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Introduction The remaining epithelial layer existent in connective tissue graft (CTG) harvested from the hard palate, which underwent de-epithelization outside the oral cavity, can be histologically detected unless it is completely removed. Its presence may cause adverse esthetics results, affecting thus the color and texture of the receptor site, and an increased risk of presence of scar tissues after surgical procedures. The proposal of this study was to evaluate the CTG (histological and morphometrically) collected from the hard palate using the Harris' technique, removing the epithelial layer outside the mouth, assessing the remaining presence of epithelial tissue. Case series Fourteen patients (14CTGs) were included in the present case series study, therefore there were two dropouts. A small part of the tip of the graft was harvested and fixed in formalin solution for histological processing, staining, and then to be morphometrically analyzed. The epithelial tissue and CTGs were assessed by three calibrated and double-blinded professionals. All information was compiled, and the statistical analysis was performed. CTGs obtained had a width average of 1224.26 μm. There was no presence of any residual of the epithelium in three samples, whereas only one had the epithelium tissue covering the entire connective layer. Furthermore, seven samples (approximately 50%) had the presence of epithelium. Conclusion Within the limitation of this study, there was incomplete removal of the epithelial layer after harvesting the CTG using the Harris' technique (44.32%), most likely due to its histological persistency, suggesting to be inaccurate the clinical removal.

Aims Investigate the effect of a novel cell-based therapy with skeletal muscle-derived mononuclear cells (SMDMCs) in a rat model of stress urinary incontinence. Methods Male Wistar-Kyoto rats' hind limb muscles were enzymatically dissociated, and SMDMCs were isolated without needing expansion. The cell population was characterized. Twenty female rats underwent urethrolysis. One week later, 10 rats received periurethral injection of 106 cells (SMDMC group), and 10 rats received saline injections (Saline group). Ten rats underwent sham surgery (Sham group). Four weeks after injection, animals were euthanized and the urethra was removed. The incorporation of SMDMCs in the female urethra was evaluated with fluorescence in situ hybridization for the detection of Y-chromosomes. Hematoxylin and eosin, Masson's trichrome staining, and immunohistochemistry for actin and myosin were performed. The muscle/connective tissue, actin and myosin ratios were calculated. Morphological evaluation of the urethral diameters and fractional areas of the lumen, mucosa, and muscular layer was performed. Results SMDMCs population was consistent with the presence of muscle cells, muscle satellite cells, perivascular cells, muscle progenitor cells, and endothelial cells. SMDMCs were incorporated into the urethra. A significant decrease in the muscle/connective tissue ratio was observed in the Saline group compared with the SMDMC and Sham groups. The proportions of actin and myosin were significantly decreased in the Saline group. No differences were observed in the morphometric parameters. Conclusions SDMSC were incorporated into the rat urethra and promoted histological recovery of the damaged urethral sphincter, resulting in decreased connective tissue deposition and increased muscle content.

As doenças cardiovasculares continuam sendo a primeira causa de morte no Brasil (32%) representando a terceira maior causa de internação hospitalar. Apesar dos avanços terapêuticos das últimas décadas, estudos epidemiológicos consideram o infarto agudo do miocárdio (AMI) uma das maiores causas de morbidade e mortalidade, sendo a maioria, ligados a realização de terapias não adequadas, dos quais 50% das mortes ocorrem nas primeiras 2 horas do ocorrido e 14% morrem antes de receber atendimento médico. O objetivo deste estudo foi de avaliar a técnica de infusão transpericárdica de células mononucleares de medula óssea (CMMO) em suínos. Três suínos fêmeas, pesando 25Kg foram induzidas ao AMI, com auxilio de cateter balão colocado no 1° ramo diagonal da artéria coronária interventricular por 45 minutos, seguido por infusão de 1x108 CMMO marcadas com Hoechst® pela via transpericárdica. O grupo controle foi composto por 3 animais, os quais receberam infusão de 1x108 CMMO marcadas com Hoechst® através da mesma técnica. Os resultados revelaram distribuição homogênea das CMMO no miocárdio, concentrando-se especialmente na área infartada, enquanto que o grupo controle apresentou distribuição homogênea ao longo do miocárdio. Nós concluímos que a técnica transpericárdica é viável para infusão de CMMO em processos de isquemia cardíaca. Cardiovascular illnesses continue to be the first cause of death in Brazil (32%), representing the third major reason of hospital internment. Although the therapeutics advances in the last decades, epidemiologic studies considered the acute myocardium infarct (AMI) to be one of the most causes of morbidity and mortality (30%), most of, related to the institution of non-adequate therapy, thereby 50% of deaths in the early two hours of the event and 14% dying before any medical assistance. Currently therapies include stent angioplasty, Thrombolytic medication and aortic-coronary venous grafting; while in experimental area the cellular therapy has been largely investigated being the cells infusion technique investigation the most enthusiastic issue. This study aimed to evaluate the transepicardic infusion technique of bone marrow mononuclear cells (BMMC) in swine. Three female swine, averaging 25 kg, were induced to AMI, with the aid of a balloon catheter displaced on the first interventricular diagonal branch of the coronary artery for 45 minutes, following to the infusion of 1x108 BMMC stained with Hoescht® by the transepicardic technique. Sham operation was carried out in three animals (Control group), which received infusion of 1x108 BMMC stained with Hoescht® by the same technique. The results revealed an inhomogeneous distribution of the BMMC in the myocardium, being more concentrated in the infarcted area, while the control group presented a homogeneous distribution along the myocardium. We concluded the transpericardic technique would be acceptable to the infusion of BMMC in cardiac ischemic processes.

Em vista das limitações éticas em torno da obtenção de células-tronco de fetos humanos, o cão é uma alternativa para estes estudos. Além disso, a terapia celular proporciona novas expectativas para o tratamento na espécie. Realizamos o estudo comparativo das células isoladas de saco vitelino, fígado e medula óssea de fetos caninos. As células foram analisadas microscopicamente e ultra estruturalmente. O imunofenótipo das células foi avaliado através de marcadores. Caracterizamos a plasticidade, o cariótipo e o potencial teratogênico destas células. Após expansão as células progenitoras formaram colônias com morfologia fibroblastóide. As células progenitoras do saco vitelino e medula óssea são compostas por: células com alta proporção núcleo-citoplasma e células com citoplasma rico em organelas, enquanto que as células progenitoras do fígado eram semelhantes à célula epitelial e células ricas em organelas. As células-progenitoras dos três tecidos fetais foram positivas para os anticorpos nestina e vimentina, mas negativas para CD45 e CD13. Células progenitoras de medula óssea foram positivas para o marcador CD44. Células progenitoras do fígado e medula óssea expressaram a proteína citoqueratina-18, enquanto as do saco vitelino expressaram ve-caderina. Células positivas para Oct3/4 foram detectadas em todas as células progenitoras. As células-progenitoras do saco vitelino e medula óssea diferenciaram-se em tecidos ósseo, cartilaginoso e muscular; já as do fígado para tecido ósseo e muscular. Nenhum tipo celular diferenciou-se em adipócitos. As células progenitoras da medula óssea diferenciaram em células semelhantes a neurônios. Sugere-se a presença de progenitores semelhantes a células mesenquimais e epiteliais. Todas as células mantiveram o cariótipo estável e não formaram tumores. Células progenitoras de medula óssea apresentaram maior capacidade de proliferação e diversidade de diferenciação. Sugere-se que estas células são possíveis candidatas para a terapia celular. The use the human fetuses for stem cells isolation have ethical limitations. In this context the dog is an excellent candidate to fetal stem cells. Furthermore, these cells can be used in cell therapy of canine diseases We aimed at isolation and comparative characterization of progenitor cells from yolk sac, liver and bone marrow of canine fetuses. Cells were characterized using stem cells antibodies. Differentiation assays as well as karyotype analysis were performed. Teratogenic properties this cells were evaluated. After establishment of primary culture, best proliferation potential was observed in bone marrow progenitor cells. Bone marrow and liver progenitor cells were more efficient in CFU-F assay, then yolk sac progenitor cells. Evidenced by TEM cells with a high nuclear-to-cytoplasmic ratio and cells with cytoplasm rich in organelles. Cells isolated from liver showed epithelial-like morphology and cytoplasm rich in organelles. The yolk sac, liver bone marrow cells reacted positively with nestin and vimentin, being negative to CD45 and CD13 antibodies. Additionally bone marrow progenitor cells were positive to CD44. Bone marrow and liver progenitor cells reacted positively with cytokeratin 18. Yolk sac progenitor cells were positive to ve-cadherin. A few Oct3/4 positive cells were found in yolk sac, liver and bone marrow. Yolk sac and bone marrow progenitor cells showed successful osteogenic, chondrogenic, myogenic differentiation. Differentiation liver progenitor cells were able to bone and muscle cells. The bone marrow progenitor cells were able to produce neuron-like cells. None of progenitor cells showed adipogenic differentiation. The study suggests the presence of mesenchymal-like and epithelial-like progenitor cells. All the karyotype remained and failed to induce the formation of tumors. Stem cells from bone marrow showed high diversity of differentiation than other cell types. It is suggested that these cells are possible candidates for cell therapy.

Esse projeto tem como objetivo estabelecer técnicas de cultivo, isolamento, expansão e quantificação de células mesenquimais e hematopoéticas de sangue de cordão umbilical (SCU) de cães de raças variadas para aplicação em terapias celulares. Atualmente, vários tipos de células-tronco têm sido estudados devido a sua capacidade de diferenciação em diversos tecidos (HERZOG et al., 2003). O isolamento, quantificação e expansão dessas células permitem que a terapia celular seja utilizada na tentativa de tratamento em patologias (BARKER et al., 2003). Entre as fontes de células-tronco adultas (medula óssea, sangue periférico, tecido subcutâneo) mais precisamente as células progenitoras mesenquimais está o sangue de cordão umbilical. Sua principal função é o transporte de nutrientes e preencher as crescentes demandas de oxigênio da mãe para o feto (ALMEIDA et al., 2000). Neste estudo foi utilizado o sangue do cordão umbilical de cães ao nascimento, de 11 cadelas gestantes pesando entre 15 e 35 Kg. As células mesenquimais foram isoladas através da separação por gradiente de densidade com o reagente FICOLL, posteriormente o sangue foi diluído em PBS e centrifugado. O pellet contendo células foi retirado e essas então acondicionadas em meio DMEM contendo 10% de soro fetal bovino e antibiótico, homogeneizadas e contadas através da câmara de Newbauer. As células foram então plaqueadas de acordo com o ensaio que seriam submetidas. Através do ensaio de CFU-F pudemos observar uma população de células aderentes isoladas da porção mononuclear do sangue de cordão umbilical que apresentou-se altamente heterogênea. Foram identificados, pelo menos, quatro tipos morfológicos distintos: células epitelióides grandes e pequenas; células de aspecto fusiforme; e células de aspecto estrelado e quando em confluência, as células apresentaram padrão de crescimento similar ao de miofibroblastos, normalmente observado em culturas de músculo liso e de estroma da medula óssea hematopoética. Na maioria das análises as colônias se apresentaram em número desejável, havendo uma variação de baixo a alto. Esse ensaio apresentou grande eficácia no isolamento, quantificação e caracterização morfológica das células mesenquimais obtidas a partir do sangue de cordão umbilical canino. This project has as objective established cultivation techniques, isolation, expansion and quantification of mesenchymal cells from umbilical cord blood (UCB) of dogs of varied breeds for application in cellular therapies. Nowadays, several cell-trunk types have been studied due to his/her differentiation capacity in several woven (HERZOG et al., 2003). The isolation, quantification and expansion of those cells allow the cellular therapy to be used in the treatment attempt in pathologies (BARKER et al., 2003). Among the adult cell-trunk sources (bone marrow, peripheral blood, subcutaneous tissue) more precisely the progenitors mesenchymal cells are the umbilical cord blood. Her main function is the transport of nutrients and to fill out the crescents demands of the mother\'s oxygen for the fetus (ALMEIDA et al., 2000). In this study, the blood was used from the umbilical cord of dogs to the birth, of 11 pregnant female dogs weighing between 15 and 35 Kg, the mesenchymal cells were isolated through the separation for density gradient with the reagent FICOLL, later the blood was diluted in PBS and centrifuged according to protocol, the pellet containing cells was removed and those then conditioned in half DMEM containing 10% of bovine fetal serum and antibiotic, homogenized and counted through the camera of Newbauer. They were plated then in agreement with the rehearsal that they would be submitted. Through the rehearsal of CFU-F (Colony Forming Unit - Fibroblast) we could observe a population of isolated adherent cells of the mononuclear population of the umbilical cord blood that came highly heterogeneous. They were identified, at least, four different morphologies: big and small epithelial cells; aspect fusiforme; and cells of starry aspect and when in confluence, the cells presented pattern of similar growth to the of miofibroblastos, usually observed in cultures of flat muscle and of stromal of the hematopoietic bone marrow. In most of the analyses the colonies came in desirable number, having a variation of low the high. That rehearsal presented great effectiveness in the isolation, quantification and morphologic characterization of the mesenchymal cells obtained starting from the umbilical cord blood of canine.

A terapia de perdas ósseas constitui um dos grandes desafios da área médico-odontológica. Apesar de o enxerto autógeno ainda ser considerado o \"gold standart\", apresenta limitações como a necessidade de um segundo sítio cirúrgico, limitação da quantidade de osso disponível e falta de previsibilidade. A Bioengenharia surgiu como um elemento promissor no tratamento de defeitos ósseos críticos conjugando três elementos principais: as células do paciente, um material carreador e moléculas sinalizadoras. Os materiais carreadores devem ser biocompatíveis, possibilitar a proliferação e diferenciação dos tipos celulares desejados, além de apresentar baixo potencial em desencadear resposta inflamatória. O colágeno tipo I constitui um dos principais produtos da matriz extracelular (MEC) do tecido ósseo sendo, posteriormente, mineralizada. O objetivo deste trabalho foi avaliar o potencial carreador de matrizes de colágeno, obtidas de pericárdio bovino submetido a diferentes tempos de hidrólise (24h e 48h). A hipótese foi de que os tempos de hidrólise, a que foram submetidos os materiais, gerando matrizes colagênicas aniônicas, pudessem interferir nos processos de proliferação, diferenciação e mineralização. O potencial em desencadear resposta inflamatória foi avaliado após 24h, 48h e 72h de contato entre as diferentes membranas e macrófagos humanos. As dosagens de mediadores inflamatórios (IL-10, TNF-alfa, TGF-beta e Óxido Nítrico -NO), demonstraram aumento significativo de TGF-beta, em relação ao controle, que desativa macrófagos e estimula proliferação de células e cicatrização. A análise ao microscópio eletrônico de varredura (MEV) e ao microscópio de luz (ML) mostrou que as células osteoprogenitoras, provenientes do endósteo de fêmur de camundongo Balb/c (FOST), proliferaram ao longo dos tempos experimentais sobre a superfície das matrizes colagênicas estudadas. Entretanto, não foram observadas células no interior destas ao longo de todos os tempos experimentais estudados. A observação de picos na dosagem da atividade da fosfatase alcalina (membrana nativa, 28 dias para membrana hidrolisada por 24h e por 48h) e dosagem da presença de cálcio nas membranas (28 dias para membrana nativa e 21 dias para as hidrolisadas), assim como a observação por MET de vesículas compatíveis com o processo de mineralização (21 dias), indicam que houve a diferenciação das células osteoprogenitoras ao longo dos tempos experimentais estudados. Verificou-se maior produção de fosfatase alcalina e cálcio nos materiais hidrolisados em relação à membrana não hidrolisada. A análise por infravermelho por transformada de Fourier (FTIR) mostrou a presença de ligações fosfato em membranas sem células, independente do tempo experimental, indicando que podem ser decorrentes do tratamento químico necessário para sua obtenção e não do processo de mineralização. A avaliação da capacidade biomimética por espectroscopia de energia dispersiva (EDS) das membranas indicou presença de P e Ca nas membranas avaliadas, mas estes podem não estar associados ao processo de mineralização fisiológica. Concluiu-se que as membranas de pericárdio bovino são biocompatíveis, permitem a proliferação de células osteoprogenitoras em sua superfície. Há indícios de diferenciação celular, mineralização e nucleação de Ca e P. Entretanto, a deficiência na formação de uma porosidade interconectada nas matrizes colagênicas limita a indicação dessas matrizes como carreadores para a bioengenharia óssea. The therapy of bone defects constitutes one of the great challenges of the medicine and dentistry. Although autogenous is considered \"gold standart\", presents limitations as the necessity of second surgical site, limitation of bone availability and lack of previsibility. Tissue bioengineering appeared as a promising treatment for critical bone defects conjugating three main elements: the patient\'s cells, a scaffold and sinalizing molecules. The biomaterials used as scaffolds must be biocompatible to possibilite proliferation and differentiation of desired cell types, besides presenting low potential to promote inflammatory response. Collagen type I constitutes one of the main products of the extracellular matrix (ECM) of bone tissue, being later mineralized. The objective of this work was to evaluate the carrier potential of anionic collagen matrices, obtained of bovine pericardium submitted to different hydrolysis times (24h and 48h). The hypothesis was that the hydrolysis times, could influence the processes of proliferation, differentiation and mineralization. The potential activation of inflammatory response was evaluated after 24h, 48h and 72h of contact among the different membranes and human macrophages. The quantification of inflammatory mediators (IL-10, TNF-alpha, TGF-beta and NO-2) demonstrated significant TGF-beta increase in relation to the control, that disactivates macrophages and stimulates proliferation of cells and wound healing. Scaning electron microscope (SEM) and light microscope (LM) showed that osteoprogenitor cells, obtained from the endosteum of femur of Balb/c mouse (FOST), had proliferated on the surface of the studied collagenic matrices. However, at the studied experimental times, cells were not observed in the pores of these matrices. The peaks in the quantification of alkaline phosphatase (native membrane, 28 days for matrices hydrolysed for 24h e48h) and quantification of calcium in the membranes (28 days for native membrane and 21 days for the hydrolysed), and vesicles compatible with the mineralization process by transmission electron microscopy - TEM (21 days) indicated differentiation of the FOST cells at the studied experimental times. It was verified higher amount of alkaline phosphatase and calcium in hydrolysed materials in relation to the not hydrolysed. The analysis of Fourier transformed infra red (FTIR) showed phosphate peaks in cell-free membranes, independent of the experimental time, indicating that phosphate could be from membrane chemical treatment and not by mineralization process. The evaluation of biomimetic capacity of the membranes by spectroscopy of dispersive energy (EDS) indicated presence of P and Ca in the evaluated membranes, but they can not be associate to biomineralization. It was concluded that the biomembranes are biocompatible and allow FOST proliferation in its surface. There was an indication of cellular differentiation, mineralization and nucleation of Ca and P. However, the deficience in interconnected porosity in the collagenic matrices limits its indication as scaffolds for tissue bioengineering.