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L’ostéoarthrose (OA) est une pathologie qui touche les articulations principalement chez les personnes âgées. Il devient capital de mieux cerner cette pathologie à cause des coûts économiques qu’elle engendre mais surtout à cause du vieillissement de la population. Cette maladie se caractérise par une dégradation du cartilage articulaire, une sclérose osseuse, une inflammation de la membrane synoviale ainsi que la présence d’ostéophytes. L’étiologie de cette pathologie est restée nébuleuse car la recherche sur la maladie touchait principalement le cartilage articulaire. Toutefois, le rôle clé de l’os sous-chondral dans l’OA est maintenant reconnu. L’obésité étant un facteur de risque de l’OA, nous avons émis l’hypothèse que la leptine, une adipocytokine clé dans l’obésité, joue un rôle important dans l’OA. En effet, la leptine modifie le phénotype des ostéoblastes (Ob) normaux humain et puisque les Ob OA humains ont un phénotype altéré, notre objectif était de déterminer le rôle potentiel de la leptine dans ces cellules. Pour ce faire, nous avons préparé des cultures primaires d’Ob issus de la plaque sous-chondral du plateau tibial de patients OA et d’individus normaux (N). L’expression de la leptine et de son récepteur actif (OB-Rb) ont été mesurées par RT-PCR en temps réel, et leur production a été mesurée par ELISA et immunobuvardage (IB). La prolifération des Ob OA a été déterminée par incorporation de BrdU. La phosphorylation de p42/44 MAPK dans les Ob OA a été déterminée par IB. Le phénotype des Ob fut déterminé par la mesure de l’activité de la phosphatase alcaline (ALP) et la sécrétion d’ostéocalcine (OC), en présence ou non de leptine. De plus, les effets des ARNs d’interférences (SiRNA) anti-leptine et anti OB-Rb sur le phénotype des Ob OA furent déterminés via leur impact sur l’activité de l’ALP et sur la sécrétion d’OC. L’effet dose-réponse de la leptine sur les expressions d’OB-Rb, du facteur de croissance TGF-1 ou encore sur sa propre expression furent déterminées par RT-PCR en temps réel. Pour terminer, la signalisation de la leptine a été étudiée en évaluant l’effet dose réponse de celle-ci sur la production des protéines JAK2 et STAT3 phosphorylées par IB. Les résultats obtenus ont montrés que les Ob OA expriment et produisent plus de leptine que les Ob N. Au niveau phénotypique, ces Ob OA possèdent une activité de l’ALP ainsi qu’une sécrétion d’OC plus importante que celles observées chez les Ob N. L’ajout d’anticorps inactivant l’interaction leptine et OB-Rb ou d’inhibiteurs chimiques comme tyrphostin ou piceatannol diminuèrent l’activité de l’ALP ainsi que la sécrétion d’OC dans les Ob OA. Par contre, l’ajout de leptine exogène aux Ob OA augmenta l’activité de l’ALP sans pour autant faire varier la sécrétion d’OC. La leptine à des doses de 1ng/ml à 10mg/ml stimula la prolifération des Ob OA ainsi que la phosphorylation de p42/44 MAPK. La leptine exogène diminua l’expression de TFG-1 tandis qu’elle stimula la phosphorylation de JAK2 et STAT3 ou encore sa propre expression de manière dose-dépendante. Cependant, l’expression d’OB-Rb diminua de manière dose-dépendante. Enfin, le traitement des Ob OA avec des Si leptine ou Si OB-Rb diminua l’activité d’ALP, la sécrétion d’OC, l’expression de la leptine, l’expression d’OB-RB ainsi que l’expression du facteur TGF-1. L’ensemble de ces données démontre que la leptine endogène des Ob OA est sous contrôle des facteurs de croissance et qu’elle contribue à maintenir le phénotype anormal de l’os sous-chondral OA. De plus, ceci suggère que la leptine serait un acteur important dans la régulation du remodelage osseux. / Osteoarthritis (OA) is a disease which mainly affects the joints in the elderly. It becomes essential to better understand this disease because of the economic costs it brings, but mainly because of population aging. This disease is characterized by a deterioration of cartilage, bone sclerosis, inflammation of the synovial membrane and the presence of osteophytes. The knowledge of its etiology has remained incomplete because research on this disease focused mainly on the articular cartilage. However, the key role of subchondral bone in OA is now recognized. Obesity is a risk factor for OA, then we hypothesized that leptin, a key adipocytokine in obesity plays an important role in OA. Indeed, leptin alters the phenotype of osteoblasts (Ob) and human Ob has altered phenotype in OA patients, our objective was to determine the potential role of leptin in OA Ob. To do this, we prepared primary cultures of Ob from the sub-chondral plate of the tibial plateaus of OA patients and normal individuals (N). The expression of leptin and its receptor active (OB-Rb) were measured by RT-PCR in real time, and their production was measured by ELISA and western blot (WB). The proliferation of Ob OA was determined by BrdU incorporation. The phosphorylation of p42/44 MAPK was evaluated by WB. The phenotype of Ob was determined by measuring the activity of alkaline phosphatase (ALP) and the secretion of osteocalcin (OC), in the presence or absence of leptin. Moreover, the effects of small interference RNAs (siRNAs) anti-leptin and anti OB-Rb on the phenotype of OA Ob were determined through their impact on the activity of the ALP and the secretion of OC. The dose-response effect of 1eptin on its own expression or the expressions of OB-Rb, the growth factor TGF-β1 were determined by RT-PCR in real time. Finally, signalisation of leptin in OA Ob was studied by evaluating the dose-response effect of this on the production of JAK2 and STAT3 protein phosphorylated by WB. The results showed that the OA Ob express and produce more leptin than N. Moreover, these Ob OA have an activity of the ALP and a secretion OC higher than those observed in N Ob. The addition of antibodies inactivating interaction leptin and OB-Rb or chemical inhibitors such as tyrphostin or piceatannol diminished the activity of the ALP and the secretion of OC in OA Ob against by the addition of exogenous leptin to Ob OA increased the activity of the ALP without influencing the secretion of OC. Leptin at doses of 1ng/ml to 10mg/mL stimulated the proliferation of OA Ob and the phosphorylation of p42/44 MAPK. Exogenous leptin decreased the expression of TGF-β1 while it stimulated the phosphorylation of JAK2 and STAT3 and expression of its own in dose-dependent manner. However, the expression of OB-Rb decreased in dose-dependent. Finally, the treatment of OA Ob with Si leptin or Si OB-Rb decreased activity of ALP, the secretion of OC, the leptin expression, expression of OB-Rb and the expression of TGF-β1 factor. All these data show that endogenous leptin Ob OA controls the growth factors and contributes to maintaining the abnormal phenotype of the subchondral bone OA. Moreover, this suggests that leptin is an important player in the regulation of bone remodelling

Sulfated glycosaminoglycans (GAG) are components of the bone marrow stem cell niche and to a minor extent of mature bone tissue with important functions in regulating stem cell lineage commitment and differentiation. We anticipated that artificial extracellular matrices (aECM) composed of collagen I and synthetically oversulfated GAG derivatives affect preferentially the differentiation of osteoblast-precursor cells and early osteoblasts. A set of gradually sulfated chondroitin sulfate and hyaluronan derivatives was used for the preparation of aECM. All these matrices were analysed with human bone marrow stromal cells to identify the most potent aECM and to determine the influence of the degree and position of sulfate groups and the kind of disaccharide units on the osteogenic differentiation. Oversulfated GAG derivatives with a sulfate group at the C-6 position of the N-acetylglycosamine revealed the most pronounced proosteogenic effect as determined by tissue nonspecific alkaline phosphatase activity and calcium deposition. A subset of the aECM was further analysed with different primary osteoblasts and cell lines reflecting different maturation stages to test whether the effect of sulfated GAG derivatives depends on the maturation status of the cells. It was shown that the proosteogenic effect of aECMwasmost prominent in early osteoblasts. [ABSTRACT FROM AUTHOR]

Einen Aspekt dieser Arbeit stellt die Analyse der Stimulation von Ratten-Calvaria-Osteoblasten (RCA) mit den beiden Wachstumsfaktoren TGF-b1 und BMP-4 während der Proliferations- sowie Differenzierungs- und Mineralisierungsphase dar. Hierfür soll die Phosphorylierung und Aktivierung von Erk1 und Erk2, sowie von Smad1 und Smad2 mit Hilfe eines Kinase-Aktivitätsassays sowie der Westernblot-Analyse untersucht werden. Im Rahmen dieser Arbeit soll weiterhin untersucht werden, welche Bedeutung die Wachstumsfaktoren TGF-b1 und BMP-4 auf die Aktivität der alkalischen Phosphatase (ALP), einem wichtigen Differenzierungsmarker in Osteoblasten, ausüben. Enzymatische Aktivitätsbestimmungen und zytochemische Färbung aktiver ALP sollen darüber Aufschluss geben. Weiterhin soll der Gehalt an ALP-mRNA durch PCR bestimmt werden. Ein weiteres wichtiges Ziel dieser Arbeit ist die Analyse der Bedeutung von Erk1, Erk2, Smad1 und Smad2 auf die Aktivität der ALP. Dafür sollen Inhibitoren eingesetzt werden. Die enzymatische Aktivitätsbestimmung soll darüber aufklären. Außerdem soll mit Hilfe von kurzen, doppelsträngigen RNA-Molekülen (siRNA) ein knock down der Kinasen herbeigeführt werden und dessen Auswirkung auf die Aktivität der ALP enzymatisch bestimmt werden. Dafür muss zunächst die Wirksamkeit der siRNA auf RNA-Ebene mittels PCR und auf Proteinebene mittels Westernblot-Analysen überprüft werden. Zusätzlich soll die Bedeutung der Wachstumsfaktoren und der Kinasen Erk1 und Erk2 auf die Mineralisierung der RCA analysiert werden. Dafür wird die Menge des zellassoziierten Kalziums und Phosphats experimentell bestimmt, wodurch der Mineralisationsgrad der Zellen wiedergegeben werden kann.

The thesis is based on articles describing the fundamental research of carbon based nanomaterials for their possible utilization in biomedicine. The aim of this thesis was to describe the way how human osteoblasts (SAOS-2 cell line) and primary human mesenchymal stem cells (hMSC) adhere, grow and behave on surfaces made of several carbon allotropes - nanocrystalline diamond (NCD), single walled carbon nanotubes (SWCNTs) films and graphene. The utilization of carbon as the basic material promised good biocompatibility and possibility of useful surface modifications. The NCD had modified surface nanotopography (nanoroughness and nanostructuring prepared by dry ion etching). All the materials had modified surface atomic termination with oxygen and hydrogen which changes the surface electrical conductivity, surface charge and wettability. It was hypothesized that the surface termination can also influence the cell adhesion and growth. It turned out that all the studied materials were suitable as substrates for cultivation of mentioned cell types. Various nanoroughnesses of NCD surface had different effect on the cell adhesion and cell metabolic activity. Nanostructuring of the NCD influenced the formation of focal adhesions. The surface terminations of NCD and the other studied nanomaterials in...

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontic procedures are an effective treatment option for immature teeth with infected necrotic pulps to allow for healing and potential continued root development, yet challenges to ideal treatment outcomes remain. Consistent development of root length and width of dentin remains a challenge, as does development of the pulp-dentin complex. Previous in vitro studies have assessed the role of different growth factors and bioactive molecules in combination with scaffolds to potentially facilitate continued development of the pulp-dentin complex using dental pulp stem cells (DPSCs). The proline-rich tyrosine kinase 2 (Pyk2) is linked with osteoblast activity and the regulation of bone mass. Further, the Pyk2 inhibitor PF-4618433 (PF-46) has been shown in previous studies to enhance osteoblast activity and mineral deposition in vitro. However, whether Pyk2 targeting promotes the osteogenic differentiation of DPSCs remains unknown. Objective: The purpose of this study was to investigate the effect of a Pyk2 inhibitor, PF-46, on the proliferation, differentiation, and mineralization of human DPSCs. Materials and Methods: Human DPSCs were cultured in 24-well plates with α-MEM with 10% FBS, and containing 0 μM (vehicle control) or 0.1 μM, 0.3 μM, or 0.6 μM PF-46. Fresh media and treatments were replaced every 2-3 days. After 1 day incubation, cytotoxic effects were evaluated by using an MTS proliferation assay. After 4 days of treatment, direct cell counting was performed. To induce osteogenic differentiation, ascorbic acid and β-glycerol phosphate were added to the culture media and the DPSCs were cultured with PF-46 for 14 days. Then, an alkaline phosphatase (ALP) assay and mineral deposition assay were performed. Differences between treatment groups were analyzed by a one-way ANOVA followed by pair-wise tests conducted using Tukey’s multiple comparisons procedure with a 5% significance level. Results: The 0.6 μM PF-46 group had a significantly higher cell count, ALP activity and mineral deposition when compared to 0 μM PF-46. The 0.1 and 0.3 μM PF-46 groups also had significantly higher ALP activity compared to the 0 μM PF-46 group after 14 days of incubation. There was a general trend of increased differentiation and mineral deposition as the concentration of PF-46 increased from 0.1 μM to 0.6 μM. Conclusion: There was a general concentration-dependent increase in cell count, differentiation, and mineral deposition by human DPSCs as the concentration of PF-46 increased from 0 μM up to 0.6 μM, with the highest activity observed with 0.6 μM PF-46. Although further research is needed, these results suggest that strategies that target Pyk2 may potentially be used to improve the osteogenic differentiation of DPSCs to aid endodontic regeneration.

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PhD (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Tulbaghia violacea Harv. has found extensive uses in traditional medicine for the treatment of numerous ailments among which are tuberculosis, oesophageal cancer, diabetes and cardiovascular diseases. Current reports show that cardiovascular diseases are now the primary cause of mortality worldwide. Thus, the potential of T. violacea plant extracts against cardiovascular diseases should be explored. The objectives of this study were, (i) to conduct qualitative and quantitative preliminary phytochemical screening of T. violacea aqueous leaf extracts, (ii) to conduct Gas chromatography–mass spectrometry (GC-MS) analysis for screening of compounds present in the plant extract, (iii) to evaluate the antioxidant activity of the T. violacea crude extracts using the DPPH:1.1-diphenyl-2-picrylhydrazyl and ABTS: 2,2-azino-bis 3-ethylebenzthiazoline-6-sulfonic acid assays, (iv) to evaluate the antimicrobial activity of the T. violacea crude extracts using disk diffusion and Minimum inhibitory concentration/Minimum bactericidal concentration (MIC/MBC), (v) to evaluate the antithrombogenic properties of T. violacea crude extracts on polystyrene, (vi) to fabricate polycaprolactone (PCL) and PCL-T. violacea incorporated scaffolds, (vii) to evaluate the antithrombogenic properties of T. violacea crude extracts on the fabricated PCL and PCL-T. violacea fabricated scaffolds and, (viii) to evaluate the growth and differentiation of adipose derived stem cells (ADSCs) on the fabricated scaffolds. The qualitative and quantitative phytochemical screening was conducted using standard procedures. Folin-Ciocalteu method was used to evaluate both total phenolic content (TPC) and total tannin content (TTC), the Aluminium chloride method was used for total flavonoid content (TFC) and GC-MS was used to screen for compounds present in the plant extract. The antioxidant activity was evaluated using DPPH and ABTS and the antimicrobial activity was evaluated using disc diffusion and MIC/MBC assays. The antithrombogenic properties of the T. violacea aqueous leaf extracts was then evaluated using platelet activation and whole blood clotting kinetics on polystyrene discs which have been reported to induce platelet activation. The experiment was performed in the absence and presence of 100 and 1000 μg/ml T. violacea plant extracts for both the platelet activation study which used blood plasma and the whole blood clotting kinetics assay which used fresh whole blood. Platelet adhesion was evaluated using fluorescence microscopy and a scanning electron microscope (SEM) was used to evaluate their morphology. Three scaffolds designated as PCL, 10% Tvio and 15% Tvio were fabricated which consisted of a 10% PCL powder and 10% as well as 15% T. violacea aqueous plant extract with respect to the PCL powder weight. The scaffolds were then characterized using Fourier-transform infrared spectroscopy (FTIR) and Energy-dispersive x-ray spectroscopy (EDS). The scaffolds were then evaluated for their antithrombogenic properties in the presence and absence of 100 and 1000 μg/ml T. violacea plant extracts. Platelet adhesion was evaluated using a fluorescent microscope and the morphology was evaluated using SEM. For the cell study, adipose derived stem cells (ADSCs) were cultured on the designed scaffolds and evaluated for their toxicity, viability, adhesion, proliferation, morphology and differentiation into osteoblasts over a period of 3 weeks. Lactate dehydrogenase (LDH) assay was used for toxicity studies, alamar blue assay was used for viability, fluorescence microscopy was used to evaluate cellular adhesion and proliferation while the alkaline phosphate (ALP) assay was used to evaluate differentiation of the cells into osteoblasts. Cell morphology was evaluated using SEM. Phytochemical screening of the prepared T. violacea aqueous extract revealed the presence of terpenoids, flavonoids, cardiac glycosides, saponins, protein, phenols, tannins, carbohydrates and amino acids. This is the first study that has identified the presence of carbohydrates and amino acids in T. violacea aqueous leaf extracts. Different concentrations of 0.1, 1.0 and 10 mg/ml of plant extract were used to conduct the quantitative phytochemical screening assays. There was a concentration dependent increase in the amount of phenols, tannins and flavonoids as the concentration of the plant extracts increased. This was the first study that evaluated the total tannic content of T. violacea plant extracts. The amount of total phenols was higher than that of flavonoids and tannins at every concentration range studied followed by the total flavonoids and lastly total tannins. The GC-MS analysis showed the presence of 33 compounds among which were 2,4 – Dithiapentate - 2,2-dioxide, Cannabidiol, 2,4,5,7 –Tetrathiaoctane and 2,4,5,7 - Tetrathiaoctane 2-dioxide. The presence of sulphur compounds support the characteristic garlic-like smell as well as some of the biological activities of T. violacea plant extracts. The antioxidant activities based on DPPH (0.49 mg/ml) and ABTS (0.24 mg/ml) suggest that T. violacea can be used as potential antioxidant agents. For the antimicrobial activity using disc diffusion, the extracts exhibited appreciable antibacterial activities against Bacillus subtilis, Serratia marcescens, Staphylococcus aureus and S. epidermidis. The highest zone of inhibition was observed for S. epidermidis at 19.50 ± 0.87 mm. The MIC results revealed that the plant extract of T. violacea was moderately active against B. subtilis, S. aureus, S. epidermidis, E. coli, and S. marcescens with MIC value of 2.5 mg/ml. However, the antimicrobial effect of the extract on S. epidermidis was bactericidal when compared to the bacteriostatic effect on the other active microorganisms. The antithrombogenic results on the polystyrene discs showed a significant reduction in the number of platelets that adhered on the polystyrene surfaces treated with plasma mixed with 100 μg/ml of plant extract when compared to the untreated control and the 1000 μg/ml treatment. For the 1000 μg/ml treatment, there was a significant increase in the number of platelets that adhered to polystyrene surfaces. These results were confirmed by the fluorescence and SEM results which showed a higher platelet count for the 1000 μg/ml treatment when compared to the other groups. The whole blood clotting kinetics study showed delayed blood clotting with the 100 μg/ml treatment over a period of 60 min when compared to the untreated control and the 1000 μg/ml treatment. These results correspond with the lower platelet adhesion observation and thus confirm the anticlotting properties of T. violacea aqueous leaf extracts at lower concentrations. The mean diameter of the scaffolds was recorded on the SEM as 275.60 ± 60.65 nm, 193 ± 30 nm and 537 ± 138 nm for the PCL, 10% Tvio and 15% Tvio scaffolds, respectively. The FTIR spectrum revealed the presence of amide groups as well hydroxyl O–H stretching groups which were the characteristic groups for the presence of T. violacea plant extracts in the polycaprolactone. The EDS results showed the presence of potassium, chlorine and sulphur compounds which were only present in the T. violacea scaffolds in addition to the carbon, oxygen and silicon observed in the PCL scaffold. The fabricated scaffolds were then used to evaluate platelet adhesion and activation on blood plasma in the absence and presence of 100 and 1000 μg/ml T. violacea aqueous leaf extracts. The results showed that the 10% Tvio scaffold was more effective in inhibiting platelet adhesion and activation at every treatment group especially when plasma was used in the absence of T. violacea plant extracts. A similar observation to the polystyrene study was observed were addition of 1000 μg/ml of plant extract resulted in the highest number of activated platelets. The study suggests the potential of the 10% Tvio scaffold in the prevention of platelet adhesion and aggregation. The in vitro cell adhesion, proliferation and differentiation of adipose derived stem cells (ADSCs) on the fabricated T. violacea loaded PCL nanofibers was then evaluated. The LDH assay illustrated less activity on the 10% Tvio scaffold when compared to PCL and 15% Tvio scaffolds however, none of the scaffolds were considered as toxic. The alamar blue assay was used for viability after 4 and 7 days of culture. The results showed a significant increase in cell viability for all scaffolds from day 4 to day 7 with the 10% Tvio scaffold having the highest overall cell viability for both day 4 and day 7 of cell cultures. Immunofluorescence staining was then used to count the number of cells using DAPI (4′,6-diamidino-2-phenylindole) stained images and illustrated that the T. violacea incorporated scaffolds supported better cell growth compared to the PCL scaffold. Cell morphology on the T. violacea scaffolds was denser and spread out into cellular extensions when compared to the PCL scaffold after 7 days of cell culture, supporting the higher number of adhered cells from the fluorescence results. For the long term cell study after week 1 and 3, the ALP results showed a significant difference in ALP activity between week 1 and week 3 for all scaffolds. The highest ALP activity was observed for the 15% Tvio scaffolds which is a marker for initial phase of bone matrix deposition. The designed T. violacea scaffolds supported better cell growth compared to the PCL scaffold and their morphology was more spread out and covered the entire surface of the scaffolds after week 3. Lastly, the cell count and osteocalcin differentiation was more prominent on 10% Tvio scaffold indicating higher levels of the protein marker for bone formation. Thus, supporting the use of the 10% Tvio scaffold for long-term cell studies. In conclusion, the results of this study indicated that the aqueous extract of T. violacea is rich is phytochemicals and also possess a broad range of pharmaceutically important compounds which may be attributed to the high antioxidant and antimicrobial activities identified. The results from this study suggest that T. violacea aqueous extracts have antithrombogenic properties at lower concentrations. Scaffolds fabricated with the incorporation of T. violacea plant extract also confirm the potential antiplatelet activity of the fabricated 10% Tvio scaffold. The results also suggest the potential of the fabricated 10% Tvio scaffold to enhance cell adhesion, proliferation and differentiation over long-term cell studies. It can thus be recommended that T. violacea may be useful for tissue engineering applications and bone repair with prospects of preventing cardiovascular diseases associated with bone defects. This research study has provided the foundation for clinical evaluation and outlined the potential effects of T. violacea aqueous leaf extracts as a clinical drug.

Despite the continued development of specialized immunosuppressive therapies in the form of monoclonal antibodies, glucocorticoids remain a mainstay in the treatment of rheumatological and auto-inflammatory disorders. Therapeutic glucocorticoids are unmatched in the breadth of their immunosuppressive properties and deliver their anti-inflammatory effects at unparalleled speed. However, long-term exposure to therapeutic doses of glucocorticoids decreases bone mass and increases the risk of fractures – particularly in the spine – thus limiting their clinical use. Due to the abundant expression of glucocorticoid receptors across all skeletal cell populations and their respective progenitors, therapeutic glucocorticoids affect skeletal quality through a plethora of cellular targets and molecular mechanisms. However, recent evidence from rodent studies, supported by clinical data, highlights the considerable role of cells of the osteoblast lineage in the pathogenesis of glucocorticoid-induced osteoporosis: it is now appreciated that cells of the osteoblast lineage are key targets of therapeutic glucocorticoids and have an outsized role in mediating their undesirable skeletal effects. As part of this article, we review the molecular mechanisms underpinning the detrimental effects of supraphysiological levels of glucocorticoids on cells of the osteoblast lineage including osteocytes and highlight the clinical implications of recent discoveries in the field.

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