Los estímulos mecánicos son uno de los factores que afectan a la diferenciación celular en el proceso de regeneración del tejido óseo, por lo tanto, en el desarrollo de andamios para ingeniería de tejidos, se pueden aplicar las cargas mecánicas con el fin de inducir la actividad de las células. Cuando se aplican cargas mecánicas, los estímulos mecánicos específicos transmitidos a las células a nivel microscópico pueden estudiarse mediante técnicas numéricas. El objetivo de esta tesis fue estudiar la mecanoregulación de la diferenciación de tejido en andamios de fosfato de calcio utilizando modelos de elementos finitos basados en micro tomografía axial computarizada.Dos muestras de materiales porosos basados en fosfato de calcio fueron utilizadas. Se desarrollaron mallas de elementos finitos congruentes, discretizando la fase sólida y los macro poros interconectados, con el fin de tener en cuenta la morfología irregular de los andamios.En primer lugar, se estudió la distribución de los estímulos mecánicos. La fase sólida y el fluido intersticial se simularon como material elástico lineal y como fluido Newtoniano, respectivamente. Se simuló una compresión del 0.5% en el sólido y un fluido con velocidades de entrada de 1, 10 y 100 µm/s en los poros. Se encontraron distribuciones de deformación similares en las paredes ambos materiales, con valores máximos de 1.6% en compresión y de 0.6% en tracción. En algunos poros, la velocidad del fluido aumentó a 100 y 1000 veces la velocidad de entrada. Este estudio mostró como estímulos mecánicos macroscópicos pueden causar distintos niveles de estímulos mecánicos microscópicos dentro los andamios, debido a la morfología.A continuación se realizó un estudio en el tiempo de la diferenciación de tejido en un andamio sometido a condiciones in vitro. La compresión y la perfusión se modelaron como en el estudio anterior. Se simularon una compresión del 0.5% y una velocidad de entrada de fluido constante de 10 µm/s o una presión de entrada de fluido constante de 3 Pa. La deformación cortante octaédrica y el esfuerzo cortante del fluido se utilizaron como estímulos mecano-regulatorios basándose en la teoría de Prendergast et al. (1997). Al aplicar velocidad constante, se predijeron fluctuaciones entre los estímulos equivalentes a la formación de tejido y a la muerte celular, debido al aumento en el esfuerzo cortante del fluido cuando el tejido comienza a llenar los poros. Sin embargo, al aplicar presión constante, se predijo estímulo equivalente a la diferenciación de tejido óseo en la mitad del volumen de los poros. Estos resultados sugieren que para permitir la diferenciación de tejido, la velocidad del fluido debe disminuirse cuando el tejido empieza a mineralizarse.Finalmente, se llevó acabo un estudio en el tiempo de la angiogénesis y de la diferenciación de tejido en un andamio bajo condiciones in vivo. La deformación cortante octaédrica y la velocidad relativa del fluido se utilizaron como estímulos mecano-regulatorios. Las fases sólida y porosa fueron tratadas como materiales poroelásticos. Se simuló la actividad individual de las células. Compresiones de 0.5 y 1% fueron simuladas. La mayoría de los vasos crecieron en los poros de la periferia del andamio y se bloquearon por las paredes. Se formaron redes capilares similares independientemente de la magnitud de deformación utilizada. Al aplicar 0.5% de compresión, estímulos correspondientes a la formación de hueso se predijeron en el 70% del volumen de los poros, sin embargo, sólo el 40% del volumen se llenó de osteoblastos debido a la falta de oxigeno. Este estudio mostró el efecto de la falta de vascularización en el centro del andamio en la diferenciación de tejido.Ese tipo de estudios, combinados con estudios in vitro, deberían contribuir a la comprensión del proceso de diferenciación de los tejidos dentro de los andamios y por lo tanto a la mejora de los métodos de diseño de andamios. / Mechanical stimuli are one of the factors that affect cell differentiation in the process of bone tissue regeneration; therefore, in the development of scaffolds for tissue engineering, mechanical loads can be applied in order to induce cell activity. The specific mechanical stimuli transmitted to cells at a microscopic level when mechanical loads are applied can be studied using numerical techniques. The objective of this thesis was to study the mechanoregulation of tissue differentiation within calcium phosphate scaffolds using micro computed tomographed based finite element models.Two samples of porous calcium phosphate based materials were used. Congruent finite element meshes, with the solid phase and the interconnected pores discretized, were developed in order to account for the scaffold irregular morphology.First, a study of the distribution of mechanical stimuli was performed. The solid phase and the fluid flow within the pores were modeled as linear elastic solid material and Newtonian fluid respectively. Compressive strains of 0.5% of total deformation applied to the solid and interstitial fluid flows with inlet velocities of 1, 10 and 100 µm/s applied to the pores were simulated. Similar strain distributions for both materials were found, with compressive and tensile strain maximal values of 1.6% and 0.6% respectively. For the fluid flow models, the fluid velocity in some of the scaffold pores increased to 100 and 1000 times the inlet velocity. This study showed how mechanical loads and fluid flow applied to the scaffolds caused different levels of mechanical stimuli within the samples according to the morphology of the materials.Next, a study of the mechanoregulation of tissue differentiation over time in a scaffold subjected to in vitro loads was performed. The solid phase and the fluid flow were modeled as in the study described above. Compressive strain of 0.5% and fluid flow with constant inlet velocity of 10 µm/s or constant inlet pressure of 3 Pa were applied. Octahedral shear strain and fluid shear stress were used as mechano-regulatory stimuli based on the theory of Prendergast et al. (1997). When a constant velocity was simulated, fluctuations between stimuli equivalent to tissue formation and cell death were predicted due to the increase in the fluid shear stress when tissue started to fill the pores. However, when constant pressure was applied, stimuli equivalent to bone formation were predicted in about half of the pore volume. These results suggest that in order to allow tissue differentiation within a scaffold, the fluid velocity should be decreased when tissue starts mineralizing.Finally, a study of the angiogenesis and the mechanoregulation of tissue differentiation over time in a scaffold subjected to in vivo conditions was performed. Octahedral shear strain and relative fluid velocity were used as mechano-regulatory stimuli. The solid and pore phases were treated as poroelastic materials. Individual cell activity was simulated within the pore domain. Compressive strains of 0.5 and 1% of total deformation were simulated. Most vessels grew in the pores at the periphery of the scaffolds and were blocked by the scaffold walls. Similar capillary networks were formed independently of the magnitude of the mechanical strain applied. When 0.5% of strain was applied, 70% of the pore volume was affected by mechano-regulatory stimuli corresponding to bone formation; however, because of the lack of oxygen, only 40% of the volume was filled with osteoblasts. This study showed the effect of the lack of vascularization in the center of the scaffold on the tissue differentiation.Such kind of studies, combined with in vitro studies, should contribute to the understanding of the process of tissue differentiation within the constructs and therefore to the improvement of scaffold design methods.

Background: Regenerative therapies based on autologous mesenchymal stem cells (MSC) as well as stem cells in general are still facing an unmet need for non-invasive sampling, availability, and scalability. The only known adult source of autologous MSCs permanently available with no pain, discomfort, or infection risk is the outer root sheath of the hair follicle (ORS). Methods: This study presents a non-invasively-based method for isolating and expanding MSCs from the ORS (MSCORS) by means of cell migration and expansion in air–liquid culture. Results: The method yielded 5 million cells of pure MSCORS cultured in 35 days, thereby superseding prior art methods of culturing MSCs from hair follicles. MSCORS features corresponded to the International Society for Cell Therapy characterization panel for MSCs: adherence to plastic, proliferation, colony forming, expression of MSC-markers, and adipo-, osteo-, and chondro-differentiation capacity. Additionally, MSCORS displayed facilitated random-oriented migration and high proliferation, pronounced marker expression, extended endothelial and smooth muscle differentiation capacity, as well as a paracrine immunomodulatory effect on monocytes. MSCORS matched or even exceeded control adipose-derived MSCs in most of the assessed qualities. Conclusions: MSCORS qualify for a variety of autologous regenerative treatments of chronic disorders and prophylactic cryopreservation for purposes of acute treatments in personalized medicine.

Einleitung<br /> Säugetiere haben zwei verschiedene Arten von Fettgewebe: das weiße Fettgewebe, welches vorwiegend zur Lipidspeicherung dient, und das braune Fettgewebe, welches sich durch seine Fähigkeit zur zitterfreien Thermogenese auszeichnet. Weiße und braune Adipozyten sind beide mesodermalen Ursprungs. Die Mechanismen, die zur Entwicklung von Vorläuferzellen in den weißen oder braunen Fettzellphenotyp führen, sind jedoch unbekannt. Durch verschiedene experimentelle Ansätze konnte gezeigt werden, daß diese Adipocyten vermutlich durch die Differenzierung zweier Typen unterschiedlicher Vorläuferzellen entstehen: weiße und braune Preadipozyten. Von dieser Hypothese ausgehend, war das Ziel dieser Studie, die Genexpression weißer und brauner Preadipozyten auf Unterschiede systematisch zu analysieren.<br /> <br /> Methoden<br /> Die zu vergleichenden Zellen wurden aus primären Zellkulturen weißer und brauner Preadipozyten des dsungarischen Zwerghamsters gewonnen. „Representational Difference Analysis“ wurde angewandt, um potentiell unterschiedlich exprimierte Gene zu isolieren. Die daraus resultierenden cDNA Fragmente von Kandidatengenen wurden mit Hilfe der Microarraytechnik untersucht. Die Expression dieser Gene wurde in braunen und weißen Fettzellen in verschiedenen Differenzierungsstadien und in braunem und weißem Fettgewebe verglichen.<br /> <br /> Ergebnisse<br /> 12 Gene, die in braunen und weißen Preadipozyten unterschiedlich exprimiert werden, konnten identifiziert werden. Drei Komplement Faktoren und eine Fettsäuren Desaturase werden in weißen Preadipozyten höher exprimiert; drei Struktur Gene (Fibronectin, Metargidin und a Actinin 4), drei Gene verbunden mit transkriptioneller Regulation (Necdin, Vigilin und das „small nuclear ribonucleoprotein polypeptide A“) sowie zwei Gene unbekannter Funktion werden in braunen Preadipozyten höher exprimiert. Mittels Clusteranalyse (oder Gruppenanalyse) wurden die gesamten Genexpressionsdaten charakterisiert. Dabei konnten die Gene in 4 typischen Expressionsmuster aufgeteilt werden: in weißen Preadipozyten höher exprimierte Gene, in braunen Preadipozyten höher exprimierte Gene, während der Differenzierung herunter regulierte Gene und während der Differenzierung hoch regulierte Gene.<br /> <br /> Schlußfolgerungen<br /> In dieser Studie konnte gezeigt werden, daß weiße und braune Preadipozyten aufgrund der Expression verschiedener Gene unterschieden werden können. Es wurden mehrere Kandidatengene zur Bestimmung weißer und brauner Preadipozyten identifiziert. Außerdem geht aus den Genexpressionsdaten hervor, daß funktionell unterschiedliche Gruppen von Genen eine wichtige Rolle bei der Differenzierung von weißen und braunen Preadipozyten spielen könnten, wie z.B. Gene des Komplementsystems und der extrazellulären Matrix. / Introduction<br /> Mammals have two types of adipose tissue: the lipid storing white adipose tissue and the brown adipose tissue characterised by its capacity for non-shivering thermogenesis. White and brown adipocytes have the same origin in mesodermal stem cells. Yet nothing is known so far about the commitment of precursor cells to the white and brown adipose lineage. Several experimental approaches indicate that they originate from the differentiation of two distinct types of precursor cells, white and brown preadipocytes. Based on this hypothesis, the aim of this study was to analyse the gene expression of white and brown preadipocytes in a systematic approach. <br /> <br /> Experimental approach<br /> The white and brown preadipocytes to compare were obtained from primary cell cultures of preadipocytes from the Djungarian dwarf hamster. Representational difference analysis was used to isolate genes potentially differentially expressed between the two cell types. The thus obtained cDNA libraries were spotted on microarrays for a large scale gene expression analysis in cultured preadipocytes and adipocytes and in tissue samples.<br /> <br /> Results<br /> 4 genes with higher expression in white preadipocytes (3 members of the complement system and a fatty acid desaturase) and 8 with higher expression in brown preadipocytes were identified. From the latter 3 coded for structural proteins (fibronectin, metargidin and a actinin 4), 3 for proteins involved in transcriptional regulation (necdin, vigilin and the small nuclear ribonucleoprotein polypeptide A) and 2 are of unknown function. Cluster analysis was applied to the gene expression data in order to characterise them and led to the identification of four major typical expression profiles: genes up-regulated during differentiation, genes down-regulated during differentiation, genes higher expressed in white preadipocytes and genes higher expressed in brown preadipocytes.<br /> <br /> Conclusion<br /> This study shows that white and brown preadipocytes can be distinguished by different expression levels of several genes. These results draw attention to interesting candidate genes for the determination of white and brown preadipocytes (necdin, vigilin and others) and furthermore indicate that potential importance of several functional groups in the differentiation of white and brown preadipocytes, mainly the complement system and extracellular matrix.

Le système musculo-squelettique se compose des muscles, du squelette et du tissu conjonctif qui comprend, entre autres, les tendons et le tissu conjonctif musculaire. Le tissu conjonctif musculaire contribue à l'élasticité et à la rigidité des muscles, alors que les tendons transmettent les forces musculaires à l'os nécessaires aux mouvements du corps. Contrairement au muscle et au squelette, la mise en place et la formation du tissu conjonctif restent à ce jour peu étudiées. Afin d'identifier les mécanismes moléculaires sous-jacents à la formation du tissu conjonctif au cours du développement du membre, cinq facteurs de transcription à doigt de zinc ont été examinés : OSR1, OSR2, EGR1, KLF2 et KLF4. Ces facteurs de transcription sont exprimés dans différents sous-compartiments du système musculo-squelettique et leur surexpression influence la différentiation des cellules mésenchymateuses du membre. Afin d'élucider leurs rôles au niveau de la régulation génique, plusieurs stratégies à haut-débit (RNA-seq, ChIP-seq) ont été mises en place. Ces stratégies ont permis : (i) d'identifier que les facteurs de transcription partagent des fonctions régulatrices communes liées à la transduction du signal, à la communication cellulaire et à l'adhésion cellulaire ; (ii) de révéler que les gènes différentiellement exprimés étaient enrichis pour des signatures d'activation et de répression chromatiniennes, suggérant qu'ils sont dynamiquement régulés ; (iii) de distinguer les gènes cibles directs des cibles indirectes. Ces résultats fournissent ainsi une base pour des travaux futurs visant à mieux comprendre l'inter-connectivité entre les différents composants de l'appareil locomoteur. / The musculoskeletal system is composed of muscles, skeletal elements and connective tissues such as tendon and muscle connective tissue. Muscle connective tissue contributes to the elasticity and rigidity of muscles, while tendons transmit forces generated by muscles to the bone to allow body motion. In contrast to muscle and skeleton, connective tissue patterning and formation remain poorly investigated. In order to identify molecular mechanisms underlying connective tissue formation during limb development, five zinc-finger transcription factors were investigated: OSR1, OSR2, EGR1, KLF2 and KLF4. These transcription factors are expressed in distinct subcompartments of the musculoskeletal system and influence the differentiation of limb mesenchymal cells upon overexpression. To further investigate their roles at the molecular level, several genome-wide strategies (RNA-seq, ChIP-seq) were employed. These strategies enabled: (i) to identify that the transcription factors share common regulatory functions and positively regulate biological processes related to signal transduction, cell communication and biological adhesion; (ii) to reveal that the differentially expressed genes were enriched for both active and repressive chromatin signatures at their promoters, suggesting that they are dynamically regulated; (iii) to distinguish between indirect and direct target genes. Altogether, these results provide a framework for future investigations to better understand the interconnectivity between components of the musculoskeletal system.

Im Rahmen der Biomechanik werden der Einfluss mechanischer Bedingungen auf die Heilung biologische Gewebe, wie zum Beispiel Knochen und Knorpeln untersucht. Die vorliegende Arbeit bestimmte zum Einen am Beispiel des Humerus das mechanische Verhalten von intakten und frakturierten Knochen mit verschiedener Knochenqualität (Osteoporose versus gesunden Knochen) unter verschiedenen physiologischen Belastungen. Dazu wurde ein Finite Elemente Modell des entsprechenden Knochens erstellt. Die Knochenqualität erwies sich für die Heilung als wichtigerer Parameter, als die jeweilige physiologische Belastung. Künftige Therapien der Osteoporose sollten daher die jeweils individuelle Dichteverteilung des entsprechenden Knochens explizit berücksichtigen. Zum zweiten wurde ein biphasisches, linear-elastisches Gewebedifferenzierungsmodell entwickelt, mit dem durch iterative Berechnung der Elastizität die Heilung eines osteochondralen Defektes verfolgt werden konnte. Damit konnten die Steifigkeiten und die Orte im und um den ursprünglichen Defekt, an denen sich während der osteochondralen Heilung die verschiedenen Gewebearten neu bilden, quantitativ und qualitativ (Vergleich mit Tierexperimentation) ermittelt werden. Der Erfolg dieses Modells erlaubte die Antwort auf verschiedene Fragestellungen: Einfluß der Defekt- und Gelenkgeometrie auf die Häufigkeit des Auftretens osteochondraler Defekte und ihre Heilungschancen, sowie die Wahl der Steifigkeit eines optimalen Biomaterials zur Defektausfüllung. Osteochondrale Defekte scheinen in konkaver Geometrie etwas besser zu heilen, weil dort mehr hyaliner Knorpel gebildet wird. Grafts mit derselben Steifigkeit des ursprünglichen Knochens bilden kalzifizierenden Knorpel um mehr hyaliner Knorpel am Ende des Heilungsprozesses und sind daher weicheren Biomaterialien vorzuziehen. / In biomecanics the influence of mechanical conditions on healing of biological tissues as bones or soft tissues are analysed. In the frame of this work the mechanical behavior of intact and fractured bones with different bone qualities (osteoporotic versus normal) has been examined in a proximal humerus. Therefore a finite element model of the bone was constructed. It was found that the bone quality has a stronger impact on healing than the actual physiological loading condition does. Hence, for a future therapy of osteoporosis the precise density distribution of each individual bone must be considered. In a second step a biphasic, linear-elastic model for tissue differentiation was developed, where osteochondral healing was simulated by iterative calculation of the elastic modulus of Young within the joint region. By using this model it was possible to predict in which order in all regions of the joint the osteochondral healing took place. The stiffnesses of the newly differentiated tissues agreed well to the derived quantities of animal experimentation. Hence, this tissue differentiation model could be used to analyse some questions concerning the geometry and healing success of osteochondral defects. In concave geometry more hyaline cartilage was formed, which has better mechanical properties than fibrous one. Moreover, the stiffness of an optimal biomaterial could be determined: grafts with the same stiffness as the original bone will lead to the formation of calcified bone and more hyaline cartilage, which is favourable compared to a less stiffer biomaterial.