bone in exostosis

Exostosis is the most frequent benign tumor in children and adults. Isolated exostosis is frequent but multiple exostosesareobservedinseveraltypesofMultipleHered- itaryExostosis(MHE).MHEisanautosomalgeneticdisease due to mutation in the Golgi-associated heparin sulfate polymerases(EXT1,EXT2orEXT3)[58].Althoughthepatho- genesisofisolatedandMHEarenotfullyunderstood,these tumorssharecommoncharacteristics:theyarecoveredbya capeofproliferationcartilage,theypossessashellofcorti- calbonewhichsurroundamoreorlessdevelopednetworkof trabecularbonecontainingbonemarrow.Wehaverecently reportedaseriesofthirtypatientswitheitherisolatedor multiple exostosis (three cases of MHE) [19]. Histological analysisoftheundecalcifiedtumorrevealedthepresenceof aluminuminthebonematrixofthetumorin2/3ofpatients.

Surprisingly,ironwasalsodepositedinthesameareasin1/3 ofthepatients(Fig.8).Becauseadysregulationoftheosteo- progenitorcellhasbeendescribedinthisdisease,itismost probablethatmetabolicchangesintheosteoblastfunction areresponsible for the abnormaldepositionof these two metals.

The origin of aluminum in bone

Itisnowwellrecognizedthataluminumentersinthebody from different sources. As mentioned above, aluminum is present in foods and beverages (the additive E173 is aluminumoxide)butotherroutesofpenetrationinthebody have been identified: trans or percutaneous absorption (from vaccines and anti-sweating or antiperspirant prod- ucts, e.g.alum stone)and locally released frommetallic biomaterials. In the body, 95% of ingested aluminum is eliminatedin thefeces andthe5% remaining circulatein theblood mainlyboundtotransferrinandalbumin (Fig.9

Figure9 Metabolismofaluminuminhumans.Greenarrows:

inputsourcesare either byingestats (water,food, drugs...), ofcutaneousorigin(vaccines,deodorant,antiperspirant...)or internalreleasefrom TA6Vtitanium devices.Redarrow: 95%

oftheingestedaluminumisexcretedinthefeces.Pinkarrow:

4%oftheabsorbedaluminumcirculatesinthebloodboundto transferrin(80%),albumin(10%)andotherproteins(10%).Yel- lowarrow:83%ofthecirculatingaluminumisexcretedinurine.

Blueorgans:aluminumcanbedepositedinthreemaintissues:

centralnervoussystem,spleenandbone.

Métabolismedel’aluminiumchezl’homme.Lesflèchesvertes indiquentlessourcesd’entréedel’aluminiumdansl’organisme soitàpartirdesingestats (eau,nourriture,médicament...), voie cutanée (vaccin, déodorant ou antiperspirant...) ou endogènesparrelargagedel’aluminiumàpartirdebiomatéri- auxentitane.Flècherose:5%del’aluminiumabsorbécircule dansle sang, lié à la transferrine(80 %), àl’albumine (10

%) et à d’autres protéines (10 %). Flèche rouge : 95 % de l’aluminium ingéré est excrété dans les fèces. Flèche jaune :83 % del’aluminium circulantest excrété danslesurines.

Les organesen bleus représententles cibles danslesquelles l’aluminiumnon éliminépeut sedéposer :système nerveux central,rateetmatriceosseuse.

summarizes the aluminum metabolism in the body). The circulatingaluminum canmainlybefixedin twopreferen- tialorgans: brainand bone. The metalis recognizedasa neurotoxicinAlzheimerandParkinsondiseaseswhereitcan alsocolocalize withiron.Inbone, aluminumis acauseof bonelossinlaboratoryanimalsandhumansandhighdoses causeosteomalacia.Finally,thereareanumberofstrange coincidences concerning the metabolism of these two metalsin the body:same transporters,cytotoxic activity,

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impairmentof osteoblasticactivity atlowdose, inhibition ofbonemineralizationathighdoses.

Conclusion

Aluminumcan bind tothe phosphategroups of HAof the calcified bone matrix. Histochemical methods are by far superiortospectroscopicmethods (EDSandWDS) because aluminumandironareidentifiedinthesamelocationofthe bonematrix. Although thepathophysiologicalmechanisms arenotfullyunderstoodatthecellularandmolecularlev- els,colocalizationofaluminumandironiscertainlynotthe fruitof chanceandmucheffort isstillneeded toimprove ourknowledgeonthetoxicactivityofmetals.

Disclosure of interest

Theauthorsdeclarethattheyhavenocompetinginterest.

Acknowledgments

Thisreviewwaspresentedasanoralcommunicationatthe KeeleMeetinginLille(February—March2015).Manythanks toMrs.Lechatforsecretarialassistance.Wealsothankall surgeonsor clinicianswho addressedbone samplestoour laboratoryandespeciallyD.Moukoko,N.Henric,V.Steiger, P.LeNay,J.M.Frin,C.DeBodman,D.Mulleman,M.Audran, E. Legrand, B. Bouvard. This researchwasfunded bythe Ministèredel’EnseignementSupérieuretdelaRecherche.

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