Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

Made available in DSpace on 2014-10-09T12:33:14Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:16Z (GMT). No. of bitstreams: 0 / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:33:14Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:16Z (GMT). No. of bitstreams: 0 / Por mais de 30 anos, a utilização do meio de cultura, enriquecido com soro bovino, e de fibroblastos murinos, com a taxa de proliferação controlada por irradiação ou por ação de drogas anti-cancerígenas, vem desempenhando com sucesso o seu papel de auxiliar no desenvolvimento dos queratinócitos em cultura, para fins clínicos. Porém, atualmente há uma preocupação crescente acerca da possibilidade de transmissão de príons e virose animais, aos pacientes transplantados. Levando em conta esta preocupação, o presente trabalho tem como objetivo cultivar fibroblastos humanos em meio enriquecido com lisado de plaquetas humanas e determinar a dose de irradiação dessas células, para obtenção da camada de sustentação na cultura de células da epiderme. Para realização do objetivo proposto, padronizamos a lise das plaquetas, utilizamos este lisado para cultivar os fibroblastos humanos e verificamos a dose de irradiação suficiente para inibir sua duplicação. Queratinócitos humanos foram cultivados nestas camadas de sustentação, em meio de cultura suplementado com o lisado. Com os resultados obtidos concluímos que o lisado de plaquetas a 10% promoveu uma melhor adesão e proliferação dos fibroblastos humanos e em todas as doses testadas (60 a 300 Gy), estes tiveram as suas atividades mitóticas inativadas pela radiação ionizante, sendo que as camadas de sustentação obtidas com doses de 70 a 150 Gy foram as que proporcionaram o melhor desenvolvimento dos queratinócitos em meio contendo 2,5% de lisado de plaquetas humanas. Portanto, foi possível padronizar, tanto o cultivo dos fibroblastos humanos, quanto sua inativação para utilização como camada de sustentação na cultura de queratinócitos, de maneira a eliminar os componentes xenobióticos. / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 5 uppsatser.

Thesis (M.S.)--Duquesne University, 2004. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p. 55-60) and index.

Introdução: A escolha de parâmetros mais eficientes na aplicação terapêutica de um recurso de eletroterapia garante uma efetividade clínica maior ao tratamento realizado. O modo contínuo de aplicação do ultrassom vem sendo pouco estudado nas fases agudas das lesões. Objetivo: Avaliar os efeitos do ultrassom contínuo nas fases inflamatória, proliferativa e de remodelação no processo de regeneração do tecido conjuntivo muscular de ratos. Material e Métodos: Trinta ratos foram submetidos à lesão muscular nos músculos tibial anterior de ambos os membros traseiros e divididos em dois grupos, sendo o grupo Lesado (L) composto pelos membros traseiros esquerdos dos animais e o grupo lesado tratado com ultrassom (LUS), composto pelos membros traseiros direitos. Cada grupo foi subdividido em seis pequenos grupos (n = 5), que foram formados de acordo com o número de dias após a lesão em que os animais foram mortos, sendo no 3º, 7º, 14º, 21º, 28º e 35º dias. Após 24 horas da realização da lesão cirúrgica, o Grupo LUS começou a receber a aplicação do ultrassom no modo contínuo, área de radiação efetiva de 1cm2, com freqüência de transdutor de 1MHz, intensidade de 0,2W/cm2, por 1 minuto, uma vez ao dia. Os membros traseiros esquerdos submetidos à lesão cirúrgica e não tratados serviram como grupo controle (L). Para análise morfológica das fibras musculares foram realizados cortes seriados de 10 ?m de espessura, obtidos através de em um micrótomo criostato. Os músculos foram congelados em uma posição onde os cortes pudessem fornecer imagens longitudinais das fibras musculares e da fenda incisional. Cada série de corte foi obtida a partir da região superficial do músculo, a cada 100 ?m de profundidade e forneceu duas lâminas que foram coradas com a técnica Hematoxilina Eosina. A contagem de células foi feita em uma área de 0,625mm2 (área de um campo microscópico), com auxílio de uma câmera acoplada ao microscópio e monitor de vídeo e tendo como ponto de referência os limites da fenda incisional, ou seja, a região com fibras musculares lesionadas de um lado e as células infiltradas na fenda incisional de outro. A determinação da densidade de leucócitos polimorfonucleares e mononucleares, e de fibroblastos foi feita através da contagem média de 10 campos por amostra, sendo cinco campos representativos do tecido muscular seccionado e cinco campos da fenda incisional, totalizando 300 campos por grupo experimental (30 ratos ao todo). As células com morfologia de células satélites ou mioblastos foram ignoradas. Resultados: A análise da contagem de células revelou diferenças significativas (p <= 0,05) entre o número de células polimorfonucleares no 3º, 7º, 21º e 28º dias de tratamento com as seguintes xiii médias: L3 = 27,0; L7 = 29,6; L21 = 20,0 e L28 = 12,8 e LUS3 = 43,2; LUS7 = 29,2; LUS21 = 8,8 e LUS28 = 3,6. O número de fibroblastos foi significativamente maior (p<=0,05) apenas no 21º dia com L21 = 14,2 e LUS21 = 27,0. Conclusão: O ultrassom contínuo promoveu um aumento na densidade de células polimorfonucleares e fibroblastos durante o processo de regeneração do músculo tibial anterior de ratos, incrementando a recuperação deste tecido. / Introduction: The choice of more efficient parameters in the ultrasound application allows a major clinical effectivity to the performed treatment. The therapeutic continuous ultrasound has been being poorly studied in injury acute phases. Objective: To assess the effects of the continuous ultrasound in the inflammatory, proliferative and remodeling phases of rats\' conjunctive muscular tissue regeneration process. Material and Methods: Thirty rats were submitted to muscular lesion in tibialis anterior muscle of both rear limbs. They were divided into two groups, the lesion group (L) consisting of the rats left rear limbs, and the lesion group treated with ultrasound (LUS) consisting of the right rear limbs. Each group was subdivided into six small groups (n = 5), which were formed according to period of time (in days) after the lesion in which the rats were killed, on the 3rd, 7th, 14th, 21st, 28th and 35th days. Twenty four hours after performing the surgical lesion, the LUS group received the first 1MHz continuous ultrasound treatment with effective radiating area = 1cm2, intensity of 0.2W/cm2, for one minute, once a day. The left rear limbs submitted to the surgical lesion and not treated at all composed the control group (L). To perform the muscle fibers morphological analysis, a series of 10?m thick slices were made using a microtome with criostate. The muscles were frozen in a position which the slices could provide longitudinal images of the muscle fibers and incision gap. A series of samples were collected from the lesion area surface, and further two deeper series, at 100?m distance from one another. Two slices were thus provided and stained with hematoxilin-eosin. Cell count was made on an area of 0.625 mm2, by means of a camera coupled to the microscope and to a video monitor. The reference point were the limits of the incision gap, which is a region with injured muscle fibers in one side and infiltrated cells of the incision gap on the other. The determination of the density of polymorphonuclear and mononuclear leukocytes, and fibroblasts was made by the exam of 10 fields per sample (five fields representing the cut muscular tissue and more five fields of the incision gap), totaling 300 fields per group. Cells with myoblast or satellite cells morphology were ignored. Results: The cells counting analysis revealed significant differences (p <= 0.05) in the density of polymorphonuclear cells on the 3rd, 7th, 21st and 28th treatment days and the means are: L3 = 27; L7 = 29.6; L21 = 20; L28 = 12.8 and LUS3 = 43.2; LUS7 = 29.2; LUS21 = 8.8; LUS28 = 3.6. The density of fibroblasts was significant greater (p <= 0.05) only in the 21st day with means of L21 = 14.2 and LUS21 = 27. Conclusion: The continuous ultrasound increased the density of polymorphonuclear and fibroblasts cells during the anterior tibialis muscle regenerating process of rats, enhancing the recovery of this tissue.

Introdução: A escolha de parâmetros mais eficientes na aplicação terapêutica de um recurso de eletroterapia garante uma efetividade clínica maior ao tratamento realizado. O modo contínuo de aplicação do ultrassom vem sendo pouco estudado nas fases agudas das lesões. Objetivo: Avaliar os efeitos do ultrassom contínuo nas fases inflamatória, proliferativa e de remodelação no processo de regeneração do tecido conjuntivo muscular de ratos. Material e Métodos: Trinta ratos foram submetidos à lesão muscular nos músculos tibial anterior de ambos os membros traseiros e divididos em dois grupos, sendo o grupo Lesado (L) composto pelos membros traseiros esquerdos dos animais e o grupo lesado tratado com ultrassom (LUS), composto pelos membros traseiros direitos. Cada grupo foi subdividido em seis pequenos grupos (n = 5), que foram formados de acordo com o número de dias após a lesão em que os animais foram mortos, sendo no 3º, 7º, 14º, 21º, 28º e 35º dias. Após 24 horas da realização da lesão cirúrgica, o Grupo LUS começou a receber a aplicação do ultrassom no modo contínuo, área de radiação efetiva de 1cm2, com freqüência de transdutor de 1MHz, intensidade de 0,2W/cm2, por 1 minuto, uma vez ao dia. Os membros traseiros esquerdos submetidos à lesão cirúrgica e não tratados serviram como grupo controle (L). Para análise morfológica das fibras musculares foram realizados cortes seriados de 10 ?m de espessura, obtidos através de em um micrótomo criostato. Os músculos foram congelados em uma posição onde os cortes pudessem fornecer imagens longitudinais das fibras musculares e da fenda incisional. Cada série de corte foi obtida a partir da região superficial do músculo, a cada 100 ?m de profundidade e forneceu duas lâminas que foram coradas com a técnica Hematoxilina Eosina. A contagem de células foi feita em uma área de 0,625mm2 (área de um campo microscópico), com auxílio de uma câmera acoplada ao microscópio e monitor de vídeo e tendo como ponto de referência os limites da fenda incisional, ou seja, a região com fibras musculares lesionadas de um lado e as células infiltradas na fenda incisional de outro. A determinação da densidade de leucócitos polimorfonucleares e mononucleares, e de fibroblastos foi feita através da contagem média de 10 campos por amostra, sendo cinco campos representativos do tecido muscular seccionado e cinco campos da fenda incisional, totalizando 300 campos por grupo experimental (30 ratos ao todo). As células com morfologia de células satélites ou mioblastos foram ignoradas. Resultados: A análise da contagem de células revelou diferenças significativas (p <= 0,05) entre o número de células polimorfonucleares no 3º, 7º, 21º e 28º dias de tratamento com as seguintes xiii médias: L3 = 27,0; L7 = 29,6; L21 = 20,0 e L28 = 12,8 e LUS3 = 43,2; LUS7 = 29,2; LUS21 = 8,8 e LUS28 = 3,6. O número de fibroblastos foi significativamente maior (p<=0,05) apenas no 21º dia com L21 = 14,2 e LUS21 = 27,0. Conclusão: O ultrassom contínuo promoveu um aumento na densidade de células polimorfonucleares e fibroblastos durante o processo de regeneração do músculo tibial anterior de ratos, incrementando a recuperação deste tecido. / Introduction: The choice of more efficient parameters in the ultrasound application allows a major clinical effectivity to the performed treatment. The therapeutic continuous ultrasound has been being poorly studied in injury acute phases. Objective: To assess the effects of the continuous ultrasound in the inflammatory, proliferative and remodeling phases of rats\' conjunctive muscular tissue regeneration process. Material and Methods: Thirty rats were submitted to muscular lesion in tibialis anterior muscle of both rear limbs. They were divided into two groups, the lesion group (L) consisting of the rats left rear limbs, and the lesion group treated with ultrasound (LUS) consisting of the right rear limbs. Each group was subdivided into six small groups (n = 5), which were formed according to period of time (in days) after the lesion in which the rats were killed, on the 3rd, 7th, 14th, 21st, 28th and 35th days. Twenty four hours after performing the surgical lesion, the LUS group received the first 1MHz continuous ultrasound treatment with effective radiating area = 1cm2, intensity of 0.2W/cm2, for one minute, once a day. The left rear limbs submitted to the surgical lesion and not treated at all composed the control group (L). To perform the muscle fibers morphological analysis, a series of 10?m thick slices were made using a microtome with criostate. The muscles were frozen in a position which the slices could provide longitudinal images of the muscle fibers and incision gap. A series of samples were collected from the lesion area surface, and further two deeper series, at 100?m distance from one another. Two slices were thus provided and stained with hematoxilin-eosin. Cell count was made on an area of 0.625 mm2, by means of a camera coupled to the microscope and to a video monitor. The reference point were the limits of the incision gap, which is a region with injured muscle fibers in one side and infiltrated cells of the incision gap on the other. The determination of the density of polymorphonuclear and mononuclear leukocytes, and fibroblasts was made by the exam of 10 fields per sample (five fields representing the cut muscular tissue and more five fields of the incision gap), totaling 300 fields per group. Cells with myoblast or satellite cells morphology were ignored. Results: The cells counting analysis revealed significant differences (p <= 0.05) in the density of polymorphonuclear cells on the 3rd, 7th, 21st and 28th treatment days and the means are: L3 = 27; L7 = 29.6; L21 = 20; L28 = 12.8 and LUS3 = 43.2; LUS7 = 29.2; LUS21 = 8.8; LUS28 = 3.6. The density of fibroblasts was significant greater (p <= 0.05) only in the 21st day with means of L21 = 14.2 and LUS21 = 27. Conclusion: The continuous ultrasound increased the density of polymorphonuclear and fibroblasts cells during the anterior tibialis muscle regenerating process of rats, enhancing the recovery of this tissue.

Mesenchymal stromal cells (MSC) are adherent, clonogenic, fibroblast-like cells endowing with unique multipotent differentiation potential and immunosuppressive properties. They are considered as promising candidates for regenerative medicine and immunotherapy. <p>MSC can be isolated from different tissue sources including bone marrow (BM), adipose tissue (AT) and Wharton’s Jelly (WJ). Although fulfilling the ISCT criteria required to be recognized as MSC, MSC from these different sources could disclose some differences taking into account their different anatomical origin and ontogeny as well.<p>In the present work, we investigated the influence of MSC source on their immunosuppressive as well as differentiation properties. We further extended our study to the role of the microenvironment (infection and inflammation) on these features. <p>We show that BM-MSC express Toll-like receptors (TLR) from TLR1 to TLR6. In an inflammatory environment, TLR2, 3 and 4 are significantly upregulated. By upregulating TLR3 and TLR4 transcription, inflammation increases BM-MSC responsiveness to LPS (TLR4 ligand) and poly(I:C) (TLR3 ligand) leading to a pro-inflammatory shift of their cytokine profile. The effect of TLR ligation on BM-MSC osteogenic potential is donor dependent. Inflammation as well as stimulation with LPS and poly(I:C) result in a decrease of BM-MSC immunosuppressive capabilities. <p>We further observed that BM-, AT- and WJ-MSC do not have the same pattern of TLR expression and consequently do not respond the same way to bacterial or viral infection. WJ-MSC do not express TLR4 and although TLR3 is present at the protein level it is not functional as its ligation do not trigger cytokine expression. Inflammation modulates this TLR pattern expression by upregulating TLR3 in all three MSC types and TLR4 only in BM-MSC. TLR ligation increases the production of inflammatory cytokines in BM- and AT- but not in WJ-MSC and augments anti-inflammatory cytokines in AT-MSC. Although inflammation increases in all MSC types the secretion of inflammatory cytokines, additional TLR triggering does not further affect WJ-MSC. The immunosuppressive potential of WJ-MSC on mixed leucocytes reaction (MLR) is not affected either by inflammation or by TLR triggering.<p>On the differentiation side, WJ-MSC has the lower potential to differentiate into osteoblast as compared to BM- and AT-MSC, as revealed by alkaline-phosphatase (ALP) activity and by measuring extracellular Ca2+ deposits. However, inflammation is able to strongly increase the osteogenic differentiation of WJ-MSC as calcification and ALP activity appears as early as at day 7. However this latter enzymatic activity remains much lower than that disclosed by BM-MSC. TLR3 or TLR4 triggering does not affect the osteogenesis of WJ-MSC while it increases it in AT- and also, although to lesser extent, in BM-MSC.<p><p>Our work establishes that the source from which MSC is derived is of major importance for the design of MSC based immunointervention. WJ-MSC appear to be the most attractive cell type when an immunosuppressive action is required in an inflammatory or infectious context. Although WJ-MSC are poorly osteogenic, a complete osteogenic differentiation can be obtained under inflammatory conditions. Taking into account their easy accessibility as well as their huge proliferative potential, these data open an avenue for using these cells in regenerative medicine particularly in clinical settings where chronic inflammation or infection have to be considered. <p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Actinobacillus actinomycetemcomitans is present in elevated proportions and numbers in dental bacterial biofilms of patients with localized aggressive periodontitis. This variant of periodontal disease, occurring in adolescents and young adults, is characterized by rapid and severe destruction of the connective tissues and bone supporting the teeth, eventually culminating in tooth loss. The cytolethal distending toxin (Cdt) is a newly discovered bacterial protein toxin, uniquely present in A. actinomycetemcomitans among all known to-date oral bacterial species. The Cdt has the capacity to inhibit mammalian cell growth, but its putative role in the pathogenesis of the disease is unclear. The aim of this in vitro work has been to study the effects of A. actinomycetemcomitans on periodontal connective tissue cell cultures, and to evaluate the possible involvement of its Cdt. A. actinomycetemcomitans inhibited the proliferation of gingival and periodontal ligament fibroblasts, as a result of a combined arrest at the G1 and G2/M phases of the cell cycle. This growth inhibition was non-lethal and the cells remained metabolically active, although their DNA synthesis was reduced. The intoxicated cells exhibited increased size and irregular structure, characterized by distension and elongation. This cellular enlargement occurred in both G1 and G2/M phase arrested cells. The Cdt of A. actinomycetemcomitans was responsible for the observed growth inhibition, as well as the concomitant morphological alterations. The possible induction of inflammatory cytokines related to bone resorption was investigated in response to A. actinomycetemcomitans, and the involvement of Cdt was evaluated. Extensive focus was given to the study of receptor activator of NF-κB ligand (RANKL) expression, a membrane-bound ligand that signals osteoclast progenitors to differentiate and fuse into mature osteoclasts, activating bone resorption. It was demonstrated that A. actinomycetemcomitans induced RANKL mRNA and protein expression in the cells studied, but did not affect the expression of its decoy receptor, osteoprotegerin. This induction was solely attributed to its Cdt, as demonstrated by the use of a cdt-knockout A. actinomycetemcomitans strain, purified recombinant Cdt, and antibodies blocking the Cdt. In addition, this event was not mediated by pro-inflammatory cytokines known to stimulate RANKL. Interleukin-6 mRNA and protein expression were also enhanced by A. actinomycetemcomitans, but Cdt had limited involvement in this enhancement. In conclusion, two distinct mechanisms by which A. actinomycetemcomitans Cdt may be involved in the pathogenesis of localized aggressive periodontitis are proposed. Firstly, the growth arrest of the resident fibroblasts may impair the physiological connective tissue remodelling equilibrium and lead to connective tissue attachment loss. Secondly, the induction of RANKL by these cells, residing in the proximity of the alveolar bone, may locally stimulate osteoclastogenesis and promote alveolar bone resorption. This work also provides further insights to the understanding of Cdt mechanisms of action, contributing to the global characterization of the toxin’s virulence.

Células mesenquimais indiferenciadas humanas foram obtidas por digestão enzimática e centrifugação do produto de lipoaspiração, expandidas in vitro, e implantadas no tecido subcutâneo de camundongos atímicos. No grupo I, cada animal recebeu o implante de uma membrana de 0,25cm2 de ácido glicólico e carbonato de trimetileno semeada com 1 x 106 destas células .No grupo II, cada um recebeu a injeção de 0,2ml de gel de ácido hialurônico reticulado contendo o mesmo número destas células. Com três semanas de implante, células humanas e vasos foram identificados nos dois carreadores. Entretanto, com oito semanas, somente no gel de ácido hialurônico as células humanas e os vasos estavam presentes / Human undifferentiated mesenchymal cells were obtained by enzymatic digestion and centrifugation of the product of liposuction. These cells were expanded, in vitro, and implanted subcutaneously in athymic mice. In group I, each animal received the implant of a 0,25cm2 membrane of glycolic acid and trimethylene carbonate, seeded with 1 x 106 of these cells. In group II, each one received 0,2 ml of cross-linked hyaluronic acid gel containing the same amount of these cells. With three weeks of implantation, human cells and vessels were identified in both carriers. However, with eight weeks of implantation, only in hyaluronic acid gel human cells and vessels were present