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Salamanna F.,Rizzoli Research Innovation Technology | Martini L.,Rizzoli Research Innovation Technology | Martini L.,Rizzoli Orthopaedic Institute | Pagani S.,Rizzoli Research Innovation Technology | And 7 more authors.
Acta Histochemica | Year: 2013

Breast cancer frequently metastasizes to the skeleton thus interrupting the normal bone remodeling process and causing bone degradation. Having suitable in vitro and in vivo models is important for understanding the pathogenesis and developing treatment strategies for bone metastasis in humans.In order to improve and characterize an in vitro model of bone metastasis from breast cancer an MRMT-1 rat breast carcinoma cell line or their conditioned medium were directly co-cultured with rat monocytes. To confirm the in vitro results, an in vivo model, in which MRMT-1 cells were inoculated into the proximal surface of the tibia, was also adopted. Osteoclast viability, activity and differentiation showed a significant increase (p< 0.05, p< 0.0005, p< 0.0005, respectively) between co-culture with MRMT-1 cells and the other culture conditions. Moreover, scanning electron microscopy analysis, phalloidin staining and 4'-6-diamidino-2-phenylindole (DAPI) nuclear acid staining confirmed that co-culture with MRMT-1 cells also induced a greater differentiation in osteoclast structure and morphology. Finally, the in vivo outcome at 3 weeks showed the presence of a severe osteolytic lesion, thus confirming the effectiveness of the present in vitro results. These results demonstrated an improvement of an in vitro model of bone metastases from breast cancer in which co-culture with MRMT-1 cells was shown to be a dynamic system that closely mimics the in vivo situation. The present study may help improve our understanding of the complex " vicious cycle" between osteoblasts, osteoclasts and tumor cells. © 2012 Elsevier GmbH.


Salamanna F.,Rizzoli Research Innovation Technology | Maglio M.,Rizzoli Orthopaedic Institute | Giavaresi G.,Rizzoli Research Innovation Technology | Giavaresi G.,Rizzoli Orthopaedic Institute | And 6 more authors.
Age | Year: 2015

Bone loss occurs insidiously and initially asymptomatically; therefore, osteoporosis is frequently diagnosed only after the first clinical fracture. The aim of this study was to test the hypothesis is that by simply observing the behavior of cultured peripheral monocytes, it might be possible to diagnose altered bone remodeling and, therefore, limit the complications associated with osteoporosis, especially fractures. Monocytes isolated as mononuclear precursors from healthy and ovariectomized rats were cultured both in basal and differentiation medium for up to 3 weeks. Viability and differentiation capability towards the osteoclastic phenotype was checked by light microscopy at early times, whereas differentiation state and synthetic activity (tartrate-resistant acid phosphatase (TRAP) staining; phalloidin, fluorescin isothiocynate (FITC) staining, cathepsin K, metalloproteinase 7 and 9, MMP-7 and MMP-9) were measured at 1, 2, and 3 weeks. Compared to their controls, monocytes isolated from ovariectomized rats proliferate and lean toward the osteoclastic phenotype in the absence of differentiating factors. In both culture conditions, osteoclasts from ovariectomized rats showed significantly higher productions of cathepsin K, MMP-7, and MMP-9 than those of cells isolated from healthy rats, steadily over time. These results obtained in an animal osteoporotic model, if confirmed by clinical studies, open up the possibility to assess the presence of an alteration in bone remodeling with a simple in vitro diagnostic test requiring a small blood sample and less than 48 h. This might allow to early select patients with a spontaneous viability and differentiation of monocytes to osteoclasts for further diagnostic techniques. © 2015, American Aging Association.


PubMed | Rizzoli Research Innovation Technology
Type: Journal Article | Journal: Age (Dordrecht, Netherlands) | Year: 2015

Bone loss occurs insidiously and initially asymptomatically; therefore, osteoporosis is frequently diagnosed only after the first clinical fracture. The aim of this study was to test the hypothesis is that by simply observing the behavior of cultured peripheral monocytes, it might be possible to diagnose altered bone remodeling and, therefore, limit the complications associated with osteoporosis, especially fractures. Monocytes isolated as mononuclear precursors from healthy and ovariectomized rats were cultured both in basal and differentiation medium for up to 3 weeks. Viability and differentiation capability towards the osteoclastic phenotype was checked by light microscopy at early times, whereas differentiation state and synthetic activity (tartrate-resistant acid phosphatase (TRAP) staining; phalloidin, fluorescin isothiocynate (FITC) staining, cathepsin K, metalloproteinase 7 and 9, MMP-7 and MMP-9) were measured at 1, 2, and 3 weeks. Compared to their controls, monocytes isolated from ovariectomized rats proliferate and lean toward the osteoclastic phenotype in the absence of differentiating factors. In both culture conditions, osteoclasts from ovariectomized rats showed significantly higher productions of cathepsin K, MMP-7, and MMP-9 than those of cells isolated from healthy rats, steadily over time. These results obtained in an animal osteoporotic model, if confirmed by clinical studies, open up the possibility to assess the presence of an alteration in bone remodeling with a simple in vitro diagnostic test requiring a small blood sample and less than 48 h. This might allow to early select patients with a spontaneous viability and differentiation of monocytes to osteoclasts for further diagnostic techniques.


PubMed | Rizzoli Research Innovation Technology
Type: Journal Article | Journal: Acta histochemica | Year: 2012

Breast cancer frequently metastasizes to the skeleton thus interrupting the normal bone remodeling process and causing bone degradation. Having suitable in vitro and in vivo models is important for understanding the pathogenesis and developing treatment strategies for bone metastasis in humans. In order to improve and characterize an in vitro model of bone metastasis from breast cancer an MRMT-1 rat breast carcinoma cell line or their conditioned medium were directly co-cultured with rat monocytes. To confirm the in vitro results, an in vivo model, in which MRMT-1 cells were inoculated into the proximal surface of the tibia, was also adopted. Osteoclast viability, activity and differentiation showed a significant increase (p<0.05, p<0.0005, p<0.0005, respectively) between co-culture with MRMT-1 cells and the other culture conditions. Moreover, scanning electron microscopy analysis, phalloidin staining and 4-6-diamidino-2-phenylindole (DAPI) nuclear acid staining confirmed that co-culture with MRMT-1 cells also induced a greater differentiation in osteoclast structure and morphology. Finally, the in vivo outcome at 3 weeks showed the presence of a severe osteolytic lesion, thus confirming the effectiveness of the present in vitro results. These results demonstrated an improvement of an in vitro model of bone metastases from breast cancer in which co-culture with MRMT-1 cells was shown to be a dynamic system that closely mimics the in vivo situation. The present study may help improve our understanding of the complex vicious cycle between osteoblasts, osteoclasts and tumor cells.

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