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Bioactive glasses undergo dynamic changes in vivo to produce an apatite layer permitting a strong bond with living tissues including both bone and soft tissues, and their compositions can be modified and tailored. The aim of this project was to generate high phosphate low fluoride containing bioactive glasses and explore their bioactivity and biological performances in vitro. Bioactive glasses (0-7% F- content, constant 6.33% P2O5 in Mol.%) were produced and the particles immersed in Tris Buffer solution or cell culture medium (α-MEM) to determine apatite formation and ion (Ca, P, Si and F) release. Bioactive glass conditioned medium was used to treat pre-osteoblasts MC3T3-E1 for cytotoxicity, pre-osteogenic and pro-angiogenic responses, and to human oral fibroblasts and epithelial cells for proliferation. Antibacterial ability was explored by incubating supra- and sub-gingival bacteria with bioactive glass particulates. Rapid apatite formation was observed in F- containing bioactive glasses after only 2 h immersion in Tris buffer solution, while it was not detectable until 72 h in the F- free bioactive glass. Alkaline phosphatase activity, cell number, collagen formation, bone-like mineral nodules and osteogenic gene expression of MC3T3-E1 cells were significantly promoted in low F- bioactive glass (P6.33F1) conditioned medium. MC3T3-E1 VEGF gene expression was increased, and protein production was dose-dependently promoted with F- containing bioactive glass conditioned medium, which also promoted human oral fibroblast proliferation, but suppressed epithelial cell numbers. After incubation with glass particulates, the growth of L. casei, S. mitis, A. actinomycetemcomitans and P. gingivalis, was significantly inhibited; the antibacterial activity being dependent on the F- content of the bioactive glasses. As a potential bone graft substitute in vivo, such novel bioactive glasses would be expected to stimulate bone formation and overcome problems associated with infection and the poor vascularisation in large bone graft sites. Additionally, they could reduce the need for further clinical intervention, and in particular, will be advantageous for the periodontal soft tissue regeneration.

Polycaprolactone (PCL) is a biodegradable synthetic polymer that is currently used in a number of biomedical applications. A number of concerns have been raised over the toxicity of initiators commonly employed for the synthesis of PCL. Therefore, more biocompatible initiators have been studied. The biocompatibility of PCL, itself, is adequate; however, improved bioactivity is desirable for several applications. Copolymerisation, and incorporation of bioactive fillers can both be used as ways of enhancing the bioactivity of PCL. Therefore, the global objective of this project was to enhance the bioactivity of PCL by copolymerisation of PCL with poly(ethylene glycol) (PEG) using a biocompatible calcium-based initiator. This calcium-initiator was expected to leave potentially bioactive calcium-initiator residues in the synthesised copolymers. A study of the ring-opening polymerisation of epsilon-caprolactone (CL) in the presence of a poly(ethylene glycol) (PEG) / calcium hydride (CaH2) co-initiation system was performed. Polymerisation kinetics were monitored by following the degree of conversion of CL by Fourier transform-Raman (FT-Raman) spectroscopy and 1H nuclear magnetic resonance spectroscopy (NMR). Resultant PCL-b-PEG-b-PCL (PCL/PEG/PCL) triblock copolymers were analysed by NMR and gel permeation chromatography (GPC). The observed rates of polymerisation for the synthesis of PCL/PEG/PCL triblock copolymers using the PEG / CaH2 co-initiator were much lower than expected. 1H NMR and Raman microspectroscopy analysis showed that the concentration of the active calcium-PEG alkoxide was much lower than the initial feed concentration of PEG. Even so, the molecular weight of PCL/PEG/PCL triblock copolymers could be predicted from the CL : PEG feed ratio. This was found to be due to a fast reversible transfer process. Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) analysis of solutions containing acid digested, pure PCL/PEG/PCL copolymers showed calcium concentrations at equal to or greater than 77 % of the calcium feed concentration. These calcium-initiator residues were isolated and their structures confirmed by Fourier transform infrared-attenuated total reflectance spectroscopy (FTIR-ATR). They were found to be a mixture of calcium hydroxide (Ca(OH)2) and calcium carbonate (CaCO3). The effect of calcium-initiator residues on the in vitro mineralisation of PCL/PEG/PCL triblock copolymers, as well as the same effect on a model calcium-salt-doped PCL homopolymer system, was studied by immersion in simulated body fluid (SBF). In the model studied, PCL homopolymer was doped with low concentrations (0.2 - 2 w / w % Ca) of Ca(OH)2, or CaCO3. Results from the model study showed calcium phosphate (CaP) mineral deposition on Ca(OH)2-doped PCL, and not on CaCO3-doped PCL. This was attributed to the higher solubility of Ca(OH)2, compared to CaCO3. Minimal CaP deposition was observed on PCL/PEG/PCL triblock copolymers. This was attributed to the low Ca(OH)2 concentration in these samples. For all mineralised samples in the SBF studies, the formation of carbonated HAP was observed. Overall, the synthesis of PCL/PEG/PCL copolymers using the PEG / CaH2 co-initiator was found to be a suitable method for preparing reproducible materials. The calcium-based initiator was also found to have potential for increasing the bioactivity of PCL-based materials.

No description available.

Myeloid-derived suppressor cells (MDSC), emerging during tumor growth or chronic inflammation play a critical role in regulating T cell function. However, mechanisms governing the generation of these cells remain unclear, and need to be further defined. Using a DSS-induced colitis and recovery model, we characterized the dynamic changes within myeloid compartments and the emergence of MDSC during active and resolution phases of inflammation. We show that the immature myeloid compartment expands in bone marrow (BM) specifically at the resolution phase of inflammation during colitis transition to recovery. Additionally, we found enhanced levels of IL-17 in the serum of colitis mice tightly correlates with expansion of the IMC compartment, and is likely the factor responsible for expansion of these cells. Our study also determined that the expanded population of myeloid cells underwent a functional reprogramming event. In particular, two major functional changes occurred when colitic mice were allowed to recover: 1) CD11b+Gr-1+ myeloid cells in bone marrow and spleen acquired T cell suppressive functions, and 2) acquired the ability to enter into circulation from BM, confirming previously reported characteristics of MDSC. Additionally, we determined that acquired migratory capability in the low density myeloid cells isolated from resolution time points was due to enhanced surface expression of chemokine receptor CXCR2. Furthermore, we determined that after mobilization of MDSC from the bone marrow, these cells collected in the T cell-rich spleens, where they effectively functioned to suppress T cell proliferation. Through these acquired functions, our study determines a protective role for MDSC during the recovery phase of post-acute inflammation during persistent DSS-induced colitis.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A reparaÃÃo tecidual consiste em uma importante caracterÃstica de prevenÃÃo do processo inflamatÃrio; alguns fatores podem reduzir, retardar ou impedir este processo. Existe um interesse constante em relaÃÃo ao uso de correntes de baixa intensidade aliado da suplementaÃÃo com nutracÃuticos (alta relaÃÃo &#969;9:&#969;6 e baixa relaÃÃo &#969;6:&#969;3). O objetivo desse trabalho foi avaliar os efeitos do prÃ-condicionamento com misturas de Ãleos contendo &#969;3/&#969;6 e &#969;9/&#969;6 e correntes de baixa intensidade MENS( Microcurrent ElÃtric Neuromuscular Stimulation) sobre a reparaÃÃo tecidual apÃs incisÃes em pele de ratos. Foram utilizados 108 ratos Wistar, distribuidos em 3 grupos: G-1 controle (soro fisiolÃgico) n= 36 subdivididos em dois subgrupos: nÃo estimulado e estimulado; G-2 controle neutro (Ãleo de milho e soja) n=36, subdivididos em dois subgrupos: nÃo estimulado e estimulado e G-3 ALA (Ãleo de oliva+linhaÃa+cÃnola), n=36 subdivididos em dois subgrupos: nÃo estimulado e estimulado. As misturas de Ãleos foram administradas por via orogÃstrica, e a aplicaÃÃo da corrente elÃtrica, ocorreram 1 hora antes da incisÃo cirÃrgica. Os animais foram sacrificados no sÃtimo, dÃcimo quarto e vigÃsimo primeiro dia. Amostra de pele de 1cm/1cm foram coletadas entre o segundo e terceiro ponto da sutura. As amostras foram analisadas a partir de lÃminas histopatolÃgicas coradas com hematoxilina-eosina (HE) e forneceram a anÃlise geral dos cortes de tecido com espessura de 5Âm. Os dados obtidos pela tÃcnica de (HE) foram classificados de acordo com a intensidade inflamatÃria e transformados em variÃveis quantitativas mediante atribuiÃÃo de Ãndice para os achado histolÃgicos segundo (VIDINSK, 2006). Os dados estatÃsticos foram analisados utilizando-se o programa graphpad prisma for Windows, versÃo 6,0 para avaliaÃÃo conjunta dos efeitos dos grupos G-1,.G-2 e G-3, e dos subgrupos nÃo estimulado e estimulado, e dos dias (7Â, 14Â e 21Â). As variÃveis da avaliaÃÃo histolÃgica foram analisadas pelo teste nÃo paramÃtrico de Mann-Whitney e pÃs-teste de Dunn. O nÃvel de significÃncia foi de 5% (p<0,05). Os resultados obtidos mostram que a eletroestimulaÃÃo prÃ-condicionante promoveu elevaÃÃo do exsudato e reduÃÃo da reepitelizaÃÃo no 7Â dia. O uso dos prÃ-condicionantes Ãleo 1(milho e soja) e 2 (oliva+canÃla+linhaÃa) reduziram a vascularizaÃÃo no 7Â e 14Â dias respectivamente.Ambos os grupos dos Ãleos 1 e 2 induziram a reduÃÃo da fibrose no 14Â dia. O prÃcondicionamento com Ãleo1 levou a elevaÃÃo da expressÃo do NFkB no 7Â dia. Conclui-se que a eletroestimulaÃÃo promoveu elevaÃÃo de marcadores da fase inflamatÃria com consequente aceleraÃÃo desse processo e o uso dos Ãleos 1 e 2 reduziram a fibrose e a vascularizaÃÃo na fase proliferativa. O prÃcondicionamento com o Ãleo 1(milho e soja) promoveu mais inflamaÃÃo no 7Âdia. / The tissue repair consists in an important feature of the inflammatory process prevention, some factors may reduce, delay or prevent this process. There is a continued interest in the use of currents with low intensity combined by supplementation with nutraceuticals (high ratio w9:w6 and low ratio w6:w3). The objective of this work was to evaluate the effects of preconditioning with oil blends containing (w3/w6) and (w9/w6), and currents of low intensity MENS on the tissue repair after incisions in mice skin. One hundred and eight (108) Wistar rats were used , divided into three groups: G-1 control (saline) n = 36 subdivided into two subgroups: unstimulated and stimulated; G-2 neutral control (corn and soybean oil) n= 36, subdivided into two subgroups: unstimulated and stimulated and G-3 ALA (olive oil + flaxseed + canola), n= 36 subdivided into two subgroups unstimulated and stimulated. The oil mixtures were administered by orogastric, and the application of electric current directly into mouse skin occurred 1 hour before surgical incision. The animals were sacrificed on the seventh, fourteenth and twenty-first day. Skin sample of 1cm/1 cm were collected between the second and third suture point. The samples were analyzed from surgical specimens stained with hematoxylin-eosin (HE) and provided a general analysis of tissue sections with a thickness of 5 um. The data obtained by the (HE) technique were classified according to the inflammatory intensity and transformed into quantitative variables by assigning index for the hestological finding according to (VIDINSK, 2006). Statistical data were analysed using the GraphPad Prism for Windows, version 6.0, for joint assessment of the effects of the groups G-1, G-2 e G-3, and the subgroups unstimulated and stimulated, and days (7, 14, 21). The variables of histological evaluation were analysed by nonparametric Mann-Whitney. The significance level was 5 % (p<0,05). The results show that the preconditioning electrical stimulation caused the elevation of exudate and reduced the reepitheliazation on the 7th day. The use of preconditons oil 1 (corn and soybeans) and oil 2 (olive+ canola + flaxseed) reduced the vascularization on the 7th and 14th days respectively. Both groups, 1 and 2 of oils, induced the reduction of fibrosis on the 14th day. The preconditioning with oil 1 led to elevated expression of NFKB on the 7th day. It is concluded that the electrical stimulation caused the elevation of markers of inflammatory phase with consequent acceleration of this process and the use of oils 1 and 2 reduced the fibrosis and vascularization on proliferative phase. The preconditioning with the oil 1 (corn and soybeans) caused more inflammation on the 7th day.

Orientador: Adriana Aigotti Haberbeck Brandão / Banca: Adriana Aigotti Haberbeck Brandão / Banca:Ana Lia Anbinder / Banca: Rosilene Fernandes da Rocha / Resumo: A osteoporose, doença caracterizada pela perda de massa óssea, tem sido alvo de estudos nos últimos anos. Fraturas decorrentes da osteoporose são muito comuns e podem apresentar consolidação mais lenta. O objetivo deste trabalho foi avaliar o efeito do risedronato (medicamento alopático) e da Calcarea phosphorica 6CH (medicamento homeopático) no processo de reparo ósseo em ratos machos com osteoporose induzida por castração. Para tanto, foram utilizados oitenta e quatro ratos de três meses de idade separados em quatro grupos de vinte e um animais, sendo três grupos submetidos à castração e um grupo a falsa cirurgia (sham). Um mês após, foram realizadas lesões ósseas monocorticais na tíbia de todos os animais e a partir do dia seguinte, os respectivos tratamentos foram iniciados de acordo com os seguintes grupos: CR - castrado/ risedronato (1mg/kg/dia); CCp - castrado/Calcarea phosphorica 6CH (três gotas/dia); CP - castrato/placebo e SP - sham/placebo que receberam apenas três gotas ao dia de água destilada. Os animais foram sacrificados aos sete, catorze e vinte e oito dias após o início do tratamento e as tíbias removidas. Radiografias digitais foram realizadas e avaliadas pelo programa Image Tool para obter a densidade óptica na área do defeito. Então as tíbias foram descalcificadas e processadas para a realização das análises histológicas. A histomorfometria mediu a porcentagem de osso formado utilizando um retículo graduado colocado no centro da lesão através do programa Image J. Para a análise estatística, os dados foram submetidos aos testes de ANOVA, Tukey e Dunnett, ao nível de 5%. Segundo a análise da densidade óptica, o grupo CCp apresentou os melhores resultados aos sete e catorze dias, mas foi superado pelo grupo CR aos vinte e oito dias. Segundo a histomorfometria o grupo SP apresentou o melhor resultado aos sete dias, ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Osteoporosis, a disease characterized by progressive bone loss, has been the target of several studies lately. It results in a much higher risk for fractures and might cause slower bone consolidation. The aim of this work was to study the effects of Risedronate (allopathic medicine) and Calcarea phosphorica 6CH (homeopathic medicine) to repair bone lesions in male rats with osteoporosis, induced by castration. For this, eighty-four three-months-old rats were used divided in four groups of twenty-one animals. Three groups where castrated and one group was submitted to Sham surgery. One month later, monocortical lesions were made in all animals' tibiae and after one day they began the different treatments according to the following groups: CR - castrated/Risedronate (1mg/kg/day); CCp - castrated/Calcarea phosphorica 6CH (3 drops/day); CP - castrated/placebo and SP - Sham/placebo that received 3 drops/day of distilled water. The animals were sacrificed at seven, fourteen and twenty-eight days after the beginning of treatment and had their tibiae removed. The tibiae's digital XR was analyzed in order to evaluate optical density, using the Image Tool program. Then, they were decalcified and processed for histologic analysis. Histomorphometric measures of bone percentual formation were evaluated using a graticule in the central area of the lesion, with the Image J program. Data was submitted to ANOVA, Tukey and Dunnett tests (5% level). According to the optical density, the CCp group showed the best results at seven and fourteen days, but it was surpassed by the CR group at the 28th day. The histomorphometrical analyses showed that the SP group had the best result at seven days but the CR group formed more bone than all the other groups at 14 and 28 days.Measuring the bony callus, the CR group had the thicker callus at seven and 28 days. It... Complete abstract, click electronic address below) / Mestre

Healing wound as a model for the study of cell interactions Abstract Galectins play an important role in the processes of cell proliferation, differentiation, migration and extracellular matrix formation. Furthermore, galectins are able to transfer cellular signals and to participate in cell interaction. It has been proven that galectins play an important role in the microenvironment formation of a tumor and/or healing wound. This study demonstrated significant role of galectins, in particular Galectin-1, in wound healing and cell interactions (endothelial cells, fibroblasts and keratinocytes) forming a part of the granulation tissue and tumor stroma. We have demonstrated that the extracellular matrix rich on Galectin-1 creates a suitable environment for the cultivation of keratinocytes. Galectin-1 also induces differentiation of fibroblasts into myofibroblasts. The knowledge of above mentioned processes is important to better understand the complexity of cancer biology and its parallel to wound healing. Key words: tissue repair, regeneration, galectin, tumor

Healing wound as a model for the study of cell interactions Abstract Galectins play an important role in the processes of cell proliferation, differentiation, migration and extracellular matrix formation. Furthermore, galectins are able to transfer cellular signals and to participate in cell interaction. It has been proven that galectins play an important role in the microenvironment formation of a tumor and/or healing wound. This study demonstrated significant role of galectins, in particular Galectin-1, in wound healing and cell interactions (endothelial cells, fibroblasts and keratinocytes) forming a part of the granulation tissue and tumor stroma. We have demonstrated that the extracellular matrix rich on Galectin-1 creates a suitable environment for the cultivation of keratinocytes. Galectin-1 also induces differentiation of fibroblasts into myofibroblasts. The knowledge of above mentioned processes is important to better understand the complexity of cancer biology and its parallel to wound healing. Key words: tissue repair, regeneration, galectin, tumor