Elucidating the Role if Integrin-extracellular Matrix Protein Interactions in Regulating Osteoclast Activity

Millions of people around the world suffer from the debilitating effects of inflammatory bone diseases characterized by excessive bone loss due to an increase in osteoclast formation and activity. Osteoclasts are multinucleated cells responsible for bone resorption in health and disease. Arthritic joints also have elevated levels of extracellular matrix proteins affecting the disease progression. The interaction between osteoclasts and the external milieu comprised of extracellular matrix proteins through integrins is essential for modulating the formation and activity of osteoclasts. The focus of this thesis was to elucidate how the interaction between the extracellular matrix proteins and osteoclasts regulates osteoclast formation and activity and the role of alphavbeta3 in this process. In primary rabbit osteoclast cultures, blocking the integrin alphavbeta3 using Vitaxin, an anti-human alphavbeta3 antibody, decreased osteoclast resorption by decreasing osteoclast attachment. Vitaxin’s inhibitory effect on osteoclast attachment was enhanced when osteoclasts were pretreated with M-CSF, a growth factor known to induce an activated conformation of the integrin alphavbeta3. Using the RAW264.7 cell line, the effects of the matrix proteins fibronectin and vitronectin on osteoclast activity were compared to those of osteopontin. Both fibronectin and vitronectin decreased the number of osteoclasts formed compared to osteopontin. Fibronectin’s effect on osteoclastogenesis was through decreasing pre-osteoclast migration and/or fusion but not through inhibiting their recruitment. In contrast, fibronectin induced resorption through increasing resorptive activity per osteoclast in comparison to vitronectin and osteopontin. These stimulatory effects were accompanied by an increase in the pro-inflammatory cytokines nitric oxide and IL-1beta Crosstalk between the signalling pathways of nitric oxide and IL-1betawas suggested by the ability of the nitric oxide inhibitor to decrease the level of IL-1beta which occurred exclusively on fibronectin. Osteoclasts on fibronectin also had a compact morphology with the smallest planar area while vitronectin increased the percentage of osteoclast with migratory morphology and osteopontin induced osteoclast spreading. The increase in compact morphology on fibronectin was associated with a decrease in extracellular pH. Low extracellular pH was found to increase the total time osteoclasts spend in a compact phase. These results show that matrix proteins differentially regulate osteoclast formation, activity and morphology.Ph

A fast and reproducible method to quantify magnetic nanoparticle biodistribution

The quantification of nanoparticles, particularly superparamagnetic iron oxide nanoparticles (SPIONs), both in vitro and in vivo has become highly important in recent years. Some methods, such as induced coupled plasma (ICP) spectroscopy and UV-visible chemical titration using Prussian Blue (PB), already exist however they consist of the titration of the whole iron content. These standard methods need sample preparations leading to their destruction and long measurement time. In this study, we used magnetic susceptibility measurements (MSM) to titrate the concentration and biodistribution of magnetic particles in the organs of rats. The advantages of the MSM SPION quantification technique are presented and compared to widely used methods of iron oxide titration such as ICP and PB UV-visible titration. We have demonstrated that MSM is a simpler, faster (1 second per measurement), more reproducible and highly sensitive technique for SPION detection with minimal detection around 2 μgFe mL(-1) without being influenced by neither the SPION coating nor their surrounding environment. Moreover, MSM is a more robust method as it is not affected by endogenous iron facilitating the distinction of SPIONs (iron present as nanoparticles) from background iron in tissues. This advantage allows the decrease of control samples needed in biological studies. In conclusion, we have demonstrated that MSM is a standard method that can be easily setup to determine the biodistribution of SPIONs regardless of their environment

Accumulation of amino-polyvinyl alcohol-coated superparamagnetic iron oxide nanoparticles in bone marrow: implications for local stromal cells

First, it will be investigated if amino-polyvinyl alcohol-coated superparamagnetic iron oxide nanoparticles (A-PVA-SPIONs) are suitable for MRI contrast enhancement in bone marrow. Second, the impact of A-PVA-SPION exposure in vivo on the viability and key functions of local bone marrow stromal cells (BMSCs) will be investigated

Improved dynamic response assessment for intra-articular injected iron oxide nanoparticles

The emerging importance of nanoparticle technology, including iron oxide nanoparticles for monitoring development, progression, and treatment of inflammatory diseases such as arthritis, drives development of imaging techniques. Studies require an imaging protocol that is sensitive and quantifiable for the detection of iron oxide over a wide range of concentrations. Conventional signal loss measurements of iron oxide nanoparticle containing tissues saturate at medium concentrations and show a nonlinear/nonproportional intensity to concentration profile due to the competing effects of T₁ and T₂ relaxation. A concentration calibration phantom and an in vivo study of intra-articular injection in a rat knee of known concentrations of iron oxide were assessed using the difference-ultrashort echo time sequence giving a positive, quantifiable, unambiguous iron signal and monotonic, increasing concentration response over a wide concentration range in the phantom with limited susceptibility artifacts and high contrast in vivo to all other tissues. This improved dynamic response to concentration opens possibilities for quantification due to its linear nature at physiologically relevant concentrations

Ex situ evaluation of the composition of protein corona of intravenously injected superparamagnetic nanoparticles in rats

It is now well recognized that the surfaces of nanoparticles (NPs) are coated with biomolecules (e.g., proteins) in a biological medium. Although extensive reports have been published on the protein corona at the surface of NPs in vitro, there are very few on the in vivo protein corona. The main reason for having very poor information regarding the protein corona in vivo is that separation of NPs from the in vivo environment has not been possible by using available techniques. Knowledge of the in vivo protein corona could lead to better understanding and prediction of the fate of NPs in vivo. Here, by using the unique magnetic properties of superparamagnetic iron oxide NPs (SPIONs), NPs were extracted from rat sera after in vivo interaction with the rat's physiological system. More specifically, the in vivo protein coronas of polyvinyl-alcohol-coated SPIONs with various surface charges are defined. The compositions of the corona at the surface of various SPIONs and their effects on the biodistribution of SPIONs were examined and compared with the corona composition of particles incubated for the same time in rat serum

Amino-polyvinyl alcohol coated superparamagnetic iron oxide nanoparticles are suitable for monitoring of human mesenchymal stromal cells in vivo

Mesenchymal stromal cells (MSCs) are promising candidates in regenerative cell-therapies. However, optimizing their number and route of delivery remains a critical issue, which can be addressed by monitoring the MSCs' bio-distribution in vivo using super-paramagnetic iron-oxide nanoparticles (SPIONs). In this study, amino-polyvinyl alcohol coated (A-PVA) SPIONs are introduced for cell-labeling and visualization by magnetic resonance imaging (MRI) of human MSCs. Size and surface charge of A-PVA-SPIONs differ depending on their solvent. Under MSC-labeling conditions, A-PVA-SPIONs have a hydrodynamic diameter of 42 ± 2 nm and a negative Zeta potential of 25 ± 5 mV, which enable efficient internalization by MSCs without the need to use transfection agents. Transmission X-ray microscopy localizes A-PVA-SPIONs in intracellular vesicles and as cytosolic single particles. After identifying non-interfering cell-assays and determining the delivered and cellular dose, in addition to the administered dose, A-PVA-SPIONs are found to be non-toxic to MSCs and non-destructive towards their multi-lineage differentiation potential. Surprisingly, MSC migration is increased. In MRI, A-PVA-SPION-labeled MSCs are successfully visualized in vitro and in vivo. In conclusion, A-PVA-SPIONs have no unfavorable influences on MSCs, although it becomes evident how sensitive their functional behavior is towards SPION-labeling. And A-PVA-SPIONs allow MSC-monitoring in vivo