Oncostatin m, an inflammatory cytokine produced by macrophages, supports intramembranous bone healing in a mouse model of tibia injury.

Different macrophage depletion strategies have demonstrated a vital role of macrophages in bone healing, but the underlying molecular mechanisms are poorly understood. Here, with the use of a mouse model of tibia injury, we found that the cytokine oncostatin M [OSM or murine (m)OSM] was overexpressed during the initial inflammatory phase and that depletion of macrophages repressed mOSM expression. In Osm(-/-) mice, by micro-computed tomography and histology we observed a significant reduction in the amount of new intramedullar woven bone formed at the injured site, reduced number of Osterix(+) osteoblastic cells, and reduced expression of the osteoblast markers runt-related transcription factor 2 and alkaline phosphatase. In contrast, osteoclasts were normal throughout the healing period. One day after bone injury, Stat3, the main transcription factor activated by mOSM, was found phosphorylated/activated in endosteal osteoblastic cells located at the hedge of the hematoma. Interestingly, we observed reduced activation of Stat3 in Osm(-/-) mice. In addition, mice deficient in the mOSM receptor (Osmr(-/-)) also had reduced bone formation and osteoblast number within the injury site. These results suggest that mOSM, a product of macrophages, sustains intramembranous bone formation by signaling through Osmr and Stat3, acting on the recruitment, proliferation, and/or osteoblast differentiation of endosteal mesenchymal progenitor cells. Because bone resorption is largely unaltered, OSM could represent a new anabolic treatment for unconsolidated bone fractures.

Dynamitin affects cell-surface expression of voltage-gated sodium channel Nav1.5.

The major cardiac voltage-gated sodium channel Nav1.5 associates with proteins that regulate its biosynthesis, localization, activity and degradation. Identification of partner proteins is crucial for a better understanding of the channel regulation. Using a yeast two-hybrid screen, we identified dynamitin as a Nav1.5-interacting protein. Dynamitin is part of the microtubule-binding multiprotein complex dynactin. When overexpressed it is a potent inhibitor of dynein/kinesin-mediated transport along the microtubules by disrupting the dynactin complex and dissociating cargoes from microtubules. The use of deletion constructs showed that the C-terminal domain of dynamitin is essential for binding to the first intracellular interdomain of Nav1.5. Co-immunoprecipitation assays confirmed the association between Nav1.5 and dynamitin in mouse heart extracts. Immunostaining experiments showed that dynamitin and Nav1.5 co-localize at intercalated discs of mouse cardiomyocytes. The whole-cell patch-clamp technique was applied to test the functional link between Nav1.5 and dynamitin. Dynamitin overexpression in HEK-293 (human embryonic kidney 293) cells expressing Nav1.5 resulted in a decrease in sodium current density in the membrane with no modification of the channel-gating properties. Biotinylation experiments produced similar information with a reduction in Nav1.5 at the cell surface when dynactin-dependent transport was inhibited. The present study strongly suggests that dynamitin is involved in the regulation of Nav1.5 cell-surface density.

3D cell culture and osteogenic differentiation of human bone marrow stromal cells plated onto jet-sprayed or electrospun micro-fiber scaffolds.

A major limitation of the 2D culture systems is that they fail to recapitulate the in vivo 3D cellular microenvironment whereby cell-cell and cell-extracellular matrix (ECM) interactions occur. In this paper, a biomaterial scaffold that mimics the structure of collagen fibers was produced by jet-spraying. This micro-fiber polycaprolactone (PCL) scaffold was evaluated for 3D culture of human bone marrow mesenchymal stromal cells (MSCs) in comparison with a commercially available electrospun scaffold. The jet-sprayed scaffolds had larger pore diameters, greater porosity, smaller diameter fibers, and more heterogeneous fiber diameter size distribution compared to the electrospun scaffolds. Cells on jet-sprayed constructs exhibited spread morphology with abundant cytoskeleton staining, whereas MSCs on electrospun scaffolds appeared less extended with fewer actin filaments. MSC proliferation and cell infiltration occurred at a faster rate on jet-sprayed compared to electrospun scaffolds. Osteogenic differentiation of MSCs and ECM production as measured by ALP, collagen and calcium deposition was superior on jet-sprayed compared to electrospun scaffolds. The jet-sprayed scaffold which mimics the native ECM and permits homogeneous cell infiltration is important for 3D in vitro applications such as bone cellular interaction studies or drug testing, as well as bone tissue engineering strategies.

Bone tissue formation with human mesenchymal stem cells and biphasic calcium phosphate ceramics: the local implication of osteoclasts and macrophages.

Human mesenchymal stem cells (hMSC) have immunomodulative properties and, associated with calcium phosphate (CaP) ceramics, induce bone tissue repair. However, the mechanisms of osteoinduction by hMSC with CaP are not clearly established, in particular the role of osteoclasts and macrophages. Biphasic calcium phosphate (BCP) particles were implanted with or without hMSC in the paratibial muscles of nude mice. hMSC increased osteoblastic gene expression at 1 week, the presence of macrophages at 2 and 4 weeks, osteoclastogenesis at 4 and 8 weeks, and osteogenesis at 4 and 8 weeks. hMSC disappeared from the implantation site after 2 weeks, indicating that hMSC were inducers rather than effectors of bone formation. Induced blockage of osteoclastogenesis by anti-Rankl treatment significantly impaired bone formation, revealing the pivotal role of osteoclasts in bone formation. In summary, hMSC positively influence the body foreign reaction by attracting circulating haematopoietic stem cells and inducing their differentiation into macrophages M1 and osteoclasts, thus favouring bone formation.

Osteoblastic and osteoclastic differentiation of human mesenchymal stem cells and monocytes in a miniaturized three-dimensional culture with mineral granules.

The pathologies of the skeleton have a significant socioeconomic impact on our population. Although therapies have improved the treatment of osteosarcoma and osteoporosis, their efficacy still remains limited. In this context, we developed a miniaturized 3-D culture model of bone cells on calcium phosphate ceramics. Human bone marrow mesenchymal stem cells (MSCs) were three-dimensionally cultured on particles of biphasic calcium phosphate (BCP, 125-200µm) in osteogenic media. The MSCs seeded on the BCP particles adhered and proliferated, producing abundant collagenous extracellular matrix (ECM). Light and confocal laser scanning microscopy showed that the MSCs created bridges between the BCP particles and formed a 3-D structure. Energy dispersive X-ray analysis in a scanning electron microscope confirmed the mineralization of the collagen matrix. The 96-well sized bone constructs were tested by immunohistology and transcription analysis, proving cell differentiation. Both techniques corroborated the osteoblastic differentiation with high production of bone sialoprotein and osteocalcin. Peripheral blood CD14-positive monocytes (MOs) were pre-differentiated into osteoclasts prior to seeding on the 3-D constructs. Multinucleated and tartrate-resistant acid phosphatase-positive cells were also identified at the surface of the 3-D constructs after 90days of culture. In addition, cell viability within these constructs was measured by flow cytometry. In summary, we have developed a miniaturized 3-D culture of bone cell precursors with osteoblasts and osteoclasts. This 3-D culture may make it possible to test the effects of new drugs for bone healing, osteoporosis and osteosarcomas, in more appropriate cell-cell and cell-matrix interactions than conventional 2-D cultures.

Liposomal clodronate inhibition of osteoclastogenesis and osteoinduction by submicrostructured beta-tricalcium phosphate.

Bone graft substitutes such as calcium phosphates are subject to the innate inflammatory reaction, which may bear important consequences for bone regeneration. We speculate that the surface architecture of osteoinductive ß-tricalcium phosphate (TCP) stimulates the differentiation of invading monocyte/macrophages into osteoclasts, and that these cells may be essential to ectopic bone formation. To test this, porous TCP cubes with either submicron-scale surface architecture known to induce ectopic bone formation (TCPs, positive control) or micron-scale, non-osteoinductive surface architecture (TCPb, negative control) were subcutaneously implanted on the backs of FVB strain mice for 12 weeks. Additional TCPs samples received local, weekly injections of liposome-encapsulated clodronate (TCPs + LipClod) to deplete invading monocyte/macrophages. TCPs induced osteoclast formation, evident by positive tartrate resistant acid phosphatase (TRAP) cytochemical staining and negative macrophage membrane marker F4/80 immunostaining. No TRAP positive cells were found in TCPb or TCPs + LipClod, only F4/80 positive macrophages and foreign body giant cells. TCPs stimulated subcutaneous bone formation in all implants, while no bone could be found in TCPb or TCPs + LipClod. In agreement, expression of bone and osteoclast gene markers was upregulated in TCPs versus both TCPb and TCPs + LipClod, which were equivalent. In summary, submicron-scale surface structure of TCP induced osteoclastogenesis and ectopic bone formation in a process that is blocked by monocyte/macrophage depletion.