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Made available in DSpace on 2014-06-11T19:31:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-04Bitstream added on 2014-06-13T20:02:12Z : No. of bitstreams: 1 paulo_ao_dr_arafo.pdf: 1640675 bytes, checksum: 92907baf8888322bc6931c68024509f2 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo deste estudo foi avaliar a associação de células derivadas de periósteo autógeno (CDPA) e xenoenxerto de hidroxiapatita e colágeno I (HA/Col) no reparo de defeito crítico de crânio de ratos. CDPA de 10 ratos Wistar foram semeadas na densidade de 1,0x106 células sobre discos de HA/Col (8x2mm) em DMEM:HAMF12 com 10% soro fetal bovino e dexametazona, ácido ascórbico e glicerofosfato por 6 dias. Ensaio funcional comparou efeito de coágulo sanguíneo (G1), osso autógeno (G2), HA/Col (G3) e HA/Col+CDPA (G4) preenchendo defeito de 8 mm de crânio de ratos (n=40) em 1 e 3 meses. A análise radiográfica não exibiu variação temporal, tendo G1 e G2 discreto novo osso marginal; radiopacidade dos materiais em G2, G3 e G4 impediu confirmar osteogênese central. A análise histológica dos grupos mostrou em G1 tecido conjuntivo denso e ilhas de ossificação (1-3m), em G2 coalescência do material e osteogênese (1-3m), em G3 ossificação intramembranosa (1m) e novo osso homogêneo ao redor e substituindo HA (3m) e em G4 abundante tecido conjuntivo frouxo permeando material (1m) e novo osso heterogêneo (3m); não houve necrose, inflamação crônica ou exuberância de células gigantes tipo corpo estranho. As análises histomorfométrica e estatística mostraram diferença signif icativa (p<0,05) para biomaterial (1m) de G2 (23,3%) com G3 (44,6%) e G4 (47,5%) e para ganho ósseo (3m) de G2 (10,9%) com G1 (4,6%) e G4 (5,1%), tendo G3 (8,5%) neoformação óssea próxima a G2. Conclui-se que CDPA não aumentam formação óssea se associadas a HA/Col. / The aim of this study was evaluate the association of autogenous periosteum derived cells (APDC) and xenograf t made up hydroxyapatite and collagen I (HA/Col) in repair of critical size defect in rat calvaria. APDC of 10 Wistar rats were seeded with density of 1,0x106 cells above discs of HA/Col (8x2mm) in DMEM:HAMF12 with 10% fetal bovine serum and dexamethasone, ascorbic acid and glicerophosphate until 6 days. Functional assay compared ef fect of blood clot (G1), autogenous bone (G2), HA/Col (G3) and HA/Col+CDPA (G4) f illing the 8mm-defect in rat skull (n=40) at 1 and 3 months. Radiographic analysis did´t have time variation, with G1 and G2 showing mild peripheral new bone; radiopacity of materials in G2, G3 and G4 didn’t enable to conf irm central osteogenesis. Histologic analysis of groups showed in G1 dense connective tissue and bone islets (1-3m), in G2 fusion of material and osteogenesis (1-3m), in G3 intramembranous ossification (1m) and homogeneous new bone around and substitute HA (3m) and in G4 abundant loose connective tissue permeating the material (1m) and heterogeneous new bone (3m); there is not necrosis, chronic inf lammation or exuberance of giant cell like foreign body reaction. Histomorphometric and statistic analysis showed signif icative dif ferences (p<0.05) for biomaterial (1m) f rom G2 (23,3%) to G3 (44,6%) and G4 (47,5%) and for bone gain (3m) f rom G2 (10,9%) to G1 (4,6%) and G4 (5,1%), with G3 (8,5%) showing new bone formation closer to G2. It’s possible to conclude that APDC don’t increase bone formation if associate to HA/Col.

Resumo: O objetivo deste estudo foi avaliar a associação de células derivadas de periósteo autógeno (CDPA) e xenoenxerto de hidroxiapatita e colágeno I (HA/Col) no reparo de defeito crítico de crânio de ratos. CDPA de 10 ratos Wistar foram semeadas na densidade de 1,0x106 células sobre discos de HA/Col (8x2mm) em DMEM:HAMF12 com 10% soro fetal bovino e dexametazona, ácido ascórbico e glicerofosfato por 6 dias. Ensaio funcional comparou efeito de coágulo sanguíneo (G1), osso autógeno (G2), HA/Col (G3) e HA/Col+CDPA (G4) preenchendo defeito de 8 mm de crânio de ratos (n=40) em 1 e 3 meses. A análise radiográfica não exibiu variação temporal, tendo G1 e G2 discreto novo osso marginal; radiopacidade dos materiais em G2, G3 e G4 impediu confirmar osteogênese central. A análise histológica dos grupos mostrou em G1 tecido conjuntivo denso e ilhas de ossificação (1-3m), em G2 coalescência do material e osteogênese (1-3m), em G3 ossificação intramembranosa (1m) e novo osso homogêneo ao redor e substituindo HA (3m) e em G4 abundante tecido conjuntivo frouxo permeando material (1m) e novo osso heterogêneo (3m); não houve necrose, inflamação crônica ou exuberância de células gigantes tipo corpo estranho. As análises histomorfométrica e estatística mostraram diferença signif icativa (p<0,05) para biomaterial (1m) de G2 (23,3%) com G3 (44,6%) e G4 (47,5%) e para ganho ósseo (3m) de G2 (10,9%) com G1 (4,6%) e G4 (5,1%), tendo G3 (8,5%) neoformação óssea próxima a G2. Conclui-se que CDPA não aumentam formação óssea se associadas a HA/Col. / Abstract: The aim of this study was evaluate the association of autogenous periosteum derived cells (APDC) and xenograf t made up hydroxyapatite and collagen I (HA/Col) in repair of critical size defect in rat calvaria. APDC of 10 Wistar rats were seeded with density of 1,0x106 cells above discs of HA/Col (8x2mm) in DMEM:HAMF12 with 10% fetal bovine serum and dexamethasone, ascorbic acid and glicerophosphate until 6 days. Functional assay compared ef fect of blood clot (G1), autogenous bone (G2), HA/Col (G3) and HA/Col+CDPA (G4) f illing the 8mm-defect in rat skull (n=40) at 1 and 3 months. Radiographic analysis did't have time variation, with G1 and G2 showing mild peripheral new bone; radiopacity of materials in G2, G3 and G4 didn't enable to conf irm central osteogenesis. Histologic analysis of groups showed in G1 dense connective tissue and bone islets (1-3m), in G2 fusion of material and osteogenesis (1-3m), in G3 intramembranous ossification (1m) and homogeneous new bone around and substitute HA (3m) and in G4 abundant loose connective tissue permeating the material (1m) and heterogeneous new bone (3m); there is not necrosis, chronic inf lammation or exuberance of giant cell like foreign body reaction. Histomorphometric and statistic analysis showed signif icative dif ferences (p<0.05) for biomaterial (1m) f rom G2 (23,3%) to G3 (44,6%) and G4 (47,5%) and for bone gain (3m) f rom G2 (10,9%) to G1 (4,6%) and G4 (5,1%), with G3 (8,5%) showing new bone formation closer to G2. It's possible to conclude that APDC don't increase bone formation if associate to HA/Col. / Orientador: Idomeo Bonetti Filho / Coorientador: José Mauro Granjeiro / Banca: Mário Tanomaru Filho / Banca: Fábio Luiz Camargo Villela Berbert / Banca: Manoel Eduardo de Lima Machado / Banca: Arlindo Di Spagna Souza / Doutor

L'objectif de ce travail de thèse est de dégager des méthodes et des outils permettant de mieux comprendre le rôle joué par les phénomènes de transport (cellulaire, hydraulique et chimique) dans la mise au point de stratégies thérapeutiques de réparation osseuse. Pour cela, nous avons choisi d'associer deux approches : la réalisation d'études expérimentales et la mise au point de modèles numériques. Nous avons ainsi pu, lors d'une première étude présentée dans le chapitre 2 de ce document, relier la perméabilité intrinsèque d'un milieu poreux, paramètre déterminant dans l'étude du transport de fluide en son sein, à la structure géométrique de ses pores. Nous avons également mis en évidence l'importance des interactions électrochimiques lors de la progression d'une solution ionique (telle que les fluides physiologiques) à travers le tissu osseux, en raison de la structure poreuse et de la composition chimique (présence de fibres de collagènes chargées par exemple) de ce dernier. Ces outils ont ensuite permis d'analyser, en première approche, les résultats expérimentaux obtenus lors de la réalisation de tests de perméabilité sur des échantillons de périoste fémoral ovin, dans le but d'identifier les phénomènes physico-chimiques à l'origine du comportement particulier de cette membrane (chapitre 5). Nous nous sommes par ailleurs intéressés au développement d'implants osseux associant un substrat minéral biocompatible et des cellules souches mésenchymateuses, afin de favoriser une reconstruction tissulaire en volume des lésions de grande taille. Nous avons ainsi pu mettre en place, dans le chapitre 3, un dispositif expérimental permettant de réaliser de manière reproductible un test d'ensemencement cellulaire et d'évaluer le nombre, la répartition et le taux de viabilité des cellules greffées sur le biomatériau utilisé. A partir des résultats expérimentaux issus des tests d'ensemencement cellulaire, nous avons ensuite développé un modèle numérique dans le chapitre 4, pour dégager un ensemble de critères à respecter dans l'élaboration d'un substitut osseux qui favoriserait un développement tissulaire homogène contrôlé lors des premières étapes de la culture in vitro de ce type d'implants. Ce modèle constitue une première étape dans la détermination d'un cahier des charges géométrique de tels substrats / This study aims to set up methods and tools to improve our understanding of the role played by transport phenomena (transport of cells, fluid and chemical species) in the development of new therapeutic protocols for bone reconstruction, using a double approach: experimental studies and numerical simulations. Hence, in the second chapter of this document, we have been able to link the intrinsic permeability of a porous medium – a key parameter regarding fluid transport through porous media – to the geometric structure of its pores. We have also highlighted the influence of electrochemical interactions on the flow of an ionic solution (such as physiologic fluids) through cortical bone, due to its porous structure and its chemical composition (presence of electrically charged fibers). These tools have then enabled us to analyze, at first glance, the experimental results of permeability tests conducted on ovin femoral periosteum, to identify the chemical-physical phenomena responsible for the specific behavior of this membrane (chapter 5). We also focused on the development of large bone implants coupling a mineral substitute and mesenchymal stem cells to enhance a volumic reconstruction of critical-sized bone defects. We have therefore designed, in chapter 3, a custom experimental set up that allows one to perform a reproducible cell seeding test on a porous scaffold and quantify the number of seeded cells as well as their viability rate. The experimental results provided by these tests have then initiated the numerical model exposed in chapter 4, that aims to highlight criteria to meet regarding the design of new bone substitutes that would enhance a homogeneous volumic tissue growth during the first stages of the extit [in vitro} development of coupled implants

Bakgrund. Vid tumörresektion i de orofaryngeala vävnaderna, utförs mandibulotomi för att skapa åtkomst och möjliggöra kirurgiskt avlägsnande. Allvarliga komplikationer har rapporterats i litteraturen och bidragande orsaker är den kirurgiska tekniken, strålningsterapin samt en inskränkt näringstillförsel till mandibelkroppen.Syfte. Syftet med denna studie var att undersöka om bevarande av periost vid utförande av mandibulotomi, leder till färre patienter med komplikationer relaterade till mandibulotomin..Metod. Patienter som genomgick mandibulotomi mellan 2003-2012 vid Skånes Universitetssjukhus granskades retrospektivt. Trettiosex patienter inkluderades fördelade på 18 i en supraperiostal- (locking plates) och 18 i en subperiostal grupp (non-locking plates). Den kliniska uppföljningen var 12 månader och den röntgenologiska varierade mellan 10 till 17 månader efter mandibulotomin. Komplikationerna delades in i mindre allvarliga; abscess, fistel och gingival nekros samt mer allvarliga; osteoradionekros, ben- och plattblotta, non-union och mikrovaskulärlambåinfektion.Resultat. Antalet patienter med komplikationer under 12 månaders uppföljning var 14 (38,9 %). Subperiostala gruppen hade en incidens på 9 fall medan den supraperiostala hade en incidens på 5. Antalet patienter som uppvisade komplikationer vid 12 månaders uppföljning var 9 (25 %), med endast en patient representerad i den supraperiostala gruppen.Slutsats. Enligt denna retrospektiva studie har patienter, som genomgått mandibulotomi med supraperiostal placering av osteosyntesplattorna, färre persisterande komplikationer av allvarlig grad efter ett år, jämfört med en subperiostal placering. Det förefaller därför fördelaktigt att använda supraperiostal placeringsteknik, när man applicerar osteosyntesplattor efter mandibulotomi på strålade patienter. / Background. Mandibulotomy is frequently used to gain access at resection of tumours in the oropharyngeal space. Severe complications have been reported in the literature related to the surgical technique, radiation and nutritional deficiency of the mandibular body.Objective. The purpose of this study is to investigate if preserving the periosteum, while performing a mandibulotomy, leads to fewer patients with complications related to the mandibulotomy.Method. Patients undergoing mandibulotomy between 2003 and 2012 at Skåne University Hospital were reviewed retrospectively. Thirty six patients were included, 18 in the supraperiosteal group (locking plates) and 18 patients in the subperiosteal group (non-locking plates). Clinical follow up was 12 months and radiographic follow up was 10 to 17 months post-surgery. Complications were divided into minor; abscess, fistula, gingival necrosis and major; osteoradionecrosis, bone and plate exposure, non-union and micro vascular flap infection.Results. The summation of patients with complications during the 12 months follow up was 14 (38 %). The subperiosteal group was represented in 9 cases and the supraperiosteal group in 5. The total number of patients with complications persisting at 12 months after the operation were 9 (25 %), with only one patient represented in the supraperiosteal group.Conclusion. Fewer patients, undergoing mandibulotomy with the supra- compared to the subperiosteal placement of the osteosynthetic plates, showed persisting complications at one year postoperatively (p <0.05). It seems to be preferable to use a supraperiosteal fixation method when placing the osteosynthetic plates after a mandibulotomy in irradiated patients.

La périostine est une protéine matricellulaire préférentiellement exprimée aux sites de contraintes mécaniques, notamment le périoste, et dans le stroma associé à de nombreux types de cancers. En premier lieu, nous nous sommes attachés à évaluer la pertinence de cette protéine en tant que biomarqueur du métabolisme osseux et de la réaction stromale dans les métastases osseuses. Nous avons développé le premier dosage ELISA de la périostine circulante chez la souris présentant des caractéristiques analytiques (spécificité, précision) conformes aux exigences réglementaires. Ce dosage nous a permis de préciser l’implication de la périostine dans le métabolisme osseux et les métastases osseuses de cancer du sein. Nos données in vitro et in vivo suggèrent que la périostine n’est pas un indice direct du remodelage osseux, contrairement aux marqueurs biologiques conventionnels, mais une composante de l’ossification primaire. Nous avons aussi montré dans les métastases osseuses d’origine mammaire que la périostine est surexprimée par les cellules stromales de la métastase, comme cela a pu être observé au niveau des tumeurs primaires. Enfin, nous avons confirmé par une approche bioinformatique la relation étroite entre périostine et réaction stromale dans la plupart des tumeurs chez l’Homme. La périostine et d’autres protéines conjointement exprimées pourraient donc constituer un panel de marqueurs biologiques de la progression tumorale, certains pouvant se révéler comme nouvelles cibles thérapeutiques en oncologie. / Periostin is a matricellular protein preferentially expressed at sites subjected to mechanical constraints, including the periosteum, and in the stroma associated to several tumor types. We first aimed to evaluate the relevance of periostin as a biomarker of bone metabolism or stromal reaction in bone metastases. We developed the first ELISA for serum periostin in mouse with analytical characteristics (specificity, precision) that are in accordance with regulatory standards. This ELISA allowed us to specify further the involvement of periostin in bone metabolism and breast cancer bone metastases. Our in vitro and in vivo data suggested that periostin is a component of primary ossification rather than a direct index of bone remodeling, unlike conventional bone markers. In breast cancer bone metastases, we also showed that periostin is overexpressed by stromal cells associated with bone metastasis, in agreement with its localization in the stroma of primary tumors. Finally, using bioinformatics analyses of large datasets from various tumors in human, we confirmed the close relationship between periostin and the stromal reaction. Periostin and other co-expressed proteins could therefore constitute a set of biological markers of cancer progression, and/or appear as potential therapeutic targets.

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