Contexte : l'aldostérone (Aldo) est impliquée dans la rigidité artérielle et l'insuffisance cardiaque (IC), mais les mécanismes sous-jacents restent méconnus. La galectine-3 (Gal-3), une lectine se liant aux bêta-galactoside, joue un rôle important dans la fibrose et l'IC. Dans cette étude, nous avons recherché si la Gal-3 était impliquée dans la fibrose vasculaire induite par l'Aldo. Méthodes et résultats : Des cellules musculaires lisses vasculaires de rat (CMLVs) ont été stimulées avec de l'Aldo en combinaison avec des antagonistes du récepteur minéralocorticoïde (MR) ou des inhibiteurs de la Gal-3. L'Aldo régule l'expression de la Gal-3 via le MR dans les CMLVs. De plus, la surexpression de la Gal-3 augmente spécifiquement la synthèse de collagène de type I. Les inhibiteurs de la Gal-3 ou sa sous-expression (siRNA) bloquent la synthèse de collagène de type I induite par l'Aldo. Des rats ont été traités avec de l'Aldo + sel combiné avec du spironolactone ou de la pectine de citron modifiée (MCP) pendant 3 semaines. Les rats hypertensifs traités à l'Aldo ont présenté une hypertrophie vasculaire, une fibrose et une augmentation de l'expression aortique de Gal-3. Les traitements avec le spironolactone ou le MCP préviennent tous ces effets. Des souris sauvages (WT) et mutées pour la Gal-3 (KO) ont été traitées avec de l'Aldo pendant 6 heures. Le bolus d'Aldo augmente l'expression de la Gal-3 et du collagène de type I dans l'aorte des souris WT alors qu'aucun changement ne se produit dans les souris KO pour la Gal-3. Conclusions : Nos donnés indiquent que la Gal-3 est indispensable à la réponse fibrotique de l'Aldo dans les CMLVs in vitro et in vivo, suggérant un rôle clef pour la Gal-3 dans la fibrose vasculaire / Background. Aldosterone (Aldo) is involved in arterial stiffness and heart failure (HF), but the mechanisms have remained unclear. Galectin-3 (Gal-3), a beta-galactoside-binding lectin, plays an important role in fibrosis and HF. We here investigated whether Gal-3 is involved in Aldo-induced vascular fibrosis. Methods and Results. Rat vascular smooth muscle cells (VSMCs) were stimulated with Aldo combined with mineralocorticoid receptor (MR) antagonists and Gal-3 inhibitors. Aldo upregulated Gal-3 expression via MR in VSMCs. Moreover, Gal-3 over-expression specifically enhanced collagen type I synthesis. Gal-3 inhibitors or Gal-3 silencing (siRNA) blocked Aldo-induced collagen type I synthesis. Rats were treated with Aldo-salt combined with spironolactone or modified citrus pectin (MCP) for 3 weeks. Hypertensive Aldo-treated rats presented vascular hypertrophy, fibrosis and increased aortic Gal-3 expression. Spironolactone or MCP treatment reversed all the above effects. Wild type (WT) and Gal-3 knock-out (KO) mice were treated with Aldo for 6 hours. Aldo bolus increased aortic Gal-3 and collagen type I expression in WT mice whereas no changes occurred in Gal-3 KO mice. Conclusions. Our data indicate that Gal-3 is required for the fibrotic response to Aldo in VSMCs in vitro and in vivo, suggesting a key role for Gal-3 in vascular fibrosis

Le microenvironnement tumoral via les macromolécules matricielles est connu pour jouer un rôle clé dans la réponse des cellules cancéreuses à la chimiothérapie en favorisant leur survie et leur prolifération. L'impact du collagène de type I, protéine matricielle majeure du microenvironnement, a été évalué au niveau des capacités migratoires des cellules tumorales et de leur réponse aux agents anticancéreux, doxorubicine et metformine. Cette approche a étémenée chez des cellules humaines HT1080 hautement invasives au moyen de systèmes de culture par coating 2D ou en matrice 3D. Les effets de modifications post-traductionnelles du collagène comme la carbamylation et de la glycation ont été également étudiées. Les résultats montrent que le collagène 3D inhibe l'activité anti-migratoire de la doxorubicine. Cette protection met en jeuune préservation des niveaux d'activation de FAK et RhoA impliquées dans la formation des fibres de stress d'actine et des plaques d'adhésion focales. Le collagène glyqué 2D et dans une moindre mesure le carbamylé inhibent l'adhésion, la migration des cellules tumorales et désorganisent leur cytosquelette d'actine via des modifications de distribution de la vinculine, de FAK et des intégrines 1. Cet impact de la glycation a été aussi mis en évidence en matrice 3D après modification du processus de glycation. Enfin, la glycation exerce un effet protecteur vis-àvis des capacités anti-prolifératives et anti-migratoires de la doxorubicine et de la metformine. En conclusion, nous mettons en évidence une nouvelle forme de résistance CAM-DR dirigée contre l'activité anti-invasive de médicaments ; cet effet pouvant être généré par une protéine matricielle native ou modifiée lors de situations physiopathologiques associées au cancer. / The tumor microenvironment via the extracellular matrix plays an important role in cancer cell response to chemotherapy by promoting their survival and proliferation. In this work, we studied the impact of collagen type I, a major matrix protein of tumor microenvironment, on the migration capacities of tumor cells and on their response to anticancer drugs such as doxorubicin and metformin. This approach was performed with the highly invasive human cell line HT1080,by means of 2D coating or 3D matrix cell culture systems. The effects of collagen posttranslational modifications such as carbamylation and glycation were also assessed. The results show that the 3D collagen inhibits the anti-migratory effect of doxorubicin. This protection is carried out through the preservation of the activation states of FAK and RhoA, which are involved in the formation of actin stress fibers and focal adhesions. On 2D coating, the glycated collagen and at a lesser extent the carbamylated one decrease the adhesion, the migration oftumor cells and, disorganize the actin cytoskeleton via a modified distribution of vinculine, FAK and beta1 integrins. This impact is also demonstrated by using 3D matrices, after adaptation of the glycation process. In addition, we reported that the glycated collagen protects against the antiproliferative and the anti-migratory effects of doxorubicin and metformin. In conclusion, we highlighted a new form of CAM-DR resistance that targets the drugs anti-invasive activity. This impact could be induced by the native form of matrix proteins or the modified one found inpathological situations which are associated to cancer.

Η σεργλυκίνη (SG) είναι μια πρωτεογλυκάνη που εκφράζεται και εκκρίνεται από το σύνολο σχεδόν των αιμοποιητικών κυττάρων, ενώ αποτελεί την κύρια πρωτεογλυκάνη η οποία εκκρίνεται από τις κυτταρικές σειρές πολλαπλού μυελώματος (ΠΜ). Έχει βρεθεί ότι η SG συμμετέχει στη ρύθμιση πληθώρας παραγόντων που εμπλέκονται σε αντιδράσεις φλεγμονής. Τα αποτελέσματά μας δείχνουν ότι η SG που εκκρίνεται από τα μυελωματικά κύτταρα έχει την ικανότητα να αναστέλλει τόσο την κλασσική όσο και τη λεκτινική οδό του συστήματος του συμπληρώματος, ενώ δε φαίνεται να έχει καμία επίδραση στο εναλλακτικό μονοπάτι. Επιπρόσθετα η SG δεν έχει την ικανότητα να προκαλεί την ενεργοποίηση κάποιου από τα τρία μονοπάτια του συμπληρώματος. Βρέθηκε ότι η ανασταλτική δράση της SG εκδηλώνεται μέσω της αλληλεπίδρασής της με τους παράγοντες C1q και MBL. Οι γλυκοζαμινογλυκανικές (GAGs) αλυσίδες της SG είναι υπεύθυνες για τη δέσμευση με την κολλαγονούχα ουρά του παράγοντα C1q, ενώ στη δέσμευση με την MBL πρωτεΐνη πέρα από τη συμμετοχή των GAG αλυσίδων απαιτείται και ο πρωτεϊνικός κορμός της SG. Επιπλέον βρέθηκε ότι αλυσίδες CS-E ελαττώνουν την ικανότητα δέσμευσης της SG με τα μόρια C1q και MBL. Οι αλληλεπιδράσεις της SG με τον C1q και την MBL βρέθηκε ότι είναι ιοντικού χαρακτήρα και σε αντίθεση με τη δέσμευση της SG με την MBL, που εξαρτάται από την παρουσία των ιόντων Ca2+, η αλληλεπίδραση της SG με τον C1q είναι ανεξάρτητη των ιόντων αυτών. Αν και τα επίπεδα της SG στον ορό των ασθενών με ΠΜ εμφανίζονται αυξημένα σε σχέση με τους φυσιολογικούς μάρτυρες, δεν εντοπίστηκαν στατιστικά σημαντικές διαφορές στην δραστικότητα της κλασσικής και της εναλλακτικής οδού του συμπληρώματος στους ασθενείς με ΠΜ και στους φυσιολογικούς δότες. Ωστόσο ενδιαφέρον αποτελεί το γεγονός ότι στους ασθενείς με ΠΜ τα υψηλά επίπεδα SG στον ορό εμφάνισαν τάση συσχέτισης με ελαττωμένη δραστικότητα της κλασσικής οδού του συμπληρώματος. Επιπρόσθετα, η SG που εκκρίνεται απ τα μυελωματικά πλασματοκύτταρα έχει την ικανότητα να προστατεύει τα κύτταρα αυτά από την επίδραση του συμπληρώματος, μετά την ενεργοποίησή του με τη χρήση φαρμάκων. Φαίνεται ότι και η SG της μεμβράνης των μυελωματικών κυττάρων παρουσιάζει προστατευτική δράση έναντι του συμπληρώματος, μιας και κύτταρα που δεν εκφράζουν SG στην επιφάνειά τους είναι δύο με τρείς φορές περισσότερο ευαίσθητα στη δράση του συμπληρώματος, σε σχέση με κύτταρα που την εκφράζουν. Προτείνουμε ότι τόσο η εκκρινόμενη όσο και η δεσμευμένη στην κυτταρική επιφάνεια SG προστατεύουν τα μυελωματικά πλασματοκύτταρα κατά την ανοσοθεραπεία και συμβάλουν στην επιβίωσή τους. Στην παρούσα μελέτη δείξαμε ότι η SG αλληλεπιδρά με το κολλαγόνο τύπου Ι, την πιο άφθονη μορφή κολλαγόνου στο οστό. Επιπλέον βρέθηκε ότι η SG της κυτταρικής επιφάνειας συμμετέχει στην προσκόλληση των μυελωματικών κυττάρων στο κολλαγόνο τύπου Ι. Η αλληλεπίδραση αυτή φαίνεται ότι επάγει την έκφραση από τα μυελωματικά πλασματοκύτταρα των ΜΜΡ-2 και ΜΜΡ-9. Επιπρόσθετα, υπολογίστηκαν τα επίπεδα των ΜΜΡ-2 και ΜΜΡ-9 στον ορό και στο μυελό ασθενών με ΠΜ. Βρέθηκε ότι στον ορό των ασθενών με ΠΜ τα επίπεδα των δύο ενζύμων εμφανίζονται ελαττωμένα σε σχέση με τα φυσιολογικά δείγματα, ενώ αντίθετα στον μυελό των ασθενών η ΜΜΡ-2 βρέθηκε σημαντικά αυξημένη σε σχέση με τους φυσιολογικούς δότες. Καμία μεταβολή δεν παρατηρήθηκε στα επίπεδα της ΜΜΡ-9. Τέλος τα επίπεδα της ΜΜΡ-2 του μυελού ασθενών με ΠΜ βρέθηκε ότι σχετίζονται τόσο με τα επίπεδα του ενζύμου στον ορό, όσο και με τον δείκτη οστικής απορρόφησης ΝΤx, υποδηλώνοντας τη συμμετοχή της στην παθοβιοχημεία της οστικής νόσου που εμφανίζεται στο ΠΜ. Αντιθέτως καμία συσχέτιση δεν βρέθηκε για την ΜΜΡ-9, δηλώνοντας ότι ίσως το ένζυμο αυτό να εμπλέκεται σε άλλες διεργασίες που επιτελούνται στο ΠΜ. / Serglycin (SG) is a proteoglycan expressed by hematopoietic cells and is constitutively secreted by multiple myeloma (MM) cells. SG participates in the regulation of various inflammatory events. We found that SG secreted by human MM cell lines inhibits both the classical and lectin pathways of complement, without influencing alternative pathway activity. It was also shown that SG could not initiate any activation of the complement system. The inhibitory effect of SG is due to direct interactions with C1q and mannose binding lectin (MBL). C1q-binding is mediated through the glycosaminoglycan moieties of SG, whereas binding to MBL requires the presence of SG protein core. Interactions between SG and C1q as well as MBL are diminished in the presence of chondroitin sulfate type E. In addition, we localized the SGbinding site to the collagen-like stalk of C1q. Interactions between SG and C1q as well as MBL are ionic in character and only the interaction with MBL was found to be partially dependent on the presence of calcium. Although we found the serum levels of SG to be elevated in patients with MM compared to healthy controls, no statistical significant differences were observed for classical and alternative pathway activity in sera between MM patients and healthy donors. Despite that, it is proved that increase levels of SG show a tendency to correlate with decreased levels of classical pathway activity in serum of MM patients. Moreover, we found that SG expressed from myeloma plasma cells protects these cells from complement activation induced by treatment with anti-thymocyte immunoglobulins. It is also demonstrated that SG on the surface of MM cells inhibits complement deposition on the membrane of these cells. Cells which do not express SG on its’ surface are 2-3 times more sensitive to complement attack compared with cells expressing high levels of SG. This might protect myeloma cells during immunotherapy and promote survival of malignant plasma cells. Moreover, it is shown that SG from MM cells is capable to interact with collagen type I, the most abundant collagen in bone. Furthermore, SG is present on the surface of myeloma plasma cells and it is involved in the adhesion of MM cells to collagen type I in bone marrow microenvironment. In addition, it is shown that the interaction of myeloma plasma cells to collagen type I, mediated by cell surface SG, induces expression and secretion of both MMP-2 and MMP-9 from MM cells. Along the process, we have investigated levels of MMP-2 and MMP-9 in serum and marrow of patients with MM. Decreased levels of both MMP-2 and MMP- 9 in serum of MM patients have been observed compared to healthy donors. Instead, MMP-2 was found to be increased in bone marrow of MM patients compared to control samples, while no differences observed for MMP-9. Finally, marrow levels of MMP-2 seem to correlate with serum levels of the enzyme along with a marker of bone resorption, NTx. This might indicate the implication of MMP-2 in the pathogenesis of bone disease in MM. Even though, no correlation of MMP-9 with NTx was observed, proving that MMP-9 may play a significant role in different pathogenetic mechanism occurring in MM.

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

Este estudo avaliou a efetividade de uma nova membrana bioabsorvível com propriedades mecânicas e químicas mais adequadas para o uso em tendões. A avaliação foi realizada em coelhos submetidos a reconstrução da porção gastrocnêmia do tendão calcâneo. Foi feita uma avaliação prévia das propriedades mecânicas da membrana com uso de dinamômetro digital que demonstrou capacidade de deformação elástica mínima de 100%. Todos os coelhos foram submetidos a tenotomia e reparo da porção gastrocnêmia do tendão direito, após isso foram randomicamente separados em grupos envelopado com membrana e controle. A extração foi realizada nos seguintes períodos: sete, 14 e 28 dias. A avaliação foi realizada através da macroscopia, histologia, mensuração objetiva do colágeno à luz polarizada pelo image-J®, mensuração de glicosaminoglicanos sulfatados e expressão gênica de proteoglicanos. Na avaliação macroscópica o grupo com membrana apresentou menos aderência e melhor direcionamento das fibras e tecido mais homogêneo em 14 e 28 dias, p=0,02 e 0,03 respectivamente. Na histologia a Classificação de Watkins modificada apresentou as seguintes médias: 14,67±0,42 membrana e 12,67±0,56 sem membrana, p=0,03 em 14 dias, e 19,88±0,83 membrana e 17,25±0,62 sem membrana, p=0,02 em 28 dias. Na mensuração do colágeno as médias dos valores de cinza(mvc) o colágeno tipo III foram de 17,97±1,83 membrana e 12,63±1,07 sem membrana p=0,03 em 14 dias. Para o colágeno tipo I as médias foram de 2,41±0,33mvc membrana e 1,31±0,18mvc sem membrana p=0,01 em 14 dias e 7,30±0,63mvc membrana e 2,92±0,32mvc sem membrana p < 0,0001 em 28 dias. A média dos GAGs foi avaliada em três porções do tendão, proximal, central e distal, em ug/mg de tecido seco. Em sete dias apresentou diferença significativa apenas na porção distal 0,80±0,04 com e 0,38±0,04 sem membrana para condroitin-sulfato em 14 dias não apresentou diferenças entre os grupos. O dermatan-sulfato apresentou diferença estatisticamente significante em 7 dias apenas na porção central 0,42±0,09 com membrana e 1,29±0,67 sem membrana p=0,02. Em 14 dias não foram observadas diferenças entre os grupos. Houve grande variabilidade na expressão gênica no teste das amostras com beta-Actina e GAPDH levando a resultados inconclusivos ou não variação entre os grupos que pode sugerir não variabilidade na expressão gênica dos GAGs no período de 28 dias. Os dados fornecidos por esse trabalho mostram que a envelopagem com a membrana bioabsorvível promoveu aceleração dos processos cicatriciais da porção gastrocnêmia do tendão calcâneo de coelhos / This study assessed the effectiveness of a new absorbable membrane, that presents mechanical and chemical features more suitable to tendons, in rabbit tendons. Before the animal model assessments a mechanical study of the membrane was carried out demonstrating that the minimal capability for elastic deformation of the membrane was more than 100%. All rabbits underwent to tenotomy and reconstruction of the right gastrocnius tendons, thereafter they were randomly divided in tendon wrapped by the membrane and control groups. Extraction was performed in the following periods of time: seven, 14 and 28 days. Assessments used macroscopy, histology, objective collagen assessment by using polarized light and Image-J® program in mean of gray values(mgv), sulphated glycosaminoglycans, genetic expression of proteoglycans. In the macroscopic 14 and 21-day assessments the membrane group presented less adherences p=0.02 and p=0.03 respectively. The modified Watkins classification: 14,67±0,42 membrane and 12,67±0,56 without membrane p=0,03 for 14 days; 19,88±0,83 membrane and 17,25±0,62 without membrane p=0,02 for 28 days. The type III collagen were 17,97±1,83 membrane and 12,63±1,07 without membrane p=0,029 for 14 days. For type I collagen were 2,41±0,33 membrane and 1,31±0,18 without membrane p=0,01 for 14 days and 7,30±0,63 memebrane and 2,92±0,32 without membrane p < 0,0001 for 28 days. The glycosaminoglycans were measured in 3 tendon portions, distal, central and proximal, by using ug/mg of dry tissue. In seven days just the distal part presented statistical differences 0,80±0,04 membrane and 0,38±0,04 without membrane, for 14 days no differences were found for Chondroitin-Sulphate. For Dermatan-Sulphate the central part of the tendon 0,42±0,09 and 1,29±0,67 p=0,02, for 14 days no differences were found. There was high variability for beta-actin and GAPDH for the samples in 28 days with inconclusive results that may mean no variability in gene expression of GAGs at this time period. Results as mentioned above demonstrated that the wrapped tendons by the new membrane presented acceleration in the healing processes for gastrocnemius tendons of New Zealand Rabbits

Os eventos celulares e extracelulares que ocorrem durante o processo de osseointegração do titânio (Ti) são bastante influenciados por suas propriedades de superfície, como morfologia, topografia e composição química. A modificação bioquímica da superfície do Ti consiste em imobilizar proteínas ou peptídeos nessa superfície com a finalidade de induzir respostas celulares e teciduais específicas na interface osso-implante que acelerem ou aumentem a osseointegração. O objetivo deste estudo foi avaliar o desenvolvimento do fenótipo osteoblástico em culturas de células crescidas sobre Ti revestido com colágeno tipo I. Para tanto, células osteoblásticas derivadas de fragmentos ósseos do processo alveolar de humanos foram cultivadas sobre discos de Ti usinados revestidos (Ti-col) ou não (Ti-usinado) com colágeno tipo I e foram avaliados os seguintes parâmetros: adesão, morfologia e proliferação celulares, síntese de proteína total, atividade de fosfatase alcalina (ALP), formação de matriz mineralizada, e expressão de genes marcadores do fenótipo osteoblástico por reação em cadeia da polimerase em tempo real (PCR em tempo real). O Ti-col alterou o crescimento e a expressão gênica das culturas e não teve efeito na adesão e morfologia celulares, síntese de proteína total, atividade de ALP e formação de matriz mineralizada comparado ao Ti-usinado. Esses resultados indicam que a superfície Ti-col pode favorecer um maior crescimento da cultura durante a fase proliferativa e um aumento e/ou aceleração da diferenciação, como indicado por alterações na expressão gênica de marcadores do fenótipo osteoblástico. Portanto, essa modificação de superfície pode ter um impacto nos processos de reparo e remodelação do tecido ósseo adjacente a implantes, favorecendo a ocorrência de maior formação óssea. / Cellular and extracellular events that occur during titanium (Ti) osseointegration process are highly influenced by its surface properties, such as morphology, topography and chemical composition. The objective of biochemical modification of Ti is to immobilize proteins or peptides on its surface in order to induce specific cellular and tissue responses at the boneimplant interface in order to accelerate or enhance osseointegration. The aim of this study was to evaluate the osteoblastic phenotype development in cells grown on collagen type I-coated Ti surface. Osteoblastic cells from human alveolar bone fragments were cultured on turned Ti either coated with collagen type I (col-Ti) or not (turned-Ti) and the following parameters were assessed: cell adhesion, morphology, and proliferation, total protein content, alkaline phosphatase (ALP) activity, bone-like formation and gene expression of osteoblastic markers by real-time polymerase chain reaction (real-time PCR). Col-Ti altered culture growth and gene expression of osteoblastic markers without affecting cell adhesion, morphology, protein synthesis, ALP activity, and matrix mineralization. These results demonstrated that col-Ti favours cell growth during the proliferative phase and osteoblastic differentiation, as demonstrated by changes in mRNA expression profile during the matrix mineralization phase, suggesting that this Ti surface modification may affect the processes of bone healing and remodelling.

Osteogenesis imperfecta is an inherited disorder caused mainly by collagen type I genes mutations, COL1A1 and COL1A2. These mutations affect especially connective tissue. Disease is characterized by fragile bones, deformations and increased frequency of fractures. It's worldwide extensive disorder regardless of age, sex, nationality or races. The incidence is 1: 16 - 20 000 births. Currently, we described nine clinically distinct forms of Osteogenesis imperfecta. Only the first four types OI, type I-IV, are caused by collagen type I genes mutations . In these nine types there are distinguished mild and severe forms. Type II and III are lethal forms, death occur offen during prenatal period or in the first days of the life affected individuals. Characteristic clinical features of collagen forms OI are an increased incidence of fractures, deformations of bones, blue sclera, hearing loss, Dentinogenesis imperfecta small or subnormal growth (Marini, 2010). This study alignment is mainly the description of the clinical forms, exploring the molecular basis of disease and determine the relationship between the type and position of the mutation and the resulting phenotype of affected individuals. We have analysed exons 31-40, including associated non-coding regions, of the COL1A1 gene (so-called MLBR =...

Yes / Studies have been conducted to highlight the anti-inflammatory and immunosuppressive properties of synthetic cannabinoids as well as their potential for cartilage repair. Various wound healing techniques can be used to investigate the mechanisms of chondrocyte repair in monolayers or three dimensional tissues constructs. In this work the effect of WIN55, 212-2 (WIN-2) on nitric oxide (NO) and matrix metalloproteinase-2 (MMP-2) expressed by wounded chondrocyte monolayers was investigated. Moreover, expression of collagen type-I and type-II, fibronectin and S100 proteins were detected using immunofluorescence and quantitatively verified using ELISA based techniques following treatment with 1 μM and 2 μM of WIN-2. Treating chondrocytes with 1 μM of WIN-2 significantly increased expression of collagen type-II, fibronectin and S100, and significantly reduced collagen type-I expressions as compared to the control groups. On the other hand, both concentrations of WIN-2 significantly reduced the expression of the inflammation markers NO and MMP-2 in a dose dependent manner. These findings highlight the potential use of the synthetic cannabinoids for improving cartilage healing properties as well as acting as an anti-inflammatory agent which could be used to enhance tissue engineering protocols aimed at cartilage repair.

Osteogenesis imperfecta (OI) is a heterogeneous disease of connective tissue, the cardinal symptom being fractures and severity ranging from mild to lethal. Dominant mutations in collagen I, encoded by COL1A1 and COL1A2, cause &gt;90% of cases. To delineate genotype-phenotype correlations and pharmaco-genetic response, collagen I was sequenced in 150 unrelated Swedish families and clinical data were collected in Paper I. Mutation type, gene affected, and N- to C-terminal location correlated with phenotype and severity. Bisphosphonate response assessed by calculated yearly change in lumbar spine bone mineral density (BMD) was inversely related to age and BMD at treatment initiation. Mutations associated with a more severe phenotype exhibited an increased response after 2 years; however, all types of OI responded well. To investigate the effect of naturally occurring variations in collagen I, the only common coding single nucleotide polymorphism (rs42524 in COL1A2) was genotyped in 2004 healthy men in Paper II. Heterozygous genotype was associated with decreased BMD and an increased risk of stroke. An adolescent with repeated fractures despite a markedly high BMD harbored a unique C-terminal procollagen cleavage-site mutation in COL1A1, which motivated extensive investigations in concert with a similar COL1A2 case in Paper III. The probands were found to have impaired procollagen processing, incorporation of collagen with retained C-propeptide in matrix and increased mineral to matrix ratio, which demonstrates that C-propeptide cleavage is crucial to normal bone mineralization and structure. Bisphosphonate therapy has insufficient effect in OI, and as classical OI is a dominant disorder severe cases would benefit from silencing of the mutated allele. In Paper IV and V small interfering RNAs (siRNAs) were used to allele-specifically target primary human bone cells heterozygous for I) a coding polymorphism in COL1A2 and II) insertion/deletions in the 3’UTR of COL1A1 and COL1A2. Results were promising with altered allele ratios and decreased mRNA levels in the predicted fashion. To summarize, this thesis found that collagen I is crucial to bone and connective tissue and that collagen I mutations create markedly diverse phenotypes. Age, BMD and pharmaco-genetic effects influence the response to bisphosphonate therapy in individuals with OI; however, novel approaches are needed. Utilizing allele-specific siRNAs may be a way forward in the treatment of severe OI.