Introduction: Currently, there are no specific pharmacological treatments for sepsis-induced acute respiratory distress syndrome (ARDS). And mesenchymal stem cells (MSCs) have shown reparative potential in both sepsis and ARDS. Objectives: To determine the role of MSC administration in the modulation of pulmonary host-responses to sepsis via differential regulation of regulatory microRNAs (miRNAs/miRs). Methods: MicroRNA and mRNA profiling was performed to identify differential expression. Quantitative real time polymerase chain reaction (qRT-PCR), trans-endothelial electrical resistance (TEER) measurements, and luciferase activity assay were used. Results: MicroRNA expression was examined in Human Pulmonary Microvascular Endothelial Cells (HPMECs). One miRNA – miR-193b-5p, targets occludin, a tight junction protein associated with endothelial leakage. A specific regulatory relationship between miR-193b-5p and occludin was identified. The loss in endothelial integrity was rescued when miR-193b-5p inhibitor was transfected. Conclusion: miR-193b-5p is a suppressor of occludin. Studying transcriptional changes allows identification of therapeutically relevant mediators for ARDS/ALI treatment.

Introduction: Currently, there are no specific pharmacological treatments for sepsis-induced acute respiratory distress syndrome (ARDS). And mesenchymal stem cells (MSCs) have shown reparative potential in both sepsis and ARDS. Objectives: To determine the role of MSC administration in the modulation of pulmonary host-responses to sepsis via differential regulation of regulatory microRNAs (miRNAs/miRs). Methods: MicroRNA and mRNA profiling was performed to identify differential expression. Quantitative real time polymerase chain reaction (qRT-PCR), trans-endothelial electrical resistance (TEER) measurements, and luciferase activity assay were used. Results: MicroRNA expression was examined in Human Pulmonary Microvascular Endothelial Cells (HPMECs). One miRNA – miR-193b-5p, targets occludin, a tight junction protein associated with endothelial leakage. A specific regulatory relationship between miR-193b-5p and occludin was identified. The loss in endothelial integrity was rescued when miR-193b-5p inhibitor was transfected. Conclusion: miR-193b-5p is a suppressor of occludin. Studying transcriptional changes allows identification of therapeutically relevant mediators for ARDS/ALI treatment.

The regenerative potential of adult stem cell populations within the human body bears great promises for their use in regenerative medicine. The bone marrow (BM) harbours two different types of adult stem cells, haematopoietic stem and progneitor cells (HSPCs) and multipotent mesenchymal stromal cells (MSCs), which are tightly regulated in their distinct anatomically defined niches by multiple cues such as cytokines, cell-cell contacts, the extracellular matrix (ECM) and the physical microenvironment. The ex vivo expansion of these cells for applications in regenerative therapies is of great interest and several biomaterial approaches attempt to mimic the natural BM niche and its components to control stem cell maintenance and differentiation. However, as of now the complexity of such stem cell niches is hard to recapitulate. Towards this goal, this work was focussing on the ECM environment of BM stem cells and was set out to engineer improved in vitro culture systems. MSC themselves are one of the most important cell types within the BM that secrete and construct ECM-networks and thereby shape the microenvironment of the residing cells. The potential of primary human BM-MSC to secrete ECM in vitro has been exploited to generate niche-like ECM surrogates in a robust and versatile format. Application of decellularisation regimes allowed the fabrication of complex matrices which demonstrated suprastructural, compositional and physicochemical properties compareable to those of the native BM-ECM environment. Reliable stability and reproduciblity was achieved by a dedicated procedure of maleic anhydride co-polymer-mediated covalent binding of fibronectin and subsequent anchorage of cell-secreted ECM molecules. As a result of the high reproducibility, a complete proteomic register of ECM molecules was obtained in combination with determining the complex fibrillar and soft gel-like characteristics of MSC-derived matrices. Based on the established BM niche-like substrate, the impact of extracellular matrices on MSC and HSPC ex vivo behavior has been explored. Both cell types demonstrated strong adhesion to ECM substrates and depicted a changed cellular morphology upon contact with native ECM structures compared to standard culture substrates or simple ECM protein coatings, indicating an intense interplay between the cell and the microenvironment. MSC that re-grew into their own matrices have shown advantageous proliferation and cytokine secretion levels as well as enhanced differentiation intensity (upon differentiation induction) compared to MSC that were cultured on less complex substrates. Similarly, HSPC were also instructed for enhanced expansion on MSC-derived matrices without exhaustion of stem cell-marker expressing progenitor cells. The efficiency of these matrices was related to their ability to mimic the native composite suprastructure, ligand nano-topography, molecular composition and physical properties of natural BM ECM environments. The data obtained within this thesis set the ground for a more rational design of artificial stem cell niches with defined and distinct properties, offering exciting options for the in-depth analysis and understanding of stem cell regulation by exogenous cues.

Die Nicht-alkoholische Steatohepatitis (NASH), gehörig zu der Gruppe der chronischen Lebererkrankungen als eine schwere Form der Nicht-alkoholischen Fettleber-erkrankungen (NAFLD), nimmt in ihrer Prävalenz ständig zu. Gründe dafür sind u.a. eine gesteigerte Nahrungsaufnahme sowie Veränderungen der Nahrungszusammen-setzung. Es kommt zur Ausbildung einer Steatose, die sich unter Mitwirkung verschie-dener Einflussfaktoren zur Steatohepatitis weiterentwickeln kann, wobei die Pathoge-nese noch nicht genau verstanden ist. Die Nicht-alkoholische Steatohepatitis geht oft einher mit Insulinresistenz und starkem Übergewicht. Die Folgen für die Leber sind Funktionseinschränkungen und –verlust, hervorgerufen durch eine massive Akkumula-tion von Triglyzeriden in den Hepatozyten, Entzündungsprozesse sowie einem fibro-tischen Umbau der Leber. Im fortgeschritten Stadium wird eine Lebertransplantation unausweichlich, die jedoch aufgrund des zunehmenden Mangels an Spenderorganen oft nicht möglich ist. Eine Alternative bietet die Transplantation mesenchymaler Stammzellen (MSC). MSC können in vitro in leberzellähnliche Zellen differenziert wer-den und weisen dabei essentielle hepatozytäre Eigenschaften auf, wodurch sie als möglicher Ersatz bzw. als Überbrückungstherapie bis zur Lebertransplantation in Frage kommen. Die vorliegende Arbeit beschäftigte sich mit dieser Fragestellung. Dazu wur-de ein Tiermodell der NASH mittels Methionin-Cholin-defizienter Diät (MCD-Diät) etab-liert und die Transplantation von hepatozytär differenzierten MSC durchgeführt. An-hand spezifischer zellulärer und biochemischer Marker der NASH konnte die Wirkung des Zelltransplantats auf die Empfängerleber analysiert werden. Es hat sich gezeigt, dass die MSC einen anti-inflammatorischen, anti-fibrotischen und pro-proliferativen Einfluss auf das Empfängerparenchym hatten und somit zur Verbesserung der Symptomatik der NASH beitrugen.

Dental and orthopaedic implants are currently the solutions of choice for teeth and joint replacements with success rates continually improving, but they still have undesirable failure rates in patients who are compromised by disease or age, and who in many cases are the ones most in need. The success of titanium (Ti) implants depends on their ability to osseointegrate with the surrounding bone and this, in turn, is greatly dependent on the surface characteristics of the device. Advancements in surface analysis and surface modification techniques have improved the biological performance of metallic implants by mimicking the hierarchical structure of bone associated with regular bone remodeling. In this process, damaged bone is resorbed by osteoclasts, which produce resorption lacunae containing high microroughness generated after mineral dissolution under the ruffled border, as well as superimposed nanoscale features created by the collagen fibers left at the surface. Indeed, increasing Ti surface roughness at the micro and sub-microscale level has been shown to increase osteoblast differentiation in vitro, increase bone-to-implant contact in vivo, and accelerate healing times clinically. Recently, the clinical application of surface nanomodification of implants has been evaluated. Still, most clinically-available devices remain smooth at the nanoscale and fundamental questions remain to be elucidated about the effect of nanoroughness on the initial response of osteoblast lineage cells. Another property that could be used to control osteoblast development and the process of osseointegration is the electrical surface charge of implants. The presence of endogenous electrical signals in bone has been implicated in the processes of bone remodeling and repair. The existence of these native signals has prompted the use of external electrical stimulation to enhance bone growth in cases of fractures with delayed union or nonunion, with several in vitro and in vivo reports confirming its beneficial effects on bone formation. However, the use of electrical stimulation on Ti implants to enhance osseointegration is less understood, in part because of the lack of in vitro models that truly represent the in vivo environment. In addition, an aspect that has not been thoroughly examined is the electrical implication of implant corrosion and its effect on the surrounding tissue. Implants are exposed to extreme conditions in the body such as high pH during inflammation, and cyclic loads. These circumstances may lead to corrosion events that generate large electrochemical currents and potentials, and may cause abnormal cell and tissue responses that could be partly responsible for complications such as aseptic loosening of implants. Consequently, Ti implants with tailored surface characteristics such as nanotopography and electrical polarization, could promote bone healing and osseointegration to ensure successful outcomes for patients by mimicking the biological environment of bone without the use of systemic drugs. The objective of this thesis is to understand how surface nanostructural and electrical characteristics of Ti and Ti alloy surfaces may affect osteoblast lineage cell response in vitro for normal tissue regeneration and repair. Our central hypothesis is that combined micro/nanostructured surfaces, as well as direct stimulation of Ti surfaces with fixed direct current (DC) potentials, can enhance osteoblast differentiation.

Low back pain is a common cause of disability and work inability, often associated with intervertebral disc degeneration. The current understanding of disc degeneration is limited and none of the available treatments is entirely effective. The work described herein investigates potential strategies for the biological herapeutic restoration of disc degeneration. Firstly, an in vitro study to investigate the effects of BMP-7 on human discal cellular viability was performed. Cultured cells were treated with TNF-a or H202 to induce apoptosis, resulting in the down regulation of extracellular matrix proteins, decreased cell viability, morphological changes and activation of caspase-3; however, addition of BMP-7 alone prevented the observed effects, demonstrating the ability of BMP-7 to prevent apoptosis of human disc cells in vitro. Secondly, the differentiation potential of stem cells towards disc-like cells was studied. Rodent mesenchymal stem cells (rMSCs) were cultured alone or co-cultured with rat disc tissue. Differentiation potential of rMSCs was evaluated by mRNA and protein expression, cellular function and morphological studies. The co-culture conditions led to the expression of chondrocytic markers in rMSCs, whereas rMSCs cultured alone did not express the chondrocytic markers. Cellular contact between the co-cultured rMSCs and the discal tissue were observed. This study demonstrated that rMSCs can differentiate into functional disc-like cells in a tissue influenced co-culture environment. Finally, the survival and differentiation of CD34+ or CD34?? bone marrow (hBM) cells, in an intra-discal xenogeneic transplantation rat model was investigated. Human CD34+ or CD34?? cells were isolated, fluorescent-labelled and injected into rat coccygeal discs. The survival of transplanted cells was confirmed by fluorescent positive cells as well as a human nuclear specific marker. Interestingly, CD34?? cells survived until day 42 in the injected discs, and differentiated into cells ex:pressing a chondrocytic phenotype. In contrast, CD34+ cells could not be detected by day 21. This data suggests that transplanted hBM CD34?? cells, in contrast to CD34+ cells, were able to survive and differentiate within the intervertebral disc. Together, the results of these studies can both encourage and contribute to the basis of potential biological therapies in the restoration of intervertebral disc degeneration.

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

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 143 pages. Includes Vita. Includes bibliographical references.

Articular cartilage plays a vital role in load-bearing joints, providing an almost frictionless surface to articulating bones. However, the avascular nature and low cell density of the tissue means that following injury, there is limited potential for regeneration and repair. With the ageing population, the prevalence and economic burden associated with osteoarthritis (OA) is increasing rapidly, but as of yet there are no fully effective ways to treat the condition. Research into novel therapies has therefore become a popular avenue of investigation, and human mesenchymal stem/stromal cells (hMSCs) have been highlighted as particularly promising targets. However, current, methods for inducing the chondrogenic differentiation of hMSCs, which typically employ the use of transforming growth factor beta 1 or 3 (TGFβ1/3), result in the production of hypertrophic rather than hyaline tissue, hampering translational progress. Growth differentiation factor 5 (GDF5) belongs to the TGFβ superfamily of proteins and is vital for skeletal formation, however its use in cartilage tissue engineering (TE) strategies has been somewhat neglected. Here we demonstrate that GDF5 significantly increases aggrecan gene expression (a marker of articular cartilage), without affecting collagen type X expression (a marker of chondrocyte hypertrophy), in chondrocyte pellet cultures derived from hMSCs, making it a promising target for the formation of permanent articular cartilage. The therapeutic application of growth factors is, at present, limited due to their expense, susceptibility to proteolytic degradation, and rapid clearance, leading to large quantities being required to get anywhere near the desired outcome. The highly sulfated glycosaminoglycan (GAG), heparin, is already extensively used in the clinic as an anticoagulant, and is also able to bind and potentiate the activity of a wide range of growth factors. As such, researchers are now using it to enhance stem cell expansion/ differentiation protocols, as well as to improve the delivery/ activity of growth factors in TE strategies. Here, we identify GDF5 as a novel heparin/heparan sulfate (HS)-binding protein, and show that endogenous HS proteoglycans (HSPGs) are vital for localizing GDF5 to the cell surface, but are not required for its signalling activity. Importantly, we report that clinically relevant doses of heparin (≥ 10 nM), but not equivalent concentrations of HS, inhibit GDF5’s biological activity, in both hMSC-derived chondrocyte pellet cultures, and in the skeletal cell line ATDC5. We demonstrate that these inhibitory effects are due to heparin (but not HS) inhibiting both GDF5 binding to endogenous HSPGs and GDF5-induced induction of Smad 1/5/8 signalling. This study may therefore explain the variable (and disappointing) results seen with heparin-loaded biomaterials for skeletal TE, and the adverse skeletal effects, such as osteoporosis, that have been reported in the clinic following long-term heparin treatment. Together, our results caution the use of heparin in the clinic and in TE applications, and prompt the transition to using more specific GAGs (e.g. HS derivatives or synthetics), with better-defined structures and fewer off-target effects, if optimal therapy is to be achieved. In the case of GDF5, we have used a variety of developed techniques to begin uncovering important structural and functional information regarding the HS-GDF5 interaction, which are hoped to ultimately pave the way towards achieving this aim. Although further analysis is necessary, our data indicate that relatively long HS sequences are required for binding, and that both ionic and non-ionic interactions play a role in the interaction. In addition we suggest that low- rather than high-affinity HS variants may be key to potentiating the activity of this growth factor.

Magnetic hyperthermia (MH) is an anti-cancer treatment which exploits the heat produced by tumour-targeted magnetic nanoparticles (MNPs) subjected to an alternating magnetic field (AMF). A problem limiting the clinical use of MH, however, is the inability to adequately localise the MNPs at the tumour site. A cellular approach using mesenchymal stem cells (MSCs) as carriers has been proposed as these cells are believed to home to sites of tissue injury and tumour growth, however problems with MNPs uptake and toxicity retard progress and need to be overcome. The aim of this project was to find an alternative approach in MH treatment, creating engineered human MSCs able to biosynthesise MNPs. To achieve this goal, MSCs were transfected with either, or both, M. magneticum AMB-1 mms6 and mmsF genes. M. magneticum AMB-1 is a genus of magnetotactic bacteria, containing magnetosomes, which are lipidic organelles containing single crystals of magnetite. M. magneticum-AMB1 mms6 and mmsF genes are important for final crystal morphology and are known to play a role in crystal synthesis and growth respectively. The originality of this study was in using mms6 and mmsF genes, which were codon-optimized for mammalian expression, alone or in combination, for transfection of human MSCs, which have known tumour homing capacity. The transfected MNPs-bearing MSCs, able to migrate into the tumour tissue, were subjected to AMF in MH experiments in an attempt to induce cancer cell death. mms6 and mmsF gene expression, following transfection, was investigated in the human osteosarcoma cell line MG63 by reverse transcription polymerase chain reaction (RT-PCR). The cellular ultrastructure of transfected MG63 cells was investigated by transmission electron microscopy (TEM), revealing the presence of nanoparticles. The magnetism of transfected MG63 cells was proved by superconducting quantum interference device (SQUID) and supported by in vitro MH experiments. Then, human MSCs were transfected with mms6 and mmsF genes, alone or in combination. The effect of transfection experiments and MNPs synthesis on MSCs markers of stemness, cell proliferation and differentiation ability were investigated. The MTB genes expression in human MSCs was assessed by RT-PCR and cell magnetism was confirmed by SQUID, in vitro MH experiments and by magnetic force microscopy (MFM). Then, in vitro studies of MH were undertaken to establish whether mms6 transfected MSCs expressing MNPs supported a MH effect when exposed to an AMF. Cells were initially exposed to an AMF of 565.3 kHz frequency in monolayers and in 3D arrangements and cell death/viability was assessed. Subsequently, the effect of the same AMF on 3D models of mixed populations of mms6-expressing MSCs and cancer cells was assessed. The results indicate that viability of MNPs-expressing MSCs and adjacent cancer cells is reduced following AMF exposure. In vivo studies of MH were undertaken following intracardiac injection of mms6-expressing MSCs in tumour-bearing mice (epidermoid carcinoma). The expression of mms6-expressing MSCs inside mice organs was confirmed by RT-PCR, fluorescence microscopy and immunohistochemistry. The effect of the application of an AMF of 565.3 kHz on mice tumours was studied with different techniques (tumour size and volume measurement, multiphoton microscopy, haematoxylin and eosin staining, and activated Caspase 3 expression), to understand if MNPs created inside mms6- expressing MSCs, following AMF exposure, could lead to cancer cell death. Results indicate that mice tolerate the treatment well, however no appreciable tumour reduction or necrosis was evident. Overall the results suggest that mms6 transfection alone confers the highest magnetisation to MSCs compared to mmsF alone or mms6+mmsF co-transfected, and that mms6 expression in human MSCs does not have an adverse effect on important cell functions. mms6-expressing MSCs, when exposed to an AMF, show reduced viability and enhanced cell cytotoxicity in vitro. When co-cultured with cancer cells in 3D models in vitro, mms6-expressing MSCs are able to reduce viability of adjacent cancer cells confirming the potential applicability of mms6- expressing MSCs for MH treatment. In vivo proof of concept experiments show that mms6-expressing MSCs can locate to the tumour tissue, and mms6-expressing intracardiac injected MSCs mice exposed to AMF tolerate the treatment well. However, the number of mms6-expressing MSCs able to localize to the tumour tissue in this experiment was too low to give an appreciable tumour reduction, so more experiments are needed to enhance the experimental protocol. A number of improvements are required to progress this novel technique towards clinical application. Gene transfection and MNPs production need to be optimised, the best frequency for MH needs to be established and MSCs delivery to the tumour has to be significantly increased to allow concentration of MNPs. The study has helped to increase our knowledge on the creation of magnetic human MSCs to potentially use these cells in MH cancer treatment.