Diss. (sammanfattning) Uppsala : Uppsala univeritet, 2008. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Alabama at Birmingham, 1998. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Alabama at Birmingham, 1998. / Includes bibliographical references.

Η αιμοποιητική φωλαιά (hematopoietic stem cell niche) περιέχει οστεοβλάστες, οι οποίοι ρυθμίζουν τη φυσιολογική αιμοποίηση. Ωστόσο, λίγα στοιχεία είναι γνωστά, έως τώρα, για το ρόλο των οστεοβλαστών στη διαδικασία της αιμοποίησης σε ασθενείς με Μυελοδυσπλαστικό Σύνδρομο (ΜΔΣ). Το ΜΔΣ, αποτελεί μια ετερογενή ομάδα κλωνικών αιματολογικών διαταραχών, με αυξημένο κίνδυνο εκτροπής προς Οξεία Μυελογενή Λευχαιμία (ΟΜΛ). Μελέτες σε ex-vivo συστήματα καλλιεργειών (co-cultures) περιγράφουν την επίδραση των μεσεγχυματικών κυττάρων (“feeder cells”) στο δυναμικό πολλαπλασιασμού, στη μεταναστευτική ικανότητα, καθώς και στη διατήρηση (stemness) των αρχέγονων αιμοποιητικών κυττάρων (HSCs) φυσιολογικών δοτών. Η μελέτη αυτή στοχεύει στη διερεύνηση των βιολογικών χαρακτηριστικών των οστεοβλαστών από ασθενείς με ΜΔΣ καθώς και τις αλληλεπιδράσεις φυσιολογικών HSCs και οστεοβλαστών ασθενών με ΜΔΣ. Για το σκοπό αυτό δημιουργήθηκε ένα σύστημα δισδιάστατης καλλιέργειας (2-D culture system) χρησιμοποιώντας οστεοβλάστες που παρήχθησαν από μεσεγχυματικά κύτταρα μυελού των οστών (human marrow mesenchymal stem cells-MSCs). Τα MSCs απομονώθηκαν από το μυελό των οστών ασθενών με ΜΔΣ και υγιών δοτών και καλλιεργήθηκαν σε κατάλληλο θρεπτικό μέσο. Ακολούθησε επαγωγή της διαφοροποίησης των MSCs, μετά από συνεχόμενες καλλιέργειες σε οστεοβλάστες. Στη μελέτη χρησιμοποιήθηκαν 13 δείγματα μυελού των οστών από ασθενείς με ΜΔΣ (6 RA, 3 RAEBI, 2 RAEBII, 1 5q- και 1 υποπλαστικό MDS) και 8 δείγματα μυελού φυσιολογικών μαρτύρων όμοιας ηλικίας. Για τη μελέτη της επίδρασης των οστεοβλαστών από ασθενείς με ΜΔΣ στην αιμοποίηση χρησιμοποιήθηκαν φυσιολογικά HSCs από κινητοποιημένο περιφερικό αίμα υγιών δοτών (mPB, n=4), τα οποία τοποθετήθηκαν πάνω στους ήδη εγκατεστημένους οστεοβλάστες (osteoblast confluent monolayer cultures). Τα MSCs και οι οστεοβλάστες που αναπτύχθηκαν ελέγχθηκαν μορφολογικά και ανοσοφαινοτυπικά, με τη χρήση μικροσκοπίας και κυτταρομετρίας ροής αντίστοιχα. Μονοπύρηνα κύτταρα από δείγματα κινητοποιημένου περιφερικού αίματος υγιών δοτών τοποθετήθηκαν στο δισδιάστατο καλλιεργητικό σύστημα, σε καλλιεργητικό υλικό αιμοποιητικών κυττάρων, χωρίς την εξωγενή προσθήκη κυτταροκινών. Με τη χρήση κυτταρομετρίας ροής ελέγχθηκε η έκφραση των μορίων που σχετίζονται με την προσκόλληση των αιμοποιητικών κυττάρων στην αιμοποιητική φωλαιά καθώς και την εγκατάσταση και διατήρησή τους σε αυτή. Ο έλεγχος έγινε στις 36 ώρες και τις 7 ημέρες συγκαλλιέργειας και αφορούσε τα μόρια CXCR4, το οποίο ρυθμίζει την άμεση πρόσδεση των HSCs στην φωλαιά κατά τη διαδικασία του “homing”, CD49d (Very Late Antigen-4- VLA4) και CD49e (Very Late Antigen-5- VLA5), τα οποία παρέχουν σήματα επιβίωσης ή προάγουν την ενεργοποίηση μιας φάσης ηρεμίας (quiescence) στα HSCs μετά την είσοδο τους στη φωλαιά (localization). Η έκφραση των μορίων αυτών μελετήθηκε στους υποπληθυσμούς των CD34+, CD34+/CD38+ και CD34+/CD38- κυττάρων. Παράλληλα εκτιμήθηκε το ποσοστό (συχνότητα) των CD34+ αιμοποιητικών κυττάρων καθώς επίσης και η προσκόλλησή τους στους οστεοβλάστες. Μετά τη συγκαλλιέργεια, οι οστεοβλάστες που προήλθαν από υγιείς δότες προκάλεσαν τον πολλαπλασιασμό των CD34+ κυττάρων των φυσιολογικών αιμοποιητικών κυττάρων που τοποθετήθηκαν πάνω στο εγκατεστημένο στρώμα των οστεοβλαστών (3-fold και 9-fold αύξηση στις 36ώρες και τις 7ημ., αντίστοιχα). Αύξηση επίσης, παρατηρήθηκε (2 fold αύξηση) στα CD34+ κύτταρα στις συγκαλλιέργειες των 36h, φυσιολογικών HSCs με οστεοβλάστες που παρήχθησαν από ασθενείς με ΜΔΣ, ενώ καμία διαφορά δεν παρατηρήθηκε μεταξύ των διαφορετικών υποτύπων ΜΔΣ. Στις 7 ημέρες συγκαλλιέργειας από την άλλη, δεν 12 παρατηρήθηκε καμία διαφορά στη συχνότητα εμφάνισης ενός πιο άωρου φαινοτύπου των φυσιολογικών HSCs που αναπτύχθηκαν σε οστεοβλάστες από ασθενείς με χαμηλού κινδύνου ΜΔΣ (low risk MDS). Αντιθέτως, τα CD34+ κύτταρα αυξήθηκαν κατά πολύ (16- fold αύξηση), όταν φυσιολογικά HSCs, τοποθετήθηκαν σε οστεοβλάστες ασθενών με υψηλού κινδύνου ΜΔΣ (high risk MDS). Επιπλέον, παρατηρήθηκε αύξηση της έκφρασης των μορίων CXCR4, CD49d και CD49e στα CD34+ κύτταρα μετά από συγκαλλιέργεια φυσιολογικών HSCs και οστεοβλαστών από υγιείς δότες, συγκριτικά με τα επίπεδα έκφρασης των μορίων αυτών πριν την τοποθέτηση τους στο σύστημα συγκαλλιέργειας. Η αύξηση της έκφρασης του μορίου CXCR4 ήταν λιγότερο εμφανής στην περίπτωση συγκαλλιέργειας των φυσιολογικών HSCs με οστεοβλάστες από ασθενείς με ΜΔΣ, όπου η μεγαλύτερη διαφορά παρατηρήθηκε στο σύστημα που περιείχε τους οστεοβλάστες από ασθενείς χαμηλού κινδύνου ΜΔΣ (3- και 1,7- fold αύξηση στις 7ημέρες καλλιέργειας με οστεοβλάστες από υγιείς δότες και χαμηλού κινδύνου ΜΔΣ ασθενείς, αντίστοιχα). Το πρότυπο έκφρασης των μορίων CD49d και CD49e ήταν όμοιο στα κύτταρα που τοποθετήθηκαν τόσο σε οστεοβλάστες προερχόμενους από υγιείς δότες, όσο και οστεοβλάστες από ΜΔΣ ασθενείς. Ο φαινότυπος, τόσο όσον αφορά τα μορφολογικά όσο και τα ανοσοφαινοτυπικά χαρακτηριστικά, των MSCs ήταν ίδιος και στις δυο ομάδες μελέτης, ενώ η διαφοροποίηση των MSCs προς οστεοβλάστες ήταν όμοια τόσο στα MSCs που προήλθαν από φυσιολογικούς δότες όσο και σε αυτά που προήλθαν από ασθενείς με ΜΔΣ, δείχνοντας παρόμοια έκφραση των ειδικών οστεοβλαστικών πρωτεϊνών αλλά και της διαδικασίας της ενασβεστοποίησης. Σύμφωνα με τα αποτελέσματα που λάβαμε, οι οστεοβλάστες από υγιείς δότες προώθησαν την αύξηση του ποσοστού των προγονικών αιμοποιητικών κυττάρων και οδήγησαν στην επαγωγή της έκφρασης του μορίου CXCR4, ενός πολύ σημαντικού μορίου για τη μετανάστευση, την εγκατάσταση αλλά και την ανάπτυξη. Ωστόσο, η διαφορετική δραστηριότητα, τόσο όσον αφορά το ποσοστό των CD34+ όσο και την έκφραση του μορίου CXCR4, όταν τα φυσιολογικά αιμοποιητικά κύτταρα συγκαλλιεργήθαν με οστεοβλάστες που προήλθαν από ασθενείς με ΜΔΣ, οδηγεί στην υπόθεση ότι υπάρχει μεταβολή στη λειτουργία των οστεοβλαστών, οπότε προβλέπεται και μια επακόλουθη αλλαγή στη ρύθμιση της εγκατάστασης των HSC στην αιμοποιητική φωλαιά, στους ασθενείς με ΜΔΣ. / The hematopoietic stem cell niche contains osteoblasts that regulate normal hematopoiesis. However, little is known about the role of osteoblasts in MDS hematopoiesis so far. Myelodysplastic syndrome comprises a heterogeneous group of clonal stem cell disorders with dismal prognosis and difficulty in their therapeutic approach, which is characterized by ineffective hematopoiesis. It appears with dysplastic hematopoietic cells, peripheral blood cytopenias and high risk of evolution to acute myeloid leukemia (AML). Data derived from ex vivo co-culture systems using mesenchymal stromal cells as a feeder cell layer suggest that cell-cell contact has a significant impact on the expansion, migratory potential and “stemness” of hematopoietic stem cells. In this study, we investigated the biological characteristics of osteoblasts from MDS patients and the interactions between these cells and normal hematopoietic stem cells (HSCs). Osteoblasts were differentiated from marrow MSCs from 13 MDS patients (6 RA, 3 RAEBI, 2 RAEBII, 1 5q- and 1 hypoplastic MDS) and 8 age-matched healthy individuals. To study the effect of MDS osteoblasts on hematopoiesis, normal HSCs from mobilized peripheral blood from healthy individuals (n=4) were seeded onto osteoblast confluent monolayer cultures using a culture medium appropriate for the culture of HSCs, without the exogenous addition of cytokines. We studied the morphology and immunophenotype of MSCs and osteoblasts by microscopy and flow cytometric analysis, respectively. Cytometric analyses of homing associated molecules were performed 36h and 7d later. These molecules are CXCR4, which regulates the direct adhesion of HSCs to the bone marrow niche during “homing”, CD49d (Very Late Antigen-4- VLA4) and CD49e (Very Late Antigen-5- VLA5), which produce survival signals or promote the maintenance of a quiescent state for HSCs after entering the stem cell niche (localization). We investigated the expression of these molecules in CD34+, CD34+/CD38+ and CD34+/CD38- cell populations. Furthermore we studied the frequency of CD34+ hematopoietic cells and also their ability to adhere osteoblasts.Osteoblasts from healthy individuals increased the frequency of CD34+ cells by 3- and 9-fold increase in normal hematopoietic cells after 36h and 7d co-cultures respectively. A 2-fold increase was also seen in CD34+ cells when normal HSCs grown on MDS-osteoblasts for 36h and no difference was seen between the MDS subtypes. When the culture period was extended to 7d, there was no change in the frequency of immature phenotype of normal HSCs in osteoblast cultures from low-risk MDS patients. In contrast, CD34+ cells increased several fold (16-fold increase) when normal HSCs were cultured on high-risk MDS 14 osteoblasts, twice the values obtained in osteoblast co-cultures from healthy individuals and low risk patients. The expression of adhesion molecules CXCR4, CD49d and CD49e on CD34+ cells from normal HSCs was increased in co-cultures with osteoblasts from healthy individuals compared to the values obtained before culture (3-fold increase at 7d). The increase in CXCR4 expression was less pronounced in the presence of osteoblasts from MDS patients with the largest difference being found in low-risk MDS patients (1,7-fold increase at 7d). The expression pattern of CD49d and CD49e was identical between cells grown on MDS- and normal- osteoblast co-cultures.The morphological and immunophenotypical analysis of MSCs show the same results for the two study groups, while the differentiation of MSCs to osteoblasts was similar for both healthy individuals and MDS patients, after having similar expression of bone specific proteins and mineralization activity. According to our data, osteoblasts from healthy individuals promoted the expansion of immature hematopoietic progenitors and induced the cell surface expression of CXCR4, an important molecule in HSCs homing, retention and development. However, the different expression of CXCR4 and the change in frequency of CD34+ cells that were detected when normal HSCs co-operated with MDS-osteoblasts, suggests alteration in osteoblast function and the subsequent regulation of the HSC residency in the niche in MDS patients compared with healthy individuals.

Umbilical cord blood (UCB) is a source of hematopoietic stem and progenitor cells (HSPC) used for allogeneic transplantation. Ex vivo expansion of HSPC can improve the slow platelet and neutrophil engraftment associated with UCB transplants. HSPCs reside in niches, some of which are near the endosteal bone surface, where they can associate with immature osteoblasts. Interestingly, osteoblasts can enhance the growth of HSPC in culture and their platelet engraftment activity. Using a proteomics approach, I identified 47 differentially expressed proteins between mesenchymal stem cells and immature osteoblasts. Several of these were previously implicated in HSPC maintenance such as IGF2, IGFBP2, DCN, GAS6 and VCAM1. Moreover, several other proteins belong to the alternative and classical complement pathways. Finally, I discovered that microvesicles found in osteoblast conditioned medium may also modulate the growth of HSPC, at least in ex vivo cultures.

Made available in DSpace on 2015-06-17T19:34:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-08-26. Added 1 bitstream(s) on 2015-06-18T12:47:11Z : No. of bitstreams: 1 000829196.pdf: 397092 bytes, checksum: ae8c06f67bfaa9d589934981c550dd81 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A hipertensão arterial representa um fator de risco sistêmico para várias doenças incluindo redução da massa óssea por aumentar a reabsorção e diminuir a formação óssea. Considerando que a formação óssea é resultante da atividade de osteoblastos, o objetivo deste estudo foi avaliar as características fenotípicas e genotípicas de células osteoblásticas diferenciadas de CTMs (células-tronco mesenquimais) de SHR. A partir de medulas ósseas de fêmures de SHR, CTMs foram obtidas por adesão ao plástico de cultura de células e diferenciadas em osteoblastos por cultura em meio osteogênico até que atingissem a subconfluência. Em seguida, as células foram subcultivadas na densidade de 2x104 células/poço em placas de 24 poços. Células obtidas de Wistar foram utilizadas como controle. Para a avaliação das respostas celulares foram realizados ensaios de proliferação celular, atividade de fosfatase alcalina (ALP), formação de matriz mineralizada e expressão gênica dos marcadores osteoblástico: 1) runt-related transcription fator 2 (RUNX2), 2) Osterix (OSX), 3) Fosfatase alcalina (ALP), 4) Proteína óssea morfogenética 2 (BMP2); 5) Sialoproteína óssea (BSP), 6) Osteocalcina (OC), 7) Osteopontina (OPN), 8) e Colágeno (COL), além dos receptores β1 e β2 adrenérgicos. Os dados foram comparados pelo teste ANOVA seguido do teste de Tukey, e o nível de significância adotado foi de 5% (p≤0,05). Células osteoblásticas de SHR apresentaram alterações no fenótipo osteoblástico, exibindo, em todos os períodos avaliados, menor proliferação celular, atividade de ALP e formação de matriz mineralizada. A expressão gênica de todos os marcadores ósseos também foi menor em SHR quando comparado com Wistar na maioria dos períodos avaliados. A expressão gênica do receptor β2 adrenérgico foi maior em SHR em todos os períodos avaliados e não foi detectada... / Hypertension is a systemic risk factor for several diseases including reduction of bone mass by increasing bone resorption and reducing bone formation. Considering that bone formation results from osteoblast activity, the aim of this study was to assess the phenotypic and genotypic characteristics of osteoblastic cells differentiated from MSCs of SHR. Bone marrow was harvested from SHR femurs and MSCs were selected by adherence to plastic cell culture and differentiated into osteoblasts by culturing in osteogenic medium until subconfluence. Then, the cells were subcultured at a density of 2x104 cells / well in 24 well plates. Cells from Wistar rats were used as control. Cells responses were evaluated by assaying proliferation, alkaline phosphatase activity (ALP), mineralized matrix formation and the gene expression of the osteoblastic markers: runt-related 1) runt-related transcription fator 2 (RUNX2), 2) Osterix (OSX) 3) Alkaline phosphatase (ALP), 4) Bone morphogenetic protein 2 (BMP2); 5) Bone sialoprotein (BSP), 6) Osteocalcin (OC), 7) Osteopontin (OPN), 8) and Collagen type 1 alpha (COL), in addition to the gene expression of β1 and β2 adrenergic receptors. Data were compared by ANOVA followed by Tukey test and the level of significance was 5% (p ≤ 0.05). SHR derived osteoblasts showed changes in osteoblastic phenotype, exhibiting in all periods reduced cell proliferation, ALP activity and mineralized matrix formation. The gene expression of all bone markers was also lower in SHR compared with Wistar in many periods. The gene expression of the β2 adrenergic receptor was greater in SHR in all periods and it was not detected the expression of β1receptor either in SHR or Wistar. Based on these results, we conclude that osteoblasts differentiated from SHR MSCs exhibit reduced or delayed in the differentiation process. We also suggest...

Orientador: Ana Lia Anbinder / Banca: Maria Aparecida Neves Jardini / Banca: Débora Pallos / Resumo: Os tratamentos mais usados para periodontite são a raspagem e aplainamento radicular, tratamento não cirúrgico, associado ou não ao uso de antimicrobianos. No entanto, novas terapias têm sido testadas com foco na modulação da resposta do hospedeiro. Alguns micro-organismos têm efeitos benéficos na saúde dos seres humanos, pois produzem efeitos antimicrobianos e anti-inflamatórios, os chamados probióticos. O presente trabalho avaliou o efeito antimicrobiano de Lactobacillus reuteri sobre Porphyromonas gingivalis e a influência deste probiótico em sua forma viva, morta (paraprobiótico) e sobrenadante em modelo de invertebrado Galleria mellonella, infectado por P. gingivalis. Posteriormente, foi determinada a viabilidade celular, níveis de óxido nítrico e de interleucina (IL)-1βb, IL-6, IL-17 e fator de necrose tumoral (TNF)- α, através do ensaio de ELISA em osteoblastos infectados por LPS de P. gingivalis in vitro. Os dados foram submetidos ao teste estatístico ANOVA, Kruskal-Wallis ou Log-rank (Mantel-Cox), com nível de significância de 5%.L. reuteri e seu sobrenadante possuem a mesma atividade antimicrobiana. O probiótico viável e o morto apresentaram efeitos iguais na sobrevivência de G. mellonella e L. reuteri vivo foi o único que aumentou densidade hemocitária das lagartas. O probiótico e o paraprobiótico reduziram igualmente os níveis IL-1β, IL-6, TNF-α e IL-17, sendo que o paraprobiótico, diferentemente do lactobacilo vivo, reduziu significantemente as quantidades de IL-... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The most used treatments for periodontitis are scaling and non-surgical root planing, associated or not to the use of antimicrobials. However, new therapies have been tested with a focus on host response modulation. Some microorganisms have beneficial effects on human health because they produce antimicrobial and antiinflammatory effects, called probiotics. The present study evaluated the antimicrobial effect of Lactobacillus reuteri on Porphyromonas gingivalis and the influence of this probiotic in its live, inactivated (paraprobiotic) form and supernatant on Galleria mellonella invertebrate model, after infection by P. gingivalis. Later, the cell viability, nitric oxide levels and interleukin (IL)-1b, IL-6, IL-17 and tumor necrosis factor (TNF)- α, by the Elisa assay, were evaluated in osteoblasts infected with P. gingivalis LPS in vitro. Data were submitted to ANOVA, Kruskal-Wallis or Log-rank (Mantel-Cox) statistical test, with a significance level of 5%. L. reuteri and its supernatant have the same antimicrobial activity. The viable and inactivated probiotic had equal effects in G. mellonella survival and L. reuteri alive was the only one that increased the hemocyte density in the invertebrate model. Probiotics and paraprobiotics also reduced levels of IL-1β, IL-6, TNF-α and IL-17 cytokines, whereas paraprobiotic, unlike living lactobacillus, significantly reduced the amounts of IL-6 and TNF-α, compare to the LPS control group. The highest reductions of the studied cytok... (Complete abstract click electronic access below) / Mestre

A técnica do enxerto de granulação óssea tem demonstrado bons resultados na recuperação do periodonto e na melhora dos parâmetros clínicos dos dentes com comprometimento periodontal. Pouco se sabe porém, a respeito de qual tipo de tratamento de superfície radicular se faz mais condizente com o emprego dessa técnica. O objetivo dessa pesquisa foi avaliar a proliferação de células de granulação óssea sobre fragmentos radiculares com os seguintes tratamentos de superfície: Controle - somente raspagem, EDTA, terapia fotodinâmica (PDT- laser InGaAIP - 30mW, 30s, 45J/cm², 660nm + azul de toluidina), e ácido cítrico com tetraciclina. Todos os grupos teste receberam previamente tratamento com raspagem e alisamento com 20 golpes de cureta. Células de granulação óssea foram cultivadas em quadruplicata sobre os fragmentos por um período de 24, 48 e 72 horas. Após esse período de cultivo os fragmentos foram fixados para análise em microscópio eletrônico de varredura (MEV). Cinco campos por fragmento foram usados para a visualização e contagem de células aderidas a superfície radicular (centro, campo superior direito e esquerdo e campo inferior direito e esquerdo). A análise da calibração do examinador foi feita através de uma combinação de testes estatísticos como erro casual de Dahlberg, erro sistemático e correlação de Pearson (p<0,05). A análise da amostra foi realizada através do ANOVA de medidas repetidas complementado por Tukey, com nível de significância de 5% (p<0,05). Os resultados demostraram diferenças estatisticamente significantes, quanto ao numero de células, para as superfícies tratadas com terapia fotodinâmica no período de 72 h (p<0,05). Através de nossos resultados concluímos que o tratamento radicular com terapia fotodinâmica favorece a proliferação de células de granulação óssea humanas in vitro. / The osseous granulation graft has been demonstrating good results on the periodontal healing, resulting the improvement of clinical periodontal parameters. There are very few knowledge about what kind of dental surface would be more proper for the application of this technique. The aim of this study was to evaluate the proliferation of osseous granulation cells on human root fragments treated by different techniques as scaling and root planning (control), citric acid plus tetracycline, EDTA and photodynamic therapy (PDT InGaAIP, 45J/cm², 30mW, 30s, 660nm, toluidine blue O). All test groups were previously treated which 20 curette strikes. Osseous granulation cells was culture in quadruplicate on these fragments for 24h, 48h and 72h. After that, all fragments were fixed and prepared for analysis in Scanning Electron Microscopy (SEM). Aiming to counting the cells adhered on the roots, we obtained electron micrographs of 5 areas (center, upper right and left field, lower right and left field). The examiner calibration was analyzed by Dahlberg Casual Error measurement, systematic error test and Pearson correlation test (p<0.05). Statistical analysis was performed by ANOVA, followed by Tukey test, with a 5% level of significance (p<0.05). There were significant differences in cell number after 72h culture in favor of PDT group (p<0,05). We can conclude that the surface treatment of roots which PDT favor the proliferation of osseous granulation cells in vitro.

Made available in DSpace on 2014-06-11T19:23:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-12-18Bitstream added on 2014-06-13T18:51:19Z : No. of bitstreams: 1 luvizuto_er_me_araca.pdf: 1006198 bytes, checksum: 7f15315191ddd77cc08393427bd6824f (MD5) / Objetivos: Avaliar a interferência da ovariectomia (OVX) e seu tratamento com estrógeno (E2) ou com raloxifeno (RLX) no balanço entre RANKL/OPG na cronologia do processo de reparo alveolar em diferentes períodos (7, 14, 21 e 42 dias) através da imunofluorescência por co-localização e análise histomorfométrica. Materiais e Métodos: Os grupos estudados foram: sham, OVX, OVX+E2, OVX+RLX. Após obtenção dos cortes histológicos corados em hematoxilina e eosina e as reações de co-localização por imunofluorescência de RANKL/OPG, os resultados foram avaliados quantitativamente. Resultados:Aos 7 dias: menor neoformação de trabéculas ósseas,o grupo OVX+RLX apresentou menor valor médio. O grupo OVX apresentou o maior turnover ósseo representado pelas co-localizações de OPG e RANKL. Aos 14 dias o grupo OVX+RLX apresentou menor formação óssea. O grupo sham apresentou intensa atividade celular representada pela alta imunorreatividade à OPG e RANKL observada nas células. Aos 21 dias os grupos experimentais apresentaram maiores níveis de ossificação; não apresentaram diferença estatística. O grupo OVX apresentou o menor turnover ósseo. Aos 42 dias houve diferença estatística na quantidade de formação óssea entre o grupo sham comparado aos demais grupos (p<0,05) e o grupo OVX apresentou o maior turnover ósseo. Conclusão: A ovariectomia atrasou o processo de reparo alveolar e alterou o turnover ósseo. A reposição do estrógeno e o tratamento com raloxifeno melhoraram as respostas, mas não restabeleceram completamente os valores da histometria e da colocalização do grupo sham. / Objectives: To evaluate the influence of the ovariectomy (OVX), and its treatments with estrogen (E2) or with raloxifene (RLX) on the RANKL/OPG balance during the periods in the chronology of the alveolar wound healing process (7, 14, 21 end 42 pos operative days) in female rats by means of immunocolocalization and histomorphometric analysis. Methods: The studied groups were: sham, OVX, OVX with E2 replacement, OVX with (RLX) treatment. After obtaining the histological tissue pieces colored in hematoxilin and eosin and the immunocolocalization reaction for RANKL and OPG, the results were quantitatively evaluated. Results: At 7 days, was observed lesser neoformed trabeculae bone, the smaller medium value was observed to the OVX+RLX group. The OPG and RANKL immunocolocalization showed larger bone tunover to OVX group. At 14 days there was a larger quantity of neoformed trabeculae bone, the smaller medium value was observed to the OVX+RLX group, the sham group presented an intense cellular activity. At 21 days the experimental groups had a greater ossification levels; no statistical significance was observed. The OVX group had the lowest bone turnover. At 42 days there were statistically differences on the quantity of ossification within sham group compared to the other groups (p<0.05). The OVX group showed the largest bone turnover. Conclusions: Ovariectomy delays the alveolar wound healing process and interferes with the bone turnover. The E2 replacement and the RLX treatment improved the healing but not enough to reach histomorphometric and immunocolocalization valours of the sham group.

Submitted by ELISA MATTIAS SARTORI null (elisamsartori@gmail.com) on 2016-06-23T20:05:53Z No. of bitstreams: 1 Elisa Mattias Sartori.pdf: 1436534 bytes, checksum: dd0f5ca15c561bf7bd3853e437fe11ee (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-06-27T18:58:23Z (GMT) No. of bitstreams: 1 sartori_em_dr_arafo.pdf: 1436534 bytes, checksum: dd0f5ca15c561bf7bd3853e437fe11ee (MD5) / Made available in DSpace on 2016-06-27T18:58:23Z (GMT). No. of bitstreams: 1 sartori_em_dr_arafo.pdf: 1436534 bytes, checksum: dd0f5ca15c561bf7bd3853e437fe11ee (MD5) Previous issue date: 2016-04-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Proposição: Este estudo teve como objetivo avaliar a modulação dos miRNAs que afetam o potencial osteogênico de CTMs em diferentes topografias de superfície de discos de vidro. Material e Métodos: Células tronco mesenquimais humanas foram plaqueadas nas diferentes superfícies e comparadas após 3, 7 e 14 dias para atividade de fosfatase alcalina, expressão de genes (Osteocalcina, Osteopontina, Sialo Proteína Óssea, Osterix, Runx2, BMP2 e ALP) e expressão de miRNAs. Microscopia eletrônica de varredura das superfícies com células foram obtidas para avaliação de adesão celular. Resultados: Atividade de fosfatase alcalina nas diferentes superfícies foi significantemente maior na superfície com nanotopografia. Do mesmo modo, a expressão de genes relacionados com osteoblastos foi mais elevada na superfície nano. Com 14 dias foi observado um aumento de 3.5 e 9 vezes para os genes Runx2 e Osterix, respectivamente. O gene da BMP2 e ALP também apresentou um aumento de 4 e 7 vezes comparado ao controle. Utilizando a tecnologia de sequenciamento de RNA (RNA-Seq) onde todos os RNAs existentes foram sequenciados, um total de 123 miRNAs com diferença de expressão foram encontrados comparando a superfície controle (dia 7) com a superfície nano (dia 14). 48 miRNAs apresentaram uma redução na expressão e 75 apresentaram um aumento de expressão. Alguns destes apresentaram marcadores para genes osteogênicos já identificados, tais como hsa-miR-135b-5p marcador para OCN, BSP, Runx2, CO15A1 e OSX, hsa-miR-122-5p marcador para OPN, hsa-miR-196a-5p marcador para BMP4, hsa-miR-26b-5p marcador para BMP2 e hsa-miR-148b-3p marcador para OPN. Conclusão: As superfícies com nanotopografia tem o potencial de melhorar a resposta de osseointegração de maneira a reduzir o tempo de osseointegração e também aumentar a produção de tecido ósseo ao redor dos implantes favorecendo assim áreas de qualidade óssea baixa. A utilização de miRNAs para alterar a resposta de diferenciação pode também ajudar a controlar o processo de osseointegração. / Purpose: This study aimed to evaluate the modulation of miRNAs that affect the osteogenic potential of MSCs in different surface topographies of glass disks. Material and Methods: Human mesenchymal stem cells (hMSCs) were plated on different surfaces of glass disks and compared at 3,7 and 14 days for alkaline phosphatase (ALP) activity, expression of genes (Osteocalcin, Osteopontin, Bone Sialo Protein, Osterix, Runx2, BMP2 and ALP) and expression of miRNAs. Scanning electron microscopy of surfaces with cells were obtained to analyse the attachment of cells. Results: ALP activity on different surfaces was significantly greater in the nanotopography surface. At day 14 there was a 3.5-fold and a 9-fold increase for Runx2 and Osterix gene, respectively. BMP2 and ALP also increased by 4- and 7-fold compared to control. Using RNA sequencing technology (RNA-Seq) a total of 123 miRNAs were found differently expressed comparing control (day 7) to nano surface (day 14). 48 miRNAs were downregulated and 75 were upregulated. Some of them regulated osteogenic genes such as hsa-miR-135b-5p that targets OCN, BSP, RUNX2, CO15A1 and OSX, hsa-miR-122-5p wich targets OPN, hsa-miR-196a-5p targets BMP4, hsa-miR-26b-5p that targets BMP2 and hsa-miR-148b-3p that targets OPN. Conclusion: Surfaces with nanotopography have the potential to improve osseointegration response in order to reduce the time of osseointegration and also increase the production of bone tissue around the implants improving low bone quality areas. The use of miRNAs to affect differentiation response may also help control the osseointegration process. / CAPES: BEX8187/14-2