Shedding Light on Osteosarcoma Cell Differentiation: Impact on Biomineralization and Mitochondria Morphology.

Osteosarcoma (OS) is the most common primary malignant bone tumor and its etiology has recently been associated with osteogenic differentiation dysfunctions. OS cells keep a capacity for uncontrolled proliferation showing a phenotype similar to undifferentiated osteoprogenitors with abnormal biomineralization. Within this context, both conventional and X-ray synchrotron-based techniques have been exploited to deeply characterize the genesis and evolution of mineral depositions in a human OS cell line (SaOS-2) exposed to an osteogenic cocktail for 4 and 10 days. A partial restoration of the physiological biomineralization, culminating with the formation of hydroxyapatite, was observed at 10 days after treatment together with a mitochondria-driven mechanism for calcium transportation within the cell. Interestingly, during differentiation, mitochondria showed a change in morphology from elongated to rounded, indicating a metabolic reprogramming of OS cells possibly linked to an increase in glycolysis contribution to energy metabolism. These findings add a dowel to the genesis of OS giving new insights on the development of therapeutic strategies able to restore the physiological mineralization in OS cells.

Natural-like Chalcones with Antitumor Activity on Human MG63 Osteosarcoma Cells.

Osteosarcoma (OS) is a malignant disease characterized by poor prognosis due to a high incidence of metastasis and chemoresistance. Recently, Licochalcone A (Lic-A) has been reported as a promising agent against OS. Starting from chalcones selected from a wide in-house library, a new series was designed and synthetized. The antitumor activity of the compounds was tested on the MG63 OS cell line through the innovative Quantitative Phase Imaging technique and MTT assay. To further investigate the biological profile of active derivatives, cell cycle progression and apoptosis induction were evaluated. An earlier and more consistent arrest in the G2-M phase with respect to Lic-A was observed. Moreover, apoptosis was assessed by Annexin V staining as well as by the detection of typical morphological features of apoptotic cells. Among the selected compounds, 1e, 1q, and 1r proved to be the most promising antitumor molecules. This study pointed out that an integrated methodological approach may constitute a valuable platform for the rapid screening of large series of compounds.

Multifaceted activity of polyciclic MDR revertant agents in drug-resistant leukemic cells: Role of the spacer.

A small library of derivatives carrying a polycyclic scaffold recently identified by us as a new privileged structure in medicinal chemistry was designed and synthesized, aiming at obtaining potent MDR reverting agents also endowed with antitumor properties. In particular, as a follow-up of our previous studies, attention was focused on the role of the spacer connecting the polycyclic core with a properly selected nitrogen-containing group. A relevant increase in reverting potency was observed, going from the previously employed but-2-ynyl- to a pent-3-ynylamino moiety, as in compounds 3d and 3e, while the introduction of a triazole ring proved to differently impact on the activity of the compounds. The docking results supported the data obtained by biological tests, showing, for the most active compounds, the ability to establish specific bonds with P-glycoprotein. Moreover, a multifaceted anticancer profile and dual in vitro activity was observed for all compounds, showing both revertant and antitumor effects on leukemic cells. In this respect, 3c emerged as a "triple-target" agent, endowed with a relevant reverting potency, a considerable antiproliferative activity and a collateral sensitivity profile.

Calcite as a Precursor of Hydroxyapatite in the Early Biomineralization of Differentiating Human Bone-Marrow Mesenchymal Stem Cells.

Biomineralization is the process by which living organisms generate organized mineral crystals. In human cells, this phenomenon culminates with the formation of hydroxyapatite, which is a naturally occurring mineral form of calcium apatite. The mechanism that explains the genesis within the cell and the propagation of the mineral in the extracellular matrix still remains largely unexplained, and its characterization is highly controversial, especially in humans. In fact, up to now, biomineralization core knowledge has been provided by investigations on the advanced phases of this process. In this study, we characterize the contents of calcium depositions in human bone mesenchymal stem cells exposed to an osteogenic cocktail for 4 and 10 days using synchrotron-based cryo-soft-X-ray tomography and cryo-XANES microscopy. The reported results suggest crystalline calcite as a precursor of hydroxyapatite depositions within the cells in the biomineralization process. In particular, both calcite and hydroxyapatite were detected within the cell during the early phase of osteogenic differentiation. This striking finding may redefine most of the biomineralization models published so far, taking into account that they have been formulated using murine samples while studies in human cell lines are still scarce.

Analysis of Intracellular Magnesium and Mineral Depositions during Osteogenic Commitment of 3D Cultured Saos2 Cells.

In this study, we explore the behaviour of intracellular magnesium during bone phenotype modulation in a 3D cell model built to mimic osteogenesis. In addition, we measured the amount of magnesium in the mineral depositions generated during osteogenic induction. A two-fold increase of intracellular magnesium content was found, both at three and seven days from the induction of differentiation. By X-ray microscopy, we characterized the morphology and chemical composition of the mineral depositions secreted by 3D cultured differentiated cells finding a marked co-localization of Mg with P at seven days of differentiation. This is the first experimental evidence on the presence of Mg in the mineral depositions generated during biomineralization, suggesting that Mg incorporation occurs during the bone forming process. In conclusion, this study on the one hand attests to an evident involvement of Mg in the process of cell differentiation, and, on the other hand, indicates that its multifaceted role needs further investigation.

Glycyrrhetinic Acid Liposomes and Hyalurosomes on Spanish Broom, Flax, and Hemp Dressings to Heal Skin Wounds.

The focus of this work was to prepare Spanish Broom, flax, and hemp dressings impregnated with glycyrrhetinic acid (GA) liposomes or hyalurosomes to promote the healing process and protect the skin wounds. Vesicles were prepared by the film hydration method and characterized in terms of size, particle size distribution, ζ potential, encapsulation efficiency, in vitro release, and biocompatibility on 3T3 fibroblasts. Loaded liposomes and hyalurosomes showed nanometric size (355 ± 19 nm and 424 ± 32 nm, respectively), good size distribution (lower than 0.3), and appropriate encapsulation efficiency (58.62 ± 3.25% and 59.22 ± 8.18%, respectively). Hyalurosomes showed good stability during the storage period, which can be correlated to the negative ζ potential, and allowed a fast and complete release of GA. Preliminary biological studies revealed that both kinds of loaded vesicles were not cytotoxic and that hyalurosomes could exert a slight stimulating effect on fibroblast proliferation. Finally, in vitro release studies from the different dressings impregnated with the loaded vesicles demonstrated that a high amount of GA could be reached at the wound site after 60 min from application. In conclusion, the results suggested that the developed dressings, especially those impregnated with hyalurosomes, can be efficiently used to promote the healing process.

Fluorescence lifetime imaging of intracellular magnesium content in live cells.

The first detailed analysis of FLIM applications for Mg cell imaging is presented. We employed the Mg-sensitive fluorescent dye named DCHQ5, a derivative of diaza-18-crown-6 ethers appended with two 8-hydroxyquinoline groups, to perform fluorescence lifetime imaging in control and Mg deprived SaOS-2 live cells, which contain different concentrations of magnesium. We found that the lifetime maps are almost uniform all over the cells and, most relevantly, we showed that the ratio of the amplitude terms is related to the magnesium intracellular concentration.

Magnesium Is a Key Regulator of the Balance between Osteoclast and Osteoblast Differentiation in the Presence of Vitamin D3.

Magnesium (Mg) is crucial for bone health. Low concentrations of Mg inhibit the activity of osteoblasts while promoting that of osteoclasts, with the final result of inducing osteopenia. Conversely, little is known about the effects of high concentrations of extracellular Mg on osteoclasts and osteoblasts. Since the differentiation and activation of these cells is coordinated by vitamin D3 (VD3), we investigated the effects of high extracellular Mg, as well as its impact on VD3 activity, in these cells. U937 cells were induced to osteoclastic differentiation by VD3 in the presence of supra-physiological concentrations (>1 mM) of extracellular Mg. The effect of high Mg concentrations was also studied in human bone-marrow-derived mesenchymal stem cells (bMSCs) induced to differentiate into osteoblasts by VD3. We demonstrate that high extra-cellular Mg levels potentiate VD3-induced osteoclastic differentiation, while decreasing osteoblastogenesis. We hypothesize that Mg might reprogram VD3 activity on bone remodeling, causing an unbalanced activation of osteoclasts and osteoblasts.

Single cell versus large population analysis: cell variability in elemental intracellular concentration and distribution.

The quantification of elemental concentration in cells is usually performed by analytical assays on large populations missing peculiar but important rare cells. The present article aims at comparing the elemental quantification in single cells and cell population in three different cell types using a new approach for single cells elemental analysis performed at sub-micrometer scale combining X-ray fluorescence microscopy and atomic force microscopy. The attention is focused on the light element Mg, exploiting the opportunity to compare the single cell quantification to the cell population analysis carried out by a highly Mg-selective fluorescent chemosensor. The results show that the single cell analysis reveals the same Mg differences found in large population of the different cell strains studied. However, in one of the cell strains, single cell analysis reveals two cells with an exceptionally high intracellular Mg content compared with the other cells of the same strain. The single cell analysis allows mapping Mg and other light elements in whole cells at sub-micrometer scale. A detailed intensity correlation analysis on the two cells with the highest Mg content reveals that Mg subcellular localization correlates with oxygen in a different fashion with respect the other sister cells of the same strain. Graphical abstract Single cells or large population analysis this is the question!

Magnesium Deprivation Potentiates Human Mesenchymal Stem Cell Transcriptional Remodeling.

Magnesium plays a pivotal role in energy metabolism and in the control of cell growth. While magnesium deprivation clearly shapes the behavior of normal and neoplastic cells, little is known on the role of this element in cell differentiation. Here we show that magnesium deficiency increases the transcription of multipotency markers and tissue-specific transcription factors in human adipose-derived mesenchymal stem cells exposed to a mixture of natural molecules, i.e., hyaluronic, butyric and retinoid acids, which tunes differentiation. We also demonstrate that magnesium deficiency accelerates the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. We argue that magnesium deprivation generates a stressful condition that modulates stem cell plasticity and differentiation potential. These studies indicate that it is possible to remodel transcription in mesenchymal stem cells by lowering extracellular magnesium without the need for genetic manipulation, thus offering new hints for regenerative medicine applications.

Where is it and how much? Mapping and quantifying elements in single cells.

The biological function of a chemical element in cells not only requires the determination of its intracellular quantity, but also the spatial distribution of its concentration. Different strategies can be employed to quantify and map the intracellular concentration of elements in single cells. The assessment of the intracellular elemental concentration, which is the relevant information, requires the measurement of cell volume. This challenging and demanding task requires combining different techniques allowing gathering of both morphological and compositional information on the same cell. Moreover, the need to analyse samples more similar to their natural state requires complex hardware equipment, and supplementary efforts in preparation protocols. Nevertheless, the response to the question: "where is it and how much?" is worth all these efforts. This review aims at providing an insight into the recent and most advanced techniques and strategies for quantifying and mapping chemical elements in single cells. We describe and discuss indirect detection techniques (label based) which make use of fluorescent dyes, and direct ones (label free), such as particle induced X-ray emission, proton backscattering spectrometry, scanning transmission ion spectrometry, nano-secondary ion mass spectrometry, X-ray fluorescence microscopy, complemented by X-ray imaging.

Quantitative chemical imaging of the intracellular spatial distribution of fundamental elements and light metals in single cells.

We report a method that allows a complete quantitative characterization of whole single cells, assessing the total amount of carbon, nitrogen, oxygen, sodium, and magnesium and providing submicrometer maps of element molar concentration, cell density, mass, and volume. This approach allows quantifying elements down to 10(6) atoms/µm(3). This result was obtained by applying a multimodal fusion approach that combines synchrotron radiation microscopy techniques with off-line atomic force microscopy. The method proposed permits us to find the element concentration in addition to the mass fraction and provides a deeper and more complete knowledge of cell composition. We performed measurements on LoVo human colon cancer cells sensitive (LoVo-S) and resistant (LoVo-R) to doxorubicin. The comparison of LoVo-S and LoVo-R revealed different patterns in the maps of Mg concentration with higher values within the nucleus in LoVo-R and in the perinuclear region in LoVo-S cells. This feature was not so evident for the other elements, suggesting that Mg compartmentalization could be a significant trait of the drug-resistant cells.

A novel fluorescent chemosensor allows the assessment of intracellular total magnesium in small samples.

The present study investigated the analytical capabilities of a new fluorescent chemosensor, named DCHQ5, a phenyl derivative belonging to the family of diaza-crown-hydroxyquinolines, for the quantitative assessment of total intracellular Mg content. The results obtained were compared to the analytical performances of DCHQ1 - the parent probe of the series which so far was the only suitable species for the quantitative assessment of the intracellular total magnesium and showed comparable results to atomic absorption spectroscopy. Different protocols were tested in several cell lines both by flow cytometry and by steady state fluorescence spectroscopy assays. The results obtained support the possibility to use DCHQ5 to accurately quantify the intracellular total Mg in much smaller samples than DCHQ1, also displaying an increased stable intracellular staining. These features, combined with the high fluorescence enhancement upon cation binding, and the possibility to be excited both in the UV and visible region, make DCHQ5 a valuable and versatile analytical tool for Mg assessment in biological samples.

Colorectal Cancer Cell Invasion and Functional Properties Depend on Peri-Tumoral Extracellular Matrix.

We investigated how the extracellular matrix (ECM) affects LoVo colorectal cancer cells behavior during a spatiotemporal invasion. Epithelial-to-mesenchymal transition (EMT) markers, matrix-degrading enzymes, and morphological phenotypes expressed by LoVo-S (doxorubicin-sensitive) and higher aggressive LoVo-R (doxorubicin-resistant) were evaluated in cells cultured for 3 and 24 h on Millipore filters covered by Matrigel, mimicking the basement membrane, or type I Collagen reproducing a desmoplastic lamina propria. EMT and invasiveness were investigated with RT-qPCR, Western blot, and scanning electron microscopy. As time went by, most gene expressions decreased, but in type I Collagen samples, a strong reduction and high increase in MMP-2 expression in LoVo-S and -R cells occurred, respectively. These data were confirmed by the development of an epithelial morphological phenotype in LoVo-S and invading phenotypes with invadopodia in LoVo-R cells as well as by protein-level analysis. We suggest that the duration of culturing and type of substrate influence the morphological phenotype and aggressiveness of both these cell types differently. In particular, the type I collagen meshwork, consisting of large fibrils confining inter fibrillar micropores, affects the two cell types differently. It attenuates drug-sensitive LoVo-S cell aggressiveness but improves a proteolytic invasion in drug-resistant LoVo-R cells as time goes by. Experimental studies on CRC cells should examine the peri-tumoral ECM components, as well as the dynamic physical conditions of TME, which affect the behavior and aggressiveness of both drug-sensitive and drug-resistant LoVo cells differently.

Development and Characterization of Azithromycin-Loaded Microemulsions: A Promising Tool for the Treatment of Bacterial Skin Infections.

In recent years, the treatment of bacterial skin infections has been considered a major healthcare issue due to the growing emergence of antibiotic-resistant strains of Staphylococcus aureus. The incorporation of antibiotics in appropriate nanosystems could represent a promising strategy, able to overcome several drawbacks of the topical treatment of infections, including poor drug retention within the skin. The present work aims to develop microemulsions containing azithromycin (AZT), a broad-spectrum macrolide antibiotic. Firstly, AZT solubility in various oils, surfactants and co-surfactants was assessed to select the main components. Subsequently, microemulsions composed of vitamin E acetate, Labrasol® and Transcutol® P were prepared and characterized for their pH, viscosity, droplet size, zeta potential and ability to release the drug and to promote its retention inside porcine skin. Antimicrobial activity against S. aureus methicillin-resistant strains (MRSA) and the biocompatibility of microemulsions were evaluated. Microemulsions showed an acceptable pH and were characterized by different droplet sizes and viscosities depending on their composition. Interestingly, they provided a prolonged release of AZT and promoted its accumulation inside the skin. Finally, microemulsions retained AZT efficacy on MRSA and were not cytotoxic. Hence, the developed AZT-loaded microemulsions could be considered as useful nanocarriers for the treatment of antibiotic-resistant infections of the skin.

Substrate Type and Concentration Differently Affect Colon Cancer Cells Ultrastructural Morphology, EMT Markers, and Matrix Degrading Enzymes.

Aim of the study was to understand the behavior of colon cancer LoVo-R cells (doxorubicin-resistant) vs. LoVo-S (doxorubicin sensitive) in the initial steps of extracellular matrix (ECM) invasion. We investigated how the matrix substrates Matrigel and type I collagen-mimicking the basement membrane (BM) and the normal or desmoplastic lamina propria, respectively-could affect the expression of epithelial-to-mesenchymal transition (EMT) markers, matrix-degrading enzymes, and phenotypes. Gene expression with RT-qPCR, E-cadherin protein expression using Western blot, and phenotypes using scanning electron microscopy (SEM) were analyzed. The type and different concentrations of matrix substrates differently affected colon cancer cells. In LoVo-S cells, the higher concentrated collagen, mimicking the desmoplastic lamina propria, strongly induced EMT, as also confirmed by the expression of Snail, metalloproteases (MMPs)-2, -9, -14 and heparanase (HPSE), as well as mesenchymal phenotypes. Stimulation in E-cadherin expression in LoVo-S groups suggests that these cells develop a hybrid EMT phenotype. Differently, LoVo-R cells did not increase their aggressiveness: no changes in EMT markers, matrix effectors, and phenotypes were evident. The low influence of ECM components in LoVo-R cells might be related to their intrinsic aggressiveness related to chemoresistance. These results improve understanding of the critical role of tumor microenvironment in colon cancer cell invasion, driving the development of new therapeutic approaches.

Magnesium: Biochemistry, Nutrition, Detection, and Social Impact of Diseases Linked to Its Deficiency.

Magnesium plays an important role in many physiological functions. Habitually low intakes of magnesium and in general the deficiency of this micronutrient induce changes in biochemical pathways that can increase the risk of illness and, in particular, chronic degenerative diseases. The assessment of magnesium status is consequently of great importance, however, its evaluation is difficult. The measurement of serum magnesium concentration is the most commonly used and readily available method for assessing magnesium status, even if serum levels have no reliable correlation with total body magnesium levels or concentrations in specific tissues. Therefore, this review offers an overview of recent insights into magnesium from multiple perspectives. Starting from a biochemical point of view, it aims at highlighting the risk due to insufficient uptake (frequently due to the low content of magnesium in the modern western diet), at suggesting strategies to reach the recommended dietary reference values, and at focusing on the importance of detecting physiological or pathological levels of magnesium in various body districts, in order to counteract the social impact of diseases linked to magnesium deficiency.

Development of Spanish Broom and Flax Dressings with Glycyrrhetinic Acid-Loaded Films for Wound Healing: Characterization and Evaluation of Biological Properties.

The selection of an appropriate dressing for each type of wound is a very important procedure for a faster and more accurate healing process. So, the aim of this study was to develop innovative Spanish Broom and flax wound dressings, as alternatives to cotton used as control, with polymeric films containing glycyrrhetinic acid (GA) to promote wound-exudate absorption and the healing process. The different wound dressings were prepared by a solvent casting method, and characterized in terms of drug loading, water uptake, and in vitro release. Moreover, biological studies were performed to evaluate their biocompatibility and wound-healing efficacy. Comparing the developed wound dressings, Spanish Broom dressings with GA-loaded sodium hyaluronate film had the best functional properties, in terms of hydration ability and GA release. Moreover, they showed a good biocompatibility, determining a moderate induction of cell proliferation and no cytotoxicity. In addition, the wound-healing test revealed that the Spanish Broom dressings promoted cell migration, further facilitating the closure of the wound.

Assessment and Imaging of Intracellular Magnesium in SaOS-2 Osteosarcoma Cells and Its Role in Proliferation.

Magnesium is an essential nutrient involved in many important processes in living organisms, including protein synthesis, cellular energy production and storage, cell growth and nucleic acid synthesis. In this study, we analysed the effect of magnesium deficiency on the proliferation of SaOS-2 osteosarcoma cells. When quiescent magnesium-starved cells were induced to proliferate by serum addition, the magnesium content was 2-3 times lower in cells maintained in a medium without magnesium compared with cells growing in the presence of the ion. Magnesium depletion inhibited cell cycle progression and caused the inhibition of cell proliferation, which was associated with mTOR hypophosphorylation at Serine 2448. In order to map the intracellular magnesium distribution, an analytical approach using synchrotron-based X-ray techniques was applied. When cell growth was stimulated, magnesium was mainly localized near the plasma membrane in cells maintained in a medium without magnesium. In non-proliferating cells growing in the presence of the ion, high concentration areas inside the cell were observed. These results support the role of magnesium in the control of cell proliferation, suggesting that mTOR may represent an important target for the antiproliferative effect of magnesium. Selective control of magnesium availability could be a useful strategy for inhibiting osteosarcoma cell growth.

The assessment of intracellular magnesium: different strategies to answer different questions.

The role of magnesium in cell metabolism is complex and still not completely clarified. Although magnesium has been shown to modulate many phenomena in cells, its intracellular distribution and subcellular compartmentalization have not yet elucidated in detail, mainly as a consequence of the inadequacy of analytical techniques. The method usually employed to quantify total magnesium in cells or tissue are F-AAS or more sensitive techniques as graphite furnace AAS and inductively coupled plasma mass spectroscopy (MS). Thanks to the development of new specific fluorescent dyes, several progresses have been made in the comprehension of the fundamental biological process at the cellular and sub-cellular level. Moreover, the biological function of a chemical element in cells does not only require the determination of its intracellular quantity but also the spatial distribution of its concentration. Most of Mg2+-sensitive fluorescent dyes detect only the free metal ions, precluding the possibility of identifying the total pool of Mg. This review aims at giving an overview on different techniques focusing on two approaches to quantify total Mg in a small cell population or in single cells: i) Indirect Mg detection, label-based methods that represent the best choice to quantify the elemental concentration on a large cell population; ii) direct Mg detection (label-free), Synchrotron-based x-ray microscopy techniques that offer the possibility of achieving a detailed map of the intracellular concentration of a specific chemical element on single cell.