Indiana University-Purdue University Indianapolis (IUPUI) / Bone is a dynamic tissue that is constantly remodeling to repair damage and strengthen regions exposed to loads during everyday activities. However, certain conditions, including long-term unloading of the skeleton, hormonal imbalances, and aging can disrupt the normal bone remodeling cycle and lead to low bone mass and osteoporosis, increasing risk of fracture. While numerous treatments for low bone mass have been devised, dynamic mechanical loading modalities, such as axial loading of long bones and lateral loading of joints, have recently been examined as potential methods of stimulating bone formation. The effectiveness of mechanical loading in strengthening bone is dependent both on the structural and geometric characteristics of the bone and the properties of the applied load. For instance, curvature in the structure of a bone causes bending and increased strain in response to an axial load, which may contribute to increased bone formation. In addition, frequency of the applied load has been determined to impact the degree of new bone formation; however, the mechanism behind this relationship remains unknown. In this thesis, the application of mechanical loading to treat osteoporotic conditions is examined and two questions are addressed: What role does the structural geometry of bone play in the mechanical damping of forces applied during loading? Does mechanical resonance enhance geometric effects, leading to localized areas of elevated bone formation dependent on loading frequency? Curvature in the structure of bone was hypothesized to enhance its damping ability and lead to increased bone formation through bending. In addition, loading at frequencies near the resonant frequencies of bone was predicted to cause increased bone formation, specifically in areas that experienced high principal strains due to localized displacements during resonant vibration. To test the hypothesis, mechanical loading experiments and simulations using finite element (FE) analysis were conducted to characterize the dynamic properties of bone. Results demonstrate that while surrounding joints contribute to the greatest portion of the damping capacity of the lower limb, bone absorbs a significant amount of energy through curvature-driven bending. In addition, results show that enhanced mechanical responses at loading frequencies near the resonant frequencies of bone may lead to increased bone formation in areas that experience the greatest principal strain during vibration. These findings demonstrate the potential therapeutic effects of mechanical loading in preventing costly osteoporotic fractures, and explore characteristics of bone that may lead to optimization of mechanical loading techniques. Further investigation of biomechanical properties of bone may lead to the prescribing of personalized mechanical loading treatments to treat osteoporotic diseases.

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In dieser Doktorarbeit wird die Hypothese geprüft, ob Osteozyten einen direkten Einfluss auf die Knocheneigenschaften in ihrer unmittelbaren Umgebung haben. Der zentrale Experimentieransatz ist dabei die Korrelation der Organisation des Osteozytennetzwerks mit den Mineraleigenschaften des Knochens auf der Submikrometerebene. Es wird gezeigt, dass bereits die anfängliche Ausrichtung der Osteoblasten entscheidend für die Synthese von hoch ausgerichtetem Knochenmaterial ist. Die dabei entstehenden Osteozytennetzwerke sind so organisiert, dass die Osteozyten und ihre Zellfortsätze jeweils einen möglichst kleinen Abstand zum Knochenmineral haben. Deshalb wird vermutet, dass genau diese Netzwerkorganisation mitentscheidend ist, wie gut die Zellen das Mineral in ih-rer Umgebung beeinflussen können. Messungen der Knochenmineraleigenschaften auf Submikrometerebene mit Röntgenkleinwinkelstreuung bestätigen diese Vermutung. Dabei wird deutlich, dass Knochenmaterial in der Nähe der Osteozyten durch andere Mineraleigenschaften geprägt ist. Um zu klären, wie Osteozyten Mineral in ihrer direkten Umgebung verändern können, werden Mechanismen der passiven Mineralherauslösung aus der mineralisierten Oberfläche des Osteozytennetzwerks untersucht. Es wird gezeigt, dass kalziumarme ionische Lösungen unter physiologischen Bedingungen große Mengen von Kalzium-Ionen aus dem Knochen lösen und diese dann durch die Osteozytennetzwerkstrukturen diffundieren können. Zum Abschluss wurde medullärer Knochen von Hühnern als ein Modellsystem für rasanten Knochenumbau untersucht. Dieser spezielle Knochentyp dient den Hennen als labiles Kalziumreservoir und ermöglicht dadurch die tägliche Eierschalenproduktion. Experimente am medullären Knochen-material zeigen insbesondere die Bedeutung von weniger stabilen Mineralstrukturen die benötigt werden um den Knochen an den schnellen, sich wiederholenden Knochenauf- sowie Abbau optimal anzupassen. / This thesis aims to test the hypothesis whether osteocytes have a direct influence on bone material properties in their vicinity. In this regard, the concomitant ana-lysis of osteocyte network organization and bone ultrastructural properties on the submicron level is the central approach to answer this question. In this work, it is shown that already initial cell-cell alignment during the process of bone formation is crucial for the synthesis of highly organized bone. Furthermore it is proposed that the occurrence of highly ordered osteocyte networks visualized with confocal laser scanning microscopy (CLSM) has a strong impact on the ability of osteocytes to directly influence bone material properties. These highly organized networks are another consequence of initial cell-cell alignment and are found to be arranged such as to feature short mineral cell distances. Examination of sub-micron mineral properties with scanning small angle x-ray scattering (sSAXS) shows that bone material in the direct vicinity of osteocytes and their cell proc-esses shows different mineral properties compared to bone further away in the depth of the tissue. Moreover, mechanisms of passive mineral extraction from the mineralized surface of the osteocyte network, due to the treatment with calcium poor ionic solutions, are investigated. It is shown that this chemical process occurring under physiological conditions leads not only to the dissolution of considerable amounts of calcium, but also to efficient diffusion of these ions through the osteocyte network structures. Finally, medullary bone which is intended as a labile calcium source for daily egg shell formation in hens is used as a model system for rapid bone turnover rates. This bone type in particular indicates the importance of uniquely adapted, less stable mineral structures to fit the requirements for rapid bone resorption as well as reformation.

Les Fonctions biologiques sont le résultat de l'interaction de beaucoup de processus, avec differents objectives, complexités, niveaux d'hiérarchie, et changements de conditions que modi ent le comportement de systèmes. Nous utilisons des équations diferenciales ou dynamiques plus générales, et Stochastic Systèmes de Transition pour décrire la dynamique de changements des modèles. La composition, réconciliation et reutilisation des modèles nous permettent d'obtenir des descriptions de systèmes biologiques complètes et compatibles et leur combiner. Notre spéci cation de Systèmes Hybrides avec BioRica assures l'intégrité de modèles, et implement notre approche. Nous appliquons notre approche pour décrire in-silico deux systèmes: la dynamique de la fermentation du vin, et des décisions cellulaires associées à la formation de tissu d'os.

Both platelet-rich plasma (PRP) and vascular endothelial growth factor (VEGF) can promote regeneration. The aim of this study was to compare the effects of these two elements on bone formation and vascularization in combination with bone marrow stromal cells (BMSC) in a critical-size bone defect in rabbits. The critical-size defects of the radius were filled with: (1) a calcium-deficient hydroxyapatite (CDHA) scaffold + phVEGF165-transfected BMSC (VEGF group), (2) CDHA and PRP, or (3) CDHA, autogenous BMSC, and PRP. As controls served: (4) the CDHA scaffold alone and (5) the CDHA scaffold and autogenous BMSC. The volume of new bone was measured by means of micro-CT scans, and vascularization was assessed in histology after 16 weeks. Bone formation was higher in the PRP + CDHA, BMSC + CDHA, and PRP + BMSC + CDHA groups than in the VEGF group (p < 0.05). VEGF transfection significantly promoted vascularization of the scaffolds in contrast to BMSC and PRP (p < 0.05), but was similar to the result of the CDHA + PRP + BMSC group. The results show that VEGF-transfected BMSC as well as the combination of PRP and BMSC improve vascularization, but bone healing was better with the combination of BMSC and PRP than with VEGF-transfected BMSC. Expression of VEGF in BMSC as a single growth factor does not seem to be as effective for bone formation as expanded BMSC alone or PRP which contains a mixture of growth factors. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Endosseous implants have notably high success rates, yet a small percentage of implants still fail for unidentified reasons. Recent literature points to hyperglycemia, resulting from untreated or undiagnosed diabetes, as a possible contraindication in an otherwise apparently healthy population. To investigate the effect of surface design on peri-implant healing in the presence of hyperglycemia, STZ-treated rats were implanted with custom rectangular implants of two surface topographies: grit blasted (GB) and grit-blast with a calcium phosphate nanotopography (GB-DCD). Tensile testing was conducted at 5, 7, and 9 days post-operative. Results demonstrated hyperglycemia to delay early stages of the peri-implant healing. Contact osteogenesis was increased along the GB-DCD surface, even in an environment of uncontrolled hyperglycemia, and the GB-DCD surface outperformed the GB surface in both healthy and hyperglycemic animals, showing peri-implant bone matured more rapidly on nanotopographically complex surfaces, even in the presence of uncontrolled hyperglycemia.

Les fonctions biologiques sont le résultat de l'interaction de beaucoup de processus, avec différents objectifs, complexités, niveaux de hiérarchie, et changements de conditions que modifient le comportement de systèmes. Nous utilisons des équations différentielles ou dynamiques plus générales, et systèmes stochastiques de transition pour décrire la dynamique de changements des modèles. La composition, réconciliation et réutilisation des modèles nous permettent d'obtenir des descriptions de systèmes biologiques complètes et compatibles et leur combiner. Notre spécification de systèmes hybrides avec BioRica assure l'intégrité de modèles, et implémente notre approche. Nous appliquons notre approche pour décrire in-silico deux systèmes: la dynamique de la fermentation du vin, et des décisions cellulaires associées à la formation de tissu d'os. / The main aim of this thesis is to develop an approach that allows us to describe biological systems with theoretical sustenance and good results in practice. Biological functions are the result of the interaction of many processes, that connect different hierarchy levels going from macroscopic to microscopic level. Each process works in different way, with its own goal, complexity and hierarchy level. In addition, it is common to observe that changes in the conditions, such as nutrients or environment, modify the behavior of the systems. So, to describe the behavior of a biological system over time, it is convenient to combine different types of models: continuous models for gradual changes, discrete models for instantaneous changes, deterministic models for completely predictable behaviors, and stochastic or non- deterministic models to describe behaviors with imprecise or incomplete information. In this thesis we use the theory of Composition and Hybrid Systems as basis, and the BioRica framework as tool to model biological systems and analyze their emergent properties in silico.With respect to Hybrid Systems, we considered continuous models given by sets of differential equations or more general dynamics. We used Stochastic Transition Systems to describe the dynamics of model changes, allowing cofficient switches that control the parameters of the continuous model, and strong switches that choose different models. Composition, reconciliation and reusing of models allow us to build complete and consistent descriptions of complex biological systems by combining them. Compositions of hybrid systems are hybrid systems, and the refinement of a model forming part of a composed system results in a refinement of the composed system. To implement our approach ideas we complemented the theory of our approach with the improving of the BioRica framework. We contributed to do that giving a BioRica specification of Hybrid Systems that assures integrity of models, allowing composition, reconciliation, and reuse of models with SBML specification.We applied our approach to describe two systems: wine fermentation kinetics, and cell fate decisions leading to bone and fat formation. In the case of wine fermentation, we reused known models that describe the responses of yeasts cells to different temperatures, quantities of resources and toxins, and we reconciled these models choosing the model with best adjustment to experimental data depending on the initial conditions and fermentation variable. The resulting model can be applied to avoid process problems as stuck and sluggish fermentations. With respect to cell fate decisions the idea is very ambitious. By using accurate models to predict the bone and fat formation in response to activation of pathways such as the Wnt pathway, and changes of conditions affecting these functions such as increments in Homocysteine, one can analyze the responses to treatments for osteoporosis and other bone mass disorders. We think that here we are giving a first step to obtain in silico evaluations of medical treatments before testing them in vitro or in vivo.

Both platelet-rich plasma (PRP) and vascular endothelial growth factor (VEGF) can promote regeneration. The aim of this study was to compare the effects of these two elements on bone formation and vascularization in combination with bone marrow stromal cells (BMSC) in a critical-size bone defect in rabbits. The critical-size defects of the radius were filled with: (1) a calcium-deficient hydroxyapatite (CDHA) scaffold + phVEGF165-transfected BMSC (VEGF group), (2) CDHA and PRP, or (3) CDHA, autogenous BMSC, and PRP. As controls served: (4) the CDHA scaffold alone and (5) the CDHA scaffold and autogenous BMSC. The volume of new bone was measured by means of micro-CT scans, and vascularization was assessed in histology after 16 weeks. Bone formation was higher in the PRP + CDHA, BMSC + CDHA, and PRP + BMSC + CDHA groups than in the VEGF group (p < 0.05). VEGF transfection significantly promoted vascularization of the scaffolds in contrast to BMSC and PRP (p < 0.05), but was similar to the result of the CDHA + PRP + BMSC group. The results show that VEGF-transfected BMSC as well as the combination of PRP and BMSC improve vascularization, but bone healing was better with the combination of BMSC and PRP than with VEGF-transfected BMSC. Expression of VEGF in BMSC as a single growth factor does not seem to be as effective for bone formation as expanded BMSC alone or PRP which contains a mixture of growth factors. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Indiana University-Purdue University Indianapolis (IUPUI) / Signal Transducer and Activator of Transcription 3 (Stat3) is a transcription factor expressed in bone and joint cells that include osteoblasts, osteocytes, osteoclasts, and chondrocytes. Stat3 is activated by a variety of cytokines and growth factors, including IL-6/gp130 family cytokines. These cytokines not only regulate the differentiation of osteoblasts and osteoclasts, but also regulate proliferation of chondrocytes through Stat3 activation. In 2007, mutations of Stat3 have been confirmed to cause a rare human immunodeficiency disease – Job syndrome which presents skeletal abnormalities like: reduced bone density (osteopenia), scoliosis, hyperextensibility of joints, and recurrent pathological bone fractures. Changes in the Stat3 gene alter the structure and function of the Stat3 proteins, impairing its ability to control the activity of other genes. However, little is known about the effects of Stat3 mutations on bone cells and tissues. To investigate the in vivo physiological role of Stat3 in bone homeostasis, osteoblast/osteocyte-specific Stat3 knockout (KO) mice were generated via the Cre-LoxP recombination system. The osteoblast/osteocyte-specific Stat3 KO mice showed bone abnormalities and an osteoporotic phenotype because of a reduced bone formation rate. Furthermore, inactivation of Stat3 decreased load-driven bone formation, and the disruption of Stat3 in osteoblasts suppressed load-driven mitochondrial activity, which led to an elevated level of reactive oxygen species (ROS) in cultured primary osteoblasts. Stat3 has been found to be responsive to mechanical stimulation, and might play an important role in mechanical signal transduction in osteocytes. To investigate the role Stat3 plays in mechanical signaling transduction, osteocyte-specific Stat3 knockout (KO) mice were created. Inactivation of Stat3 in osteocytes presented a significantly reduced load-driven bone formation. Decreased osteoblast activity indicated by reduced osteoid surface was also found in osteocyte-specific Stat3 KO mice. Moreover, sclerostin (SOST) protein which is a critical osteocyte-specific inhibitor of bone formation, its encoded gene SOST expression has been found to be enhanced in osteocyte-specific Stat3 KO mice. Thus, these results clearly demonstrated that Stat3 plays an important role in bone homeostasis and mechanotransduction, and Stat3 is not only involved in bone-formation-important genes regulation in the nucleus but also in mediation of ROS and oxidative stress in mitochondria.