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Made available in DSpace on 2014-06-11T19:35:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-10-08Bitstream added on 2014-06-13T19:45:08Z : No. of bitstreams: 1 limaneto_jf_dr_botfmvz.pdf: 5509939 bytes, checksum: 6d585da226479576f95a5b051bde27a2 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A célula-tronco (CT) é definida como uma célula com capacidade de gerar diferentes tipos celulares e reconstituir diversos tecidos. Além disso, a CT apresenta propriedades de auto-renovação, gerando cópias idênticas a si mesma. De acordo com sua origem, as células-tronco podem ser chamadas de adultas e embrionárias. As células-tronco adultas (CTA) mais utilizadas nas clínicas de terapia celular são as células-tronco hematopoiéticas e as células tronco mesenquimais, encontradas principalmente na medula óssea, tecido adiposo e no sangue do cordão umbilical. As células-tronco embrionárias (CTE) são derivadas da massa celular interna de embriões no estágio de blastocisto. Desta maneira este trabalho teve como objetivo desenvolver uma metodologia adequada para o isolamento, cultivo e caracterização de células tronco embrionárias e mesenquimais de eqüinos, além de verificar a capacidade que as células possuem em se diferenciar in vitro em outros tipos célulares. Foi coletado sangue da medula óssea de eqüinos entre 8 e 15 anos de idade. As células tronco mesenquimais foram isoladas após a primeira e segunda passagem. As células foram caracterizadas com marcadores de superfície CD34 (mononucleares) e CD44 (mesenquimais). Após isolamento e caracterização, as células tronco mesenquimais foram diferenciadas para as linhagens osteogênica, adipogênica, condrogênica e neurogênica. A confirmação da diferenciação das células tronco foi realizada por marcadores teciduais específicos. Estas células também, foram capazes de expressarem marcadores neurais. Para o isolamento das células tronco embrionária eqüina, embriões com oito a nove dias foram coletado e a massa celular interna (MCI) isolada mecanicamente. Após o isolamento, a MCI foi transferida para a placa de cultivo previamente preparada com monocamada de fibroblastos para o desenvolvimento... / The stem cell (SC) is defined as cells with the capacity of generate different cellular types and rebuild various tissues. Moreover, the SC has a selfregenerate ability, generating identical copies of itself. According to its origins, the SC can be named as “adult” or “embryonic”. The adult stem cell (ASC) more often used in clinical trials and cellular therapy, are the hematopoietic stem cells and the mesenchymal stem cells, isolated mainly from the marrow bone, adipose tissue and umbilical cord blood. The embryonic stem cells (ESC) are obtained from the inner cell mass of embryos at the blastocyst stage. In this way the present study had as objective to develop an adequate methodology of isolation, culture and characterization of embryonic and mesechymal stem cells from horses, verifying the capacity of those cells to differentiate in vitro into different cells types. Bone marrow blood was collected from horses, aging from 8 to 15 years and filtered with a donation blood kit filter, to avoid clots. The mesenchymal stem cells were isolated after the first and the second passage. The SC were characterized using surface markers CD34 (monuclear) and CD44 (mesenchymal). After the isolation and characterization, the mesenchymal stem cells were differenced into osteogenic, adipogenic, condrogenic and neurogenic lineage. The cells differentiations were confirmed using specific tissue markers. To isolate the embryonic stem cells equine embryos with 8 to 9 days were used. The inner cell mass (ICM) were extract mechanically and transferred to a culture dish previously prepared with fibroblasts monolayer to colony formation and development. The colonies were characterized with pluripotency markers and then submitted to a differentiation process into neurogenic lineage, confirmed by specific neural tissue markers

Type 1 diabetes mellitus is a metabolic disorder characterized by an autoimmune attack against the insulin producing Beta-cells of the pancreas. Also known as insulin-dependent diabetes, patients must receive exogenous injections of insulin in order to maintain glycemic homeostasis. The necessity of monitoring one’s own blood glucose levels and self-administering insulin is a tedious routine for type 1 diabetics, and this standard treatment option fails to treat any of the underlying causes of the disease. According to van Belle et al, the prevalence of diabetes is rising worldwide amongst all age-groups, from 2.8% in 2000 to an estimated 4.4% by 2030, thus the need to find a more curative treatment approach is eminent. In the emerging field of regenerative medicine, mesenchymal stem cells have been identified as a possible therapeutic tool to replace damaged parenchymal tissue. Along with their ability to modulate the local microenvironment, the introduction of properly differentiated mesenchymal stem cells into patients with Type 1 diabetes may provide a treatment option that helps supplement the lost islet cells without provoking an immune response. Preliminary clinical trials have shown that stem cell therapy decreases the amount of exogenous insulin required daily, decreases fasting glucose levels, decreases amount of glycated hemoglobin and increases C-peptide levels. These four indicators of diabetic control suggest that mesenchymal stem cells are an effective means of helping manage Type 1 diabetes. Still, much research needs to be done to fully understand the biomechanics behind the cells’ actions in order to expand human clinical trials. Although complete insulin independence is rarely achieved in patients receiving mesenchymal stem cell treatment, the promising results shown so far suggest more studies be undertaken in hopes of finding a corrective approach to treat Type 1 diabetes.

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Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

<strong>Osteoarthritis</strong>, the most prevalent joint disease in the United Kingdom, is a progressive condition that results in end-stage full-thickness cartilage loss and has important social and economic impacts on society. Since cartilage lacks regenerative capabilities, it is essential to develop approaches to initiate and enhance cartilage regeneration. In this context, tissue engineering is emerging as an attractive approach for the regeneration of cartilage tissue damaged due to disease or trauma. A scaffold-free cartilage construct, analogous to those found during embryonic precartilage condensation, has received much attention as an alternative novel modality for cartilage <strong>tissue engineering</strong>. Cartilage repair with <strong>scaffold-free</strong> tissue more closely resembles the natural situation and mimics the features of the original tissue. Moreover, scaffold-free cartilage implants can overcome the complications caused by the use of suboptimal scaffolds by avoiding the need for a foreign scaffold at all. Culturing cells into tissue patches without the requirement for a scaffold can be achieved through <strong>cell sheet engineering</strong>, which uses thermo-responsive culture dishes. However, the high costs of the tissue culture consumables, and the relatively low cellular yield, makes this process less attractive. This thesis presents a novel method for generating shape-, size- and thickness-adjustable 3-dimensional scaffold-free cell pellet sheets for use as implantable biological cell patches for cartilage tissue engineering. This new technique of bioengineering scaffold-free cell pellet sheets proves to be reproducible, easily applicable, sizable and thickness adjustable. <strong>Stem cells</strong> have added a new thrust to tissue engineering. Their distinctive self-renewal and plasticity have not only optimized many tissue engineering developments, but also rendered feasible some applications which would otherwise be unattainable with somatic cells. Human mesenchymal stem cells (HMSCs) were used to examine the optimal condition for generating cell pellet sheets with this new method. Furthermore, the resultant differentiated pellet sheets were compared directly with HMSCs, human chondrocytes and human fibroblasts alone to evaluate the feasibility of using this cell pellet sheet for clinical applications in terms of their biological and mechanical properties. The results of this thesis suggest that the engineered scaffold-free, chondrogenic, differentiated MSC pellet sheet not only exhibits desirable biologic features similar to chondrocytes, but also demonstrates good integrative and viscoelastic potential that might offer exciting possibilities for the development of novel biologically-based clinical therapies. In summary the data presented herein indicate the following points: <table><ul style="list-style-type:square"><li>The differentiation of human MSCs into chondrogenic cells was achieved.</li> <li>A novel approach of centrifugal seeding on a PDMS surface was shown to effectively generate chondrogenic-differentiated cell pellet sheets without impairing the biological functions of chondrocytes.</li> <li>Various cell types such as human MSCs, human chondrocytes and human fibroblasts were found to respond well to the novel methodology and generated viable, cohesive, less shrinkable, and readily-detachable cell pellet sheets, the size and thickness of which could be adapted as required. The results obtained were superior to those obtained using the conventional thermo-responsive culture dish method.</li></table> This new methodology developed in this thesis provides an approach to in vitro cell pellet sheet generation which is closer to the physiological process of cartilage development and which proved valuable for the study of in vitro generation of scaffold-free cell patches as an important adjunct to many traditional cartilage restorative procedures. Future research on in vivo assessment of the cell sheet and the functional role of these sheets in repairing damaged cartilage is recommended.

Atrophic non-union is a major complication following fracture of a bone. It represents a biological failure of the fracture healing process and occurs in 5-10% of cases. A number of factors predispose to atrophic non-union including high energy injuries, open fractures, diabetes, and smoking. Atrophic non-unions cause immense patient morbidity and consume large amount of health care resources. Bone grafts taken from the iliac crest contain biologic components required for fracture healing and are considered as the gold standard treatment of aseptic atrophic non-union. However, harvesting bone grafts from the iliac crest is associated with significant patient morbidity which can reduce quality of life. Mesenchymal stem cells (MSCs) have the ability to proliferate and undergo multilineage differentiation. The emergence of MSC therapy provides an alternative strategy for treating impaired fracture healing. MSCs contribute to normal fracture healing both directly as bone progenitor cells and indirectly as mediator secreting cells. Although a number of studies have shown that MSCs can promote bone regeneration in small animal fresh critical size defects, this is not analogous to most clinical aseptic atrophic non-unions which do not have a significant bone gap. There remains therefore a clinical need for an appropriate strategy for using stem cells in atrophic non-unions. Thus, the aim of this study aim was to develop a clinically relevant strategy to promote fracture healing in an atrophic non-union model using the percutaneous injection of MSCs as a minimally invasive technique. An atrophic non-union model was established and validated. A small (1 mm) non-critical size defect was created at the mid shaft tibia and the fracture site was stabilised using an external fixator. Atrophic non-union was induced by stripping the periosteum for one bone diameter either side of the osteotomy site and curettage of the intramedullary canal over the same distance. The procedure reliably created an atrophic non-union. Fracture healing was evaluated using (1) serial radiography, (2) micro-computed tomography, (3) histomorphology and (5) biomechanical testing. Fracture scoring systems including the radiographic union scale in tibia (RUST) and the Lane & Sandhu score were validated in a preclinical model. A simple sample preparation technique for evaluating bone mechanical properties was developed and used to assess the stiffness and strength of the fracture repair. Percutaneous injection of MSCs locally into the fracture site in the early ‘post-injury’ period at three weeks after induction of atrophic non-union was found to improve the fracture healing process significantly (83% of cases), while MSCs implantation in the late ‘post-injury’ period at eight weeks after induction of atrophic non-union showed no significant improvement of fracture healing (20% of cases). Percutaneous local implantation of MSCs rescued the fracture healing process in cases destined to progress to atrophic non-union. In clinical practice, there may be an advantage using MSCs from a universal donor as the processes of MSC isolation and preparation are expensive and time consuming. To investigate the feasibility of using non-autologous cells, the atrophic non-union was used to determine the bone regenerative potential of using xenogeneic donor hMSCs in an atrophic non-union. The results demonstrated that the therapeutic effect of using hMSCs in a xenogeneic manner to promote fracture healing in the rat atrophic non-union model was comparable with rMSCs (88% of cases in both hMSCs and rMSCs) and there were neither significant clinical adverse effects nor adverse immune responses with the xenogeneic transplantation. However, MSCs did not persist at the fracture following injection. Perivascular stem cells (PSCs) taken from adipose tissue, which is an expendable source, have advantages over conventional MSCs as they are a defined and homogenous population and can be used without culture expansion. The administration of PSC using percutaneous injection improved the fracture healing process in atrophic non-union (60% of cases). This suggested that PSCs may present an appropriate choice for use in cell therapies to promote fracture healing in atrophic non-union. The results from this thesis can be applied to the development of a clinically relevant strategy using MSCs as a minimally invasive technique to promote fracture healing in atrophic non-union, in particular (1) the effectiveness of a cell therapy is likely to be highly dependent of the timing of injection relative to the stage of fracture healing, (2) hMSCs were as effective as rMSCs in promoting fracture healing, suggesting that it may be feasible to use an allogeneic strategy in humans, (3) the injected MSCs were not detectable even in case of successful repair, suggesting that they may act through a paracrine effect and (4) PSCs isolated from adipose tissue contributed to fracture healing in the atrophic non-union model, suggesting that adipose tissues can be used as an alternative cell sources for bone repair.

Despite the normally robust regenerative capacity of muscle tissue, extensive soft tissue damage often results in a functional loss that cannot be restored using classic reconstruction techniques. Although implanted biomaterials are capable of mechanically transmitting force generated from the remaining tissue, cellular repopulation, reinnervation and revascularization of the injured area is necessary to achieve complete functional restoration. Using an in vivo tissue engineering model, a 1.0 x 1.0 cm portion of the lateral gastrocnemius (LGAS) of Lewis rats was removed and replaced with a muscle-derived extracellular matrix (ECM). Constructs were seeded with bone marrow-derived (BMSCs) or adipose-derived stem cells (ADSCs) and the peroneal nerve was relocated over the implanted ECM. Creation of the defect resulted in a functional impairment of the LGAS, only capable of producing 85.1 ± 4.1% of the force generated in the contralateral LGAS following ECM implantation. A significant increase in specific tension (SPo) was seen in all groups following the nerve relocation procedure when compared to their corresponding cellular treatment without nerve relocation (p < 0.05). Histological quantification revealed significant increases in cellular content and blood vessel density in the top and bottom regions of ECM implants seeded with BMSCs (p < 0.05). The nerve relocation procedure significantly increased these same variables within the middle region of the ECM when compared to all groups lacking this treatment (p < 0.05). The presence of regenerating myofibers was immunofluorescently confirmed using antibodies against desmin, myosin heavy chain and laminin, while their developmental state was substantiated by the presence of central nuclei. These data corroborate a therapeutic effect of BMSCs on skeletal muscle regeneration within the ECM implant that was not seen following ADSC injection. Furthermore, the nerve relocation procedure stimulated an increased cellular and vascular growth within the middle region of the construct, likely the cause of improved functional output. / text

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Articular cartilage is an extraordinary tissue, allowing frictionless movements of articulated joints, and acting as a load-bearing cushion to protect joints from damage. Breakdown of articular cartilage may result in crippling diseases such as osteoarthritis (OA) and, since articular cartilage has a limited repair capacity, a greater understanding of the mechanisms of joint homeostasis and its response to injury are of great clinical need. In this project the hypothesis that endogenous mesenchymal stem cells (MSCs) may contribute to the healing process of a full-thickness articular cartilage defect was investigated by combining a mouse model of joint surface injury and repair with a nucleoside analogue labelling scheme in DBA/1 mice. Following injury, proliferative responses of nucleoside analogue-retaining cells were detected between 4 and 12 days post injury (dpi) in both the bone marrow and the synovial membrane of the knee joint. Phenotypic analysis of these label-retaining cells using immunofluorescence staining revealed an MSC-compatible phenotype (CD44+, CD105+, CD146+, PDGFRα+ and p75NGFR+), with differences observed between the two tissues in expression of CD105 and CD146. The response of the label-retaining cells to the injury was associated with early activation of Notch signalling (4 dpi), followed by BMP signalling at 8 dpi and TGF-β at 12 dpi. Conversely, canonical Wnt signalling, which was active in uninjured knee joints and in injured knee joints up to 8 dpi, was attenuated at 12 dpi. The contribution of nerve growth factor (NGF), known as a pain mediator in OA, to the repair process was then investigated in vitro. NGF was released by both cartilage explants and femoral head cultures following injury. Using a Transwell-based cell migration assay, NGF was revealed to have a chemotactic effect on human bone marrow derived MSCs, but not synovial membrane derived MSCs. High-density micromass cultures also revealed NGF had a potent stimulatory effect on the chondrogenic differentiation of mesenchymal cells. The data presented here demonstrate a contribution of endogenous MSCs to the repair of articular cartilage in vivo and suggest a possible new therapeutic strategy: stimulation of in vivo recruitment of MSCs by modulating signalling pathways activated during the healing process. Furthermore, a novel role for NGF as a factor involved in migration and the chondrogenic differentiation of MSCs is suggested.