TEZ DE DOCTORAT

Cercetri experimentale i clinice ale osteonecrozei maxilarelor dup tratamentul cu bisfosfonai

Rezumat

Doctorand Horea Artimoniu Alman

Conductor de doctorat Prof. Dr. Grigore Bciu

Cluj-Napoca, 2013

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CUPRINSINTRODUCERE 13STADIUL ACTUAL AL CUNOATERII 151. ISTORIA BISFOSFONAILOR 192. STRUCTURA BISFOSFONAILOR 193. MODUL DE ACIUNE AL BISFOSFONAILOR 203.1 RESORBIA OSOAS 203.2 ACIUNEA BISFOSFONAILOR ASUPRA PROCESELOR DE CALCIFICARE 203.3 MECANISMUL CELULAR DE ACIUNE AL BISFOSFONAILOR 213.4 UTILIZAREA CLINIC A BISFOSFONAILOR 214. TERAPIA CU BISFOSFONA I 224.1 BENEFICIILE TERAPIEI 224.2 RISCURILE TERAPIEI 225. EFECTELE SECUNDARE ALE BISFOSFONAILOR 226. CONCLUZII ASUPRA COMPUILOR BISFOSFONAI 247. TRATAMENTUL OSTEOPOROZEI CU BISFOSFONA I 248. ASPECTE CLINICE I PARACLINICE ALE OSTEONECROZEI MAXILARELOR 269. INCIDEN A OSTEONECROZEI 2810. TRATAMENTUL OSTEONECROZEI OASELOR MAXILARE 28

CONTRIBU IA PERSONAL 37IPOTEZA DE LUCRU 37STUDIUL 1 - MODIFICRI SEROLOGICE INDUSE DE ADMINISTRAREA EXPERIMENTAL INTRAMEDULAR A BISFOSFONA ILOR LA OBOLAN 411.1 INTRODUCERE 411.2 MATERIAL I METOD 421.3 REZULTATE 451.4 DISCUII 501.5 CONCLUZII 54STUDIUL 2 - EFECTELE INDUSE DE BISFOSFONAI LA 6 I 12 SPTMNI ASUPRA STRUCTURII OSOASE FEMURALE LA OBOLAN 552.1 INTRODUCERE 552.2 MATERIAL I METOD 552.3 REZULTATE 562.4 DISCUII 732.5 CONCLUZII 80STUDIUL 3 - OSTEONECROZA MAXILARELOR ASOCIAT CU UTILIZAREA BISFOSFONAILOR. DISCUIE PE BAZA A 52 DE CAZURI CLINICE 813.1 INTRODUCERE 813.2 MATERIAL I METOD 813.3 REZULTATE 823.4 OBSERVAII CLINICE 923.5 DISCUII 1043.6 IMPLICAIILE BISFOSFONAILOR N MEDICINA DENTAR 1053.7 CONCLUZII 106CONCLUZII GENERALE 109ORIGINALITATEA I CONTRIBU IILE INOVATIVE ALE TEZEI 111REFERIN E 112

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Cuvinte cheie: bisfosfonat, osteonecroz maxilar, regenerare osoas.

IntroducereSuccesul regenerrii osoase ridic multe probleme clinicienilor. Abordarea eficient i sigur a reconstruciei osoase este necesar pentru a compensa pierderea osoas cauzat de traume, ablaii chirurgicale, boala parodontal, boli osoase, defecte congenitale, malformaii. n aceast lucrare ne-am oprit la reconstrucia osoas a defectelor cauzate de tratamentul cu bisfosfonai. tiina ghidrii regenerrii osoase se afl n stadiu incipient, metodele de tratament utilizate n prezent fiind serios limitate. n chirurgia maxilo-facial este important evaluarea fiecrui caz clinic n parte cu scopul de a decide dac stimularea osoas poate avea loc dup intervenii chirurgicale complexe care implic proceduri de osteotomie sau rezecii osoase. Justificarea tiinific a problemei de cercetare

Osteoporoza este o problem major de sntate public. Bisfosfonaii pe cale oral sunt eficieni pentru reducerea riscului de fracturi osteoporotice i reprezint o opiune de tratament pentru pacienii cu risc de a dezvolta osteoporoz. n ciuda efectelor pozitive ale acestei clase de compui, exist i efecte negative care se instaleaz dup un interval de timp de la nceperea administrrii. Unul dintre cele mai importante efecte adverse este osteonecroza maxilarelor care se transform frecvent n osteomielit la pacienii cu terapie cu bisfosfonai i focare dentare. Osteomielita pe fond de osteonecroz se instaleaz dup extracii dentare, intervenii chirurgicale dento-alveolare sau dup traumatisme protetice care determin expunerea suportului osos n cavitatea oral septic. Afeciunea este caracterizat de o simptomatologie srac la debut, motiv pentru care leziunile osoase progreseaz fr a fi decelate pn n faza de supuraie a prilor moi periosoase. Expunerile osoase sunt dureroase i cu risc infecios chiar i dup ncetarea tratamentului cu bisfosfonai. Este recomandat un control stomatologic amnunit nainte de a ncepe tratamentul cu bisfosfonai. A doua mare clas de afeciuni n care sunt utilizai bisfosfonaii sunt tumorile maligne, n special n faza de metastaze osoase. Datorit creterii frecvenei metastazelor osoase i a efectelor benefice a bisfosfonailor n tratamentul bolilor maligne osoase, dar i a afeciunilor non-maligne (osteoporoza), tot mai muli pacieni vor beneficia de acest tip de tratament i tot mai muli clinicieni se vor confrunta cu aceast patologie. Din acest motiv, pentru prevenirea efectelor adverse ale bisfosfonailor trebuie s nelegem foarte bine patogeneza osteonecrozei. Dac exist o asociere ntre osteonecroz i bisfosfonai, atunci se ateapt ca prevalena bolii s fie tot mai mare cu ct folosirea bisfosfonailor se extinde, att la pacienii cu afeciuni maligne, ct i la cei cu afeciuni non maligne.Studii recente raporteaz complicaiile osteonecrozei maxilarelor asociate administrrii intravenoase i orale de bisfosfonai. Osteonecroza maxilarelor apare la pacienii cu tumori maligne tratai intravenos cu doze mari de bisfosfonai i la pacienii tratai pentru osteoporoz cu bisfosfonai pe cale intravenoas i oral. Bisfosfonaii sunt analogi stabili ai pirofosfatului. Bisfosfonaii se leag de hidroxiapatita suprafeelor minerale i sunt selectiv internalizai de osteoclaste, a cror activitate o inhib. Odat inhibat activitatea osteoclazic, aceasta va avea repercusiuni asupra activitaii osteoblastelor. Osteoblastele nu vor fi activate n situsurile de reparaie osoas, ca efect al lipsei resorbiei osoase. Astfel va fi periclitat echilibrul de resorbie/reparaie osoas de la nivelul 3

defectului osos. Acest dezechilibru va duce la vulnerabilizarea i predispoziia la microfracturi a esutului osos, acesta fiind lipsit de o reparaie osoas adecvat. Periclitarea echilibrului de resorbie/reparaie osoas poate induce necroza aseptic osoas. Bisfosfonaii sunt exceleni inhibitori ai resorbiei osoase, cu o poten variabil n concordan cu structura chimic. Tratamentul cu aceti compui modific starea de echilibru a resorbiei, n funcie de doza administrat. Bisfosfonaii inhib adeziunea i rspndirea celulelor tumorale.Bisfosfonaii administrai intravenos au fost primii folosii, dovedindu-i eficiena n tratamentul cancerului i a condiiilor asociate. Acestea din urm includ hipercalcemia malign, fenomenele scheletice asociate cu metastazele osoase n contextul tumorilor solide precum cancerul de sn, cancerul de prostat, cancerul pulmonar i n mielom multiplu. Bisfosfonaii intravenoi sunt eficieni n prevenia i reducerea hipercalcemiei, stabilizarea leziunilor osoase i prevenirea fracturilor n contextul implicrii scheletice. Bisfosfonaii administrai oral au fost aprobai pentru tratamentul osteoporozei i sunt frecvent folosii i n tratarea osteopeniei. Sunt folosii pentru o varietate de afeciuni precum boala Paget i osteogeneza imperfect a copilriei. Dar cea mai comun indicaie o reprezint osteoporoza. Riscurile terapiei: n 2003 i 2004 chirurgii oro-maxilo-faciali au fost primii clinicieni care au recunoscut i raportat cazuri de os expus, necrotic n regiunea maxilo-facial la pacienii tratai cu bisfosfonai pe cale intravenoas. Manifestrile la nivelul esuturilor cavit ii orale : Pierderile osoase i dentare sunt asociate cu deficiena de estrogen i osteoporoza. Resorbii severe ale crestelor alveolare la edentai, agravarea bolii parodontale i a pierderilor dentare au fost nregistrate n cazul pacienilor cu osteoporoz. Femeile care urmeaz o terapie cu bisfosfonai pentru combaterea osteoporozei prezint riscul de a dezvolta osteonecroz de maxilar, secundar tratamentului cu bisfosfonai. Rolul medicului dentist sau a chirurgului maxilo-facial este esenial n prevenirea osteonecrozei pe fondul tratamentului cu bisfosfonai prin evaluarea atent i asanarea corect a focarelor de infecie oro-dentare. Nu mai puin important este i rolul oncologului care conduce tratamentul cu bisfosfonai, oncolog care, informat fiind, trebuie s ndrume fiecare pacient la medicul dentist pentru evaluarea i eventual asanarea focarelor dentare nainte de nceperea tratamentului cu bisfosfonai. Pornind de la ideea c n reparaia osoas sunt implicate att rspunsuri anabolice ct i catabolice, prin intermediul acestui studiu se dorete evaluarea efectelelor administrrii bisfosfonailor (acid zoledronic) asupra esutului osos.

Justificarea utilitii rezultatelorEfectele adverse poteniale ale bifosfonailor ar trebui s fie luate n considerare n contextul fiecrui pacient i stabilite doza i modul de administrare atunci cnd se alege un bisfosfonat pentru tratamentul unei anumite afeciuni.S-a observat o legtur direct proporional ntre mrimea suprafeelor osoase expuse i durata tratamentului cu bisfosfonai. Studiile din literatur au demonstrat att corelaia ntre durata tratamentului cu bisfosfonai i distrucia osoas, ct i ntreruperea tratamentului cu remodelarea osoas.Scopurile acestei teze sunt: (1) de concepe pe baza a dou studii experimentale un algoritm de tratament profilactic i curativ al complicaiilor terapiei cu bisfosfonai asupra osului i de a elabora un studiu clinic

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pentru ameliorarea terapiei osteomielitei pe fond de osteonecroz maxilar n cursul tratamentului cu bisfosfonai i (2) de a revizui literatura de specialitate dedicat acestei patologii pentru a formula un material comprehensiv cu privire la etiologia, patogenia, clinica, profilaxia i tratamentul osteonecrozei maxilare la pacienii tratai cu bisfosfonai pentru a fi folosit de ctre medicii dentiti n practica lor curent. CONTRIBU IA PERSONAL a tezei este cuprins n trei studii (dou experimentale i unul clinic). Studiile experimentale abordeaz influena administrrii intramedulare de bisfosfonai asupra valorilor serice ale unor constante biologice care monitorizeaz fidel procesele de formare osoas i descrierea caracteristicilor histopatologice dup administrarea medicaiei bisfosfonate n defectele osoase create experimental. Studiul clinic dorete elaborarea unui algoritm pentru terapia osteomielitei pe fond de osteonecroz maxilar n cursul tratamentului cu bisfosfonai i ntocmirea unui material ghid privitor la etiologia, patogenia, clinica, profilaxia i tratamentul osteonecrozei maxilare la pacienii tratai cu bisfosfonai, util pentru practica curent. Studiul 1: Modificri serologice induse de administrarea experimental intramedular a bisfosfona ilor la obolan

Introducere: Scopul studiului este de a determina influena administrrii intramedulare (la nivelul osului femural) de bisfosfonai asupra valorilor serice ale fosfatazei alcaline, Ca total, Ca2+, proteinelor serice i osteocalcinului la un lot de obolani de experien din rasa Wistar. Material i metod: Lotul de studiu a fost compus din 15 obolani de experien din rasa Wistar, de sex masculin, n vrst de 6 sptmni, cu greutate ntre 100-200 g. obolanii au fost mprii ntr-un lot martor, de 5 obolani i un lot experimental, de 10 de obolani. obolanii Wistar s-au meninut pentru aclimatizare n biobaza Universitii de Medicin i Farmacie Iuliu Haieganu Cluj-Napoca, timp de o sptmn nainte de nceperea experimentului. Tuturor obolanilor li s-a practicat un defect osos la nivelul femurului drept. n anestezie general, dup asepsia i antisepsia cmpului operator s-a practicat o incizie la nivelul membrului inferior drept, pe faa antero-lateral superioar a coapsei. La aproximativ 0,5 centimetri de aceasta s-a efectuat un defect osos care a interesat ambele corticale, cu o frez de os cu diametrul de 1,5 mm, sub rcire continu cu ser fiziologic. La loturile experimentale s-a aplicat bisfosfonat. Lotului A, i s-a administrat 1 ml de bisfosfonat n doz unic intramedular, intraoperator. Grupului B i s-a administrat 1 ml de bisfosfonat (Zometa) n doze fracionate, zilnic 0,1 ml, timp de 10 zile. Fiecrui subiect i s-a recoltat cte 3 ml de snge din sinusul frontal, preoperator (momentul T0), la 14 zile (momentul T1) i 21 de zile (momentul T2) postoperator. S-au efectuat determinri sangvine ale proteinelor serice (g/dL), fosfatazei alcaline (U/L), Ca total (mmol/L), Ca total (mg/dL), Ca2+ (mmol/L) i osteocalcin (ng/ml) la momentul T0 (intraoperator), la momentul T1 (la 14 zile de la intervenie) i la momentul T2 (la 21 de zile de la intervenia chirurgical). Din probele sangvine s-au prelucrat fosfataza alcalin (U/L), proteinele serice (g/dL), Ca total (mmol/L; mg/dL), Ca2+ (mmol/L). Pentru determinarea osteocalcinului s-a utilizat kitul ELISA pentru obolani. Datele obinute au fost prelucrate statistic, utiliznd testul ANOVA. Protocolul de cercetare a fost aprobat ce ctre Comisia de Etic a Universitii, fiind n concordan cu principiile declaraiei de la Helsinki.Rezultate: Urmrind evoluia fosfatazei alcaline la lotul martor, s-a constatat o cretere a acesteia ntre momentul T0 i T1, pentru ca apoi s scad ntre momentul T1 i T2, dar nu mai 5

jos de valorile iniiale. Evoluia fosfatazei alcaline la loturile experimentale A i B a fost diferit fa de cea a lotului martor, n sensul creterii valorilor ntre momentul T0 i T1 i scderii ntre momentul T1 i T2, dar la momentul T2 valorile sunt sub cele de la momentul iniial T0. Proteinele au sczut att la lotul martor ct i la lotul experimental A i B de la momentul T0 la momentul T1 i T2, spre valori sub cele iniiale. Valorile calciului total (mmol/l) la loturile experimentale au sczut de la momentul T0 la momentul T1, ct i de la momentul T1 fa de T2, valorile finale aflndu-se sub valoarea iniial. Evoluia osteocalcinului la loturile experimentale A i B a artat o real diferen ntre loturi, ntre momentul T0 i T1 observndu-se att o scdere, ct i o cretere att la lotul A ct i la lotul B, iar la momentul T2 valorile devenind mai mari dect la momentul T0, n special la lotul B. Concluzii: 1. Administrarea unic de bisfosfonat a permis o reparaie osoas mai intens comparativ cu administrarea sa fracionat.2. Administrarea fracionat a bisfosfonatului de-a lungul unei perioade inhib osteoclazia semnificativ comparativ cu aceeai doz administrat n doz unic. 3. Procesele de reparaie osoas sunt periclitate direct proporional cu modalitatea de administrare i doza de bisfosfonai. 4. Bazat pe rezultatele acestui studiu, se recomand administrarea n doz unic a bisfosfonatului pentru ca avantajele s fie maxime, iar efectele adverse minime. 5. n fiecare etap de dezvoltare osoas acioneaz ntr-un mod coordonat factori multipli, care duc la intensificarea proceselor metabolice locale, cu influen att asupra proceselor osteoclazice ct i a celor osteoblastice, permind o reparaie osoas dac doza i calea de administrare a bisfosfonailor sunt bine alese.Studiul 2: Efectele induse de bisfosfonai la 6 i 12 sptmni asupra structurii osoase femurale la obolan

Introducere: Scopurile acestui studiu sunt (1) de a descrie caracteristicile histopatologice ale la 6 i respectiv 12 sptmni de la administrarea bisfosfonatului la un model animal i (2) identificarea efectelelor bisfosfonatului asupra modelrii osoase.Material i metod: Dup obinerea aprobrii Comisiei de Etic din cadrul Universitii de Medicin i Farmacie Iuliu Haieganu din Cluj-Napoca, au fost selectai 15 obolani de experien din rasa Wistar, 5 n lotul martor i 10 n lotul experimental. Toi obolanii au fost supui unei intervenii chirurgicale de creare a unui defect osos la nivelul femurului drept cu o frez cu diametrul de 1,5 mm transcortical prin canalul medular. obolanii din lotul experimental au fost grupai n dou loturi: lotul A, alctuit din 5 obolani, cruia i s-a administrat acid zoledronic, 1 ml n doz unic intramedular, intraoperator i lotul B, alctuit din 5 obolani, cruia i s-a administrat acid zoledronic 1 ml n doze separate, 0,1 ml timp de 10 zile. Grupului martor nu i s-a administrat acid zoledronic. Toate probele recoltate au fost examinate la microscopul optic (la 6 i respectiv 12 sptmni) pentru a evidenia diferenele n structura osoas. Rezultate: Rezultate la 6 sptmni de la nceperea experimentului: Examinarea microscopic a seciunilor seriate efectuate din zona de intervenie la 6 sptmni de la nceperea experimentului, au evideniat procese reparatorii osoase aflate n plin derulare, att la loturile experimentale ct i la lotul martor (defect osos experimental, netratat). Dar n ceea ce privete stadiul n care procesele reparatorii au ajuns n momentul recoltrii, exist diferene relativ mari att ntre loturile tratate ct i ntre acestea i lotul martor.

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La lotul martor, la 6 sptmni de la nceperea experimentului, zona superficial a defectului este ocupat de esut osos primar, care tinde s nchid orificiul de comunicare cu exteriorul. Osul aflat la acest nivel provine din proliferare activ de esut osos ce are ca punct de plecare periostul din vecintatea defectului, iar de aici se extinde att lateral (pe o anumit distan cu ngroarea peretelui), ct mai ales n zona defectului unde tinde s ocupe zona superficial pentru a ntrerupe continuitatea dintre canalul medular si exteriorul osului.n cazul lotului tratat cu o doz de bisfosfonat zona superficial a defectului este ocupat de esut osos primar, care tinde s nchid orificiul de comunicare cu exteriorul, iar peste el un esut conjunctiv care pare s participe activ la derularea proceselor reparatorii (conine cteva schie de trabecule). La lotul tratat cu 10 doze de bisfosfonat, zona superficial a defectului este ocupat de esut osos primar, care nchide comunicarea cu exteriorul. Grosimea acestui perete osos nou format este mai mare dect la lotul anterior, iar stadiul de organizare este mai avansat. Peretele osos proliferat n zona defectului este acoperit de un strat de esut conjunctiv, dispus n continuarea periostului. Acest esut conjunctiv particip activ la derularea proceselor reparatorii (conine schie de trabecule). Canalul medular este ocupat cu trabecule osoase aflate n plin proces de organizare. Rezultate la 12 sptmni de la nceperea experimentului: Examinarea microscopic a seciunilor seriate efectuate din zona de intervenie la 12 sptmni de la nceperea experimentului au evideniat procese reparatorii osoase aflate n stadiu evident mai avansat, la toate animalele luate n studiu. Trebuie menionat ns c ele se afl nc n plin derulare, ceea ce demonstreaz c, dei zona de intervenie este relativ bine consolidat, pn la finalizarea proceselor reparatorii trebuie s mai treac un anumit interval de timp. Exist diferene ntre loturile tratate ct i ntre acestea i lotul martor, n ceea ce privete stadiul n care au ajuns procesele de refacere i consolidare a osului din zona de intervenie i la 12 sptmni de la nceperea experimentului, dar ele sunt ceva mai reduse dect la 6 sptmni.La lotul martor, dup 12 sptmni de la nceperea experimentului, zona superficial a defectului este ocupat de esut osos primar, care nchide orificiul de comunicare cu exteriorul, iar canalul medular al osului este ocupat, n zona defectului, de os spongios (trabecular) relativ tnr.n cazul lotului tratat cu o doz de bisfosfonat zona superficial a defectului este ocupat de esut osos primar, care nchide orificiul de comunicare cu exteriorul, canalul medular este ocupat cu trabecule osoase aflate n plin proces de organizare, iar areolele dintre ele sunt ocupate de mduv hematogen.La lotul tratat cu 10 doze de bisfosfonat zona superficial a defectului este ocupat de esut osos primar, care nchide comunicarea cu exteriorul. Canalul medular este ocupat cu trabecule osoase aflate n plin proces de organizare iar areolele delimitate de ele conin mduv hematogen cu structur tipic. Concluzii: Bisfosfonatul a stimulat semnificativ procesul de vindecare osoas. Efectul stimulativ al bisfosfonatului a crescut cu numrul administrrilor. A crescut aria de esut osos nou sub influena bisfosfonatului, doza unic scurtnd perioada de vindecare osoas n comparaie cu administrrile multiple.

Studiul 3: Osteonecroza maxilarelor asociat cu utilizarea bisfosfonailor. Discuie pe baza a 52 de cazuri clinice

Introducere: Pe o perioad de patru ani (2007-2010) n Clinica de Chirurgie Cranio-Maxilofacial din Cluj-Napoca, au fost studiate o serie de cazuri de osteonecroz ale unor 7

pacieni diagnosticai cu tumori maligne i metastaze osoase. Tratamentul pe cale general a inclus administrarea de bisfosfonat, utilizat ca i agent chimioterapic. Osteonecroza maxilarelor asociat administrrii de bisfosfonai, descris ca os denudat, expus mediului oral, fr tendin la vindecare a fost diagnosticat la toi pacienii chimioterapizai. Material i metod: S-au studiat foile de observaie ale pacienilor cu osteonecroz a maxilarelor asociat tratamentului cu bisfosfonai internai n Clinica de Chirurgie Cranio-Maxilofacial din Cluj-Napoca n perioada 2007-2010. Pe o perioad de patru ani (2007-2010), au fost tratai 52 de pacieni cu osteonecroz a maxilarelor pe fond de tratament cu bisfosfonai. Tratamentul lor general a inclus utilizarea unui bisfosfonat ca agent chimioterapic (alendronat, ibandronat, zolendronat). Osteonecroza maxilarelor pe fond de tratament cu bisfosfonai a fost diagnosticat pe baza urmtoarelor criterii: prezena osului expus n cavitatea oral, denudat, rugos, aton, fr tendin de vindecare.Datele epidemiologice (sex, vrst, modul de administrare al bisfosfonatului, patologia asociat, tipul de bisfosfonat, tipul de tratament efectuat, timpul pn la apariia simptomelor n teritoriul maxilofacial) au fost prelucrate statistic utiliznd programul SPSS (SPSS Inc., Chicago, USA, 13.0 versiune pentru Windows). Procedurile i protocolul au fost aprobate de Comisia de Etic a Universitii de Medicin i Farmacie Iuliu Haieganu Cluj-Napoca, numr de certificat 172/2010. Rezultate: Din grupul studiat, 36 pacieni (69.2%) au fost de sex feminin i 16 (30.8%) de sex masculin. Vrsta medie a sexului feminin a fost de 61,61 ani (intervale de vrst: 32-87 ani, media: 64 ani, DS: 10,78). Vrsta medie a sexului masculin a fost de 69,5 ani (intervale de vrst: 30-81 ani, media: 73,5 ani, DS: 13,06). Dintre subiecii examinai, 6 pacieni au prezentat semne clinice de sinuzit cronic maxilar, avnd comunicare ntre cavitatea oral i cea sinuzal. Treizeci de pacieni (57.7%) au beneficiat de tratament cu bisfosfonat intravenos iar 22 pacieni (42.3%) au primit tratament cu bisfosfonat administrat pe cale oral. Tipul de bisfosfonat utilizat, patologia asociat care a indicat tratamentul cu bisfosfonat, timpul de tratament cu bisfosfonat, durata medie de tratament cu bisfosfonat pn la apariia simptomatologiei maxilofaciale, diagnosticul maxilofacial i tratamentul efectuat n sfera oro-maxilofacial au fost studiate i raportate. Tipul de bisfosfonat utilizat a fost acidul ibandronic (Bonviva) n 4 cazuri (7.69%), alendronate sodium (Fosamax) n 14 cazuri (26.92%) i acidul zoledronic (Zometa) n 34 cazuri (65.38%). Timpul mediu de tratament cu bisfosfonat a fost de 22,44 luni (95%CI 19.33 - 25.55), fiind 21,5 luni la femei (media=24 luni, interval 1-36 luni, DS=8,68) i 24,375 luni la sexul masculin (media=24 luni, interval 12-60 luni, DS=15,56). Timpul mediu de instalare a simptomatologiei oro-maxilofaciale dup tratamentul cu bisfosfonat a fost de 8,64 luni (95%CI 6.43 - 10.86), fiind 8,85 luni la femei (media=6 luni; interval 0.25-36 luni; DS=9.2) i 8,19 luni la brbai (media=6 luni; interval 1-12 luni; DS=4.18). Timpul mediu de tratament cu bisfosfonat a fost de 19,10 luni la subiecii care au primit bisfosfonatul pe cale intravenoas (DS=8.273) i 27 de luni la cei care au primit bisfosfonat pe cale oral (DS=13.060, p=0.018). Timpul mediu pn la apariia simptomatologiei maxilo-faciale a fost de 6 luni (interval 0,5-24 luni) la pacienii care au primit bisfosfonat pe cale intravenoas (SD=4.714) i 12,26 luni (interval 0.25-36 luni) la cei cu administrare oral (DS=9.973, p=0.011). Timpul mediu de tratament cu bisfosfonat a fost de 27 de luni la subiecii care au primit Bonviva (DS=6), 21,07 luni la cei care au primit Fosamax (DS=10.57) i 22,47 luni la cei care au primit Zometa (DS=11.92, p=0.2). Timpul mediu pn la apariia simptomatologiei oro-maxilofaciale a

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fost de 7,5 luni la subiecii care au primit Bonviva (SD=5.2), 11,36 luni la subiecii care au primit Fosamax (DS=12.15) i 7,67 luni la cei care au primit Zometa (DS=5.73). Raportul de implicare mandibul/maxilar a fost de 2/1. Simptomatologia n sfera oro-maxilofacial a inclus: durere n zona maxilarelor, os expus glbui-gri cu margini care se rulau spre interior, cu sau fr sechestrare, mobilitate dentar i dureri dentare, gingivit, infecii secundare, fistule cutanate, eritem, ulceraii, fistule cu secreii purulente, supuraii sau simptome care mimeaz o sinuzit maxilar cronic. Tratamentul chirurgical s-a aplicat pentru a ndeprta osul necrotic sechestrat, cu chiuretarea defectului n anestezie general, n combinaie cu terapie antimicrobian intravenoas. S-a efectuat examen histopatologic a osului rezecat pentru a stabili un diagnostic diferenial corect cu metastaze osoase sau tumori maligne. Defectul osos rezultat a fost nchis primar, n mai multe planuri. Defectele osoase mai mari au fost nchise utiliznd lambouri de vecintate rotate. Simptomatologia subiectiv i obiectiv s-a mbuntit la toi pacienii postoperator. Concluzii:

1. Osteonecroza maxilar indus de bisfosfonai administrai pe cale oral este rar, dar reprezint o entitate nou, care rspunde mai bine la tratament dect cea cu etiologia clasic. 2. Medicul curant care prescrie bisfosfonatul mpreun cu medicul dentist trebuie s monitorizeze pacienii n timpul tratamentului pentru a reduce riscul apariiei osteonecrozei maxilare asociat administrrii de bisfosfonat. 3. Riscul de apariie a osteonecrozei maxilare asociat administrrii de bisfosfonat este foarte mare la doisprezece luni dup administrarea oral i la ase luni dup administrarea intravenoas la pacienii care prezint un spin iritativ la nivelul maxilarelor (dini irecuperabili, extracii dentare, decubit protetic, manopere chirurgicale orale intempestive). 4. Avnd n vedere riscurile ce decurg din utilizarea medicaiei bisfosfonate, se impune o profilaxie riguroas.

CONCLUZII GENERALE1. Bisfosfonaii au fost asociai cu un risc crescut de apariie a osteonecrozei la maxilare. Bisfosfonaii sunt inhibitori puternici ai resorbtiei osoase, ai funciei i dezvoltrii osteoclastelor putnd altera mecanismul de turn-over osos normal.2. n fiecare etap de regenerare osoas bisfosfonaii induc, ntr-un mod coordonat, aciunea unor factori multipli, care duc la intensificarea proceselor metabolice locale, cu aciune att asupra proceselor osteoclazice ct i osteoblastice, permind o reparaie osoas, dac doza i calea de administrare sunt bine alese.3. Maxilarele sunt cele mai afectate oase, posibil datorit contactului ntre esutul osos i mediul bucal. Orice agresiune, traum, gingivit sau periodontit poate expune osul la microorganismele care cauzeaz infecia.4. Osteonecroza maxilar indus de bisfosfonai administrai pe cale oral este rar, dar reprezint o entitate nou, care rspunde mai bine la tratament dect cea indus de alte etiologii.5. Osteonecroza maxilar indus de bisfosfonai prezint o mare varietate imagistic la analiza CBCT i RMN, ajutnd la delimitarea extensiei bolii, dei nu reprezint o imagine caracteristic, specific acesteia. 6. Procedurile dentare i cele de chirurgie oral sunt factori cauzali n declanarea osteomielitei pe fondul osteonecrozei secundare tratamentului cu bisfosfonai. Aceast

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complicaie poate s apar ns i spontan, iar atunci cnd se ntmpl implic frecvent corticala lingual mandibular posterioar.7. Tratamentul profilactic al osteomielitei pe fondul tratamentului cu bisfosfonai, prin controlul focarelor de infecie dentar naintea nceperii tratamentului cu bisfosfonat, este cea mai eficient metod terapeutic.8. Medicul curant care prescrie bisfosfonatul mpreun cu medicul dentist trebuie s monitorizeze pacienii n timpul tratamentului pentru a reduce riscul apariiei osteonecrozei maxilare asociat administrrii de bisfosfonat. 9. Riscul de apariie a osteonecrozei maxilare asociat administrrii de bisfosfonat este foarte mare la doisprezece luni dup administrarea oral i la ase luni dup administrarea intravenoas la pacienii care prezint un spin iritativ la nivelul maxilarelor (dini irecuperabili, extracii dentare, decubit protetic, manopere chirurgicale orale intempestive). 10. Rezultatele obinute din studiile experimentale efectuate au relevat faptul c bisfosfonatul a stimulat semnificativ procesul de vindecare osoas din zona defectului osos experimental, vindecarea producndu-se cel puin cu 30% mai rapid la loturile experimentale comparativ cu lotul martor. 11. Referitor la numrul administrrilor, se recomand doza unic, dei zece administrri au avut un efect mai bun, diferenele nefiind att de mari nct s justifice necesarul de administrare a mai multor doze. O singur administrare este efectuat intraoperator, dup care defectul experimental este lsat s se vindece fr a mai fi perturbat. 12. Administrarea unic de bisfosfonat a condus la o reparaie osoas mai intens comparativ cu administrarea sa fracionat.13. Administrarea fracionat a bisfosfonatului de-a lungul unei perioade inhib osteoclazia semnificativ comparativ cu aceeai doz administrat n doz unic. 14. Procesele de reparaie osoas sunt periclitate n funcie de modalitatea de administrare i doza de bisfosfonai. 15. Bazat pe rezultatele acestui studiu, se recomand administrarea n doz unic a bisfosfonatului pentru ca avantajele administrrii produsului farmaceutic s fie maxime, iar efectele adverse minime. 16. Chiar dac beneficiile acestei terapii pot mbunti calitatea vieii pacienilor, pacienii trebuie monitorizai atent, deoarece sechelele acestui tratament pot fi grave. Educarea i formarea medicilor dentiti, a chirurgilor oro-maxilo-faciali ct i a oncologilor cu privire la aceast problem este de importan major. Este foarte important informarea i educarea pacienilor, privind precauiile pe care trebuie s le ia vis-a-vis de sntatea bucal i de faptul c trebuie s evite orice traumatism. 17. Medicul de medicin dentar are un rol definitoriu n managementul cazurilor de osteonecroz indus de bisfosfonai datorit faptului c o asanare anterior aplicrii chimioterapiei bisfosfonate duce la o prevenie a acestei patologii cu peste 90%. Dac aceast profilaxie nu poate s fie fcut, este de dorit ca orice manoper sngernd care ar putea s lase n urma ei os denudat s fie fcut sub protecie antibiotic i s se realizeze acoperirea osului cu un lambou mucozal. n condiii de spitalizare n clinici de chirurgie maxilofacial, la pacienii cunoscui sub tratament cu bisfosfonai, lucrrile dentare (reconstituiri prin lucrri conjuncte sau adjuncte) trebuie fcute la distan de esutul muco-gingival i osos.

ORIGINALITATEA I CONTRIBU IILE INOVATIVE ALE TEZEI 10

Lucrarea aduce un aport stiinific important, noutatea fiind sustinut de mai multe aspecte. In primul rand este realizat n premier naional investigarea pe baza a dou studii experimentale a unui algoritm de tratament profilactic i curativ al efectelor terapiei cu bisfosfonai asupra osului. n al doilea rnd a fost elaborat i un studiu clinic de conceptie personal al osteomielitei pe fond de osteonecroz maxilar n cursul tratamentului cu bisfosfonai. Subiectul este de actualitate n literatura de specialitate. Descrierea influenei adiminstrrii intramedulare de bisfosfonai asupra valorilor serice ale unor constante biologice care monitorizeaz fidel procesele de formare osoas constituie de asemenea premier naional. Elemente valoroase de apreciere a vindecrii locale sunt oferite i de studiul caracteristicilor histopatologice dup administrarea medicaiei bisfosfonate asupra defectelor osoase create experimental, contribuind la nelegerea mai aprofundat a etiologiei, patogeniei, clinicii, profilaxei i tratamentului osteonecrozei maxilare la pacienii tratai cu bisfosfonai. Studiul clinic dorete elaborarea unui algoritm pentru terapia osteomielitei pe fond de osteonecroz maxilar n cursul tratamentului cu bisfosfonai i ntocmirea unui material ghid privitor la etiologia, patogenia, clinica, profilaxia i tratamentul osteonecrozei maxilare la pacienii tratai cu bisfosfonai, util n practica curent. Dezbaterea public a rezultatelor studiilor va asigura perfecionarea n domeniu a tuturor clinicienilor implicai n ngrijirea acestor bolnavi: oncologi, endocrinologi, medici interniti, dentiti, chirurgi maxilofaciali.

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PhD Thesis

Experimental and clinical research of bisphosphonate-associated jaw osteonecrosis

Abstract

PhD Candidate Horea Artimoniu Alman

Scientific supervisor Prof. Dr. Grigore Bciu

Cluj-Napoca, 2013

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CONTENTSINTRODUCTION 13ACTUAL STATE OF KNOWLEDGE 151. THE HISTORY OF BISPHOSPHONATES 192. THE STRUCTURE OF BISPHOSPHONATES 193. THE COURSE OF ACTION OF BISPHOSPHONATES 203.1 BONE RESORBTION 203.2 THE ACTION OF BISPHOSPHONATES ON THE CALCIFICATION PROCESSES 203.3 THE CELLULAR MECHANISM OF ACTION OF THE BISPHOSPHONATES 213.4 THE CLINICAL USE OF BISPHOSPHONATES 214. THE THERAPY WITH BISPHOSPHONATES 224.1 THERAPY BENEFITS 224.2 THERAPY RISKS 225. SIDE EFFECTS OF BISPHOSPHONATES 226. CONCLUSIONS ABOUT BISPHOSPHONATES 247. TREATMENT OF OSTEOPOROSIS FOLLOWING BISPHOSPHONATES 248. CLINICAL AND PARACLINICAL ASPECTS OF JAW OSTEONECROSIS 269. THE INCIDENCE OF OSTEONECROSIS 2810. THE TREATMENT OF THE OSTEONECROSIS OF THE JAWS 28

PERSONAL RESEARCH 37THE WORKING HYPOTHESIS 37STUDY1 - SERUM CHANGES INDUCED BY INTRAMEDULLAR EXPERIMENTAL ADMINISTRATION OF BISPHOSPHONATES 411.1 INTRODUCION 411.2 MATERIAL AND METHODS 421.3 RESULTS 451.4 DISCUSSION 501.5 CONCLUSIONS 54STUDY 2 - INDUCED EFFECTS OF BISPHOSPHONATES AT 6 AND 12 WEEKS ON THE FEMORAL BONE STRUCTURE IN RATS 552.1 INTRODUCTION 552.2 MATERIAL AND METHODS 552.3 RESULTS 562.4 DISCUSSION 732.5 CONCLUSIONS 80STUDY 3 - OSTEONECROSIS OF THE JAWS ASSOCIATED WITH THE USE OF BISPHOSPHONATES. DISCUSSION OVER 52 CASES 813.1 INTRODUCTION 813.2 MATERIAL AND METHODS 813.3 RESULTS 823.4 CLINICAL OBSERVATION 923.5 DISCUSSION 1043.6 THE IMPLICATIONS OF BISPHOSPHONATES IN DENTISTRY 1053.7 CONCLUSIONS 106GENERAL CONCLUSIONS 109ORIGINAL ELEMENTS AND INNOVATIVE CONTRIBUTIONS OF THE THESIS 111REFERENCES 112

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Key words: bisphosphonate, jaw osteonecrosis, bone regeneration.

IntroductionThe success of bone regeneration raises many issues to clinicians. Effective and safe approach of bone resonstruction is needed to compensate for bone loss caused by trauma, surgical ablation, periodontal disease, congenital defects, deformities. The knowledge of bone regeneration is at an early stage, the current treatment aproaches being seriously limited. In maxillofacial surgery it is of clinical importance to assess each case individually in order to decide if bone stimulation can occur after complex surgical procedures involving bone resection or osteotomies. Scientific basis of the research problem

Osteoporosis is a major public health problem. Oral bisphosphonates are effective for reducing the risk of osteoporotic fractures and are an important treatment option for patients at risk of developing osteoporosis.Despite the positive effects of of this class of compounds, there are also negative effects which can occure after a period from the start of the administration. One of the most important side effects is osteonecrosis of the jaws which often turns into osteomyelitis in patients with bisphosphonates therapy and dental foci. Osteomyelitis occurs after tooth extraction, dento-alveolar surgery or trauma that cause exposure of the bone into the oral cavity. The disease is caracterized by poor symptom onset, which is why bone lesions progress rapidly. Bone exposure is painful and there is an infectious risk even after the end of the therapy with bisphosphonates. A detailed dental control is recommended before starting treatment with bisphosphonates. The second major class of disease in which bisphosphonates are used are malignant tumors, especially in the phase of bone metastases. Due to increased frequency of bone metastases and due to the beneficial effects of bisphosphonates in treating malignant bone disease, and also non-malignant diseases (osteoporosis), more patients will benefit from this type of treatment and more clinicians will be confronted with this pathology. Therefore, to prevent adverse effects of bisphosphonates we have to understand very well the pathogenesis of osteonecrosis. If there is an association between osteonecrosis and bisphosphonates, then disease prevalence is expected to be growing since the use of bisphosphonates increases, both in patients with malignant and non-malignant diseases.Recent studies report complications such as jaw osteonecrosis after intravenous and oral bisphosphonates administration. Osteonecrosis of the jaws occurs in patients with malignant tumors treated with high doses of intravenous bisphosphonates and in patients treated with intravenous and oral bisphosphonates for osteoporosis. Bisphosphonates are stable analogues of pyrophosphates. Bisphosphonates bind to the hydroxyapatite of the mineral surfaces and are selectively internalized by osteoclasts, whose activity they inhibit. Once the osteoclasitc activity is inhibited it will have an impact on the osteoblaststic activity. Osteoblasts will not be activated in the sites of bone repair, due to the lack of bone resorption. This will endanger the balance of bone resorption/bone repair of the bone defect. This imbalance will lead to a vulnerability of the bone and to susceptibility to microfractures, lacking of an adequate bone repair. Endangering of the balance of bone resorbtion/bone repair may lead to aseptic bone necrosis. Bisphosphonates are excellent inhibitors of bone resorption with variable potency according to the chemical structure.

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Treatment with these compounds changes resorption depending on the dose. Bisphosphonates inhibit tumor cell adhesion and spreading.Intravenous bisphosphonates were firstly used, proving its effectiveness in the treatment of cancer. The latter include malignant hypercalcemia, bone metastases, breast cancer, prostate cancer, lung cancer and multiple myeloma. Intravenous bisphosphonates are effective in preventing and reducing hypercalcemia, bone lesions stabilization and prevention of fractures. Oral bisphosphonates have been approved in treating osteoporosis and are commonly used in treatment of osteopenia. They are used for a variety of conditions such as osteogenesis imperfecta and Paget's disease. But the most common indication is represented by osteoporosis. Therapy risks: In 2003 and 2004 oro-maxillofacial surgeons were the first clinicians to recognize and report cases of exposed, necrotic bone in the maxillo-facial region in patients receiving intravenous bisphosphonates.Characteristics at the oral cavity tissues: bone and tooth loss are associated with estrogen deficiency and osteoporosis. Resorptions of the alveolar ridge in edentulous patients, worsening of periodontal disease and tooth loss were recorded in patients with osteoporosis. Women who are treated with bisphosphonates for osteoporosis have increased risk of developing osteonecrosis of the jaws, secondary to treatment with bisphosphonates.The role of the dentist and of the maxillofacial surgeon is essential in preventing osteonecrosis due to therapy with bisphosphonates by careful and proper monitoring of oro-dental foci. Not less important is the role of the oncologist, who needs to guide each patient to the dentist for proper evaluation and cure of dental foci before beginning any treatment with bisphosphonates.Starting from the idea that the bone repair processes involves both anabolic and catabolic responses, the aim of this study is to assess the effects of administering bisphosphonates (zoledronic acid) on bone.

Justification of the results utility Potential adverse effects of bisphosphonates should be considered in the context of each patient and dose and method of administration established when selecting a bisphosphonate for treatment of certain diseases. There is a relation between the size of exposed bone defect and duration of treatment with bisphosphonates. Studies have shown a correlation between duration of treatment with bisphosphonates and bone destruction as well as bone remodeling with treatment disruption.The aims of this thesis are: (1) to design, based on two experimental studies, a treatment algorithm for prophilactic and curative treatment of the complications of the bisphosphonates therapy upon bone and the development of a clincal study for improving the treatment of the osteomyelitis during treatment with bisphosphonates and (2) to to review the literature in order to formulate a comprehensive material on the etiology, pathogenesis, clinical aspects, prevention and treatment of osteonecrosis of the jaws in patients treated with bisphosphonates in order to be used by dentists in their current practice.PERSONAL CONTRIBUTION of the thesis is included in three studies (two experimental and one clinical). The experimental studies describe the influence of intramedullary administration of bisphosphonates on serum constants that closely monitor bone formation processes and the description of the histopathology after bisphosphonate medication in experimentally created 15

bone defects. The clinical trial intends to describe an algorithm for osteomyelitis therapy of the jaws during therapy with bisphosphonates and preparation of "guide" regarding the etiology, pathogenesis, clinical aspects, prevention and treatment of osteonecrosis of the jaws in patients treated with bisphosphonates, useful for clinical practice.Study 1: Serum changes induced by intramedullar experimental administration of bisphosphonates

Introduction: The aim of the study was to to determine serum alkaline phosphatase, proteins, total calcium, Ca2+ and osteocalcin in a group of rats Wistar, who underwent a bone defect in the femur, which was inserted with a product based on bisphosphonates (Zometa). Material and methods: The study group was composed of 15 Wistar rats, male, six weeks old, weighing 100200 g. The rats were divided into a control group of five rats and an experimental group of 10 rats. Wistar rats were kept for acclimatization in the biobase for a week before the start of the experiment. All rats underwent a right femoral bone defect. After obtaining an aseptic operative field, we performed a line of incision in the thigh; plans were off to reveal skin and muscular and femur. Under continuous cooling, bone notching was performed using a 1.5 mm diameter bone-bur. An intra-medullar hole at 0.5 cm from the thighbone joint was drilled, in which bisphosphonate was injected to the experimental groups (Zometa). Group A received a single dose of 1 mL bisphosphonate (Zometa) intramedullar, intraoperatively. Group B received 1 mL of bisphosphonate (Zometa) in divided doses, 0.1 mL daily for 10 days. From each subject 3 mL of blood were collected from the frontal sinus, preoperative (T0 time), after 14 days (T1 time) and after 21 days (T2 time) postoperatively. The wound was closed in two planes (muscle and skin). There have been determinations of blood protein serum [g/dL], alkaline phosphatase [U/L], total Ca [mmol/L], total Ca [mg/dL], Ca2+ [mmol/L] and osteocalcin [ng/mL] at time T0 (intraoperative), time T1 (14 days after intervention) and at time T2 (21 days after surgery). From the blood samples alkaline phosphatase [U/L], serum proteins [g/dL], total Ca [mmol/L, mg/dL], Ca2+ [mmol/L] levels were processed. For determination of rat osteocalcin ELISA kit was used. The data were statistically analyzed using the ANOVA test. The research protocol was approved by the Ethics Committee of the University, according to the principles of the Declaration of Helsinki.Results: Following the evolution of alkaline phosphatase in the control group, its growth has occurred between the time T0 and T1 and then decreases between the time T1 and T2, but not below the baseline. Evolution of alkaline phosphatase in the experimental groups A and B was different from that of the control group, increasing the time between T0 and T1 values and decreasing the time between T1 and T2, but at the time of T2 values are below the baseline T0. Both proteins decreased in the control group and the experimental group A and B from the time T0 to time T1 and T2, to values below the baseline. The values of total calcium [mmol/L] in experimental groups decreased from time T0 to time T1, and from time T1 to T2, final values falling below baseline. Evolution of osteocalcin in the both experimental groups showed a difference between groups. Between T0 and T1 there were both a decrease and an increase in groups A and B. In T2 values were higher than in T0, especially in group B.

Conclusions1. Single administration of a bisphosphonate has shown increased bone repair compared with fractional administration.2. Fractioned administration of bisphosphonates over a certain period significantly inhibits bone resorption compared with the same dose administered as a single dose.16

3. Bone repair processes are compromised directly proportional to bisphosphonate pattern and administration dose.4. Based on the results of this study, we recommend administration of a single dose of bisphosphonates for maximum advantages and minimum adverse effects.5. At each stage of bone development act in a coordinated manner multiple factors that lead to the intensification of the local metabolic processes. They influence both osteoclastic and osteoblastic activity allowing bone repair if the dose and route of administration are well chosen.

Study 2 - Histological changes in the femural bone structure induced at six and twelve weeks by experimental administration of bisphosphonates in rats Introduction: The aims of this study were (1) to describe the histopathologic features at six and twelve weeks after bisphosphonate administration in a rat model and (2) to identify the effects of bisphosphonate upon bone remodelling.

Material and methods: Following approval from the Institutional Ethical Committee (the procedures and protocol were approved by the institutional review board at the university and by the Ethics Committee, certificate number 172/2010); we selected fifteen Wistar rats of experience, five in the control group and ten in the experimental group. All rats underwent surgery to create a bone defect with a 1.5 mm diameter bone-bur at the right femur transcortical through the Haversian canal. Rats from experimental group were divided into two subgroups: group A, who received zoledronic acid, a common used bisphosphonate on the jaw bone, 1 ml single dose in the bone marrow, during surgery, and group B, who received zoledronic acid 1 ml in divided doses daily, 0.1 ml for 10 days. The control group did not receive any bisphosphonate. All samples were subjected to optic microscopic (Olympus BX41) examination at six and twelve weeks to evaluate differences in bone structure.Results: Results at 6 weeks from the beginning of the experiment: Microscopic examination of serial sections made from the intervention at 6 weeks after starting the experiment showed bone reparative processes underway in both the experimental and control group (experimental bone defect, untreated). But regarding the stage in which the reparative processes reached the point of harvest, there were relatively large differences between treatment groups and between them and the control group.In the control group at 6 weeks of the start of the experiment, the surface region of the defect was occupied by primary bone, which tends to close communication with the outside hole. Bone located at this level comes from active proliferation of bone that has as its starting point near the periosteum defect, and from here extends both sides (a certain distance with wall thickening), and especially in the area where the defect tends to occupy surface region to interrupt the continuity of the medullary canal and exterior bone.In the group treated with a dose of bisphosphonate the surface region of the defect was occupied by primary bone, which tended to close communication with the outside hole and connective tissue upon him seemed to actively participate in the reparative processes (contained several drafts of trabeculae).In the group treated with 10 doses of bisphosphonate, the surface region of the defect was occupied by primary bone, which closes communication with the outside. This newly formed bone wall thickness is greater than that of the previous group, and is in a more advanced stage of organization. Proliferated bone the defect is covered by a layer of connective tissue, ordered to continue periosteum. This tissue participated actively in the reparative processes (contains sketches of trabeculae). The medullary canal was filled with bone trabeculae. 17

Results at 12 weeks from the beginning of the experiment: Microscopic examination of serial sections of the intervention performed at twelve weeks after the start of the experiment showed bone repair processes in all studied animals. These were still developing which showed that the intervention area was relatively well consolidated. Pending completion of the repair process, a certain period of time must pass. There were differences between the experimental groups and between them and the control group, regarding the stages reached by the recovery and consolidation processes of bone repair from of the intervention area. In the control group, the surface area of the defect was occupied by primary bone, which closed the communication with the exterior and the Haversian canal was occupied in the defect area by relatively young spongious bone. The junction area between the surface of the bone defect and the new formed bone could be assessed quite well. In the experimental group treated with a dose of bisphosphonate the surface area of the defect was occupied by primary bone that closed the communication with the exterior, and the Haversian canal was occupied by bone trabeculae in process of bone restructuring, the areola between them being occupied by bone marrow. In the experimental group treated with ten doses of bisphosphonates the surface area of the bone defect was occupied by the primary bone, which closed the external communication. Haversian canal was filled with bone trabeculae being in process of repair and areola separated from them contained bone marrow with typical structure. Conclusions: Bisphosphonate had a positive influence upon bone formation. The stimulatory effect of the bisphosphonate increased with the number of administered doses. New bone area increased under the influence of bisphosphonate, a single administration shortening the healing bone period compared to multiple dose administration.

Study 3: Osteonecrosis of the jaws associated with the use of bisphosphonates. Discussion over 52 cases

Introduction: During a period of four years (20072010), 52 cases of patients with malignant tumors or osteoporosis and bisphosphonate associated osteonecrosis of the jaws have been treated in our Clinic of Cranio-Maxillofacial Surgery. Their general treatment included the use of a bisphosphonate as a chemotherapeutic agent. The bisphosphonate related osteonecrosis of the jaws has been diagnosed according to the presence of bare bone, exposed to the oral environment without healing tendency. Material and methods: The files of all patients with a bisphosphonate related osteonecrosis of the jaws of the Clinic of Cranio-Maxillofacial Surgery from Cluj-Napoca were reviewed. During a period of four years (20072010), 52 cases of patients with malignant tumors or osteoporosis and bisphosphonate associated osteonecrosis of the jaws have been treated in our Clinic of Cranio-Maxillofacial Surgery. Their general treatment included the use of a bisphosphonate as a chemotherapeutic agent. The bisphosphonate related osteonecrosis of the jaws has been diagnosed according to the presence of bare bone, exposed to the oral environment without healing tendency. The data obtained were statistically processed using Statistical Package for the Social Sciences (SPSS Inc., Chicago, USA, 13.0 version for Windows). The procedures and protocol were approved by the institutional review board at the university and by the Ethics Committee, certificate number 172/2010.Results: Thirty-six patients (69.2%) were females and 16 (30.8%) were males. The average age of females was 61.61 years (age range 3287 years, median 64 years, SD=10.78). The average age of males was 69.5 years (age range 3081 years, median 73.5 years, SD=13.06). 18

Of these, six had clinical signs of maxillary chronic sinusitis, having variable communication between the oral cavity and the maxillary sinus. Thirty patients (57.7%) received an intravenous bisphosphonate and 22 patients (42.3%) received an oral bisphosphonate. The bisphosphonate type, the condition requiring bisphosphonate treatment, the treatment duration with bisphosphonates, the treatment duration until onset of maxillofacial symptoms, the cranio-maxillofacial diagnosis and the cranio-maxillofacial treatment have been studied and reported. Bisphosphonate type used was ibandronic acid (Bonviva) in four cases (7.69%), alendronate sodium (Fosamax) in 14 cases (26.92%) and zoledronic acid (Zometa) in 34 cases (65.38%). The mean bisphosphonate treatment period was 22.44 months (95%CI 19.33 to 25.55), being 21.5 months in females (median 24 months; range 136 months; SD=8.68) and 24.375 months in males (median 24 months; range 1260 months; SD=15.56). The mean time until maxillofacial accuse after the bisphosphonate treatment was 8.64 months (95%CI 6.43 to 10.86), being 8.85 months in females (median 6 months; range 0.2536 months; SD=9.2) and 8.19 months in males (median 6 months; range 112 months; SD=4.18). The mean bisphosphonate treatment period was 19.1 months in subjects with intravenous bisphosphonate administration (SD=8.273) and 27 months in subjects who received bisphosphonates orally (SD=13.060, p=0.018).The average period until occurrence of maxillofacial symptoms was 6 months (range 0.524 months) in subjects with intravenous bisphosphonate administration (SD=4.714) and 12.26 months (range 0.2536 months) in subjects who received bisphosphonates orally (SD=9.973, p=0.011). The mean bisphosphonate treatment period was 27 months in subjects who received Bonviva (SD=6), 21.07 months in subjects who received Fosamax (SD=10.57) and 22.47 months in subjects who received Zometa (SD 11.92, p=0.2). The average period until occurrence of maxillofacial symptoms was 7.5 months in subjects who received Bonviva (SD=5.2), 11.36 months in subjects who received Fosamax (SD=12.15) and 7.67 months in subjects who received Zometa (SD=5.73). The ratio of jaw involvement was mandible/maxilla 2/1.Presenting symptoms included pain in the jaws area, exposed yellow-white bone with margins that vary from smooth to ragged with or without sequestration, tooth mobility and tooth pain, gingivitis, secondary infection of lesion sites, cutaneous fistulae, erythema and ulceration, fistula or purulent discharge, bulge that suggested a dental abscess, suppuration or symptoms that suggested a chronic maxillary sinusitis.Surgery was performed in order to remove the necrotic bone after sequestration with curettage of the defect under general anesthesia, in combination with intravenous antimicrobial therapy. Histopathological examination was performed to the resected bone in order to establish proper differential diagnosis with malignant tumor or distance metastases. The resulting bone defect was closed primarily, in layers. Broad substance defects were closed using proximity rotated flaps. Subjective and objective symptoms improved in all patients postoperatively.Conclusions: 1. Osteonecrosis of the jaws induced by orally administered bisphosphonates is rare, but represents a new entity, which responds better to treatment than that of classical etiology.2. The bisphosphonates prescribing practitioner and the dentist should monitor patients during and after treatment to reduce the risk of bisphosphonate related osteonecrosis of the jaws. 19

3. The risk of bisphosphonate related osteonecrosis of the jaws is very high twelve months after oral administration and six months after intravenous administration in patients who have a trigger point in the jaws area (irrecoverable teeth foci, dental extractions, prosthetic sores, and insensate surgical manipulations). 4. A proper monitoring of patients with risk of osteonecrosis can prevent complications and treat with curative intent osteomyelitis in the early stages.GENERAL CONCLUSIONS

1. Bisphosphonates have been associated with an increased risk of osteonecrosis of the jaws. They are potent inhibitors of bone resorption, osteoclast function and development and can alter the normal mechanism of bone turn-over. 2. At each stage of bone regeneration, bisphosphonates induce, in a coordinated manner, the action of multiple factors that can increase local metabolic processes, acting both on osteoclasic and osteoblastic processes, allowing bone repair, if the dose and route of administration are well chosen. 3. The jaw bones are the most affected, possibly due to contact between the bone and the oral environment. Any aggression, trauma, gingivitis or periodontitis can expose the bone micro-organisms that cause infection. 4. Osteonecrosis of the jaws induced by orally administered bisphosphonates is rare, but is a "new" entity which responds better to treatment than that induced by other etiologies.5. Osteonecrosis of the jaws induced by bisphosphonates has a wide variety of CBCT and MRI imaging, helping to delimit the extension of the disease, although is has not a characteristic image.6. Dental procedures and oral surgery are causal factors in triggering secondary osteomyelitis due to treatment with bisphosphonates. This complication may occur spontaneously, however, and when it happens it often involves the posterior mandibular lingual area.7. Prophylactic treatment of osteomyelitis due to treatment with bisphosphonates, by the dental infection control before starting bisphosphonates treatment, is the most effective therapeutic method.8. The physician prescribing bisphosphonates together with the dentist have to monitor patients during treatment to reduce the risk of bisphosphonates associated osteonecrosis of the jaws.9. The risk of bisphosphonate related osteonecrosis of the jaws is very high twelve months after oral administration and six months after intravenous administration in patients who have a trigger point in the jaws area (irrecoverable teeth foci, dental extractions, prosthetic sores, and insensate surgical manipulations). 10.The results of the performed experimental studies have revealed that bisphosphonates significantly stimulated the healing process of the bone defect healing occurring at least 30% faster in the experimental groups than control group. 11.Bisphosphonate had a positive influence upon bone formation. The stimulatory effect of the bisphosphonate increased with the number of administered doses. New bone area increased under the influence of bisphosphonate, a single administration shortening the healing bone period compared to multiple dose administration. 12.Single administration of bisphosphonate has lead to a more intense bone repair than fractional admonistration.

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13.Fractionated administration of bisphosphonates over a period significantly inhibits bone resprbtion compared with the same dose as a single dose. 14. Bone repair processes are endangered depending on the way and dose administration of bisphosphonates.15.Based on the results of this study, we recommend a single dose administration of the bisphosphonate, so that pharmaceutic advantages of the product to be maximal and side effects minimal. 16.Even if the benefits of this therapy can improve the quality of life of these patients, they should be closely monitored as sequelae of this treatment can be severe. Education and training of dentists, oral-maxillofacial surgeons and oncologists on this issue is of major importance. It is very important to inform and educate patients regarding the precautions to be taken regarding oral health and about the fact that they have to avoid any injury. 17. The dentist has a huge role in the management of cases of bisphosphonates related osteonecrosis of the jaws, because an early drainage before the bisphosphonate therapy may lead to a prevention of this disease by over 90%. If this prophylaxis can not be made, it is desirable that any bleeding procedure that may result in bare bone to be done with antibiotic protection and to achieve bone coverage with a mucosal flap. In terms of hospitalization in maxillofacial surgery clinic, in known patients with bisphosphonates treatment, dental procedures should be made away from the muco-gingival tissue and bone.

ORIGINAL ELEMENTS AND INNOVATIVE CONTRIBUTIONS OF THE THESISThe work brings important scientific contribution, the novelty being supported by several aspects. Firstly, the investigation, based on two experimental studies of the prophilactic and curative treatment algorithm of the effects of bisphosphonates upon bone is a national premiere. Secondly, it has been developed a clinical study, based on personal conception of the osteomielytis of the jaws during treatment with bisphosphonates. The subject is very actual. Description of the influence of the intramedullary administration of bisphosphonates upon biologic serum constants that closely monitor biological processes of bone formation is also a national premiere. Valuable items of appreciation of the local healing are offered also by the study of the histological characteristics after bisphosphonates administration in experimental bone defects. This contributes to a deeper understanding of the etiology, pathogenesis, clinic, prophylaxis and treatment of osteonecrosis of the jaw in patients treated with bisphosphonates. The clinical trial aims to elaboreate an algorithm for treating osteomyelitis of the jaws associated with bisphosphonate therapy and to prepare a "guide" regarding the etiology, pathogenesis, clinical, prevention and treatment of osteonecrosis of the jaws in patients treated with bisphosphonates useful in daily practice.Public debate of the results of the studies in this field will ensure improvement of all clinicians involved in the care of these patients: oncologists, endocrinologists, internists, dentists, maxillofacial surgeons.

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