Mesenchymal stem cells (MSCs) are the most widely used source for bone tissue engineering due to their capability of multipotent differentiation. The use of nanotechnology in biomedical applications and therapy has increased in recent years provides an elegant alternative in comparison to current tissue engineering methods. Magnetic nanowires have a high potential in the medical field, as they are biocompatible, are simple to fabricate, possess low cytotoxic effects and can be operated wirelessly via magnetic fields. A nanowire substrate (NW) can provide a surface with tunable elastic properties. Therefore, magnetic nanowires have many promising applications such as in cell therapy, cell separation, cancer treatment, and as a scaffold for cell culture. This thesis explores the effects of alternating magnetic field (AMF) as a biophysical stimulator of osteogenic differentiation of MSCs by culturing the stem cells on a magnetic iron (Fe) NW. To this end, Fe nanowires were fabricated through electrodeposition and interactions between the NW and cells were analysed by electron microscopy. An AMF was applied to the NW in order to induce a vibration. MSCs were exposed to different magnetic field intensities, 250 mT and 50 mT, for different application times, 12 hours on followed by 12 hours off for two days and 24 hours on followed by 12 hours off. Differentiation was determined through the assessment of osteogenic markers at the mRNA level by RT-PCR and at the protein level by flow cytometry and fluorescence microscopy. Different effects were observed on MSCs grown on Fe NWs following exposure to different magnetic field intensities and duration applications. MSC differentiation towards the osteogenic lineage increased with increased field intensities. The most enhanced osteogenic differentiation of MSCs was observed at 250 mT AMF for 12 hours, as evidenced by elevated osteogenic markers at mRNA level compared to that of an AMF free control. Based on these results, we proposed that culturing MSCs on magnetic nanomaterials has the potential to control and promote osteogenesis under magnetic field and without the addition of external differentiation factors. These findings provide a new tool for stem cell research as an effective technology for bone tissue engineering and regenerative medicine.

There are well over 100,000 Americans on the kidney transplant list with a median wait time of 3.6 years. However, about 17 American die each day waiting for a kidney transplant, with vascular calcification being one of the most common causes [1, 2]. One vessel that is highly susceptible to vascular calcification is the aorta leading to negative cardiovascular outcomes that are secondary to kidney disease [3]. Therefore, understanding the effects that kidney disease has on disrupting the physiology of the vasculature, and finding potential therapeutic options, are imperative to those waiting for a life-saving kidney transplant. The present study aimed to test two hypotheses: (1) Aortic calcification leads to a decrease in stress in the thoracic and abdominal aorta of a young adult mouse. (2) The attenuated aortic stress seen in aortic calcification is due to the decreased expression of smooth muscle ⍺-actin (SM⍺-Actin). To test these hypotheses, calcification was induced in the ex vivo mouse aorta, followed by histological staining for calcium deposits, immunoblots for SM⍺-Actin, and measurements of aortic stress. The results of this study support the hypotheses in that calcification impairs aortic stress and it does so by decreasing the expression of SM⍺-Actin. The present study is the first to show the effect of phosphate-induced calcification on stress and expression of SM⍺-Actin in an ex vivo mouse aorta. This study is relevant to researchers as it shows key differences between studying vascular calcification in vitro compared to ex vivo. Therefore, investigating the mechanisms of aortic calcification using an ex vivo model, may be more applicable to human patients. / 2024-05-17T00:00:00Z

Osteoporosis is a disease of the bone metabolism which is characterised with a decrease of bone substance. The cause of this disease is the imbalance between the creation of a new bone substance by osteoblasts and the resorption of a bone tissue by osteoclasts, in favour of the bone resorption. The risk group of the development of this disease are women after menopause, who naturally register a decline of the estrogen hormone. Estrogen operates as an inhibitor of proosteoclastic factors such as receptor activator of NFκB ligand (RANKL), interleukin (IL)-1, IL-6 or TNF-α. The imbalance of the bone metabolism can also be caused by a disbalance in the production of Prostaglandin E2 (PGE2) and 1α,25-dihydroxyvitamin D3. They are strong mediators which can both stimulate and inhibit an osteoclastogenesis in vitro in concordance with the conditions of the culture/co-culture. This thesis focuses on the examination of an influence of those mediators (PGE2 in the concentration of 10-6 M and 10-8 M; 1α,25-dihydroxyvitamin D3 in the concentration of 10-8 M and 10-9 M) on the osteoclastogenesis from the rat PBMC at the presence of osteoblasts, with or without the combination of proosteoclastic factors macrophage colony-stimulating factor (M-CSF) and RANKL. Osteoclastogenesis was stimulated if PGE2 and...

Statins, hydroxymethylglutaryl-coenzyme-A reductase inhibitors (HMG-Co-A), are known to reduce plasma cholesterol levels. Interestingly, Simvastatin was previously reported to have a positive effect on the proliferation and odontoblastic differentiation of human dental pulp cells. However, the biocompatibility of Simvastatin has not been studied thoroughly. The purpose of this study was to further compare the effectiveness of different concentrations of Simvastatin on the attachment, proliferation, differentiation, toxicity, mineralization, and flow cytometry of human dental pulp cells (HDPCs) and osteoblasts. HDPCs and osteoblasts were cultured with Simvastatin at various concentrations of 1, 10, 25, 50, 75, 100 μmol/L, and 0 μmol/L was used as a control. The cell attachment was evaluated at 16 hours for HDPCs and 9 hours for osteoblasts. The proliferation rate, differentiation, cytotoxicity, and mineralization were investigated at 7, 14 and 21 days. Cell cycle and apoptosis were assessed at 1 and 3 days. Statistical analysis was performed using ANOVA. P-values ≤0.05 were considered statistically significant. The results showed that 25 μmol/L demonstrated the highest cell attachment efficiency when compared to the control in HDPCs (P<0.05). There was no statistical significance (P>0.05) amongst the groups in the cell attachment efficiency in osteoblasts. All tested concentrations showed a significant decrease in the proliferation rate and mineralization (P<0.001) and an increase in cytotoxicity and cytostasis (P<0.001) in both cell types. ALP levels increased in HDPCs and osteoblasts (P<0.001). DSP and RUNX2 levels decreased in HDPCs (P<0.001). OSC levels were increased in osteoblasts, but RUNX2 was decreased (P<0.001). Cell cycle and apoptosis significantly increased as time increased (P<0.001) in both cell types. In conclusion, the present findings showed that Simvastatin adversely affects the proliferation, cell viability of HDPCs and osteoblasts by inducing apoptosis, which were confirmed by flow cytometry results. There was an increase in the odontogenic and osteogenic markers hinting at early differentiation, which decreased as time increased. / 2025-07-10T00:00:00Z

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An increase in degenerative bone disease in an ageing population, combined with a rise in the number of patients suffering from bone defects caused by physical trauma, makes the repair of bone an issue of growing clinical relevance. Current treatments such as autografts and allografts have major drawbacks, including donor site morbidity, limited availability, disease transmission and immune rejection. To overcome these issues synthetic bone grafts have been developed to mimic the mineral phase of bone. Given the significant roles of silicon in bone growth and development there has been great interest in introducing silicon into synthetic bone grafts to enhance their bioactivity. Calcium phosphate based silicate containing grafts have demonstrated enhanced bioactivity, improved physical properties, enhanced protein adsorption and greater bone formation, when compared to non-silicated calcium phosphates such as hydroxyapatite. However, is not clear whether the increased bone formation associated with these materials is the result of greater osteoblast activity or a rise in numbers of osteoblasts resulting from activation and differentiation of stem/ progenitor cells. To answer this question, multipotent stem cells were cultured on silicate substituted calcium phosphate (Si-CaP) and hydroxyapatite (HA). Si-CaP promoted greater cell adhesion and enhanced proliferation when compared to HA. Cells differentiated along the osteogenic lineage on both substrates as evidenced by up regulation of osteoblast specific genes and proteins. However, cells on Si-CaP showed earlier and greater gene expression of all osteoblast genes examined, and greater protein production as detected by immunohistochemistry. Integrin gene expression analysis revealed up regulation of α an d β subunits on both substrates during differentiation. Integrins α5 and β1 expression were greater on Si-CaP than on HA, suggesting preferential binding of fibronectin. The implication of these findings for tissue engineering is clear, suggesting these substrates may be utilized to control stem cell fate in vivo and in vitro without the need for osteogenic supplementation. Furthermore, the increased rate of differentiation seen on Si-CaP may enable the development of novel substrates for osteogenic differentiation of MSC, which may have significant impact in regenerative medicine.

Osteogenic differentiation of mesenchymal stem cells (MSCs) would be possible to induce by creating of a cell bioactive scaffold that mimic the properties of bone extracellular matrix (ECM). This induction will be not only due to the addition of osteogenic supplements, but also due to the addition of differentiation peptides. These peptides activate signaling pathways leading to cell differentiation. The aim of this study was to evaluate the effect of selected peptides on adhesion, metabolic activity, proliferation and osteogenic differentiation of porcine MSCs. Four peptides with amino acid sequences of DGEA, IAGVGGEKSGGF, GQGFSYPYKAVFSTQ and KIPKASSVPTELSAISTLYL were selected. These peptides were derived from receptor binding sequences of collagen I, collagen III, BMP-7 and BMP-2 respectively. Scaffolds were prepared from a biocompatible and biodegradable poly-ε-caprolactone (PCL) polymer, suitable for cell cultivation. Cells were cultured on scaffolds for three weeks. Various concentration of differentiation peptides were added to the culture medium. As observed in the experiment of cells cultured in basal medium supplemented with differentiation peptides no effect on adhesion, proliferation or metabolic activity of porcine MSCs was observed. In groups treated with peptides derived from BMP-2...

Os leucotrienos (LTs) são mediadores inflamatórios derivados da via 5- lipoxigenase (5-LO), com contribuição relevante na reabsorção óssea. Neste estudo investigamos o papel dos LTs na diferenciação osteogênica e o seu impacto na osteoclatogênese. Assim, foi avaliado o perfil ósseo dos camundongos 129/Sv (WT) e 5-LO Knockout (5-LO KO) por meio de microtomografia computadorizada, evidenciando maior densidade óssea vertebral e trabéculas mais espessas em machos 5-LO KO. Após isso, osteoblastos primários (OBL) foram isolados e cultivados para determinar a atividade de fosfatase alcalina (ALP) e o potencial de mineralização. Resultados mostraram que OBL KO possui maior atividade de ALP e mineralização, em todos os períodos quando comparados com WT. Em adição, o tratamento com os LTs B4 e D4 inibiu a deposição de cálcio. Os inibidores da síntese de LTs e os antagonistas do BLT1/2 foram efetivos em recuperar a formação dos nódulos mineralizados. A cinética do Alox5 apresentou um aumento da expressão nos períodos de maior diferenciação celular em OBL WT. Além disso, a expressão de OCN, MMPs 2 e 9 e RANKL foram aumentadas em células 5-LO KO em quase todos os períodos avaliados. Em geral, o estímulo com LTs, seus inibidores e antagonistas diminuiu a expressão de Sp7, Col1a1, Opg e MMP-9 e aumentou RANKL em células KO. A sinalização por meio de segundos mensageiros também foi avaliada. Células 5-LO KO apresentam menor concentração de cálcio intracelular (Ca2+i) em relação ao WT. No período de 14 dias, o estímulo com LTD4 inibiu a liberação Ca2+i independente da linhagem, em relação ao controle. Os níveis de cAMP foram menores em OBL 5- LO KO, em todos os grupos tratados ou controle. LTD4 diminuiu a concentração de cAMP, mas não LTB4, em OBL 5-LO KO. O estudo também quantificou a produção de LTB4 e outros eicosanoides em osteoblastos mostrando a sua capacidade de síntese. A análise proteômica revelou 89 proteínas com expressão diminuída em OBL 5-LO KO, de um total de 154, sendo a maioria relacionada ao citoesqueleto e ao metabolismo energético. Também foram identificadas 59 proteínas exclusivas em OBL 5-LO KO e 06 unicamente expressas em células WT, revelando as diferenças intrínsecas de cada animal. O perfil osteoclastogênico de camundongos WT vs. 5-LO KO mostrou diferenças significativas na análise fenotípica, TRAP e na expressão gênica de células derivadas da linhagem monocítica-macrofágica. Após o estímulo com M-CSF e RANKL, as células WT apresentaram osteoclastos gigantes multinucleados, porém, células 5-LO KO apresentaram uma população de células com formas e tamanhos variáveis, e menor grau de maturação. Em adição, os LTsexógenos não modularam a atividade da TRAP. O meio condicionado proveniente dos OBL WT e KO, retardaram o processo de formação dos osteoclastos. A análise da expressão gênica em osteoclastos mostrou diminuição da expressão de Alox5, Il- 1b, Il-6 e TNFa em células 5-LO KO. BLT1/2, CysLt1 e os marcadores da diferenciação Acp5, Ctsk e Nfact1 não apresentaram diferenças entre os animais. Em adição, o LTB4 diminuiu a expressão do Alox5 e a Il-1b foi aumentada em osteoclastos WT. Assim, os resultados demonstram que os LTs são capazes de modular o metabolismo ósseo, e a ausência do gene da 5-LO está relacionada ao maior perfil osteogênico. / Leukotrienes (LTs) are inflammatory mediators derived from the 5-lipoxygenase (5-LO) pathway, with a relevant contribution in bone resorption. In this study we investigated the role of LTs in osteogenic differentiation and its impact on osteoclastogenesis.Thus, the bone profile of the 129/Sv (WT) and 5-LO Knockout mice (5-LO KO) was evaluated by computerized microtomography, showing higher vertebral bone density and thicker trabeculae in 5-LO KO males. After that, primary osteoblasts (OBL) were isolated and cultured to determine alkaline phosphatase activity (ALP) and mineralization potential. Results showed that OBL KO has higher ALP activity and mineralization, in all periods when compared with WT. In addition, the treatment with LTB4 and LTD4 inhibited calcium deposition. Inhibitors of LT synthesis and BLT1/2 antagonists were effective to recover the mineralized nodules formation. The kinetics of Alox5 showed an increase in expression during cellular differentiation period in WT OBL. In addition, expression of OCN, MMPs 2 and 9 and RANKL were increased in 5- LO KO cells in almost all evaluated periods. In general, the stimulation with LTs, their inhibitors and antagonists decreased the expression of Sp7, Col1a1, Opg and MMP- 9. But it increased the RANKL expression in KO cells. The second messengers signaling was also evaluated. 5-LO KO cells showed lower concentration levels of intracellular calcium (Ca2+ i) when compared to WT cells. In the 14-day period, the LTD4 treatment inhibited the Ca2+i independent of the murine lineage, relative to the control. cAMP levels were lower in OBL 5-LO KO, in all treated or control groups. LTD4 decreased the concentration of cAMP, but not LTB4, in KO cells. The study also quantified the production of LTB4 and other eicosanoids in osteoblasts showing their ability to synthesize those metabolites. The proteomic analysis revealed 89 downregulated proteins in OBL KO, out of a total of 154, most of them related to cytoskeleton and energy metabolism. Also 59 identified proteins were unique in OBL 5-LO KO and 06 exclusively expressed in WT cells, revealing the intrinsic differences of each strain. The osteoclastogenic profile of WT vs. 5-LO KO showed significant differences in phenotypic analysis, TRAP and in the gene expression of cells derived from the monocyte-macrophage-lineage. After M-CSF and RANKL stimulation, WT cells showed multinucleated giant osteoclasts. However, 5-LO KO cells presented a population of cells with variable shapes and sizes, and a lower maturation stage. In addition, exogenous LTs did not modulate TRAP activity. The conditioned medium from OBL WT and 5-LO KO delayed the formation process of osteoclasts. Gene expression analysis in osteoclasts showed decreased expression of Alox 5, Il-1b, Il-6 and TNF in 5-LO KO cells. BLT1/2, CysLt1 and the osteoclast differentiation markers Acp5, Ctsk and Nfact1 showed no differences between the strains. In addition, LTB4 decreased the expression of Alox5, and IL-1b was increased in WT osteoclasts. Thus, the results demonstrate that the LTs are able to modulate the bone metabolism, and the absence of the 5-LO gene is related to the greater osteogenic profile.