1,720,977 research outputs found
Carbonic anhydrase IX inhibition is an effective strategy for osteosarcoma treatment
No full textObjective: Hypoxia-inducible factor 1, a regulator of CA IX activity, is often overexpressed in human osteosarcoma (OS) but not in normal tissues, and its expression levels correlate with prognosis. In this study, we investigated the therapeutic potential of newly synthesized CA IX sulfonamide inhibitors in OS.
Methods: CA IX expression was evaluated in OS cell lines and bone marrow stromal cells (BMSC). After treatment with CA IX inhibitors, cell proliferation, apoptosis, cell cycle, extracellular and cytosolic pH changes were evaluated both in vitro and in mouse OS xenografts.
Results: CA IX expression levels were significantly higher in OS than in BMSC. Accordingly, CA IX inhibitor 3 induced remarkable cytotoxicity on OS cells without affecting BMSC proliferation. This activity was increased under hypoxia, and was mediated by cell cycle arrest and by the modulation of cytosolic and extracellular pH. In vivo, CA IX inhibitor 3 reduced tumor growth by inducing significant necrosis.
Conclusions: Our results provide a strong rationale for the clinical use of the newly synthesized CA IX inhibitor 3 in human OS
Mesenchymal Stem Cells‐Derived Small Extracellular Vesicles and Their Validation as a Promising Treatment for Chondrosarcoma in a 3D Model in Vitro
Full text linkChondrosarcomas (CHS) constitute approximately 20% of all primary malignant bone tumors, characterized by a slow growth ratewith initial manifestation of few signs and symptoms. These malignant cartilaginous neoplasms, particularly those with dediffer-entiated histological subtypes, pose significant therapeutic challenges, as they exhibit high resistance to both radiation andchemotherapy. Ranging from relatively benign, low‐grade tumors (grade I) to aggressive high‐grade tumors with the potential forlung metastases and a grim prognosis, there is a critical need for innovative diagnostic and therapeutic approaches, particularly forpatients with more aggressive forms. Herein, small extracellular vesicles (sEVs) derived from mesenchymal stem cells are presentedas an efficient nanodelivery tool to enhance drug penetration in an in vitro 3D model of CHS. Employing high‐pressure homoge-nization (HPH), we achieved unprecedented encapsulation efficiency of doxorubicin (DXR) in sEVs derived from mesenchymal stemcells (MSC‐EVs). Subsequently, a comparative analysis between free DXR and MSC‐EVs encapsulated with DXR (DXR‐MSC‐EVs)was conducted to assess their penetration and uptake efficacy in the 3D model. The results unveiled a higher incidence of necroticcells and a more pronounced toxic effect with DXR‐MSC‐EVs compared to DXR alone. This underscores the remarkable ability ofMSC‐EVs to deliver drugs in complex environments, highlighting their potential application in the treatment of aggressive CHS
Mesenchymal stroma drives axonogenesis and nerve-induced aggressiveness in osteosarcoma
Full text linkBackground: Osteosarcoma (OS), the most common primary bone malignancy, is a leading cause of cancer-related mortality in children and adolescents. Besides genomic abnormalities, several features of tumour microenvironment (TME), including cancer-associated mesenchymal stromal cells (MSC), have been recognized to play a key role in OS progression. The pathogenetic function of de novo innervation in TME has been extensively studied in carcinomas but is still an unexplored area of investigation in sarcomas, including OS. Methods: We evaluated nerve infiltration in tissue samples from a small cohort of human OS (n = 5) and from canine OS (n = 11), a translational model for the human disease, by βIII-tubulin immunostaining. We then analysed nerve-stroma-tumour crosstalk using direct and indirect co-cultures of dorsal root ganglion (DRG) neurons with OS/tumour-associated mesenchymal stromal cells (MSC and cancer-associated fibroblasts, CAF), both under standard and microfluidic conditions. In particular, we investigated the effects of tumour and stromal cells on axonal tropism and outgrowth by measuring neurite recruitment, length, and branches and, vice versa, the impact of neuron-derived secretome on OS cell proliferation and migration. Finally, we assessed the secretion of pro-neurotrophic mediators, including brain-derived neurotrophic factor (BDNF), interleukin-6 (IL-6), and nerve growth factor (NGF), by MSC, CAF, and OS cells. The functional roles of IL-6 and BDNF were also verified by the blocking antibody Tocilizumab (TCZ) and the neutralizing Anti-BDNF antibody. Results: We provided evidence of OS innervation within and surrounding the tumour in association with mesenchymal stroma that also corresponded to the most proliferative area of the tumour (Ki-67+). In vitro, both MSC and, to a lesser extent, OS cells promoted axonal growth through cytokine (IL-6) and neuromodulator (BDNF) secretion. Extracellular acidosis – a hallmark of OS aggressiveness – amplified IL-6 release by stromal cells, and its pro-neurogenic effect was prevented by IL-6 blockade. In turn, tumour-associated innervation stimulated OS cell proliferation and migration, eventually driving tumour aggressiveness. Conclusions: We showed, for the first time, that bone-associated nerves, fostered by the OS microenvironment, promote tumour aggressiveness. Interfering with the nerve-tumour axis, particularly with the signalling associated with mesenchymal stroma, offers novel opportunities for OS treatment
3D Printing and Bioprinting to Model Bone Cancer: The Role of Materials and Nanoscale Cues in Directing Cell Behavior
No full textBone cancer, both primary and metastatic, is characterized by a low survival rate. Currently, available models lack in mimicking the complexity of bone, of cancer, and of their microenvironment, leading to poor predictivity. Three-dimensional technologies can help address this need, by developing predictive models that can recapitulate the conditions for cancer development and progression. Among the existing tools to obtain suitable 3D models of bone cancer, 3D printing and bioprinting appear very promising, as they enable combining cells, biomolecules, and biomaterials into organized and complex structures that can reproduce the main characteristic of bone. The challenge is to recapitulate a bone-like microenvironment for analysis of stromal–cancer cell interactions and biological mechanics leading to tumor progression. In this review, existing approaches to obtain in vitro 3D-printed and -bioprinted bone models are discussed, with a focus on the role of biomaterials selection in determining the behavior of the models and its degree of customization. To obtain a reliable 3D bone model, the evaluation of different polymeric matrices and the inclusion of ceramic fillers is of paramount importance, as they help reproduce the behavior of both normal and cancer cells in the bone microenvironment. Open challenges and future perspectives are discussed to solve existing shortcomings and to pave the way for potential development strategies
Bone on-a-chip: a 3D dendritic network in a screening platform for osteocyte-targeted drugs
Full text linkAge-related musculoskeletal disorders, including osteoporosis, are frequent and associated with long lasting morbidity, in turn significantly impacting on healthcare system sustainability. There is therefore a compelling need to develop reliable preclinical models of disease and drug screening to validate novel drugs possibly on a personalized basis, without the need of in vivo assay. In the context of bone tissue, although the osteocyte (Oc) network is a well-recognized therapeutic target, current in vitro preclinical models are unable to mimic its physiologically relevant and highly complex structure. To this purpose, several features are needed, including an osteomimetic extracellular matrix, dynamic perfusion, and mechanical cues (e.g. shear stress) combined with a three-dimensional (3D) culture of Oc. Here we describe, for the first time, a high throughput microfluidic platform based on 96-miniaturized chips for large-scale preclinical evaluation to predict drug efficacy. We bioengineered a commercial microfluidic device that allows real-time visualization and equipped with multi-chips by the development and injection of a highly stiff bone-like 3D matrix, made of a blend of collagen-enriched natural hydrogels loaded with hydroxyapatite nanocrystals. The microchannel, filled with the ostemimetic matrix and Oc, is subjected to passive perfusion and shear stress. We used scanning electron microscopy for preliminary material characterization. Confocal microscopy and fluorescent microbeads were used after material injection into the microchannels to detect volume changes and the distribution of cell-sized objects within the hydrogel. The formation of a 3D dendritic network of Oc was monitored by measuring cell viability, evaluating phenotyping markers (connexin43, integrin alpha V/CD51, sclerostin), quantification of dendrites, and responsiveness to an anabolic drug. The platform is expected to accelerate the development of new drug aimed at modulating the survival and function of osteocytes
Synthesis, characterization and biological activity of hydroxyl-bisphosphonic analogs of bile acids
No full textBisphosphonates (BPs) are now the most widely used drugs for diseases associated with increased bone
resorption, such as osteoporosis, and tumor bone diseases. A significant drawback of the BPs is their poor
oral absorption that is enhanced by the presence of bile acid substituents in the bisphosphonate
framework, with no toxic effects. A straightforward synthesis of bile acid-containing hydroxybisphosphonates
and a full characterization of these pharmaceutically important molecules, including an
evaluation of affinity and the mechanism of binding to hydroxyapatite, is presented. The biological
activity of bile acid-containing bisphosphonate salts was determined using the neutral-red assay on the
L929 cell line and primary cultures of osteoclasts. The bioactivity of the new compounds was found
superior than bisphosphonates of established activit
Cancer-associated mesenchymal stroma fosters the stemness of osteosarcoma cells in response to intratumoral acidosis via NF-κB activation
Full text linkThe role of mesenchymal stem cells (MSC) in osteosarcoma (OS), the most common primary tumor of bone, has not been extensively elucidated. We have recently shown that OS is characterized by interstitial acidosis, a microenvironmental condition that is similar to a wound setting, in which mesenchymal reactive cells are activated to release mitogenic and chemotactic factors. We therefore intended to test the hypothesis that, in OS, acid-activated MSC influence tumor cell behavior. Conditioned media or co-culture with normal MSC previously incubated with short-term acidosis (pH 6.8 for 10 hr, H+-MSC) enhanced OS clonogenicity and invasion. This effect was mediated by NF-κB pathway activation. In fact, deep-sequencing analysis, confirmed by Real-Time PCR and ELISA, demonstrated that H+-MSC differentially induced a tissue remodeling phenotype with increased expression of RelA, RelB and NF-κB1, and downstream, of CSF2/GM-CSF, CSF3/G-CSF and BMP2 colony-promoting factors, and of chemokines (CCL5, CXCL5 and CXCL1), and cytokines (IL6 and IL8), with an increased expression of CXCR4. An increased expression of IL6 and IL8 were found only in normal stromal cells, but not in OS cells, and this was confirmed in tumor-associated stromal cells isolated from OS tissue. Finally, H+-MSC conditioned medium differentially promoted OS stemness (sarcosphere number, stem-associated gene expression), and chemoresistance also via IL6 secretion. Our data support the hypothesis that the acidic OS microenvironment is a key factor for MSC activation, in turn promoting the secretion of paracrine factors that influence tumor behavior, a mechanism that holds the potential for future therapeutic interventions aimed to target OS
Bioactive natural and synthetic compounds for treating bone resorption diseases
Full text linkLe terapie convenzionali per le malattie da aumentato riassorbimento osseo sono limitate dalla tossicità sistemica, bassa biodisponibilità farmacologica e scarsa aderenza alle terapie. In questo studio sono stati considerati approcci terapeutici innovativi basati su composti naturali e sintetici.
I) Valutazione dell'attività biologica di composti naturali. Evidenze sperimentali hanno dimostrato l’attività antiproliferativa ed antiapoptotica di piante della Medicina ayurvedica. Queste proprietà sono sfruttabili nel trattamento di malattie da aumentato riassorbimento osseo, come l'osteoporosi. Per chiarire i possibili effetti terapeutici di questi composti, sono stati studiati i decotti di Rubia cordifolia, Hemidesmus indicus, Emblica officinalis, ed Asparagus racemosus. Hemidesmis indicus si è dimostrato il più efficace.
II) Valutazione dell'attività biologica di composti sintetici. I bisfosfonati (BP) sono farmaci capaci di legarsi alle superfici minerali ossee e all’idrossiapatite, nei siti di rimodellamento osseo. Poiché i BP inibiscono la funzione degli osteoclasti, sono convenzionalmente impiegati nel trattamento di malattie da aumentato riassorbimento osseo, come l'osteoporosi. Tuttavia, gli elevati costi e gli effetti collaterali legati alla somministrazione determinano una scarsa aderenza al trattamento condizionandone l’efficacia. Scopo di questo studio è stato quello di valutare l'attività biologica di BP chimicamente innovativi, meno tossici e sintetizzati con strategie catalitiche semplificate ed ecocompatibili, in modo da ridurre i costi di produzione. È stato valutato l’effetto citotossico e antiosteoclastico dei composti e confrontato con quello dei BP comunemente impiegati in clinica (neridronato, pamidronato e alendronato).
I risultati sono stati considerati raggiunti qualora fossero identificati BP di nuova sintesi non citotossici e capaci di conservare almeno il 90% della capacità dei substrati di base di inibire il riassorbimento osseo. Tutti i composti di nuova sintesi sono risultati meno tossici del BP convenzionale, anche a concentrazioni più elevate ed i più efficaci sono stati un BP coniugato con acido biliare, un BP aromatico contenente azoto ed un BP alifatico contenente zolfo.Conventional therapies for bone resorption diseases are limited by systemic toxicity, low drug bioavailability, and low rates of adherence to therapies. In this study novel therapeutic approaches based on natural and newly synthetic compounds were considered.
I) Evaluation of biological activity of natural compounds. Experimental evidences showed antiproliferative and antiapoptotic activities of plant extracts commonly used in Ayurvedic medicine. These properties could be helpful in the treatment of some bone resorption diseases, including osteoporosis. In order to clarify the possible therapeutic effects of these compounds, decoctions of Rubia cordifolia, Hemidesmus indicus, Emblica officinalis, and Asparagus racemosus were evaluated. Hemidesmis Indicus had the highest biological activity.
II) Evaluation of biological activity of synthetic compounds. Bisphosphonates (BPs) are a class of drugs able to bind to bone mineral surfaces and hydroxyapatite, at sites of bone remodelling. Since BPs inhibit osteoclast function, they are currently employed for the treatment of increased bone resorption disorders, such as osteoporosis. However, the high cost of drugs and the side effects associated with their administration result in poor adherence to treatment and therefore condition its effectiveness. The purpose of this study was to evaluate the biological activity of chemically innovative BPs, potentially less toxic and synthesized with simplified and environment sustainable catalytic strategies, to greatly reduce production costs. Cytotoxic and anti-osteoclast effect of the compounds has been verified and compared with those obtained by using BPs commonly employed in clinical setting (neridronate, pamidronate and alendronate).
The results were considered to be achieved if newly synthesized BPs non-cytotoxic and able to retain at least 90% of the capacity of the base substrates to inhibit bone resorption were identified. All newly synthesized compounds were less cytotoxic than conventional BPs, even at higher concentrations and the most effective were bile acid containing hydroxyl-BP, aromatic nitrogen-containing BP and aliphatic sulfur-containing BP
Energy metabolism in osteoclast formation and activity.
Full text linkOsteoclastogenesis and osteolysis are energy-consuming processes supported by high metabolic activities. In human osteoclasts derived from the fusion of monocytic precursors, we found a substantial increase in the number of mitochondria with differentiation. In mature osteoclasts, mitochondria were also increased in size, rich of cristae and arranged in a complex tubular network. When compared with immature cells, fully differentiated osteoclasts showed higher levels of enzymes of the electron transport chain, a higher mitochondrial oxygen consumption rate and a lower glycolytic efficiency, as evaluated by extracellular flux analysis and by the quantification of metabolites in the culture supernatant. Thus, oxidative phosphorylation appeared the main bioenergetic source for osteoclast formation. Conversely, we found that bone resorption mainly relied on glycolysis. In fact, osteoclast fuelling with galactose, forcing cells to depend on Oxidative Phosphorylation by reducing the rate of glycolysis, significantly impaired Type I collagen degradation, whereas non-cytotoxic doses of rotenone, an inhibitor of the mitochondrial complex I, enhanced osteoclast activity. Furthermore, we found that the enzymes associated to the glycolytic pathway are localised close to the actin ring of polarised osteoclasts, where energy-demanding activities associated with bone degradation take place. In conclusion, we demonstrate that the energy required for osteoclast differentiation mainly derives from mitochondrial oxidative metabolism, whereas the peripheral cellular activities associated with bone matrix degradation are supported by glycolysis. A better understanding of human osteoclast energy metabolism holds the potential for future therapeutic interventions aimed to target osteoclast activity in different pathological conditions of bone
Electrospun fibers coated with nanostructured biomimetic hydroxyapatite: A new platform for regeneration at the bone interfaces
Full text link: Reconstruction of gradient organic/inorganic tissues is a challenging task in orthopaedics. Indeed, to mimic tissue characteristics and stimulate bone regeneration at the interface, it is necessary to reproduce both the mineral and organic components of the tissue ECM, as well as the micro/nano-fibrous morphology. To address this goal, we propose here novel biomimetic patches obtained by the combination of electrospinning and nanostructured bone apatite. In particular, we deposited apatite on the electrospun fibers by Ionized Jet Deposition, a plasma-assisted technique that allows conformal deposition and the preservation in the coating of the target's stoichiometry. The damage to the substrate and fibrous morphology is a polymer-dependent aspect, that can be avoided by properly selecting the substrate composition and deposition parameters. In fact, all the tested polymers (poly(l-lactide), poly(D,l-lactide-co-glycolide, poly(ε-caprolactone), collagen) were effectively coated, and the morphological and thermal characterization revealed that poly(ε-caprolactone) suffered the least damage. The coating of collagen fibers, on the other hand, destroyed the fiber morphology and it could only be performed when collagen is blended with a more resistant synthetic polymer in the nanofibers. Due to the biomimetic composition and multiscale morphology from micro to nano, the poly(ε-caprolactone)-collagen biomimetic patches coated with bone apatite supported MSCs adhesion, patch colonization and early differentiation, while allowing optimal viability. The biomimetic coating allowed better scaffold colonization, promoting cell spreading on the fibers
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