Stimulating lymphocytes with Ifn-γ, anti-CD3, and interleukin-2 promotes the proliferation of a cell population coexpressing T-lymphocyte surface antigens such as CD3, CD8a, and CD25 as well as natural killer cell markers such as NK1.1, CD49, and CD69. These cells, referred to as cytokine-induced killer cells (CIKs), display cytotoxic activity against tumour cells, even without prior antigen presentation, and offer a new cell-based approach to the treatment of malignant diseases. Because CIKs are limited in vivo, strategies to optimize in vitro culture yield are required. In the last 10 years, mesenchymal stem cells (MSCs) have gathered considerable attention. Aside from their uses in tissue engineering and as support in haematopoietic stem cell transplantations, MSCs show notable immunomodulatory characteristics, providing further possibilities for therapeutic applications. In this study, we investigated the influence of murine MSCs on proliferation, phenotype, vitality, and cytotoxicity of murine CIKs in a coculture system. We found that CIKs in coculture proliferated within 7 days, with an average growth factor of 18.84, whereas controls grew with an average factor of 3.7 in the same period. Furthermore, higher vitality was noted in cocultured CIKs than in controls. Cell phenotype was unaffected by coculture with MSCs and, notably, coculture did not impact cytotoxicity against the tumour cells analysed. The findings suggest that cell–cell contact is primarily responsible for these effects. Humoral interactions play only a minor role. Furthermore, no phenotypical MSCs were detected after coculture for 4 h, suggesting the occurrence of immune reactions between CIKs and MSCs. Further investigations with DiD-labelled MSCs revealed that the observed disappearance of MSCs appears not to be due to differentiation processes.

A artrose, também chamada de osteoartrose ou osteoartrite (OA), é a terceira doença de maior incidência no Mundo. Nesse trabalho, buscou-se criar uma lesão osteocartilaginosa em joelhos de ratos Wistar machos com seis meses de vida, objetivando constituir um modelo animal para estudo da OA humana e, a partir desse modelo, avaliar biomateriais de forma isolada ou associados entre si e avaliados quanto à sua segurança biológica e potencial de reparação tecidual. Além disto, foi analisado o efeito reparador de células-tronco mesenquimais da polpa do dente de leite humano (MSC) isoladamente e em associação com o biomaterial formado por: Titânio + Poli Vinil Álcool + Ac. Hialurônico, nesse estudo denominado de TRIGEL (TRG). O Ac. Hialurônico (HA), por suas propriedades visco elásticas, o pó de Titânio (Ti), devido às suas propriedades biológicas únicas de ostoeintegração e o polímero Poli Vinil Álcool (PVA), com suas propriedades hidrofílicas, promovendo a formação do Hidrogel, os quais associados entre si formam um compósito, o TRG, que foi aplicado sobre uma lesão padrão no joelho da pata pélvica direita de ratos Wistar machos de seis meses de idade. Para a obtenção da lesão padrão, os animais foram divididos em três grupos de cincoanimais e cada grupo foi submetido a uma intervenção cirúrgica em seus joelhos direitos, utilizando três técnicas cirúrgicas diferentes, a saber: Grupo (I): Remoção cirúrgica dos meniscos medial e lateral mais perfuração do platô tibial seguido da aspiração da medula óssea através dessa perfuração por meio de seringa e agulha. Grupo (II): Remoção dos meniscos mais perfuração, sem aspiração, Grupo (III): Apenas a perfuração. Todos os animais foram autopsiados após 30 dias. Os joelhos dos quinze animais que constituíam os três grupos foram devidamente catalogados e enviados para a empresa Histotech, para a confecção das lâminas, tendo sido eleito, por análise histológica, o Grupo (I), por demonstrar menor reparo tecidual espontâneo. Em tese, o TRG teria as seguintes propriedades: Uma fonte de reparação tecidual (visco terapia) dada pelo HA e a capacidade amortecedora e carreadora de células-tronco do polímero PVA, que se hidrata, formando o hidrogel. O Ti, pela sua propriedade de osteointegração formaria um tampão sobre as áreas de matriz óssea exposta o que possibilitaria o afluxo de novos condrócitos, que também pode ocorrer pela ação das células-tronco. Livrar a superfície articular das áreas com exposição da matriz óssea é fundamental para o bloqueio das proteases que perpetuam a fisiopatologia da OA. Após tratamento estatístico dos diversos ensaios, utilizando-se os diversos biomateriais no tratamento da lesão, o TRG foi o biomaterial que apresentou o melhor resultado de força entre os grupos. No estudo histológico, foi evidenciada a presença de tecido cartilaginoso supra- lesional, o que só ocorreu nos animais dos grupos que receberam: apenas TRG, TRG associado com células-tronco e aquele que recebeu apenas MSCs. No entanto, mais estudos, com animais de maior porte e mais velhos, devem ser realizados para melhor analisar a segurança e o potencial terapêutico do compósito Trigel. / Osteoarthritis, or osteoarthritis (OA) is the third most debilitating disease in the world. In this study, we attempted to create an osteocartilaginous lesion in the knees of six months old male Wistar rats, aiming to constitute an animal model for the study of human OA and to use this model to evaluate the therapeutic potential of biomaterials, which are already well known for their biocompatibility properties in the clinical practice. The biomaterials were used in isolation or associated with each other and then evaluated for their biological safety and tissue repair capacity. Mesenchymal stem cells, obtained from human dental pulp from deciduous teeth (MSC) were evaluated alone and in association with the biomaterial formed by: Titanium + Poly Vinyl Alcohol + Ac. Hyaluronic, here called TRIGEL (TRG). Due to its visco-elastic properties, the Ti powder, due to its unique biological properties of ostointegration and the polymer PVA, with its hydrophylic properties, forming a hydrogel, were associated to form the composite named TRIGEL, (TRG), which was applied to a standard knee injury of the right hind leg of male Wistar rats. In order to elect the standard lesion, the animals were divided into three groups with five animals each and each group underwent a surgical intervention in their right knees, with three different surgical techniques being applied, namely: Group (1): Surgical removal ofmedial and lateral meniscus plus perforation of the tibial plateau, followed by aspiration of the bone marrow through this perforation using syringe and needle. Group (2): Removal of the meniscus plus perforation, without aspiration, Group (3): Drilling only. All groups were autopsied 30 days after the procedure and all groups were autopsied at 30 days post-procedure. The knees of the 15 animals that constituted the three groups were analyzed histologically and Group (1) (meniscus removed, perforated and aspirated), was elected as the standard lesion since it demonstrated less spontaneous tissue repair. TRG has the following properties: HA is used as a source of tissue repair (visco therapy) and hydration of the polymer; PVA, forms a hydrogel\", with damping action and as a stem cells carrier, whereas Ti was used due to its ósseo-integration, which would allow coating of the exposed bone matrix and this intra-osseous osteo-integration response would form an intercalating buffer. The healthy cartilage surfaces around this structural buffer would allow the reception of new chondrocytes or the action of the cells on TRG properties. Freeing the articular surface of the areas with bone matrix exposure is critical for blocking the proteases that perpetuate the pathophysiology of OA. In the various biomaterial tests in the treatment of the standard lesions, TRG was statistically shown to be the one that better mimicked the non-injured group. The histological study demonstrated the presence of a supra-lesional cartilagenous tissue, which only occurred in the groups which received: only TRG, mesenchymal stem cells associated with TRG and that which received only MSCs. However, further studies with larger and older animals should be pursued to better assess the safety and therapeutic potential of the Trigel composite.

A artrose, também chamada de osteoartrose ou osteoartrite (OA), é a terceira doença de maior incidência no Mundo. Nesse trabalho, buscou-se criar uma lesão osteocartilaginosa em joelhos de ratos Wistar machos com seis meses de vida, objetivando constituir um modelo animal para estudo da OA humana e, a partir desse modelo, avaliar biomateriais de forma isolada ou associados entre si e avaliados quanto à sua segurança biológica e potencial de reparação tecidual. Além disto, foi analisado o efeito reparador de células-tronco mesenquimais da polpa do dente de leite humano (MSC) isoladamente e em associação com o biomaterial formado por: Titânio + Poli Vinil Álcool + Ac. Hialurônico, nesse estudo denominado de TRIGEL (TRG). O Ac. Hialurônico (HA), por suas propriedades visco elásticas, o pó de Titânio (Ti), devido às suas propriedades biológicas únicas de ostoeintegração e o polímero Poli Vinil Álcool (PVA), com suas propriedades hidrofílicas, promovendo a formação do Hidrogel, os quais associados entre si formam um compósito, o TRG, que foi aplicado sobre uma lesão padrão no joelho da pata pélvica direita de ratos Wistar machos de seis meses de idade. Para a obtenção da lesão padrão, os animais foram divididos em três grupos de cincoanimais e cada grupo foi submetido a uma intervenção cirúrgica em seus joelhos direitos, utilizando três técnicas cirúrgicas diferentes, a saber: Grupo (I): Remoção cirúrgica dos meniscos medial e lateral mais perfuração do platô tibial seguido da aspiração da medula óssea através dessa perfuração por meio de seringa e agulha. Grupo (II): Remoção dos meniscos mais perfuração, sem aspiração, Grupo (III): Apenas a perfuração. Todos os animais foram autopsiados após 30 dias. Os joelhos dos quinze animais que constituíam os três grupos foram devidamente catalogados e enviados para a empresa Histotech, para a confecção das lâminas, tendo sido eleito, por análise histológica, o Grupo (I), por demonstrar menor reparo tecidual espontâneo. Em tese, o TRG teria as seguintes propriedades: Uma fonte de reparação tecidual (visco terapia) dada pelo HA e a capacidade amortecedora e carreadora de células-tronco do polímero PVA, que se hidrata, formando o hidrogel. O Ti, pela sua propriedade de osteointegração formaria um tampão sobre as áreas de matriz óssea exposta o que possibilitaria o afluxo de novos condrócitos, que também pode ocorrer pela ação das células-tronco. Livrar a superfície articular das áreas com exposição da matriz óssea é fundamental para o bloqueio das proteases que perpetuam a fisiopatologia da OA. Após tratamento estatístico dos diversos ensaios, utilizando-se os diversos biomateriais no tratamento da lesão, o TRG foi o biomaterial que apresentou o melhor resultado de força entre os grupos. No estudo histológico, foi evidenciada a presença de tecido cartilaginoso supra- lesional, o que só ocorreu nos animais dos grupos que receberam: apenas TRG, TRG associado com células-tronco e aquele que recebeu apenas MSCs. No entanto, mais estudos, com animais de maior porte e mais velhos, devem ser realizados para melhor analisar a segurança e o potencial terapêutico do compósito Trigel. / Osteoarthritis, or osteoarthritis (OA) is the third most debilitating disease in the world. In this study, we attempted to create an osteocartilaginous lesion in the knees of six months old male Wistar rats, aiming to constitute an animal model for the study of human OA and to use this model to evaluate the therapeutic potential of biomaterials, which are already well known for their biocompatibility properties in the clinical practice. The biomaterials were used in isolation or associated with each other and then evaluated for their biological safety and tissue repair capacity. Mesenchymal stem cells, obtained from human dental pulp from deciduous teeth (MSC) were evaluated alone and in association with the biomaterial formed by: Titanium + Poly Vinyl Alcohol + Ac. Hyaluronic, here called TRIGEL (TRG). Due to its visco-elastic properties, the Ti powder, due to its unique biological properties of ostointegration and the polymer PVA, with its hydrophylic properties, forming a hydrogel, were associated to form the composite named TRIGEL, (TRG), which was applied to a standard knee injury of the right hind leg of male Wistar rats. In order to elect the standard lesion, the animals were divided into three groups with five animals each and each group underwent a surgical intervention in their right knees, with three different surgical techniques being applied, namely: Group (1): Surgical removal ofmedial and lateral meniscus plus perforation of the tibial plateau, followed by aspiration of the bone marrow through this perforation using syringe and needle. Group (2): Removal of the meniscus plus perforation, without aspiration, Group (3): Drilling only. All groups were autopsied 30 days after the procedure and all groups were autopsied at 30 days post-procedure. The knees of the 15 animals that constituted the three groups were analyzed histologically and Group (1) (meniscus removed, perforated and aspirated), was elected as the standard lesion since it demonstrated less spontaneous tissue repair. TRG has the following properties: HA is used as a source of tissue repair (visco therapy) and hydration of the polymer; PVA, forms a hydrogel\", with damping action and as a stem cells carrier, whereas Ti was used due to its ósseo-integration, which would allow coating of the exposed bone matrix and this intra-osseous osteo-integration response would form an intercalating buffer. The healthy cartilage surfaces around this structural buffer would allow the reception of new chondrocytes or the action of the cells on TRG properties. Freeing the articular surface of the areas with bone matrix exposure is critical for blocking the proteases that perpetuate the pathophysiology of OA. In the various biomaterial tests in the treatment of the standard lesions, TRG was statistically shown to be the one that better mimicked the non-injured group. The histological study demonstrated the presence of a supra-lesional cartilagenous tissue, which only occurred in the groups which received: only TRG, mesenchymal stem cells associated with TRG and that which received only MSCs. However, further studies with larger and older animals should be pursued to better assess the safety and therapeutic potential of the Trigel composite.

No description available.

Denna uppsats är gjord på uppdrag av Tarbit Shipping som år 2011 konverterade sin tankbåt M/T Bit Viking från konventionell drift på tjockolja till LNG (Liquefied Natural Gas).Uppdraget som gavs var att upprätta en tränings manual till fartyget då det är ett krav från IMO (International Maritime Organization). Manualen skrevs i 3 st huvuddelar Kategori A, B och C. Kategori A är till för att manskap ombord ska få en kännedom om gasen och säkerhet runt den, Kategori B är skriven till däcksbefäl där det krävs en större kännedom om gasen och Kategori C är till för maskinbefäl. Manualen finns nu ombord på fartyget och på rederi kontoret för utbildning av nypåmönstrad personal och fortlöpande utbildning av ordinarie personal. Manualen är ett resultat på tolkning av IMO´s IGF kod (ANNEX11. RESOLUTION MSC.285(86)) där det står riktlinjer för säkerheten ombord på fartyg med maskiner som drivs på naturgas. / This paper has been produced as a result of an assignment set by Tarbit Shipping which 2011converted one of their product tanker ships from M/T Bit Viking its original heavy fuel oil toLNG (Liquefied Natural Gas). The assignment was to establish a training manual to the shipaccording to IMO´s (International Maritime Organization) IGF code. The manual is written inthree main parts Category A, Category B and Category C. Category A is directed at ratingsand cadets and focuses on gas and safety procedures, Category B is directed at deck officersand focuses on gas, whilst Category C is directed at engine officers and, similar to Category Baims at increasing knowledge levels for engineers. The manual is now located onboard theship and at the company’s office for education of new personnel and for continued educationfor personnel onboard. The manual is a result on an interpretation of IMO´s IGF code(ANNEX11. RESOLUTION MSC.285(86)), in which guidelines are laid out for vesselsfueled by natural gas.

Background: Multipotent mesenchymal stromal cells (MSC) can be recovered from a variety of tissues in the body. Yet, their functional properties were shown to vary depending on tissue origin. While MSC have emerged as a favoured cell type for tendon regenerative therapies, very little is known about the influence of the MSC source on their properties relevant to tendon regeneration. The aim of this study was to assess and compare the expression of tendon extracellular matrix proteins and tendon differentiation markers in MSC derived from different sources as well as in native tendon tissue. MSC isolated from equine bone marrow, adipose tissue, umbilical cord tissue, umbilical cord blood and tendon tissue were characterized and then subjected to mRNA analysis by real-time polymerase chain reaction. Results: MSC derived from adipose tissue displayed the highest expression of collagen 1A2, collagen 3A1 and decorin compared to MSC from all other sources and native tendon tissue (p < 0.01). Tenascin-C and scleraxis expressions were highest in MSC derived from cord blood compared to MSC derived from other sources, though both tenascin-C and scleraxis were expressed at significantly lower levels in all MSC compared to native tendon tissue (p < 0.01). Conclusions: These findings demonstrate that the MSC source impacts the cell properties relevant to tendon regeneration. Adipose derived MSC might be superior regarding their potential to positively influence tendon matrix reorganization.

BACKGROUND: In order to better understand the multifactorial nature of osteoporosis, animal models are utilized and compared to healthy controls. Female sheep are well established as a model for osteoporosis induced by ovariectomy, calcium and vitamin D low diet, application of steroids, or a combination of these treatments. Transcriptional studies can be performed by applying quantitative real time PCR (RT-qPCR). RT-qPCR estimates mRNA-levels of target genes in relation to reference genes. A chosen set of reference genes should not show variation under experimental conditions. Currently, no standard reference genes are accepted for all tissue types and experimental conditions. Studies examining reference genes for sheep are rare and only one study described stable reference in mandibular bone. However, this type of bone differs from trabecular bone where most osteoporotic fractures occur. The present study aimed at identifying a set of reference genes for relative quantification of transcriptional activity of ovine spine bone and ovine in vitro differentiated mesenchymal stromal cells (MSC) for reliable comparability. METHODS: Twelve candidate reference genes belonging to different functional classes were selected and their expression was measured from cultured ovMSCs (n = 18) and ovine bone samples (n = 16), respectively. RefFinder was used to rank the candidate genes. RESULTS: We identified B2M, GAPDH, RPL19 and YWHAZ as the best combination of reference genes for normalization of RT-qPCR results for transcriptional analyses of these ovine samples. CONCLUSION: This study demonstrates the importance of applying a set of reference genes for RT-qPCR analysis in sheep. Based on our data we recommend using four identified reference genes for relative quantification of gene expression studies in ovine bone or for in vitro experiments with osteogenically differentiated ovine MSCs.

BACKGROUND: In order to better understand the multifactorial nature of osteoporosis, animal models are utilized and compared to healthy controls. Female sheep are well established as a model for osteoporosis induced by ovariectomy, calcium and vitamin D low diet, application of steroids, or a combination of these treatments. Transcriptional studies can be performed by applying quantitative real time PCR (RT-qPCR). RT-qPCR estimates mRNA-levels of target genes in relation to reference genes. A chosen set of reference genes should not show variation under experimental conditions. Currently, no standard reference genes are accepted for all tissue types and experimental conditions. Studies examining reference genes for sheep are rare and only one study described stable reference in mandibular bone. However, this type of bone differs from trabecular bone where most osteoporotic fractures occur. The present study aimed at identifying a set of reference genes for relative quantification of transcriptional activity of ovine spine bone and ovine in vitro differentiated mesenchymal stromal cells (MSC) for reliable comparability. METHODS: Twelve candidate reference genes belonging to different functional classes were selected and their expression was measured from cultured ovMSCs (n = 18) and ovine bone samples (n = 16), respectively. RefFinder was used to rank the candidate genes. RESULTS: We identified B2M, GAPDH, RPL19 and YWHAZ as the best combination of reference genes for normalization of RT-qPCR results for transcriptional analyses of these ovine samples. CONCLUSION: This study demonstrates the importance of applying a set of reference genes for RT-qPCR analysis in sheep. Based on our data we recommend using four identified reference genes for relative quantification of gene expression studies in ovine bone or for in vitro experiments with osteogenically differentiated ovine MSCs.

This master thesis focuses on modelling and simulation of impact tests of composite panels. Simulations and analysis were made by using Finite Element Method in software MSC Patran and Dytran. The first part of the thesis deals with describing the properties of composite panels during impact testing and other cases of impacts on composite structures. Next part deals with the used models and results from Dytran. These results are compared with experimental data from real low-velocity impact tests. Part of the thesis is devoted to impact on panels with the stacking sequences which is supposed to be used for design of air duct for airplane Aero L-39NG.

Stimulating lymphocytes with Ifn-γ, anti-CD3, and interleukin-2 promotes the proliferation of a cell population coexpressing T-lymphocyte surface antigens such as CD3, CD8a, and CD25 as well as natural killer cell markers such as NK1.1, CD49, and CD69. These cells, referred to as cytokine-induced killer cells (CIKs), display cytotoxic activity against tumour cells, even without prior antigen presentation, and offer a new cell-based approach to the treatment of malignant diseases. Because CIKs are limited in vivo, strategies to optimize in vitro culture yield are required. In the last 10 years, mesenchymal stem cells (MSCs) have gathered considerable attention. Aside from their uses in tissue engineering and as support in haematopoietic stem cell transplantations, MSCs show notable immunomodulatory characteristics, providing further possibilities for therapeutic applications. In this study, we investigated the influence of murine MSCs on proliferation, phenotype, vitality, and cytotoxicity of murine CIKs in a coculture system. We found that CIKs in coculture proliferated within 7 days, with an average growth factor of 18.84, whereas controls grew with an average factor of 3.7 in the same period. Furthermore, higher vitality was noted in cocultured CIKs than in controls. Cell phenotype was unaffected by coculture with MSCs and, notably, coculture did not impact cytotoxicity against the tumour cells analysed. The findings suggest that cell–cell contact is primarily responsible for these effects. Humoral interactions play only a minor role. Furthermore, no phenotypical MSCs were detected after coculture for 4 h, suggesting the occurrence of immune reactions between CIKs and MSCs. Further investigations with DiD-labelled MSCs revealed that the observed disappearance of MSCs appears not to be due to differentiation processes.:Inhaltsverzeichnis I Abbildungsverzeichnis III Tabellenverzeichnis IV Bibliographische Beschreibung V Abkürzungsverzeichnis VII 1 Einleitung 1 1.1 CIK-Zellen (CIK) 3 1.1.1 Merkmale von CIK-Zellen 3 1.1.2 Wirkungsmechanismen von CIK-Zellen 3 1.1.3 Studienlage 4 1.1.4 Bisherige Ansätze zur Verbesserung der Kultivierungsbedingungen 6 1.2 Mesenchymale Stammzellen (MSC) 7 1.2.1 Allgemein 7 1.2.2 Differenzierung von MSC 7 1.2.3 Heterogenität und Einflussfaktoren der MSC - Identitätsproblematik 8 1.2.4 Charakterisierung von MSC 9 1.2.5 Therapeutische Einsatzmöglichkeiten von MSC 11 2 Zielformulierung 15 3 Material und Methoden 16 3.1 Tiere 16 3.2 Materialien 17 3.2.1 Materialien für Zellkultur 17 3.2.2 Materialien für FACS-Analyse 18 3.2.3 Materialien für Zytotoxizitätsassay 19 3.2.4 Materialien für CFU-F-Assay 20 3.3 Methoden 21 3.3.1 Statistische Auswertung 21 3.3.2 Zellkultur 22 3.3.3 FACS (Fluorescence Activated Cell Sorting) 26 3.3.4 Markierung der MSC mit DiD 28 3.3.5 Zytotoxizitätsassay (LDH-Freisetzungsassay) 29 3.3.6 CFU-F-Assay 32 4 Ergebnisse 34 4.1 Beeinflussung der Wachstumskurve 34 4.1.1 Der Wachstumskurvenverlauf von CIK-Zellen (Kontrollen) 34 4.1.2 Der Wachstumskurvenverlauf von CIK-Zellen in der Kokultur mit MSC 35 4.1.3 Der Wachstumskurvenverlauf in MSC-konditioniertem Medium 37 4.1.4 Der Wachstumskurvenverlauf bei Restimulierung an Tag 14 38 4.2 Beeinflussung des Oberflächenphänotyps 40 4.2.1 Der Oberflächenphänotyp von CIK-Zellen 40 4.2.2 Vergleich Oberflächenphänotyp Kontrollen mit kokultivierten CIK 43 4.3 Beeinflussung der Vitalität 46 4.4 Beeinflussung der Zytotoxizität 48 4.5 Identifizierung der MSC 49 4.5.1 Adhärenz an Plastikoberflächen 50 4.5.2 Fibroblastenähnliche Wachstumsmorphologie 50 4.5.3 Wachstum in Colony-Forming-Units 51 4.5.4 Der Oberflächenphänotyp von MSC 53 4.6 Schicksal der MSC in der Kokultur 54 4.6.1 Der Oberflächenphänotyp der adhärenten Zellen nach Kokultur 54 4.6.2 Kokultur mit DiD gelabelten MSC 57 5 Diskussion 59 5.1 Beeinflussung der Wachstumskurve 60 5.1.1 Mechanismen der Beeinflussung des Wachstumskurvenverlaufs 60 5.1.2 Fehlerbetrachtung 68 5.2 Identifizierung der CIK sowie Beeinflussung von Phänotyp und Vitalität 69 5.3 Beeinflussung der Zytotoxizität 70 5.3.1 Vergleich Zytotoxizität Kontrollen mit Kokulturen 70 5.3.2 Fehlerbetrachtung 71 5.4 Identifizierung der MSC 72 6 Schlussfolgerung 75 7 Ausblick 77 8 Zusammenfassung 79 Literaturverzeichnis 83 Danksagung I