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Intra-articular delivery of encapsulated human mesenchymal stem cells reduces osteoarthritis progression in a rat modelMcKinney, Jay Michael 11 July 2017 (has links)
Osteoarthritis (OA) is a degenerative disease of the joint that leads to joint instability, degradation of the articular cartilage surface and eventually joint failure. Articular cartilage surfaces exhibit unique mechanical behaviors, bearing and distributing loads across joint surfaces, but have poor regenerative capacities. Human Mesenchymal Stem Cells (hMSCs) present a promising treatment to target OA, relying on their regenerative capacity and structural contributions to tissue repair, along with their immunomodulatory and anti-inflammatory properties. The multipotency of hMSCs allow these cells to differentiate towards osteogenic, chondrogenic and adipogenic lineages and directly incorporate into native tissue. hMSCs also possess the capacity to induce numerous paracrine-mediated processes including the recruitment of stem and progenitor cells, prevention of apoptosis, facilitation of beneficial remodeling and modulation of the immune response. Through encapsulating hMSCs, the effects of their paracrine action were studied directly, as the capsule presents a mechanical barrier for direct physical interaction and integration of these cells within the native tissue. The objective of this study was to utilize encapsulation of hMSCs to determine the paracrine effects of hMSCs on the progression of OA. OA was surgically induced in rats via the medial meniscus transection (MMT) surgery, which presents the phenotypical cartilage degradation associated with OA at 3 weeks. The efficacy of hMSC intervention was assessed using Lewis Rats with MMT (n=5 per group). Intra-articular injections of encapsulated hMSCs were given one day post-op and 3 weeks post-op for the 3-week and 6-week MMT studies, respectively. Animals were euthanized on the final day for both the immediate and delayed treatment studies. Micro-structural changes of the articular cartilage, osteophytes and subchondral bone of the medial tibial plateau were assessed using contrast enhanced microCT. We hypothesized that the intra-articular delivery of encapsulated hMSCs will have a positive effect, via paracrine-mediated action, on the onset and development of OA. The capsules also have the potential to improve retention and cell viability in the knee joint space. Each of these factors could contribute to enhanced therapeutic potential of the hMSC treatment. Utilizing NIR labeled sodium alginate capsules, a retention profile for the capsules yielded a tau value of 11.48 days, whereas previous studies have shown scaffold free hMSCs show complete clearance in 7 days. The 3-week MMT, run to analyze the effects of immediate treatment of encapsulated hMSCs on the onset of OA, showed a trend towards decreased cartilage thickness and a decreased surface roughness for the hMSC group in comparison to the Saline group, specifically. Additionally, the hMSC group showed a trend towards increased mineralized osteophyte volume for the hMSC group in comparison to the Saline group. Analysis of the subchondral bone yielded no differences between the hMSC and Saline groups for bone morphology. The 6 week MMT study was run to analyze the effects of a delayed treatment of encapsulated hMSCs on OA after the disease had developed. This study showed a similar result with the immediate treatment study for surface roughness, with the hMSC group showing a decrease in comparison to the Saline group. However, no differences were noted for cartilage thickness between the two respective groups. To further analyze the cartilage in the later stages of OA, exposed bone was quantified yielding a trend towards decreased exposed bone in the hMSC group in comparison to the Saline group. The mineralized osteophyte volume for the hMSC group, of the delayed treatment study, yielded a significantly higher value than all other groups. Additionally, the subchondral bone of the hMSC group trended towards a decreased porosity in comparison to the Saline group. This is one of the first studies to use sodium alginate encapsulation of hMSCs as an innovative scaffold means for intra-articular injections into the knee space. Encapsulated hMSCs permitted not only enhanced cellular retention in the knee space but showed a potential chondroprotective role of the paracrine signaling properties of hMSCs in the early stages of OA. These advantages of encapsulated hMSCs were countered by enhancements of secondary OA phenotypic changes, mainly increased mineralized osteophyte volume and a trend towards increased subchondral bone sclerosis in the later stages of OA. hMSCs have shown great potential as disease modifying drugs and through this study we have further explored the efficacy of these drugs for future treatments of OA. This study has high clinical relevance and with clinical practice running well ahead of current scientific evidence, it is imperative that these findings be considered not only in pre-clinical work but in current and future clinical trials. / 2018-07-11T00:00:00Z
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DYNAMIC CONTROL OF HYDROGEL PROPERTIES VIA ENZYMATIC REACTIONSDustin Michael Moore (6621656) 10 June 2019 (has links)
Two Systems were designed. The first permits tunable on-demand softening of a hydrogel network. The second permits reversible on demand ligand exchange within a hydrogel network. Both means were shown to be cytocompatible and their uses demonstrated in cell culture of mesenchymal stem cells and 3T3 fibroblast cells.
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Participação de quinases reguladas por sinais extracelulares na interação entre células-tronco mesenquimais e titânio durante a diferenciação osteoblástica e adipocítica / Participation of extracellular signal-regulated kinases in mesenchymal stem cells and titanium interaction during osteoblast and adipocyte differentiationSilva, Heitor Fontes da 23 September 2016 (has links)
A osseointegração de implantes de titânio (Ti) é dependente da interação entre a superfície de Ti e células, a qual é modulada por diversas vias de sinalização intracelular. Sabe-se que as quinases reguladas por sinais extracelulares (ERKs), membros da família das proteínas quinases ativadas por mitógenos (MAPKs), atuam tanto na osteogênese, quanto na adipogênese e, portanto, podem estar envolvidas no processo de osseointegração de Ti. Nesse contexto, o objetivo do presente estudo foi avaliar se a interação entre células-tronco mesenquimais (CTMs) e superfícies de Ti usinada e com nanotopografia é modulada, ao menos em parte, por ERK1/2 e o consequente efeito da inibição dessas ERKs na diferenciação osteoblástica e adipocítica. Para isso, CTMs derivadas de medula óssea de ratos foram cultivadas sobre discos de Ti usinados e com nanotopografia em condições osteogênicas e adipogênicas, na presença ou não do inibidor de ERK1/2, PD98059, em concentração previamente determinada (25 μM) e foram avaliados parâmetros relacionados à diferenciação osteoblástica e adipocítica. Os resultados mostraram que a expressão gênica dos marcadores osteoblásticos RUNX2, osterix (OSX), fosfatase alcalina (ALP) e osteocalcina (OC) foi aumentada pela inibição da via de sinalização de ERK1/2 nas células crescidas sobre Ti usinado e apenas ALP e OC, naquelas crescidas sobre Ti com nanotopografia. A expressão proteica de RUNX2 foi discretamente maior nas células crescidas sobre Ti usinado, mas não sobre Ti com nanotopografia, quando ERK1/2 foram inibidas e essa inibição não afetou a formação de matriz extracelular mineralizada, independentemente da superfície de Ti avaliada. Com relação à diferenciação adipocítica, a inibição da via de sinalização de ERK1/2 aumentou a expressão gênica dos marcadores adipocíticos PPARγ, adiponectina (ADIPOQ) e proteína ligadora de ácido graxo do adipócito ( AP2) nas células crescidas sobre ambas as superfícies de Ti, com efeito mais acentuado na superfície usinada, sem afetar a formação de acúmulo lípidico. Em conclusão, os resultados mostraram que a inibição de ERK1/2 favoreceu a diferenciação osteoblástica de CTMs crescidas sobre a superfície de Ti usinada, mas não sobre Ti com nanotopografia. Além disso, a inibição de ERK1/2 favoreceu a diferenciação adipocítica de CTMs crescidas sobre as superfícies de Ti com nanotopografia e usinada, sendo o efeito mais acentuado na usinada. Considerando aplicações terapêuticas, esses resultados são relevantes para direcionar o desenvolvimento de superfícies de biomateriais que atuem em vias de sinalização que sabidamente modulam o processo de osteogênese. / Osseointegration of titanium (Ti) implants depends on interaction between Ti surface and cells, which is modulated by several intracellular signaling pathways. Extracellular signal-regulated kinases (ERKs) are members of mitogen-activated protein kinases (MAPKs) family and act on both osteogenesis and adipogenesis and, therefore, may be involved in the process of Ti osseointegration. In this context, the aim of this study was to evaluate if the interaction between mesenchymal stem cells (MSCs) and Ti surfaces, either machined or with nanotopography, is modulated, at least in part, by ERK1/2 and the effect of ERK1/2 inhibition on osteoblast and adipocyte differentiation. Rat bone marrow MSCs were cultured on Ti discs either machined or with nanotopography under osteogenic and adipogenic conditions, in presence or not of the ERK1/2 inhibitor, PD98059, at a concentration previously determined (25 μM) and it was evaluated parameters related to osteoblast and adipocyte differentiation. The results showed that gene expression of the bone markers RUNX2, osterix (OSX), alkaline phosphatase (ALP) and osteocalcin (OC) was increased by ERK1/2 signaling inhibition in cells grown on machined Ti and only ALP and OC in cells grown on Ti with nanotopography. RUNX2 protein expression was slightly higher in cells grown on machined Ti, but not on Ti with nanotopography, when ERK1/2 signaling was inhibited and such inhibition did not affect extracellular matrix mineralization, irrespective of the evaluated Ti surface. Regarding adipocyte differentiation, ERK1/2 signaling inhibition increased gene expression of the adipose tissue markers PPARγ, adiponectin (ADIPOQ) and adipocyte fatty acid-binding protein (AP2) in cells grown on both Ti surfaces, with more prominet effect on machined one, without affecting lipid accumulation. In conclusion, our results showed that ERK1/2 signaling inhibition favored osteoblast differentiation of MSCs grown on machined Ti, but not on Ti with nanotopography. In addition, ERK1/2 signaling inhibition favored adipocyte differentiation of MSCs grown on both Ti surfaces, being more noticeable on machined one. Considering therapeutical applications, these results are relevant to drive the development of biomaterial surfaces to act on signaling pathways that regulate the process of osteogenesis.
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Preparação e caracterização de micropartículas de colágeno ou fibroína como suporte para células-tronco / Preparation and characterization of collagen or fibroin microparticles as support for stem cellsMontanha, Vanessa Camila 22 October 2012 (has links)
Diversos biomateriais podem ser aplicados na engenharia de tecidos, mas poucos são utilizados em contato direto com células-tronco na forma de suportes de micropartículas, devido à falta de adesão, espalhamento e toxicidade do material, de forma que os tornam inviáveis junto ao cultivo celular. Um biomaterial promissor para bioengenharia é a fibroína, proteína fibrosa presente no casulo do bicho da seda (Bombyx mori), devido à sua resistência mecânica, biocompatibilidade e mínima reação inflamatória, porém, suas caracteristicas são pouco conhecidas na literatura. O mesmo não ocorre com o colágeno que já é bastante estudado por pesquisadores e, assim como a fibroína, apresenta propriedades naturais que incluem baixa resposta imunológica, baixa toxicidade e habilidade de promover o crescimento celular, porém o uso do colágeno em sua maior parte é em forma de filmes, esponjas e membranas. Como existem poucos métodos relacionados para preparação e caracterização de micropartículas em formatos esféricos e porosos, este trabalho teve por objetivo desenvolver e caracterizar micropartículas à base de colágeno ou fibroína, tratadas ou não com glutaraldeído (GA), para ser utilizado como suporte para células-tronco mesenquimais e avaliar a citotoxicidade destes materiais em cultura celular.Nos resultados de Calorimetria Exploratória Diferencial para ambos os materiais, colágeno e fibroína quando submetidas a tratamento com GA, a temperatura de desnaturação e degradação aumenta, respectivamente. Na microscopia ótica, eletrônica de varredura e birrefringência, observa-se o aparecimento de rugosidade e poros e/ou bolhas de ar no interior das micropartículas em maior quantidade quando tratadas com GA, o que pode ser um fator positivo para aderência celular no suporte. A porcentagem de água absorvida é maior no colágeno devido às estruturas hidrofóbicas em maior quantidade na fibroína, porém, quando tratadas com GA, a absorção é estabilizada em um curto tempo em ambos os materiais. Os picos nos espectros de FTIR mostram as bandas amidas I, II e III dos materiais e as alterações sofridas quando em contato com GA e os testes de citotoxicidade que ambos materiais tratados ou não, são atóxicos, mas o desenvolvimento celular nas micropartículas de fibroína é mais lento e diminui quando tratados com GA, por possuir mais estruturas ordenadas na forma de -folha quando se necessita de um crescimento mais controlado das células nas micropartículas. / There are several biomaterials can that be used in tissue engineering, but few are used in direct contact with stem cells like scaffold in the microparticle form, because of the lack of adhesion, spreading and toxicity of the biomaterial, in order to make them nonviable in the cell culture. A promising biomaterial for bioengineering is fibroin, a fibrous protein present in the fibers of silkworm (Bombyx mori) cocoon, because of its mechanical strength, biocompatibility and minimal inflammatory reaction; however, little is still described in the literature. Not so with the collagen that is already well studied by researchers and as the fibroin, has natural properties that include low immune response, low toxicity and ability to promote cell growth, but the use of collagen is mostly in form of films, sponges and membranes. As there are few methods reported for preparation and characterization of microparticles in spherical shapes and porous, this study aimed to develop and characterize microparticles based on collagen or fibroin, treated or not with glutaraldehyde (GA), to be used as a support for cells mesenchymal stem cells and evaluating the cytotoxicity of these materials in cell culture.In the results of Differential Scanning Calorimetry (DSC) curves for both materials, collagen and fibroin when subjected to treatment with GA, the denaturation and degradation temperatures increases, respectively. In Optical Microscopy, MSCanning electronic Microscopy and Birefringence results, it is observed the onset of surface roughness and porosity and or air pockets within the microparticles in greater quantity when treated with GA, which may be a positive factor for cell attachment on the support. The percentage of water absorbed is greater in the collagen structures due to more hydrophobic structure than silk fibroin, but when in treated with GA, absorption is stabilized in a shorter time in both materials. The peaks in FTIR spectra show bands amide I, II and III of the materials and the changes suffered when in contact with GA and cytotoxicity tests are non-toxic to both biomaterials treated or not, however, in the growth of cells, the fibroin microparticles is slower and decreases when treated with GA, due to its more ordered structure in the form of -sheet and more spherical than collagen due to its more ordered -sheet structures, which may be very interesting when it needs a more controlled growth of cells on microspheres.
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Efeitos da fotobiomodulação na adesão e proliferação das células-tronco da papila apical humana em scaffold de quitosana com incorporação de coágulo sanguíneo. Estudo in vitro / Effects of photobiomodulation on adhesion and proliferation of stem cells from human apical papilla in chitosan scaffold with blood clot incorporation. In vitro studyAbe, Gabriela Laranjeira 06 September 2016 (has links)
Revascularização é uma técnica utilizada em dentes jovens, que apresentem rizogênese incompleta e danos irreversíveis ao tecido pulpar, necessitando de tratamento endodôntico, para formar novo tecido em lugar da polpa perdida. Clinicamente os resultados mostram a continuidade da rizogênese e a devolução da vitalidade dental. Porém, pouco se sabe sobre o novo tecido formado e não está estabelecido se este é capaz de desempenhar todas as funções da polpa dentária. Para melhorar as características do tecido formado pela técnica da revascularização, podemos utilizar ferramentas de engenharia tecidual, como célulastronco, fatores de crescimento e arcabouços de sustentação celular (scaffolds). As célulastronco (CTs) já estão presentes quando o sangue invade o canal radicular, e utilizar essa reserva de CTs que o hospedeiro possui, procedimento conhecido como homing, é uma vantagem em comparação com outras técnicas que injetam CTs obtidas por cultivo em laboratório. Entretanto os aspectos físicos do coágulo sanguíneo formado no interior do canal radicular podem ser melhorados com a adição de hidrogel de quitosana, que interage quimicamente com o sangue e forma um scaffold híbrido mais estável. Então, o objetivo deste estudo foi testar a hipótese de que o scaffold híbrido, composto por hidrogel de quitosana e sangue, ofereceria maior estabilidade física estrutural, bem como condições favoráveis à adesão e proliferação de células-tronco da papila apical humana (SCAPs; do inglês, Stem Cell from Apical Papila). Para isso, investigamos in vitro se a incorporação do sangue ao hidrogel de quitosana gera um scaffold mais estável, se este é favorável à adesão e proliferação de células-tronco da papila apical e se a fotobiomodulação potencializa essas características celulares. Para isso, SCAPs foram isoladas e caracterizadas por citometria de fluxo, tempo de dobra populacional, e contagem de unidades formadoras de colônias fibroblásticas (CFU-F; do inglês, Colony Forming Units - Fibroblastic). Ensaios de incorporação sanguínea, dissolução e embebição foram realizados para determinar o comportamento dos scaffolds híbridos. A adesão celular foi observada pela coloração PHK26® (do inglês, Red Fluorescent Cell Linker) e por microscopias eletrônicas de varredura (MEV); e a proliferação foi investigada pelo ensaio de alamarBlue®. Adicionalmente, a sobrevivência das SCAPs após a degradação do scaffold híbrido foi avaliada pela coloração Live/Dead®. A população celular estudada apresentou características de células tronco. O scaffold híbrido, constituído de densa rede alveolar com poros interconectados, embebeu e dissolveu rapidamente. De acordo com o PKH26® e alamarBlue® SCAPs aderiram e proliferaram no scaffold híbrido. SCAPs fotobiomoduladas exibiram maior taxa de proliferação e o ensaio Live/Dead® mostrou células vivas após 12 dias de cultivo. Concluiu-se que o scaffold híbrido apresenta biocompatibilidade e condições favoráveis de sobrevivência para SCAPs, que são potencializadas pela fotobiomodulação. / Revascularization is a technique used to form a new tissue, replacing the lost pulp, in young permanent teeth presenting incomplete rhizogenesis and irreversible damage, where endodontic treatment is needed. Clinically, the results show the continuity of the root formation and the return of dental vitality. However, little is known about the newly formed tissue and it has not been established if it is able to perform all functions of the dental pulp. To improve the characteristics of the newly formed tissue by the technique of revascularization, tissue engineering tools can be used, represented by stem cells, growth factors and scaffolds for cell supportting. Stem cells (SCs) are already present when the blood invades the root canal, and to use this SCs reserve that the host possesses, procedure known as homing, is an advantage compared with other techniques that inject SCs obtained by cultivation in the laboratory. However the physical aspects of blood clot in the root canal can be improved with the addition of chitosan hydrogel that chemically interacts with the blood and forms a more stable hybrid scaffold. So the aim of this study was to test the hypothesis that the hybrid scaffold, composed of hydrogel chitosan and blood clot, provides greater structural physical stability as well as favorable conditions for adhesion and proliferation of Stem Cells from Apical Papilla (SCAPs). For this, we investigated in vitro if the incorporation of blood to chitosan hydrogel, generates a more stable scaffold and if it supports the stem cell adhesion and proliferation, in addition, if photobiomodulation potentiates these cell characteristics. For this, SCAPs were isolated and characterized by flow cytometry, population doubling time, and counting colony forming units - fibroblastic (CFUF). Blood incorporation assays, dissolution and swelling were conducted to determine the behavior of hybrid scaffolds. Cell adhesion was observed by PHK26® (Red Fluorescent Cell Linker) and scanning electron microscopy (SEM); and proliferation was investigated by alamarBlue® assay. In addition, the survival of SCAPs after degradation of the scaffold was assessed by Live/Dead® staining. The cell population showed stem cell characteristics. The hybrid scaffold, constituted of dense cellular network with interconnected pores, soaked and dissolved quickly. According to PKH26® and alamarBlue® assays, the SCAPs adhered and proliferated in the hybrid scaffold. Photobiomodulation leads to SCAPs higher proliferation rate and the Live/Dead® test showed live cells after 12 days of cultivation. It was concluded that the hybrid scaffold is biocompatible and favors survival of SCAPs, which was enhanced by photobiomodulation.
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Análise da expressão gênica em células-tronco mesenquimais de medula óssea humana durante o comprometimento com a linhagem osteogênica / Gene expression analysis of mesenchymal stem cells from human bone marrow during the osteogenic commitmentFontana, Vanessa 17 March 2009 (has links)
As células-tronco são definidas como células capazes de auto-renovação e diferenciação em células especializadas. Dentre as células-tronco adultas, as mesenquimais ocupam uma posição de destaque. São células multipotentes que podem originar células de origem mesodérmica, como osteoblastos, adipócitos e condrócitos. Além disso, vários estudos demonstraram que essas células também podem se diferenciar em células de origem extra-mesenquimal, como neurônios e hepatócitos, fenômeno este denominado plasticidade. Aspectos moleculares envolvidos com a plasticidade das células-tronco adultas, bem como durante o processo de comprometimento e diferenciação em células especializadas não estão completamente elucidados. Considera-se que as células-tronco expressam, embora em baixos níveis, uma gama diversa de genes relacionados aos diferentes destinos de diferenciação. Durante a diferenciação o repertório de genes expressos seria reduzido, acompanhado do aumento nos níveis de expressão de uma coleção menor de genes que orientariam a especialização celular. Para o presente estudo adotamos essa hipótese e elaboramos um sistema-modelo experimental no qual células-tronco mesenquimais de medula óssea humana foram induzidas in vitro à diferenciação em osteoblastos. As células-tronco mesenquimais foram obtidas de três doadores saudáveis de medula óssea e induzidas quimicamente à diferenciação em osteoblastos (meio -MEM acrescido de dexametasona, ácido ascórbico e - glicerofosfato) durante 24 h, 48 h, 7 dias e 21 dias. O RNA total foi extraído das culturas de células indiferenciadas (0h) e durante o processo de diferenciação (24 h, 48 h, 7 d e 21 d). Todo o processo de diferenciação foi monitorado com ensaios bioquímicos e por imunocitoquímica. Para avaliar a expressão gênica das célulastronco e durante o processo de diferenciação utilizamos a tecnologia dos cDNA microarrays. Os dados foram analisados com auxílio de programas de bioinformática especializados. Um banco público de dados de expressão gênica (GNF Symatlas) também foi consultado, o que nos auxiliou na interpretação dos dados. Sondas de cDNA fluorescentes (Cy3) oriundas das amostras de RNA foram hibridizadas com os cDNA microarrays (4.500 seqüências alvo). Um pool de cDNAs irrelevantes marcados com Cy5 foi usada como referência na hibridização. A expressão gênica diferencial durante a diferenciação foi analisada pelo método SAM (significance analysis of microarrays) (FDR < 5%). Os genes diferencialmente expressos foram classificados segundo sua representação tecidual de acordo com os valores de expressão obtidos do banco de dados GNF Symatlas. Além disso, foi avaliada por RT-PCR semiquantitativa, a expressão de genes relacionados à osteogênese (ALPL, osteocalcina, COL1A1, RUNX2 e osteopontina), e um marcador de adipócitos, PPARG. Observamos que as células-tronco mesenquimais no seu estado tronco expressam, em níveis similares, genes característicos das linhagens osteo e adipocítica. Entretanto, ao longo do comprometimento com a linhagem osteoblástica o número de genes modulados (reprimidos e induzidos) é crescente. Revelamos que nas fases iniciais da diferenciação osteoblástica in vitro os genes que representam células-tronco adultas foram reprimidos. Entretanto, com o avanço do comprometimento com a linhagem osteoblástica observamos que certos genes foram induzidos como, por exemplo, BMP1, SMAD7, IGFBP4, ITGA5, FN1, BGN, CDH8, GDF10 e SCARB2, todos relacionados à osteogênese. Nossos resultados são importantes para um melhor entendimento das bases genético-moleculares associadas ao potencial plástico e de diferenciação das células-tronco mesenquimais. / Stem cells (SC) are defined by their property of self-renewal and differentiation into specialized cells. The adult mesenchymal stem cells (MSC) correspond to a special type of them. MSC are multipotent and can differentiate into several cell types of mesodermal origin, including osteoblasts, adipocytes and condroblasts. Also, their capacity to differentiate into extra-mesenchymal cells, such as neurons and hepatocytes, was demonstrated and this phenomenon was termed plasticity. Nevertheless, the molecular basis of adult SC plasticity, as well during their commitment and differentiation into specialized cells isnt completely elucidated. It was hypothesized that SC express, although at low levels, many genes related to the differentiation cues that they could assume. According to this hypothesis, the repertory of genes is reduced during differentiation, at the same time that upregulating the expression of specialized genes, which characterize the future tissue. In this study, we adopted this hypothesis and to test it we used an in vitro osteogenic differentiation model-system. Mesenchymal stem cells were obtained from bone marrow of three healthy donors and induced to differentiate into osteoblasts in -MEM media supplemented with dexametasone, ascorbic acid and betaglycerol phosphate during 24 h, 48 h, 7 and 21 days. The differentiation process was monitored by biochemical and immunocytochemistry assays. The total RNA was obtained from undifferentiated (0 h) and differentiated cells. To evaluate gene expression during the differentiation, we used the cDNA microarray technology. Fluorescent Cy3 cDNA probes originated from SC RNA samples were hybridized with a cDNA microarray containing 4,500 target sequences. A pool of irrelevant cDNA was labeled with Cy5 and used as reference. The differential gene expression during differentiation was analyzed by SAM (Significance Analysis of Microarrays) method (FDR < 5%). The public gene expression database (GNF Symatlas) was used to annotate the tissue representation of the modulated genes. The expression of some specific genes related to osteogenesis (ALPL, osteocalcin, COL1A1, RUNX2 e osteopontin) and an adipocyte marker (PPARG) was evaluated by semiquantitative RT-PCR. It was observed that MSC in their stem state express, at similar levels, representative genes of the osteo and adipocyte lineages. However, during the osteoblastic differentiation process the number of modulated genes (repressed or induced) increased. It was observed that at early in vitro osteoblastic differentiation, adult SC characteristics genes were downregulated. Conversely, the osteoblastic commitment of these cells displayed upregulation of specific genes related to bone formation (BMP1, SMAD7, IGFBP4, ITGA5, FN1, BGN, CDH8, GDF10 and SCARB2). These results are important to provide a better understanding of the genetic and molecular basis of the plasticity and commitment phenomenon of adult MSC.
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Infusão de células tronco mesenquimais derivadas da medula óssea em modelo experimental de nefropatia crônica induzida por lesões de podócitos. / Infusion of bone marrow mesenchymal stem cells in an experimental model of chronic nephropathy induced by podocyte injuryRamalho, Rodrigo José 27 March 2013 (has links)
Estudos com células tronco (CT) têm despertado grande interesse devido ao seu promissor potencial terapêutico. Neste contexto, as CT mesenquimais (CTm) representam uma alternativa para o tratamento de diversas patologias em diferentes órgãos, inclusive o rim e as glomerulopatias que o acometem. As doenças glomerulares constituem uma freqüente causa de doença renal crônica e se caracterizam por apresentar proteinúria. Neste processo, os podócitos são células que apresentam um papel crucial, sendo que as podocitopatias se associam com o aparecimento de proteinúria e desenvolvimento de esclerose glomerular. A obtenção de um modelo de podocitopatia através da administração de aminonucleosídeo de puromicina (PAN), permite a melhor compreensão dessas células altamente diferenciadas que não possuem potencial de proliferação ou regeneração. O presente projeto teve como objetivo estabelecer o modelo experimental de nefropatia crônica induzida por PAN associado à nefrectomia unilateral (UniNx) para induzir lesões glomerulares mais exuberantes e, neste modelo experimental, avaliar o efeito da infusão de CTm derivadas da medula óssea. Ratos Wistar (n=52) foram divididos em três grupos: Controle (UniNx), PAN (PAN+UniNx) e PAN+CTm (PAN+UniNx+CTm). As CTm foram inoculadas na região subcapsular renal no dia 0 e os animais foram sacrificados após 30 e 60 dias. O efeito da infusão das CTm no tecido renal foi avaliado através de parâmetros clínicos e laboratoriais, além de análise histológica, imunohistoquímica, microscopia eletrônica e PCR em tempo real. Paralelamente, o projeto analisou a diferenciação in vitro de CTm em podócitos através do estímulo com colágeno tipo IV e através de cocultura de glomérulos isolados de ratos com CTm. A diferenciação celular das CTm foi analisada por citometria de fluxo, imunocitoquímica e PCR em tempo real para genes de proteínas podocitárias. No modelo in vivo foi possível observar a presença de CTm até 15 dias após a inoculação na região subcapsular renal. As CTm foram capazes de diminuir significativamente a proteinúria e a albuminúria com 30 e 60 dias, assim como a pressão arterial aos 60 dias. Não houve diferença nos valores de creatinina, uréia sérica, glomeruloesclerose e fibrose intersticial entre o grupo PAN e o grupo PAN+CTm. As CTm foram responsáveis pela diminuição significativa da fusão dos pedicelos à microscopia eletrônica, com melhora da expressão relativa de WT1 aos 60 dias e melhora parcial da expressão gênica de nefrina, podocina e sinaptopodina. A expressão proteica de WT1 também foi significativamente maior no grupo PAN+CTm em comparação ao grupo PAN. Além disso, houve melhora significante da expressão relativa de IL-4 e IL-10, e diminuição de IL-1? e TNF-? no grupo tratado. Ainda, as CTm promoveram aumento significativo da expressão gênica de VEGF aos 60 dias. Nos resultados in vitro não houve diferenciação das CTm em podócitos quando cultivadas com colágeno IV, assim como a cocultura com glomérulos não proporcionou alteração na expressão de marcadores de superfície das CTm. Concluímos que a terapia celular com CTm foi capaz de induzir proteção renal caracterizada por diminuição da proteinúria, da albuminúria e da pressão arterial, associado a menor fusão dos pedicelos, maior expressão gênica de proteínas podocitárias e de expressão celular de WT1. As citocinas inflamatórias IL-1?, TNF-?, IL-4 e IL-10, em conjunto com o VEGF, foram os possíveis mediadores responsáveis por estes resultados / Stem cells (SC) have emerged as a potential therapeutic approach for several diseases. In this context, the mesenchymal SC (mSC) are considered an alternative for the treatment of kidney diseases such as glomerulopathies. Glomerular diseases are an important cause of chronic kidney disease (CKD) and are characterized by proteinuria. In this process, the podocytes are cells that have a critical role, and the podocytopathies are associated with the onset of proteinuria and glomerular sclerosis. The achievement of a podocytopathy model through administration of puromycin (PAN) allows a better understanding of these highly differentiated cells which do not have the potential for proliferation or regeneration. The aim of the present study was to establish an experimental model of chronic nephropathy induced by PAN associated with unilateral nephrectomy (UniNx), to induce early and marked glomerular lesions and, in this experimental model, to evaluate the effect of bone marrow mSC infusion. Wistar rats (n=52) were randomly divided into three groups: Control (UniNx), PAN (PAN+UniNx) and PAN+mSC (PAN+UniNx+mSC). mSC were inoculated into the subcapsular renal region on day 0, and the animals were sacrificed after 30 and 60 days. The mSC infusion effects in renal tissue were evaluated by clinical and laboratory parameters, histology, immunohistochemistry, electron microscopy and real-time PCR. In parallel, we analyzed whether mSC could differentiate in vitro into podocytes through stimulation with collagen type IV or by means of co-culture of isolated rat glomeruli with mSC. The cell differentiation was analyzed by flow cytometry, immunocytochemistry and real-time PCR. In the in vivo model, mSC were detected until 15 days after inoculation in the renal subcapsular region. mSC were able to significantly reduce the proteinuria and albuminuria with 30 and 60 days, as well as blood pressure at 60 days. There was no difference in the values of creatinine, BUN, glomerulosclerosis and interstitial fibrosis between the groups PAN and PAN+mSC. The treated group showed lower effacement of foot process by electron microscopy, with significant improvement in the relative expression of WT1 in 60 days and partial improvement of nephrin, podocyn and synaptopodin. The WT1 protein expression was also significantly higher in the PAN+mSC group compared to the PAN group. In addition, mSC treatment significantly reduced gene expression of IL-1? and TNF-?, as well as increased the expression of IL-4 and IL-10. At 60 days mSC promoted significant increase of VEGF relative expression. In vitro results, mSC cultived with collagen type IV did not show differentiation to podocytes and the co-culture with glomeruli provided no change in expression of mSC surface markers. In conclusion, mSC therapy in the PAN model was able to induce renal protection characterized by the reduction of albuminuria, proteinuria and blood pressure, associated with a lower effacement of foot process, increased gene expression of podocytes proteins and cellular expression of WT1. Inflammatory cytokines IL-1?, TNF-?, IL-4 and IL-10 associated with VEGF were the probable mediators of these results, promoting podocyte protection
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Effects of secretion factors from umbilical cord derived mesenchymal stem cells (MSCs) on MSCs multi-differentiation potentials and underlying mechanisms / CUHK electronic theses & dissertations collectionJanuary 2014 (has links)
Introduction: MSCs are multipotent progenitor cells that can differentiate into various cell lineages, such as osteoblasts, chondrocytes and adipocytes. MSCs synthesize abundant secretion factors to extracellular matrix which contain a variety of growth factors, cytokines and microRNAs. Secretion factors could stimulate the regeneration and differentiation of surrounding cells, but their underlying mechanism still remains elusive. We hypothesized that secretion factors from different tissues derived MSCs had potential to promote MSCs differentiation and musculoskeletal tissue regeneration. We also suggested that microRNAs played an essential role in the effects of secretion factors. In present study, we investigated the effects of secretion factors obtained from different tissues derived MSCs (umbilical cord, dental pulp, gingiva and adipose tissue) on multi-differentiation potentials of MSCs, including osteogenesis, chondrogenesis, tenogenesis, neurogenesis and adipogenesis. Moreover, we illustrated the effects of umbilical cord derived MSC (UCMSC) secretion on bone, cartilage and tendon tissue repair. We further revealed that microRNAs may impact the effect of secretion factors on MSCs osteogenic differentiation. / Methods: Human bone marrow MSCs (hBMSCs) were incubated with various differentiation induction media. Secretion factors were used as supplement. Different animal models of tissue repair (bone, cartilage and tendon) were employed for study of the effects of secretion factors on tissue healing. miRNA microarray was performed to find the potential effective miRNAs in secretion factors. Real time qRT-PCR, microCT, mechanical test, immunohistological analysis and various staining methods were employed as outcome measurements. / Results: We found that both UCMSC and dental pulp derived MCS secretion could initiate osteogenic differentiation of hBMSCs without osteogenic induction medium. UCMSC secretion had positive effect on chondrogenic and tenogenic differentiation of MSCs and inhibitory effect on adipogenesis of hBMSCs. Our results showed that UCMSC secretion in HA/TCP scaffolds with hBMSCs promoted ectopic bone formation in nude mice. UCMSC secretion with rat BMSCs in hyaluronic hydrogel significantly enhanced the bone repair of rat calvarial bone critical defect. To reveal the underlying mechanism, secretion factors were analyzed by miRNA microarray. Among the differentially expressed microRNAs, we found miR-1237 could promote osteogenesis while miR-3676 could inhibit osteogenic differentiation of MSCs. / Conclusions: This study indicated that among secretion factors from MSCs form four types tissues, UCMSC secretion could initiate osteogenesis of MSCs and promote bone repair. We also demonstrated that microRNAs from secretion had impact on osteogenic differentiation of MSCs. Our study showed clinical potential of UCMSC secretion in bone regeneration, and more research are needed for optimizing the preparation and delivery of the MSCs secretive factors, as well as to understand their mechanisms of action. / 前言:間充質幹細胞是具有強大分化潛能的始祖細胞。間充質幹細胞可以分化為多種細胞系,例如成骨細胞,軟骨細胞和脂肪細胞。間充質幹細胞合成并釋放大量分泌素到細胞外基質中。這些分泌素包括多種生長因子,細胞因子和微小核糖核酸。分泌素能夠刺激周圍細胞的再生和分化,但是分泌素的作用機理還不是很清楚。我們認為,不同組織來源的間充質幹細胞分泌素有可能會促進間充質幹細胞的多系分化和骨骼肌肉組織的再生,並且微小核糖核酸在分泌素的效應中發揮了重要作用。我們首先研究了臍帶,牙髓,牙齦和脂肪來源的間充質幹細胞分泌素對于間充質幹細胞的分化能力的作用。我們還對臍帶幹細胞分泌素在骨,軟骨和肌腱修復的效果做了進一步的研究。另外,我們還發現分泌素中的微小核糖核酸在間充質幹細胞的成骨分化方面有一定的效果。 / 方法:我們用人間充質幹細胞來進行誘導分化實驗。臍帶,牙髓,牙齦和脂肪來源的間充質幹細胞的分泌素用於細胞培養基的補充。在體內實驗中我們用了不同的動物模型,把填充物和分泌素一起種植在動物體內。我們利用微小核糖核酸陣列技術來檢測分泌素中的有效微小核糖核酸。我們使用了定量聚合酶鏈反應技術,微型計算機斷層掃描成像,力學測試,免疫組織分析和多種染色方法。 / 結果:我們發現臍帶和牙髓間充質幹細胞分泌素可以在沒有成骨誘導培養基的情況下啟動骨髓間充質幹細胞的成骨分化。臍帶間充質幹細胞對成軟骨和成肌腱分化起到積極作用,而且可以抑制脂肪分化。我們發現在羥基磷灰石/磷酸三鈣材料中,臍帶間充質幹細胞分泌素與人骨髓間充質幹細胞可以共同促進裸鼠的異位成骨。臍帶間充質幹細胞分泌素與鼠骨髓間充質幹細胞一起用於透明質酸水凝膠中能夠加快大鼠頭骨缺損的修復。為了揭示分泌素的作用機理,我們用微小核糖核酸陣列技術來檢測分泌素。在表達不同的微小核糖核酸之中,我們發現miR-1237可以促進間骨髓間充質幹細胞的成骨分化,而miR-3676能夠抑制骨髓間充質幹細胞成骨分化。 / 結論:本研究表明,在四種不同來源的分泌素中,臍帶間充質幹細胞分泌素可以啟動骨髓間充質幹細胞的成骨分化,同時加快骨組織修復。我們發現微小核糖核酸在分泌素的促進間骨髓間充質幹細胞成骨分化的效果中發揮了一定的作用。我們的研究表明,使用臍帶間充質幹細胞分泌素修復骨組織具有廣泛的臨床應用前景。間充質幹細胞分泌素的生產,使用過程和作用機理還有待于進一步的優化和研究。 / Wang, Kuixing. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 131-147). / Abstracts also in Chinese. / Title from PDF title page (viewed on 01, November, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.
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Differentiation of stem cells inside hybrid polymer gels made of environmentally sensitive microgels / CUHK electronic theses & dissertations collectionJanuary 2014 (has links)
Dai, Zhuojun. / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references. / Abstracts also in Chinese. / Title from PDF title page (viewed on 15, September, 2016).
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Role of Aqp1, Sm51 and GATA6 in differentiation and migration of bone marrow derived mesenchymal stem cells. / Aqp1, Sm51和GATA6在骨髓干细胞分化与迁移中的作用 / CUHK electronic theses & dissertations collection / Aqp1, Sm51 he GATA6 zai gu sui gan xi bao fen hua yu qian yi zhong de zuo yongJanuary 2013 (has links)
Meng, Fanbiao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 114-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
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