Apoptotic mesenchymal stromal cells support osteoclastogenesis while inhibiting multinucleated giant cells formation in vitro.

In bone regeneration induced by the combination of mesenchymal stromal cells (MSCs) and calcium-phosphate (CaP) materials, osteoclasts emerge as a pivotal cell linking inflammation and bone formation. Favorable outcomes are observed despite short-term engraftments of implanted MSCs, highlighting their major paracrine function and the possible implication of cell death in modulating their secretions. In this work, we focused on the communication from MSCs towards osteoclasts-like cells in vitro. MSCs seeded on a CaP biomaterial or undergoing induced apoptosis produced a conditioned media favoring the development of osteoclasts from human CD14+ monocytes. On the contrary, MSCs' apoptotic secretion inhibited the development of inflammatory multinucleated giant cells formed after IL-4 stimulation. Components of MSCs' secretome before and after apoptotic stress were compared using mass spectrometry-based quantitative proteomics and a complementary immunoassay for major cytokines. CXCR-1 and CXCR-2 ligands, primarily IL-8/CXCL-8 but also the growth-regulated proteins CXCL-1, -2 or -3, were suggested as the major players of MSCs' pro-osteoclastic effect. These findings support the hypothesis that osteoclasts are key players in bone regeneration and suggest that apoptosis plays an important role in MSCs' effectiveness.

Zoledronic acid inhibits pulmonary metastasis dissemination in a preclinical model of Ewing's sarcoma via inhibition of cell migration.

BACKGROUND: Ewing's sarcoma (ES) is the second most frequent primitive malignant bone tumor in adolescents with a very poor prognosis for high risk patients, mainly when lung metastases are detected (overall survival <15% at 5 years). Zoledronic acid (ZA) is a potent inhibitor of bone resorption which induces osteoclast apoptosis. Our previous studies showed a strong therapeutic potential of ZA as it inhibits ES cell growth in vitro and ES primary tumor growth in vivo in a mouse model developed in bone site. However, no data are available on lung metastasis. Therefore, the aim of this study was to determine the effect of ZA on ES cell invasion and metastatic properties. METHODS: Invasion assays were performed in vitro in Boyden's chambers covered with Matrigel. Matrix Metalloproteinase (MMP) activity was analyzed by zymography in ES cell culture supernatant. In vivo, a relevant model of spontaneous lung metastases which disseminate from primary ES tumor was induced by the orthotopic injection of 106 human ES cells in the tibia medullar cavity of nude mice. The effect of ZA (50 µg/kg, 3x/week) was studied over a 4-week period. Lung metastases were observed macroscopically at autopsy and analysed by histology. RESULTS: ZA induced a strong inhibition of ES cell invasion, probably due to down regulation of MMP-2 and -9 activities as analyzed by zymography. In vivo, ZA inhibits the dissemination of spontaneous lung metastases from a primary ES tumor but had no effect on the growth of established lung metastases. CONCLUSION: These results suggest that ZA could be used early in the treatment of ES to inhibit bone tumor growth but also to prevent the early metastatic events to the lungs.

Novel RANK antagonists for the treatment of bone-resorptive disease: theoretical predictions and experimental validation.

Receptor activator of nuclear factor-κB (RANK) and RANK ligand (RANKL) play a pivotal role in bone metabolism, and selective targeting of RANK signaling has become a promising therapeutic strategy in the management of resorptive bone diseases. Existing antibody-based therapies and novel inhibitors currently in development were designed to target the ligand, rather than the membrane receptor expressed on osteoclast precursors. We describe here an alternative approach to designing small peptides able to specifically bind to the hinge region of membrane RANK responsible for the conformational change upon RANKL association. A nonapeptide generated by this method was validated for its biological activity in vitro and in vivo and served as a lead compound for the generation of a series of peptide RANK antagonists derived from the original sequence. Our study presents a structure- and knowledge-based strategy for the design of novel effective and affordable small peptide inhibitors specifically targeting the receptor RANK and opens a new therapeutic opportunity for the treatment of resorptive bone disease.