No abstract

Bone metastases contribute to morbidity in patients with common cancers, and conventional therapy provides only palliation and can induce systemic side effects. The development of nanostructured delivery systems that combine carriers with bone-targeting molecules can potentially overcome the drawbacks presented by conventional approaches. We have recently developed biodegradable, biocompatible nanoparticles (NP) made of a conjugate between poly (D,L-lactide-co-glycolic) acid and alendronate, suitable for systemic administration, and directly targeting the site of tumor-induced osteolysis. Here, we loaded NP with doxorubicin (DXR), and analyzed the in vitro and in vivo activity of the drug encapsulated in the carrier system. After confirming the intracellular uptake of DXR-loaded NP, we evaluated the anti-tumor effects in a panel of human cell lines, representative for primary or metastatic bone tumors, and in an orthotopic mouse model of breast cancer bone metastases. In vitro, both free DXR and DXR-loaded NP, (58-580 ng/mL) determined a significant dose-dependent growth inhibition of all cell lines. Similarly, both DXR-loaded NP and free DXR reduced the incidence of metastases in mice. Unloaded NP were ineffective, although both DXR-loaded and unloaded NP significantly reduced the osteoclast number at the tumor site (P = 0.014, P = 0.040, respectively), possibly as a consequence of alendronate activity. In summary, NP may act effectively as a delivery system of anticancer drugs to the bone, and deserve further evaluation for the treatment of bone tumors.

The aim of the study was to obtain the highest number of multipotent adipose-derived mesenchymal stem cells (ADMSCs) by using culture conditions which favour cell expansion without loss of mesenchymal stem cells (MSC)-like properties. Based on the assumption that stem cells reside in niches characterized by hypoxic condition, we investigated if the low oxygen tension may improve the proliferation and stemness of ADMSCs. Intact adipose tissue was resected from eight subjects, and the stromal vascular fraction was obtained by using type II collagenase. The heterogeneity of cellular lineages was confirmed by immunophenotypic analysis that showed the presence of leukocytes (CD45+), endothelial cells (CD34+), and pericytes (CD140+). The immunophenotype of confluent ADMSCs was similar to that of bone marrow-derived MSCs, except for the expression of CD34, which was variable (donor-dependent) and inversely correlated to the CD36 expression. ADMSCs showed a high clonal efficiency (94.5 ± 1 %) and were able to generate osteoblastic, chondrocytic and adipocytic lineages. ADMSCs were cultured under normoxic (21 % O2) and hypoxic (1 % O2) conditions, and we found that hypoxia significantly favoured ADMSC proliferation and preserved the expression of stemness genes, i.e. Nanog and Sox2. Since hypoxia reflects the microenvironment in which ADMSCs must proliferate and differentiate, the culture in hypoxic condition allows to better understand the biology of these cells and their regenerative potential. Low oxygen concentrations promote cell proliferation and stemness, thus enriching the pool of cells potentially able to differentiate into multi-lineages, and extending the possibility of a long-term expansion.

The prepared conjugate represents a novel biomaterial that is able to provide nanoparticles, which can be further loaded with drugs, such as anticancer agents, and addressed to osteolytic or other bone diseases.

No abstract

Platelet-rich plasma is used to accelerate bone repair for the release of osteogenic growth factors from activated platelets. To date, the effects on osteoclasts have been only scarcely investigated, even though these cells are crucial for bone remodeling. The aim of this research was the evaluation of the effects of thrombin-activated platelets (PRP) on osteoclastogenesis from human blood precursors. We evaluated both the ability to influence osteoclast differentiation induced by the receptor activator of nuclear factor-kappaB ligand (RANKL), and the ability to induce osteoclast differentiation without RANKL. In both assays, the incubation with PRP supernatant at 10% did not significantly affect the formation of tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cells that were able to form the F-actin ring. However, when PRP at 25 and 50% was added to the medium without RANKL, the generation of TRACP-positive multinucleated cells was inhibited. PRP, even at 10%, reduced the osteoclast-mediated bone collagen degradation, suggesting inhibition of osteoclast activation. Similarly, after incubation with PRP supernatant, calcitonin receptor mRNA was lower than the untreated samples. In conclusion, PRP at 10% interfered with the complete differentiation process of human osteoclast precursors. At higher concentration it impaired osteoclast formation also at an early stage of differentiation. ß

Despite significant clinical improvements, conventional therapies for bone cancer treatment are limited by significant systemic toxicity and lack of specific targeting. In this study, we considered Alizarin, a natural hydroxyanthraquinone derived from madder root with high affinity to calcium and remarkable osteotropic features, as a novel approach for bone cancer treatment. Due to its antitumor properties, as demostrated in colon cancer cells, and to its tropism to bone, Alizarin may be an ideal drug to reduce bone tumor growth. We demonstrated that low dosages of Alizarin strongly inhibited the osteosarcoma (IC 50 for Saos-2, MG-63, and U-2 OS cells, 27.5, 29.0, and 69.9 mg/ml, respectively) and breast carcinoma (IC 50 for MDA-MB-231 cells, 62.1 mg/ml) cell proliferation in vitro. Importantly, Alizarin had a significantly lower inhibitory activity on normal cells (IC 50 for MSC, 828.6 mg/ml), thereby revealing a selective activity towards malignant cells. Furthermore, we found that Alizarin acted through the inhibition of ERK phosphorylation and cell cycle arrest in the S-phase. Finally, Alizarin significantly and strongly impaired both osteosarcoma and breast cancer tumorigenesis. Our results highlight a selective and effective inhibitory activity of Alizarin towards cancerous cells, laying the basis for further studies to investigate its application in bone cancer therapy.

Mesenchymal stem cells (MSC) have been widely used in orthopedics for several applications. Conventionally, MSC are maintained under 21% O2 which does not reflect the real O2 tension in vivo. Recently, it was reported that different O2 conditions can give different cellular responses. Here, we investigated whether prolonged exposure to hypoxia affects the osteogenic differentiation of adipose-derived stem cells (ASC). ASC from six individuals were cultured under "low" (2-3%) or "air" (21%) oxygen tensions, either without or with osteogenic stimuli. The effect of the O2 tension was evaluated on cell proliferation, surface antigens, stemness and bone-related genes expression, alkaline phosphatase activity (ALP), mineralization activity, and release of osteogenic growth factors. Without differentiating stimuli, hypoxia favored ASC proliferation, reduced the number of CD184+ and CD34+ cells, and preserved the expression of NANOG and SOX2. The combination of hypoxia and osteogenic medium induced a high proliferation rate, a rapid and more pronounced mineralization activity, a higher expression of genes related to the MSC differentiation, a higher release of mitogenic growth factors (bFGF, PDGF-BB), and the decrease in TGF-β secretion, an inhibitor of the early stage of the osteoblast differentiation. We demonstrated that hypoxia acts dually, favoring ASC proliferation and the maintenance of the stemness in the absence of osteogenic stimuli, but inducing the differentiation in a bone-like microenvironment. In conclusion, prolonged cell culture in hypoxic microenvironment represents a proper method to modulate the stem cell function that may be used in several applications, for example, studies on bone pathophysiology or bone-tissue engineering.