Angeborene oder erworbene Knochendefekte können infolge ihrer Häufigkeit, ihrer oft mangelhaften spontanen Regenerationsfähigkeit sowie ihrer in der Regel langen Heilungsdauer ein erhebliches medizinisches, soziales und ökonomisches Problem darstellen. Zur Lösung dieses Problems stehen standardisierte und seit langen praktizierte Möglichkeiten wie die Osteosynthese oder die Defektauffüllung mit biologischen Knochenersatzmaterialien zur Verfügung. Auch synthetische Knochenersatzmaterialien, zum Teil in Kombinationen mit regenerativmedizinischen Prinzipien, kommen immer häufiger zum Einsatz wenn aufgrund eines großen Substanzverlustes des Knochens Defekte aufgefüllt werden müssen. Ziel dieser Studie am Tiermodell Schwein war es, die Knochenheilung calvärer Knochendefekte kritischer Größe unter Einsatz zweier verschiedener synthetischer Knochenersatzmaterialien, auf ß-Tricalciumphosphat- (β-TCP; Syntricer®, MedArtis Medizinprodukte und Forschung AG, München, Deutschland) bzw. Hydroxyapatit- Basis (HA; Ostim®, Heraeus-Kulzer, Hanau, Deutschland), angewandt sowohl in reiner Form als auch in Kombination mit autologen Stammzellen bzw. autologem Knochenmark, zu optimieren. Zur Untersuchung standen 16 klinisch gesunde weibliche Schweine der Deutschen Landrasse zur Verfügung. Alle Tiere waren zu Versuchsbeginn etwa 6 Monate alt und das durchschnittliche Lebendgewicht betrug zwischen 50 und 60 kg. Sowohl die β-TCP- als auch die HA-Gruppe umfasste 8 Schweine. Diese wurden wiederum in eine Kurz- und eine Langzeitgruppe zu je 4 Schweinen unterteilt, deren Beobachtungszeitraum 6 bzw. 16 Wochen betrug. Je Schwein wurden vier standardisierte Bohrlochdefekte kritischer Größe am Os frontale gesetzt. Ein Defekt wurde leer gelassen und diente als Kontrolldefekt für die physiologische Knochenheilung. Die drei anderen Defekte wurden einmal mit reinem und die anderen beiden mit biotechnologisch modifiziertem Knochenersatzmaterial aufgefüllt. Die biotechnologische Modifikation der Basissubstanzen erfolgte für einen Defekt durch die Anreicherung mit aus dem Knochenmark entnommenen und kultivierten autologen Stammzellen und für den vierten Defekt mit intraoperativ frisch punktiertem autologem Knochenmark. Die Ergebnisse der Knochenheilung wurden mit Hilfe einer computertomographischen Verlaufskontrolle intra vitam, sowie durch eine mikrotomographische und histologische Untersuchung nach der Euthanasie, untersucht. Sowohl Tier- als auch Versuchsmodell erwiesen sich als geeignet zur Untersuchung der Knochenregeneration mit Knochenersatzmaterialien. Die Knochenregeneration mit Ersatzmaterial führte in beiden Gruppen nach 6 Wochen im Vergleich mit der physiologischen Heilung zu besseren Ergebnissen, wobei sich das HA, wenn auch nicht signifikant, dem β-TCP überlegen zeigte. Die mikrotomographische Untersuchung zeigte aufgrund der höheren Detailerkennbarkeit im Vergleich zum CT einen größeren Unterschied zwischen den beiden Gruppen. So liegen die Mittelwerte im CT für die mit HA und mit dessen biotechnologischen Modifikationen gefüllten Defekte im Durchschnitt bei 11,2 ( ) und in der β-TCP-Gruppe bei 10,3 ( ) während sie für die mikrotomographische Untersuchung im Durchschnitt mit 9,4 ( ) für die mit HA und 5,7 ( ) für die mit β-TCP gefüllten Defekte bewertet wurden. Die histologische Bewertung zeigt den Unterschied zwischen beiden Gruppen bezüglich der Knochenregeneration am deutlichsten. So zeigte sich in der β-TCP-Gruppe ein sehr variabler Anteil an neugebildetem Knochengewebe, während in der HA-Gruppe immer mindestens 75% neugebildetes Knochengewebe nachgewiesen werden konnte. Der Zusatz von frischem Knochenmarkpunktat oder Stammzellen zu den Knochenersatzmaterialien hatte keinen erkennbaren Einfluss auf die Regeneration des Knochens. Synthetische resorbierbare Knochenersatzmaterialien, sowohl auf β-TCP- als auch auf HA-Basis, können die Knochenheilung positiv beeinflussen, und führen damit zu einer schnelleren Knochenregeneration als bei der physiologischen Knochenheilung. Im vorliegenden Modell führt die Kombination mit biotechnologischen Modifikationen wie autologen Stammzellen oder frisch punktiertem Knochenmark nicht zu einer zusätzlichen signifikanten Verbesserung der Knochenregeneration. Das pastöse nanopartikuläre Hydroxyapatit erscheint aufgrund besserer Handhabung, schnellerer Resorption sowie besserer Knochenheilung als das überlegene Material. / Inherited or acquired bone defects can, due to their frequency, their low spontaneous regenerative potential, and their generally long healing trajectories, present a significant medical, social and economical problem. Currently available solutions include standardized and well-established methods such as osteosynthesis or bone grafting using biological bone substitutes. In addition, synthetic bone substitutes, sometimes in combination with regenerative medicine, are increasingly used in cases when large bone defects require significant tissue replacement. The goal of this study was to optimize the healing of calvarial bone defects in pigs through the use of two distinct synthetic bone substitutes, -Tricalciumphosphate (β-TCP; Syntricer®, MedArtis Medizinprodukte und Forschung AG, Munich, Germany) and Hydroxyapatite (HA; Ostim®, Heraeus-Kulzer, Hanau, Germany), applied either in their pure form or in combination with either autologous stem cells or autologous bone marrow. Subjects for this study were 16 clinically healthy female domestic pigs (Deutsche Landrasse). All animals were approximately 6 months of age at the beginning of the study, with live weights between 50 and 60 kg. The animals were split into two groups of 8 for the separate study of HA and β-TCP. Each of these groups was further divided into two groups of 4 pigs, for studies of short and long duration (6 and 16 weeks, respectively). Four standardized drill holes of critical size were made in the Os frontale of each pig. One hole was left untreated as a control of physiological bone healing. Among the remaining three holes, one was filled with pure bone substitute (HA or β-TCP), and the other two were filled with biotechnologically modified bone substitute. This modification of the pure substances consisted for one drill hole of enrichment with autologous stem cells, cultured from bone marrow and, for the final hole, with intraoperative freshly extracted bone marrow. The bone healing results were measured intra vitam by computed tomography (CT), and after euthanasia by microtomography and histology. Both the model animal and experimental design proved useful for this study of bone healing using bone substitutes. Bone regeneration with bone substitutes in both experimental groups was enhanced after 6 weeks when compared with the untreated physiologically healed defects, where HA was superior to β-TCP (though not significantly). Owing to a higher resolution, microtomographic analysis revealed a greater difference between the two study groups than did CT. Thus, the mean values as measured by CT for defects filled with HA and biotechnologically modified HA are 11.2, and for 10.3 for the β-TCP group, while they are 9.4 and 5.7, respectively, as measured by microtomography. The histological assessment revealed the greatest difference in bone healing between the two experimental groups. Here, the β-TCP group displayed highly variable amounts of newly formed bone tissue, whereas each subject in the HA group displayed a minimum of 75% newly formed bone tissue. The addition of freshly extracted bone marrow or stem cells to the bone substitutes had no detectable effect on bone regeneration. Synthetic resorptable bone substitutes, on a basis of both β-TCP and HA, can positively influence bone healing, and thereby lead to a faster regeneration of bone tissue than the physiological process. In the present study, biotechnological modification of bone substitutes with autologous stem cells or bone marrow did not further enhance bone regeneration. Given its easy handling, faster resorption, and better bone healing, the nanoparticulate Hydroxyapatite paste appears to be the superior material.

Knochenmarks-Stromazellen sind in den letzten Jahren in den Fokus der Forschung gerückt. Es konnte gezeigt werden, dass sie durch Bereitstellung von Überlebenssignalen essenziell für die Erhaltung hämatopoetischer Nischen sind. Stromales Interleukin-7 (IL-7) konnte dabei für T Zellen als Überlebenssignal identifiziert werden. Gemeinsam ist allen Stromazellen die Expression des Oberflächenmarkers CD106/VCAM-1. Ein effizientes Protokoll erlaubte die qualitative wie quantitative Isolation von Stromazellen aus dem murinen Knochenmark mit anschließender ex vivo Microarray-Analyse. Die auf diese Weise ermittelten Kandidaten-Marker wurden auf Proteinebene via Histologie und (Hochdurchsatz-) Durchflusszytometrie validier. Dazu gehören z.B. die Marker CD1d, gas6 oder ANXA2R. CD1d wurde als guter Interimsmarker für VCAM-1+PECAM-1- Stromazellen identifiziert, wohingegen die IL-7-Produzenten in der Population von CD200int/BP 1+/CD73+/CD105- angereichert sind. Gleiches gilt für den Transkriptionsfaktor Prrx1. CD55, BP-1 and Cadherin-11 zeigten eine Expressionsmuster in Abhängigkeit des verwendeten IL-7-Reportermaus-Haplotyps. Für BP-1 und Cadherin 11 konnte die Abwesenheit von reifen Lymphozyten als Ursache des Feedbacks ausgeschlossen werden. Die Haplotypen der Reportermaus legten auch eine monoallele Expression des IL-7 nahe. Die Ergebnisse dieser Arbeit zeigen VCAM-1+ (IL-7+/-) Stromazellen als heterogene Population, wenn es nach der Vielzahl der möglichen exprimierten Marker geht. Zwischen vielen dieser Marker gibt es aber wiederum auf Zelloberflächenebene einen großen Überlapp. Die funktionelle Relevanz dieser Oberflächenmarker-Diversität wird in weiteren Arbeiten zu klären sein, gibt aber den Stromazellen ein breites Repertoire vor, um Interaktionen mit Lymphozyten zu initiieren, modulieren und inhibieren. Abschließend ist zu erwarten, dass diese Erkenntnisse in die klinische Behandlung der Stroma-Nischen in Autoimmun-Fragestellungen einfließen. / Bone marrow stromal cells receive increasing amounts of attention lately. They have been shown to support survival of hematopoietic stem cells as well as memory lymphocytes which is of great importance when targeting the perseverance of autoimmune diseases. CD4+ memory T lymphocytes reside in the proximity of VCAM-1 expressing stromal cells which provide them with survival signals such as Interleukin-7. Herein, a protocol was developed to quantitatively obtain VCAM-1+ and VCAM-1+ IL-7+/- stromal cells via enzymatic/mechanic digestion and cytoskeleton-inhibition. Ex vivo gene expression analysis was performed from sorted, pure cells with good recovery. Candidate genes/markers were validated in (high-throughput) flow cytometry and histological analysis including subsequent semi-automated colocalization was performed. CD1d was found to be good surrogate marker for VCAM-1+PECAM-1- non-endothelial stroma while the population of CD200int/BP-1+/CD73+/CD105- stromal cells is greatly enriched in IL-7 producers which was equally true for the stromal transcription factor Prrx1. CD55, BP-1 and Cadherin-11 were found to be differentially expressed in differing IL-7 reporter mice haplotypes. The reporter mice haplotypes revealed monoallelic expression features of IL-7. All methodologies suggest that VCAM-1+ as well as IL-7+/- stromal cells are heterogeneous by marker expression yet don’t cluster extensively in flow cytometry co-stains. The functional relevance of the marker diversity described in this thesis remains to be tested but insinuates a broad repertoire for bone marrow stroma cells for new interaction pathways with lymphocyte subsets. Ultimately, this knowledge will hopefully feedback to clinical questions of autoimmunity for targeted treatment of stromal niches.

Cette thèse porte sur le développement d’une nouvelle technique de modélisation des problèmes IFS utilisant les méthodes particulaires. Ce travail s’inscrit dans la continuité des travaux de recherche de l’équipe biomécanique du LAMIH, concernant la compréhension du comportement de l’os humain dans son environnement de moelle osseuse. La méthode SPH a été utilisée pour la modélisation des travées osseuses, supposées dans une première approche comme des solides élastiques. La méthode LB a été développée pour la modélisation des écoulements de moelle considérée comme un fluide visqueux incompressible. L’efficacité et la performance de ces deux méthodes ont été démontrées grâce aux benchmarks académiques évalués et les résultats comparés à ceux de la littérature ou ceux obtenus par des logiciels commerciaux. A l’issue d’une revue de l’état de l’art des techniques de couplage fluide-structure, une approche partitionnée en temps a été choisie, permettant d’utiliser deux codes distincts basés sur des algorithmes de résolution de type dynamique explicite. La discrétisation spatiale est faite par une technique spécifique basée sur les domaines fictifs, cette technique est très efficace car elle ne nécessite pas de rediscrétisation des domaines. L’approche de couplage développée a été appliquée à des benchmarks académiques ainsi qu’à une application en biomécanique, ayant permis d’aboutir à des résultats numériques satisfaisants. Plusieurs pistes d’amélioration sont maintenant nécessaires afin d’aller vers des modélisations plus biofidèles telles que la prise en compte du contact et de l’endommagement. / The objective of this thesis is the development of a new technique for the FSI problems modelling using particulars methods. This work is in the continuity of the LAMIH biomechanics team research works, regarding the comprehension of behavior of bone in its environment of marrow. The SPH method was used for the trabeculae modelling, supposed in a first attempt as an elastic solid. The LB method was developed for the marrow flow modelling considered as a viscous incompressible liquid. The efficacy and performance of these two methods were demonstrated using academics benchmarks which were evaluated and the results were compared of those of literature and of those obtained from commercials softwares. Following a bibliographic review of FSI coupling techniques, a partitioned approach in time was chosen, allowing the use of two separates codes, both based on a dynamic explicit algorithm resolution scheme. The special discretization was done based on a specific technique of fictional domain, this technique is very efficient because it doesn’t require an additional domain discretization. The coupling approach developed was applied on academic benchmarks and on a biomechanical application, leading to satisfactory numerical results. Many Improvement track are now necessary to go towards more biofidelic modeling as taking into account the contact and the damage.

Background: The beneficial impact of mesenchymal stem cells (MSC) on both acute and chronic liver diseases has been confirmed, although the molecular mechanisms behind it remain elusive. We aim to identify factors secreted by undifferentiated and hepatocytic differentiated MSC in vitro in order to delineate liver repair pathways potentially targeted by MSC. Methods: Secreted factors were determined by protein arrays and related pathways identified by biomathematical analyses. Results: MSC from adipose tissue and bone marrow expressed a similar pattern of surface markers. After hepatocytic differentiation, CD54 (intercellular adhesion molecule 1, ICAM-1) increased and CD166 (activated leukocyte cell adhesion molecule, ALCAM) decreased. MSC secreted different factors before and after differentiation. These comprised cytokines involved in innate immunity and growth factors regulating liver regeneration. Pathway analysis revealed cytokine-cytokine receptor interactions, chemokine signalling pathways, the complement and coagulation cascades as well as the Januskinase-signal transducers and activators of transcription (JAK-STAT) and nucleotide-binding oligomerization domain-like receptor (NOD-like receptor) signalling pathways as relevant networks. Relationships to transforming growth factor beta(TGF-beta) and hypoxia-inducible factor 1-alpha (HIF1-alpha) signalling seemed also relevant. Conclusion: MSC secreted proteins, which differed depending on cell source and degree of differentiation. The factors might address inflammatory and growth factor pathways as well as chemo-attraction and innate immunity. Since these are prone to dysregulation in most liver diseases, MSC release hepatotropic factors, potentially supporting liver regeneration.

Mast cells contribute to the activities of innate and adaptive branches of the immune system. They participate in pro-inflammatory responses to a wide range of pathogens, such as parasites, bacteria, and other foreign agents. These beneficial properties are in contrast to the contribution of mast cells to certain pathologies, such as asthma, allergy, autoimmune disorders, anaphylaxis, and systemic mastocytosis. Thorough knowledge of mast cell biology in health and disease is critical for the development of new therapeutic approaches. However, molecular mechanisms that control mast cell development and function are still incompletely defined. Our preliminary data indicate that the transcription factor C/EBP is a key player in mast cell biology. Here, using in vitro and in vivo models, we determine how C/EBP regulates the commitment of hematopoietic progenitors towards mast cells, and modulates mast cells function. These efforts provide novel insights to the role of C/EBP in hematopoiesis, and contribute to a better understanding of the mechanisms governing mast cell biology. Key words Mast cells, C/EBP, transcription factors, bone marrow-derived mast cell cultures, mast cell development, Cebpg conditional knockout mice

Background: Axial spondyloarthritis (axSpA) is a chronic inflammatory rheumatic disease affecting primarily the spine and its adjacent structures. The disease is characterized not only by destructive joint changes but also by excessive osteoproduction, which can lead to gradual ankylosis of the spine and thus significantly reduce the mobility and quality of life. The pathogenesis of the disease is not yet fully understood, but a strong genetic background is suggested, along with dysregulation of tissue metabolism resulting from an imbalance of pro- and anti-inflammatory immune mechanisms. We are still lacking biomarker with sufficient sensitivity and specificity which could help to identify early diagnosis, to monitor subchondral damage, and to differentiate rapidly progressing patients. The aim of this work was to determine the levels of potential biomarkers of connective tissue metabolism, fat metabolism and new promising biomarkers for both disease subtypes, their relationship to disease activity and progressive structural changes. Results: We have shown increased serum/plasma levels of connective tissue metabolism biomarkers (especially matrix metalloproteinase mediated metabolites), which were able to differentiate patients with early and late forms of axSpA from healthy individuals (HC), were...

Effective prevention, or treatment, of invasive fungal infection in the neutropenic patient has hitherto been unsatisfactory because of either an inadequate anti-fungal spectrum of the agent or important toxicity. Itraconazole is effective against a broad spectrum of the opportunistic pathogens seen in Europe and North America. Prior problems with absorption, e.g. in the marrow transplant recipient, have been overcome with the introduction of an oral solution and an i.v. preparation. The deliberations of an expert meeting held in June, 1998 include recommendations on which patient requires one of these new preparations based on clinical trials, the dose and route. Important drug interactions are also detailed. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

As graft source for lymphoma or leukemia treatment, hematopoietic stem and progenitor cells (HSPCs) have been the focus of translational medicine for decades. HSPCs are defined by their self-renewing capacity and their ability to give rise to all mature blood cells. They are found anchored to a specialized microenvironment in the bone marrow (BM) called the hematopoietic niche. HSPCs can be enriched by sorting them based on the presence of the surface antigen CD34 before clinical or tissue engineering use. As these cells represent a minority in most graft sources and the amount of applicable cells is limited, ex vivo expansion-cultures were established using cytokine cocktails or small molecules. However, in vitro culture of HSPCs as suspension-cultures result in heterogeneous cell populations with undefined cellular identities. In the BM niche, HSPCs are not exclusively maintained by cytokines but also by cell-matrix adhesions mediated by integrins (ITGs). Thus, β1 and β2 ITGs were found to promote initial contact of HSPCs with mesenchymal stromal cells (MSCs) and ITGβ3 expression was shown to be a marker for long-term repopulating HSPCs in vivo. Consequently, ex vivo remodeling of the BM niche using co-cultures of HSPCs and niche cells like MSCs came into spotlight and was proven to be a promising tool for stem cell expansion. However, in clinical and research applications, direct contact of two cell populations necessitates HSPC post-culture purification. To address these problems, we established a novel culture method for remodeling the BM extra cellular stroma in vitro wherein we used decellularized extracellular matrix (ECM) scaffolds derived from immortalized mesenchymal stromal cells (SCP-1). Such scaffolds were found to be highly reproducible and served as in vitro niche for HSPCs by being more effective for the expansion of CD34+ cells, compared to classical suspension cultures. ECMs were shown to consist of multiple proteins including fibronectins, collagens, and a major niche chemokine responsible for BM homing and retention of HSPCs in vivo, namely, stromal derived factor 1 (SDF-1). SDF-1 is known to be secreted by MSCs and is anchored to matrix proteins. This reveals that ECM scaffolds produced by SCP-1 cells are a naïve reconstructed microenvironment. When CD34+ cells were seeded, only around 20% of the cells adhered to the provided ECM scaffold. These cells recognized SDF-1 via C-X-C chemokine receptor type 4 (CXCR-4), as shown by laser scanning confocal microscopy. Thus, adhesive sides as they are present in the BM niche are provided. However, CD34+ cells isolated from G-CSF mobilized peripheral blood of healthy donors were found to be heterogenous with respect to adhesion capacity. Nonetheless, it was similar to HSPC co-cultures with SCP-1 cells as feeder layer. Therefore, we separated and analyzed two cell fractions, the adherent (AT-cells) and the non- adherent supernatant (SN-cells) cells. Other signals provided by the BM extracellular stroma to HSPCs are physical cues that control HSPC fate. HSPCs sense these physical features through focal contacts and accordingly remodel their morphological and biomechanical properties. Using real-time deformability cytometry (RT-DC) to uncover biomechanical phenotypes of freshly isolated HSPCs, SN-cells, AT-cells, and classical suspension cultured HSPCs in plastic culture dishes (PCD) were analyzed. We found freshly isolated cells to be less deformable and small. AT-cells displayed actin polymerization to stress fibers, and exhibited a stiffer mechanical phenotype compared to PCD-cultured or SN-cells. This might constitute the first hint of functional adaptation. Integrins are known to establish mechanosensing focal contacts. Thus, we analyzed ITG surface expression and identified ITGαIIb, ITGαV, and ITGβ3 to be enriched on AT-cells compared to freshly isolated cells or SN-cells. Active integrins need to form heterodimers consisting of one α- and one β subunit. Interestingly, the identified ITGs exclusively interact with each other to form RGD peptide receptors. RGD is a tripeptide consisting of the amino acids arginine, glycine, and aspartic acid and was identified as an adhesion sequence within fibronectin and other extracellular proteins. Consequently, we could confirm an important role for ITGαVβ3 in HSPC- ECM interaction with respect to adhesion and migration. However, we also identified ITGβ3 expression on a subset of CD34+ cells either freshly isolated or ECM cultured cells, as a marker for erythrocyte differentiation. These findings demonstrate that, in vitro, the ECM compartment acts as a regulator of HSPC fate and portray mechanical recognition as a potent driver of differentiation. In this context, targeted modulation of ECM scaffolds could enhance cell-ECM interactions and accelerate stem cell expansion or differentiation. These modulations could also provide further insights into HSPC-niche regulation. We demonstrate that ECMs derived from osteogenic differentiated SCP-1 cells increase HSPC expansion but do not lead to increased cell adhesion. As ECM adhesion preliminary alters HSPC function, we aimed at developing ECM scaffolds with increased adhesion capacity. Using lentiviral transduction, we generated a stable knock down of fibulin-1 in SCP-1 cells. Fibulin-1 is an ECM protein known to form anti-adhesion sites with fibronectin. However, we failed to increase adherent cell numbers or enhance HSPC expansion in the fibulin-1 knock down ECMs. Taken together, SCP-1 cell-derived ECM protein scaffolds provide an in vitro niche for HSPCs capable of stem cell expansion. Integrin mediated signaling altered the biomechanical and functional properties of HSPCs and hints at suspension cultures as being inappropriate to study the physiological aspects of HSPCs. Targeted modulation of ECM scaffolds could theoretically generate suitable ex vivo environments with the capacity to gain detailed insight into HSPC regulation within their niche. This will enhance the functionality of new biomaterials and will lead to improved regenerative therapies like BM transplantation.:List of contents I List of figures IV List of tables VI Abbreviations VII 1 Introduction 1 1.1 The stem cell microenvironment 3 1.1.1 The cellular endosteal bone marrow microenvironment 6 1.1.1.1 Mesenchymal stem/stromal cells 7 1.1.1.2 Hematopoietic stem and progenitor cells 8 1.1.2 Extracellular bone marrow microenvironment 10 1.1.2.1 Extracellular matrix 11 Chemokines and Cytokines 12 Cell adhesion to ECM 13 1.2 Native ex vivo ECM scaffolds 16 2 Aim of the study 19 3 Materials and methods 21 3.1 Materials 21 3.1.1 Chemicals and reagents 21 3.1.2 Kits 23 3.1.3 Media 24 3.1.4 Antibodies 24 3.1.5 Primers, sh-RNA sequences, and vectors 25 3.1.6 Equipment 26 3.1.7 Software 27 3.2 Methods 27 3.2.1 Cell preparation and culture 27 3.2.1.1 Mesenchymal stromal cells 27 3.2.1.2 Hematopoietic stem cells 28 3.2.1.3 Single cell picked clone 1 (SCP-1) cells 28 3.2.2 Generation of surface immobilized ECM preparations 29 3.2.2.1 Surface functionalization 29 3.2.2.2 ECM preparation 29 3.2.3 Flow cytometry and fluorescent activated cell sorting 30 3.2.4 Cell cycle analyses 30 3.2.5 Proliferation analyses 31 3.2.6 Colony forming unit cell assay (CFU-GEMM) 31 3.2.7 Migration assays 31 3.2.7.1 Transwell migration 31 3.2.7.2 Live cell migration 32 3.2.8 Confocal laser scanning microscopy 32 3.2.9 Real-time deformability cytometry (RT-DC) 32 3.2.10 Molecular biological methods 33 3.2.10.1 RNA isolation, reverse transcription, and PCR 33 3.2.10.2 Lentiviral shRNA transduction 34 3.2.10.3 Western blot 35 3.2.10.4 ELISA 36 3.2.11 Statistical analysis 37 4 Results 38 4.1 Extracellular matrix scaffolds for HSPCs 38 4.1.1 ECM properties 39 4.1.2 HSPC survival in ECM and PCD cultures 40 4.1.3 HSPC expansion in ECM and PCD cultures 41 4.2 HSPC morphological and mechanical adaptation to ECM 44 4.2.1 Actin polymerization and polarization 45 4.2.2 Biomechanical phenotype 46 4.3 Bioactive SDF-1 is incorporated in ECM scaffolds 49 4.3.1 CXCR4 polarization towards ECM 50 4.4 HSPC integrin expression and migration 52 4.4.1 Integrin surface expression on HSPC subsets 52 4.4.2 Focal contact formation 53 4.4.3 Integrin activation via ECM adhesion 55 4.4.4 Clonogenicity of ECM cultured HSPCs 57 4.4.5 HSPC migration when attached to ECM scaffolds 60 4.4.5.1 Reduced migratory behavior via ITGαVβ3 inhibition 61 4.4.5.2 SDF-1 induces migration but not adhesion 64 4.5 Targeted modulation of ECM scaffolds 65 4.5.1 Fibulin-1 knock down in SCP-1 cells 66 4.5.2 HSPC support of fibulin-1 reduced ECM scaffolds 70 5 Discussion 73 5.1 SCP-1 cells as a source for ECM scaffold production 74 5.2 Cell adhesion and focal contact formation 75 5.3 HSPC multilineage potential 78 5.4 ECM scaffold modulation 79 6 Summary 83 7 Zusammenfassung 86 Bibliography 89 Danksagung 108 Anlagen 110 Erklärung zur Eröffnung des Promotionsverfahrens [Formblatt 1.2.1] 110 Erklärung zur Einhaltung rechtlicher Vorschriften [Formblatt 1.1] 110

Despite tremendous progress in medicine, injuries of the adult central neural system remain without satisfactory solution. Regenerative medicine employs tissue engineering, cellular therapies, medical devices, gene therapy, or growth factors with the aim to bridge the lesion, re-establish lost connections and enhance endogenous repair in order to restore neural function. The aim of my thesis was to evaluate therapeutic potential of two approaches, transplantation of human mesenchymal stromal cells (hMSCs) and biological scaffolds derived from extracellular matrix (ECM) for neural regeneration, particularly in models of spinal cord injury (SCI). First, hMSCs from various sources - bone marrow (BM), adipose tissue (AT) and Wharton's jelly (WJ) - were isolated and characterized in vitro. All cell types met the minimal criteria for MSC phenotype and displayed similar properties in terms of their surface marker expression, differentiation potential, migratory capacity, and secretion of cytokines and growth factors. On the other hand, the cell yield from WJ and AT was significantly higher, and MSCs isolated from these tissues proliferated better than from BM. Therapeutic effect of intrathecal application of hWJ-MSCs was then evaluated in SCI compression model in rats. The effect of low (0.5 million) and...

Introduction: Simultaneous detection of disseminated tumor cells (DTCs) and circulating tumor cells (CTCs) was shown to be associated with an especially poor prognosis and increased incidence of disease-related deaths in non-metastatic breast cancer patients. We analyzed the occurance of DTCs in bone marrow and CTCs in peripheral blood in patients with primary breast cancer, we evaluated the correlation of their presence with other prognostic markers and we investigated the changes in DTCs/CTCs number at different time points during treatment. Materials and methods: Blood of 50 patients with primary breast cancer were used for immunomagnetic separation and detection of circulating tumor cells using the commercial available system the AdnaTest Breast Cancer™ (AdnaGen GmbH, Langenhagen, Germany). Bone marrow aspirates from 50 patients were analyzed for DTCs by immunocytochemistry using the pancytokeratin antibody conjugated with FITC (Monoclonal Anti-Cytokeratin antibody F3418, Sigma Aldrich, USA). Results: DTCs were identified in 30% (15/50) and CTCs in 22% (11/50) of patients. We found that DTC positivity could point to a significantly high risk of larger primary tumor size (p- value 0.011) and significantly higher risk of lymph node involvement (p- value 0.002). For CTC positivity, no such...