Para testar a hipótese de que a desmineralização in situ das superfícies de contato enxerto-leito, e a forma como o enxerto é estabilizado ao leito, podem influenciar os mecanismos envolvidos na consolidação do enxerto, fragmentos ósseos de 10 mm de diâmetro foram removidos das metáfises proximais tibiais de 36 coelhos (Oryctolagus Cuniculus) e transplantados para uma área adjacente. Na tíbia esquerda dos animais, as superfícies de contato do enxerto e do leito hospedeiro foram desmineralizadas com ácido cítrico pH 1,0 por 3 minutos antes dos enxertos serem fixados ao leito. Na tíbia direita, o transplante do bloco ósseo não foi precedido de desmineralização. Metade dos enxertos foi imobilizada sobre o leito pela superposição de uma membrana não reabsorvível de politetrafluoretileno colada com cianoacrilato ao leito à distância da interface enxerto-leito. A outra metade dos enxertos foi fixada por um parafuso de titânio no centro do enxerto. Assim, foram formados 4 grupos de estudo: membrana (M), membrana + ácido (MA), parafuso (P) e parafuso + ácido (PA). Três animais de cada grupo forneceram espécimes para análise microscópica quantitativa e qualitativa aos 15, 30 e 45 dias de pós-operatório. A análise qualitativa demonstrou que não houve formação óssea na interface em nenhum espécime aos 15 dias e que nos demais períodos, em todos os grupos, a quantidade de tecido ósseo neoformado na interface e seu estágio de maturação aumentaram com o tempo. Ambos os métodos de fixação empregados foram eficientes em manter os enxertos em posição, porém a membrana promoveu menor reabsorção da estrutura do enxerto. A análise quantitativa computadorizada revelou que, aos 30 dias, os grupos MA e PA apresentaram maior área de formação óssea na interface (71,34 ± 12,03%; 56,74 ± 2,15% respectivamente) em relação aos grupos M e P (51,75 ± 11,02%; 43,95 ± 4,05% respectivamente) e superfícies de consolidação óssea mais extensas (93,41 ± 5,95%; 93,73 ± 4,96% respectivamente) do que os grupos sem tratamento ácido (73,49 ± 7,7%; 73,77 ± 11,77% respectivamente para M e P), sendo essas diferenças estatisticamente significantes (p<0,05). Aos 45 dias de pós-operatório, os grupos MA e PA (71,18 ± 8,9%; 58,97 ± 3,97% respectivamente) apresentaram resultados superiores aos grupos M e P (59,78 ± 11,28%; 46,08 ± 3,53% respectivamente) em relação à área de neoformação óssea na interface, porém essa diferença não foi significativa. Concluiu-se que a desmineralização ácida das superfícies contactantes nos enxertos ósseos autógenos em bloco na tíbia de coelhos promoveu a osteogênese na interface enxerto-leito e acelerou a consolidação dos enxertos. Além disso, quando o tratamento ácido foi associado ao uso de membrana como método de fixação, a consolidação e grau de reabsorção óssea foram otimizados. / In order to test the hypothesis that the demineralization \"in situ\" of contacting surfaces of bone graft/bone bed and the fixation method used for graft stabilization can influence the mechanisms involved in the consolidation of the graft, bone fragments of 10 mm in diameter were removed from the proximal tibial metaphysis of thirty-six male rabbits (Oryctolagus Cuniculus) and transplanted to an adjacent area. In the left tibia of the animals, the contacting surfaces of the graft and host bed were demineralized with citric acid pH 1.0 for 3 minutes before the grafts were fixed to the receptor bed. In the right tibia, the bone block transplantation was not preceded by demineralization. Half of the grafts were immobilized on the bone bed by a nonresorbable polytetrafluoroethylene membrane glued with cyanoacrylate adhesive to the host bed distant from the bone graft-bone bed interface. The other half of the grafts were fixed by a titanium screw in the center of the graft. Thus, four groups were formed: membrane (M), membrane + acid (MA), screw (P) and screw + acid (PA). Three animals from each group provided specimens for quantitative and qualitative microscopic analysis at 15, 30 and 45 days postoperatively. Qualitative analysis showed no significant bone formation at the interface in any specimen of the groups at 15 days and on the other periods in all groups, the amount of newly formed bone at the interface as well as the stage of bone maturation increased with time. Both fixation methods were effective in maintaining the graft in position, but the membrane resulted in less resorption of the graft. Quantitative analysis, performed by means of a computer program for image analysis, showed that at day 30, groups MA and PA, showed greater area of bone formation at the interface (71.34 ± 12.03%; 56.74 ± 2 15%) than groups M and P (51.75 ± 11.02%, 43.95 ± 4.05%) and more osseointegrated bone surfaces (93.41 ± 5.95%, 93.73 ± 4.96%) than those without acid treatment (73.49 ± 7.7%, 73.77 ± 11.77%), and these differences were statistically significant. At 45 days postoperatively MA and PA groups (71.18 ± 8.9%, 58.97 ± 3.97%) showed better results than the M and P groups (59.78 ± 11.28%, 46 , 08 ± 3.53%) compared to the area of new bone formation at the interface and osseointegrated surfaces, but these differences were not significant. It was concluded that the acid demineralization of contacting surfaces in autogenous onlay bone grafts in the tibia of rabbits promotes osteogenesis in bone graft-host bed interface and accelerates the consolidation of the grafts. Furthermore, the association of this surface treatment to the use of membrane/cyanoacrylate fixation method, optimizes the results regarding consolidation and degree of bone graft resorption.

Local invasion and lymph nodal spread impact in the outcome of Head and Neck squamous cell carcinoma (HNSCC) patients (pts). We determined CXCR1-5, CCR7 and CX3CR1 mRNA expression by means of RNAse protection assay in 98 HNSCC primary tumors and 91 adjacent mucosa and 26 metastatic lymph nodes, correlating this data with outcome. CXCL12 and CCL19/CCL21, ligands for CXCR4 and CCR7, were determined in 38 tumor fragments, 33 adjacent mucosas and 25 de metastatic lymph nodes, by means of Quantitative Real-Time PCR. Tumors presented higher CXCR1 (P=0.013), CXCR3 (P=0.008) and CXCR4 mRNA (P=0.025) expression as compared to mucosa. No correlations are observed neither lymph nodal status nor tumor size impacted on chemokine receptor expression. Metastatic lymph nodes expressed more CXCR4, CXCR5, CCR7 and CX3CR1 (P<0.0001) as compared to matched tumors. We found a longer overall survival (OS) (P=0.048) and a trend toward longer disease free survival (DFS) (P=0.074) in CX3CR1 negative (n=17) as compared to positive pts (n=21) only in oral subgroup. The same occurred for CCR7 negative oral SCC, in terms of OS (P=0.024) and DFS (P=0.049). We conclude that, of the chemokine receptors here studied, CCR7 and CX3CR1 mRNA expression seems to better reflect outcome in oral subsite only. In addition, CCL21, a CCR7 ligand mRNAs is more expressed in metastatic lymph nodes than tumors (P=0.059). Further studies are warranted to confirm these results. / Bone tissue alterations and vascular calcification (VC) are commonly found in patients with chronic renal failure (CKD). The importance of phosphorus (P) and parathyroid hormone (PTH) is not clear, yet. An in vitro study showed that inorganic phosphate was able to transform vascular smooth muscle cells (VSMC) into calcifying cells confirmed for up-expression of Runx2 in these cells. Besides, it has been demonstrated the in vivo expression of Runx2 in intimal and medial VSMC in calcified arteries of CKD patients. We evaluated the effect of phosphorus (P) and parathyroid hormone (PTH) on bone remodeling and on the expression of bone proteins (Runx2, Osteoprotegerin, type I Collagen, Osteocalcin, Osteopontin and NF?B) in aortic valve and heart in experimental uremia. Wistar rats were submitted to parathyroidectomy, nephrectomy (Nx) and continuous infusion of 1-34 rat PTH in physiologic or 5 times the normal values. The diet was identical, however the P content was low (LP: 0,2%) or high (HP: 1,2%). We performed biochemical, histomorphometric, imuno-histochemistry and RT-PCR analysis. Rats submitted to Nx developed renal failure. The P overload contributed to loss bone volume independent of uremia. Besides Nx animals that received high PTH doses bone loss was slight probably because of the anabolic effect of PTH, which was attenuated by the phosphorus overload toxic. VC was only observed in Nx animals that received high PTH doses independently of P overload. However, the P overload with physiologic PTH doses induced phenotypic changes in VSMC that was confirmed for the up-expression of Runx2 on aorta of these animals. The high concentrations of P and PTH promoted histological changes on expression of osteoprotegerin and type I Collagen in calcified arteries and heart. This study does not established ideal levels of PTH sufficient for the maintenance of the bone integrity and also to prevent VC when animal are submitted to different P overload.

As fraturas e perdas ósseas representam altos riscos para o Sistema público de Saúde (SUS), além de afetar a qualidade de vida do paciente, portanto é necessário o entendimento das bases moleculares que envolvem os mecanismos de reparo ósseo. Citocinas secretadas por células do sistema imune presentes no local da inflamação, como as IL-6, IL-10 e TNF&#945; atuam como fatores quimiotáticos para células mesenquimais, que proliferam e se diferenciam em osteoblastos pela ação autócrina e parácrina de Proteínas Morfogenéticas Ósseas (BMPs), principalmente a BMP2. Embora seja conhecido que a ação de BMP2 ocorra através de sua ligação nos receptores ActRI/BMPR, que ativam proteínas SMADS 1/5/8 efetoras, pouco se sabe sobre os mecanismos intracelulares que participam do processo de diferenciação osteoblástico. Neste estudo propôs-se analisar as diferenças no conteúdo de proteínas totais e de proteínas fosforiladas em células mesenquimais de pele induzidas à osteogênese pelo tratamento com BMP2 por diferentes períodos de tempo, utilizando-se de Isótopos Estáveis de Dimetila acoplado ao LC/MS. A partir de 150&#181;g de material inicial, foi possível identificar 2.264 proteínas, as quais foram quantificadas nos diferentes pontos de indução, sendo que 235 são fosforiladas. Análise de motivos de quinases mostrou que diversos substratos possuem sítios fosforilados correspondentes àqueles dos motivos de fosforilação das quinases Casein Kinase, p38, CDK e JNK. A análise da ontologia gênica mostrou um aumento de processos biológicos relacionados com sinalização e diferenciação após a primeira hora de indução com rhBMP2. Além disso, proteínas envolvidas com o rearranjo do citoesqueleto e com vias de sinalização Wnt e Ras foram encontradas como tendo fosforilação diferencial durante todos os períodos estudados. Os dados revelaram novos substratos intracelulares que são fosforilados nos primeiros momentos do comprometimento com a diferenciação osteoblástica mediada pelo tratamento com rhBMP2 em células mesenquimais derivadas da pele. Além disso, clones celulares que superexpressam as proteínas recombinantes humanas BMP2 e BMP4 foram gerados, e sua atividade verificada in vitro. Paralelamente, a rhBMP7, obtida anteriormente, foi purificada por cromatografia de afinidade utilizando-se uma coluna de Heparina-Sepharose, que foi posteriormente utilizada para ensaios in vitro e in vivo, nos quais se mostrou capaz de gerar osteoblastos e tecido ósseo, respectivamente, o que abre novas possibilidades para o uso destas proteínas como biofármacos no Brasil. / Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNF&#945;, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated human skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. From 150 &#181;g of starting material, 2,264 proteins containing two or more peptides were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during commitment to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells. Cell clones overexpressing the human BMP 2 and 4 recombinant proteins were also generated, and their biological activity was confirmed in vitro. In parallel, chromatography-affinity purified rhBMP7, obtained using heparin-Sepharose columns, was used for in vivo and in vitro assays to evaluate the ability of this purified protein to generate osteoblasts and bone tissue, respectively, opening new avenues for the use of these proteins as biopharmaceuticals in Brazil.

Made available in DSpace on 2015-03-26T13:46:46Z (GMT). No. of bitstreams: 1 texto completo.pdf: 741759 bytes, checksum: aa0908de8ca0efc968041d218848f7b4 (MD5) Previous issue date: 2005-08-26 / Centro Universitário Vila Velha / The bone morphogenetic proteins (BMPs) are growth promoters capable of inducing the formation of bone and cartilage, promoting quimiotaxy and differentiation of mesenchymal cells in the place where they were implanted, but for an effective osteoconduction they need a carrier substance. When implanted in animal tissues, the BMPs link to specific receptors located in the surface of the osteoinduced cells, they activate the SMAD cytoplasmatic proteins (mothers against decapentaplegic proteins) and, in the nucleus of the receiver cell, they begin to regulate the transcription of genes related with calcification and bone formation. In the present study, aspects of clinical-surgical, macroscopic and radioscopic of the bone repair were evaluated after the implantation of the BMPs in different concentrations, carried by autogenous bone graft, in defects created on skulls of 20 adult-young female rabbits, randomizedly separated on five experimental groups that correspond to five periods of observations (7, 15, 21, 35 and 60 days). After the exposure of bones skull, six bone defects were performed on the fronto-parietal region of each animal. The defect I wasn´t filled, the II was completed filled with approximately 3,0 mg of autogenous bone graft and the defects III, IV, V and VI were filled with autogenous bone graft associed with 0,5; 1,0; 2,0 and 5,0 mg of BMPs respectively. There wasn´t clinical complications after the surgery. In the macroscopic evaluations, post-mortem, it was observed that independently of the treatment period of the defects, the bony filling began starting from the borders to the center and from the bottom to the surface of the flaws. The bony filling of defect I was the smallest found when compared with the others defects, in all the observation periods. It was also verified that until 2,0 mg the bigger the concentration of BMPs used, better was bone cover. The radiographic assessment did not permit the classification of the grade of bone filling and through the statistic analyses, it was comproved that there are significant differences between the treatments. It was verified microscopically in the first evaluations, at the seventh day, that the bone growth started from the borders and from the bottom of the lesion, with mobilization and differentiation of cells deriving from the periosteum and of the meninges, respectively, and in the subsequent evaluations, the osteoblastic activity also derived from "ossification islands" to ossification centers, located in the center of the flaw. The trabecular formation increased proportionally with the concentration of BMPs used, and the apposition and bone organization increased proportionally with the time of observation. The presence of cartilaginous tissue was verified in almost all the flaws. The best relation of quantities of autogenous bone graft and BMPS were 3,0 mg and 2,0 mg, respectively, and the association of this protein with this graft contributed to the formation of new bone tissue, promoting larger mobilization, differentiation and cellular organization. It also abbreviated the time of bone formation, suggesting an endochondral ossification process. / As proteínas morfogenéticas ósseas (BMPs) são promotores de crescimento capazes de induzir a formação de osso e cartilagem, promovendo a quimiotaxia e diferenciação de células mesenquimais para o local onde foram implantadas, mas para uma efetiva osteocondução necessitam de uma substância carreadora. Quando implantadas em tecidos animais, as BMPs se ligam a receptores específicos existentes na superfície das células osteoinduzidas, ativam as proteínas citoplasmáticas SMAD (mothers against decapentaplegic proteins) e, no núcleo da célula receptora, passam a regular a transcrição de genes relacionados com calcificação e formação óssea. No presente estudo, foram avaliados os aspectos clínico-cirúrgicos, macroscópicos, radiográficos e microscópicos da reparação óssea, após implantação de BMPs em diferentes concentrações, carreadas por enxerto ósseo esponjoso, em falhas criadas no crânio de 20 coelhas adultas-jovens, separadas aleatoriamente em cinco grupos experimentais, que corresponderam a cinco períodos de observação (7, 15, 21, 35 e 60 dias). Após a exposição dos ossos do crânio, foram realizadas seis falhas ósseas na região fronto-parietal de cada animal. A falha I não foi preenchida, a II foi completamente preenchida com aproximadamente 3,0 mg de autoenxerto ósseo esponjoso e as falhas III, IV, V e VI foram preenchida com o autoenxerto associado a 0,5; 1,0; 2,0 e 5,0 mg de BMPs, respectivamente. Não foram observadas complicações clínicas após a cirurgia. Nas avaliações macroscópicas, postmortem, verificou-se que independentemente do período de tratamento das falhas, o preenchimento ósseo iniciava-se a partir das bordas para o centro e do fundo para a superfície das falhas. O preenchimento ósseo das falhas I foi o menor encontrado quando comparado com as demais falhas, em todos os períodos de observação. Constatou-se também que até os valores de 2,0 mg, quanto maior a concentração de BMPs utilizada, maior foi a cobertura óssea. A avaliação radiográfica não permitiu a classificação do grau de preenchimento ósseo e por meio de análises estatísticas, foi comprovado que há diferenças significativas entre os tratamentos. Microscopicamente verificou-se nas primeiras avaliações, aos sete dias, que o preenchimento ósseo iniciava-se a partir das bordas e do fundo da lesão, com mobilização e diferenciação de células provenientes do periósteo e das meninges, respectivamente e, nas avaliações subseqüentes, a atividade osteoblástica provinha também de ilhas de ossificação , semelhantes a centros de ossificação, localizadas no centro da falha. A formação trabecular aumentou proporcionalmente com a concentração utilizada de BMPs e a aposição e organização óssea aumentaram proporcionalmente com o tempo de observação. Em quase todas as falhas foi verificada a presença de tecido cartilaginoso. A melhor relação de quantidade de autoenxerto ósseo esponjoso e BMPs foi de 3,0 mg e 2,0 mg, respectivamente e a associação desta proteína com esse enxerto contribuiu para a formação de novo tecido ósseo, promovendo maior mobilização, diferenciação e organização celular e, abreviou o tempo de formação óssea, sugerindo processo de ossificação endocondral. Os trabalhos apresentados como parte integrante desta tese estão seguindo as normas de publicação do Arquivo Brasileiro de Medicina Veterinária e Zootecnia da Universidade Federal de Minas Gerais, indexada ao ISI, AGRIS, BIOSIS, CAB, CAS, MEDLARS, Referatvnyi Zhumal, Bibliografia Brasileira de Medicina Veterinária e Zootecnia e LILACS. ISSN 01020935.

As fraturas e perdas ósseas representam altos riscos para o Sistema público de Saúde (SUS), além de afetar a qualidade de vida do paciente, portanto é necessário o entendimento das bases moleculares que envolvem os mecanismos de reparo ósseo. Citocinas secretadas por células do sistema imune presentes no local da inflamação, como as IL-6, IL-10 e TNF&#945; atuam como fatores quimiotáticos para células mesenquimais, que proliferam e se diferenciam em osteoblastos pela ação autócrina e parácrina de Proteínas Morfogenéticas Ósseas (BMPs), principalmente a BMP2. Embora seja conhecido que a ação de BMP2 ocorra através de sua ligação nos receptores ActRI/BMPR, que ativam proteínas SMADS 1/5/8 efetoras, pouco se sabe sobre os mecanismos intracelulares que participam do processo de diferenciação osteoblástico. Neste estudo propôs-se analisar as diferenças no conteúdo de proteínas totais e de proteínas fosforiladas em células mesenquimais de pele induzidas à osteogênese pelo tratamento com BMP2 por diferentes períodos de tempo, utilizando-se de Isótopos Estáveis de Dimetila acoplado ao LC/MS. A partir de 150&#181;g de material inicial, foi possível identificar 2.264 proteínas, as quais foram quantificadas nos diferentes pontos de indução, sendo que 235 são fosforiladas. Análise de motivos de quinases mostrou que diversos substratos possuem sítios fosforilados correspondentes àqueles dos motivos de fosforilação das quinases Casein Kinase, p38, CDK e JNK. A análise da ontologia gênica mostrou um aumento de processos biológicos relacionados com sinalização e diferenciação após a primeira hora de indução com rhBMP2. Além disso, proteínas envolvidas com o rearranjo do citoesqueleto e com vias de sinalização Wnt e Ras foram encontradas como tendo fosforilação diferencial durante todos os períodos estudados. Os dados revelaram novos substratos intracelulares que são fosforilados nos primeiros momentos do comprometimento com a diferenciação osteoblástica mediada pelo tratamento com rhBMP2 em células mesenquimais derivadas da pele. Além disso, clones celulares que superexpressam as proteínas recombinantes humanas BMP2 e BMP4 foram gerados, e sua atividade verificada in vitro. Paralelamente, a rhBMP7, obtida anteriormente, foi purificada por cromatografia de afinidade utilizando-se uma coluna de Heparina-Sepharose, que foi posteriormente utilizada para ensaios in vitro e in vivo, nos quais se mostrou capaz de gerar osteoblastos e tecido ósseo, respectivamente, o que abre novas possibilidades para o uso destas proteínas como biofármacos no Brasil. / Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNF&#945;, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated human skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. From 150 &#181;g of starting material, 2,264 proteins containing two or more peptides were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during commitment to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells. Cell clones overexpressing the human BMP 2 and 4 recombinant proteins were also generated, and their biological activity was confirmed in vitro. In parallel, chromatography-affinity purified rhBMP7, obtained using heparin-Sepharose columns, was used for in vivo and in vitro assays to evaluate the ability of this purified protein to generate osteoblasts and bone tissue, respectively, opening new avenues for the use of these proteins as biopharmaceuticals in Brazil.

Local invasion and lymph nodal spread impact in the outcome of Head and Neck squamous cell carcinoma (HNSCC) patients (pts). We determined CXCR1-5, CCR7 and CX3CR1 mRNA expression by means of RNAse protection assay in 98 HNSCC primary tumors and 91 adjacent mucosa and 26 metastatic lymph nodes, correlating this data with outcome. CXCL12 and CCL19/CCL21, ligands for CXCR4 and CCR7, were determined in 38 tumor fragments, 33 adjacent mucosas and 25 de metastatic lymph nodes, by means of Quantitative Real-Time PCR. Tumors presented higher CXCR1 (P=0.013), CXCR3 (P=0.008) and CXCR4 mRNA (P=0.025) expression as compared to mucosa. No correlations are observed neither lymph nodal status nor tumor size impacted on chemokine receptor expression. Metastatic lymph nodes expressed more CXCR4, CXCR5, CCR7 and CX3CR1 (P<0.0001) as compared to matched tumors. We found a longer overall survival (OS) (P=0.048) and a trend toward longer disease free survival (DFS) (P=0.074) in CX3CR1 negative (n=17) as compared to positive pts (n=21) only in oral subgroup. The same occurred for CCR7 negative oral SCC, in terms of OS (P=0.024) and DFS (P=0.049). We conclude that, of the chemokine receptors here studied, CCR7 and CX3CR1 mRNA expression seems to better reflect outcome in oral subsite only. In addition, CCL21, a CCR7 ligand mRNAs is more expressed in metastatic lymph nodes than tumors (P=0.059). Further studies are warranted to confirm these results. / Bone tissue alterations and vascular calcification (VC) are commonly found in patients with chronic renal failure (CKD). The importance of phosphorus (P) and parathyroid hormone (PTH) is not clear, yet. An in vitro study showed that inorganic phosphate was able to transform vascular smooth muscle cells (VSMC) into calcifying cells confirmed for up-expression of Runx2 in these cells. Besides, it has been demonstrated the in vivo expression of Runx2 in intimal and medial VSMC in calcified arteries of CKD patients. We evaluated the effect of phosphorus (P) and parathyroid hormone (PTH) on bone remodeling and on the expression of bone proteins (Runx2, Osteoprotegerin, type I Collagen, Osteocalcin, Osteopontin and NF?B) in aortic valve and heart in experimental uremia. Wistar rats were submitted to parathyroidectomy, nephrectomy (Nx) and continuous infusion of 1-34 rat PTH in physiologic or 5 times the normal values. The diet was identical, however the P content was low (LP: 0,2%) or high (HP: 1,2%). We performed biochemical, histomorphometric, imuno-histochemistry and RT-PCR analysis. Rats submitted to Nx developed renal failure. The P overload contributed to loss bone volume independent of uremia. Besides Nx animals that received high PTH doses bone loss was slight probably because of the anabolic effect of PTH, which was attenuated by the phosphorus overload toxic. VC was only observed in Nx animals that received high PTH doses independently of P overload. However, the P overload with physiologic PTH doses induced phenotypic changes in VSMC that was confirmed for the up-expression of Runx2 on aorta of these animals. The high concentrations of P and PTH promoted histological changes on expression of osteoprotegerin and type I Collagen in calcified arteries and heart. This study does not established ideal levels of PTH sufficient for the maintenance of the bone integrity and also to prevent VC when animal are submitted to different P overload.

Made available in DSpace on 2015-07-22T20:50:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-01-26. Added 1 bitstream(s) on 2015-08-11T03:26:03Z : No. of bitstreams: 1 Publico-12529a.pdf: 1382817 bytes, checksum: 65bd98549521fbf2650836a170f9761e (MD5). Added 1 bitstream(s) on 2015-08-11T03:26:03Z : No. of bitstreams: 2 Publico-12529a.pdf: 1382817 bytes, checksum: 65bd98549521fbf2650836a170f9761e (MD5) Publico-12529b.pdf: 1113563 bytes, checksum: 3261d75b2356f849d087c69443322b57 (MD5) / As BMPs, proteínas indutoras de crescimento ósseo, desde o início de sua utilização têm sido avaliadas em diferentes modelos experimentais objetivando determinar sua eficácia. Sabemos que algumas substâncias podem interferir positiva ou negativamente quando utilizadas de forma sistêmica ou local, associadas à BMP. Objetivo: Este estudo tem por objetivo avaliar as possíveis interferências da utilização de antibioticoterapia profilática pré e pós-operatória, utilizando-se como princípio ativo a cefazolina, aplicada a um modelo experimental em coelhos. Métodos: Foram utilizados dois grupos de coelhos fêmea, neozelandeses, submetidos à artrodese intertransversa da coluna lombar, segmento L5-L6, por via posterior. No primeiro grupo foi utilizado o enxerto autólogo associado ao biocomposto (BMP bovino, 1,0mg e hidroxiapatita, 9,0mg). No segundo grupo foi realizado o mesmo procedimento e utilizado o mesmo biocomposto, porém os animais foram submetidos a antibioticoterapia profilática com cefazolina iniciada duas horas antes e mantida por 24 horas após o término do procedimento. Os animais foram acompanhados por 15 semanas, isolados em cativeiro e avaliados diariamente por veterinário sob o ponto de vista clínico e neurológico, sendo posteriormente sacrificados e retiradas as peças cirúrgicas para serem submetidas à análise radiográfica e histológica. Resultados: Para o grupo 1, a quantidade e localização do material implantado variaram entre os indivíduos, porém, na maioria dos casos (6 amostras), a quantidade de partículas de osso homólogo era insignificante e estava dispersa ao longo do tecido mole que recobre o dorso da vértebra, circundado por tecido reacional com área de necrose. Nos demais casos as partículas com reabsorção preenchiam o reduzido espaço entre os processos transversos. Para o grupo 2, a quantidade do material e sua localização também variaram entre os indivíduos. Na maioria dos casos inúmeras partículas de osso mole preenchiam o espaço entre os processos laterais cuja neoformação óssea levou ao aprisionamento de algumas dessas partículas. Todos os casos exibiram formação em maior ou menor intensidade de tecido cartilaginoso na superfície dos processos transversos. A análise radiográfica mostrou em sua freqüência relativa maior freqüência de fusão completa para o grupo 2 quando comparado ao grupo 1. Conclusão: Do ponto de vista histológico para o modelo e período experimental analisado, inferimos que, embora nenhum dos tratamentos propostos tenha promovido o completo fusionamento das vértebras por tecido ósseo, a utilização de osso homólogo + BMP bovina, associada à aplicação de cefazolina, promoveu maior formação cartilaginosa e óssea com menor índice de rejeição do material enxertado na área doadora, quando comparada ao grupo sem associação de cefazolina. Do ponto de vista radiográfico, a análise relativa também demonstrou-se superior para o grupo onde foi utilizado cefazolina. / The BMPs, the inductive proteins of bone growth since the beginning of their use have been evaluated in different experimental models aiming to determine their efficacy. We know that some substances can interfere positively or negatively when used in a systemic way or places associated with the BMP. Objective: this study objective to evaluate the possible interferences of antibiotic-therapy by using the active principle of cefazolin in an experimental model with rabbits. Methods: Two groups of female New Zealand rabbits underwent a lumbar spine inter-transverse artrodesys of segment L5-L6 using posterior approach. An homolog bone graft associated with a bio-compound (bovine BMP, 1,0mg and hydroxiapatita, 9,0mg) was used in the first group. The same procedure and bio-compound were used in the second group. However the animals were submitted to a prophylactic antibiotic-therapy with cefazolin starting two hours before the procedure and maintained for 24 hours after surgery. The animals were analyzed for 15 weeks, isolated in captivity and daily evaluated by a veterinarian under the clinical and neurological views and then euthanized, being the surgical pieces removed and submitted to a radiological and histological analysis. Results: For the first group the quantity and location of the implanted material varied among the individuals. However in most of the cases, the quantity and particles of homolog bone was insignificant and disperse along the soft tissue that covers the posterior region of the vertebrae. In the other cases, the particles with reabsorvation filled the reduced space between the transversal processes. For the second group, the quantity of material and its location also varied among the individuals. In most of the cases, several particles of homolog bone filled the space between the lateral processes whose bone neo-formation led to a trapping of these particles. All the cases showed formation in a higher or lower intensity of the cartilaginous tissue in the surface of the transverse processes. The radiological analysis showed in its relative frequency a higher frequency of complete fusion for group 2 when compared to group 1. Conclusion: Under the histological view for the model and experimental period analyzed, we inferred that, despite the fact that none of the proposed treatments had promoted a complete fusion of the vertebraes per bone tissue, the use of homolog bone + bovine BMPs associated with the use of cefazolin promoted a higher cartilaginous and bone formation with lower incidence of rejection of the material grafted in the doer area when compared to the group without the association of cefazolin. Under the radiological view, the relative analysis also showed to be superior in the group where cefazolin was used as a prophylactic antibiotic. / TEDE / BV UNIFESP: Teses e dissertações