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oc mice
S32
ABSTRACTS / Bone 40 (2007) S22–S89
status. This study aimed to evaluate the usefulness of QUS in the monitoring RS parameters. Among the patients with RS referred to the Department of Child Neurology and Psychiatry we selected a cohort of 46 females (age range 5–30 years) for whom a follow-up of at least 4 years was available. In all at baseline and every 12 months over the 4-year study period we measured serum calcium, serum phosphate, alkaline phosphatase, parathyroid hormone and 25hydroxyvitamin D. At the same times we assessed QUS parameters at phalanxes by Bone Profiler-IGEA (amplitude dependent speed of sound: AD-SoS and bone transmission time: BTT). In our institution the precision was 0.8% for ADSoS and 0.6% for BTT. The patients were divided into three groups: non-ambulatory (group 1; n = 11), with severe (group 2; n = 10) or mild-moderate (group 3; n = 25) ambulatory impairment. At baseline no significant differences in QUS parameters were found between the ambulatory and non-ambulatory patients. However, from the first year and thereafter during the whole 4-year study period, the non-ambulatory patients presented a reduction in both AD-SoS and BTT significantly (p b 0.01) higher with respect to the other 2 groups. At the end of the study the change in AD-SoS Z-score was-0.60 for group 1,0.40 for group 2 and 0.10 for group 3. At baseline and at all follow-up time points alkaline phosphatase was significantly higher in non-ambulatory group. The RS patients were divided in tertiles on the basis of the dose of anticonvulsants (carbamazepine) and we found that at baseline both AD-SoS and BTT were significantly lower in the group with the higher dose with respect to the group with the lower dose of anticonvulsants (1838 vs. 1879 and 0.50 vs. 0.74 respectively). During the study period in the higher tertile BTT showed a tendency to decrease without reaching statistical significance. In conclusion QUS parameters seem to be a valuable tool in the monitoring of the patients with RS. The ambulatory ability and the use of anticonvulsants seem to be the main determinants of bone status in patients with RS.
logical damages and other dysfunctions. HSCT may provide the bone with functional osteoclasts, but their appearance is a slow process, during which disease progression continues. We hypothesise that such outcome could be prevented if readily available committed osteoclast precursors were injected. We optimised the method to obtain cells that be suitable for therapy and preliminarily tested engraftment and phenotype improvement in animal models. We established a procedure to obtain the best yields of osteoclasts from adult PBMCs exposing the cells for 7 days to 50 ng/ml SCF, 20 ng/ml IL−3 and 20 ng/ml IL−6, and for further 7 days with 20 ng/ml GM−CSF. Cells were then cultured for 21 days in the presence of 25 ng/ml M−CSF and 30 ng/ml RANKL. This procedure was applied to PBMCs, CD14+ and CD34+ cells cultured in regular DMEM or in Invitrogen StemPro34 medium, specifically formulated for the survival and expansion of the HSC population. Best osteoclast yield and performance was obtained culturing PBMCs or CD34+ cells in StemPro34 medium, which resulted in a higher number of osteoclasts, which were also larger and resorbed bone more efficiently than osteoclasts obtained in regular DMEM. These cells could be cryopreserved at − 80 °C in a medium of 90% FBS and 10% DMSO retaining the ability to differentiate into osteoclasts with an yield indistinguishable from that of freshly isolated cells. Similar positive results were obtained cryopreserving with identical procedure human and mouse committed osteoclast precursors prepared as described above. Human osteoclasts were also fluorescein-labelled, and GFP-actin expressing osteoclasts were obtained from mouse bone marrow precursors. These cells were injected in vivo in animal models after 1-day of treatment with M-CSF and RANKL, and showed the ability to form multinucleated osteoclasts and to improve to some extent the osteopetrotic phenotype of oc/oc mice. These animals presented with 30% reduction of bone volume, improved haematopoiesis and reduced fibrosis, eruption of tooth rudiments and slightly longer survival compared to sham-treated mice. These results provide first hand information on the feasibility of an osteoclast therapy in osteopetrosis.
doi:10.1016/j.bone.2007.04.021 doi:10.1016/j.bone.2007.04.022
Inoculation of osteoclast precursors improves the phenotype of osteopetrotic oc/oc mice A. Cappariello 1, A. Del Fattore 2, A.C. Berardi 3, B. Peruzzi 2, N. Rucci 2, M. Longo 4, A. Ugazio 3, G.F. Bottazzo 3, A. Teti 2 1 Università dell’Aquila and Ostedale Pediatrico Bambino Gesù, L’Aquila/Rome, Italy 2 Università dell’Aquila, L’Aquila, Italy 3 Ostedale Pediatrico Bambino Gesù, Rome, Italy 4 Università dell’Aquila, Rome, Italy Osteopetrosis is a genetic disease characterised by defective osteoclasts. The autosomal recessive (ARO) malignant form is fatal, and patients who survive with intermediate forms have poor life quality. Haematopoietic Stem Cell Transplantation (HSCT) cures b 50% of cases but often leaves severe neuro-
Bone mass and architecture in children with and without fractures W. Carlino 1, T.N. Hangartner 2, H.Z. Blades 1, Y. Vickers 3, N.J. Bishop 1 1 Academic Child Health, University of Sheffield, Sheffield, United Kingdom 2 Biomedical Imaging Laboratory, Wright State University, Dayton, OH 3 Radiology, Sheffield Children’s Hospital, Sheffield, United Kingdom We have shown previously that children who fracture have reduced bone area and bone mass for body size. We hypothesised altered bone architecture should be demonstrable in children with a fracture compared to those without.
ABSTRACTS / Bone 40 (2007) S22–S89
status. This study aimed to evaluate the usefulness of QUS in the monitoring RS parameters. Among the patients with RS referred to the Department of Child Neurology and Psychiatry we selected a cohort of 46 females (age range 5–30 years) for whom a follow-up of at least 4 years was available. In all at baseline and every 12 months over the 4-year study period we measured serum calcium, serum phosphate, alkaline phosphatase, parathyroid hormone and 25hydroxyvitamin D. At the same times we assessed QUS parameters at phalanxes by Bone Profiler-IGEA (amplitude dependent speed of sound: AD-SoS and bone transmission time: BTT). In our institution the precision was 0.8% for ADSoS and 0.6% for BTT. The patients were divided into three groups: non-ambulatory (group 1; n = 11), with severe (group 2; n = 10) or mild-moderate (group 3; n = 25) ambulatory impairment. At baseline no significant differences in QUS parameters were found between the ambulatory and non-ambulatory patients. However, from the first year and thereafter during the whole 4-year study period, the non-ambulatory patients presented a reduction in both AD-SoS and BTT significantly (p b 0.01) higher with respect to the other 2 groups. At the end of the study the change in AD-SoS Z-score was-0.60 for group 1,0.40 for group 2 and 0.10 for group 3. At baseline and at all follow-up time points alkaline phosphatase was significantly higher in non-ambulatory group. The RS patients were divided in tertiles on the basis of the dose of anticonvulsants (carbamazepine) and we found that at baseline both AD-SoS and BTT were significantly lower in the group with the higher dose with respect to the group with the lower dose of anticonvulsants (1838 vs. 1879 and 0.50 vs. 0.74 respectively). During the study period in the higher tertile BTT showed a tendency to decrease without reaching statistical significance. In conclusion QUS parameters seem to be a valuable tool in the monitoring of the patients with RS. The ambulatory ability and the use of anticonvulsants seem to be the main determinants of bone status in patients with RS.
logical damages and other dysfunctions. HSCT may provide the bone with functional osteoclasts, but their appearance is a slow process, during which disease progression continues. We hypothesise that such outcome could be prevented if readily available committed osteoclast precursors were injected. We optimised the method to obtain cells that be suitable for therapy and preliminarily tested engraftment and phenotype improvement in animal models. We established a procedure to obtain the best yields of osteoclasts from adult PBMCs exposing the cells for 7 days to 50 ng/ml SCF, 20 ng/ml IL−3 and 20 ng/ml IL−6, and for further 7 days with 20 ng/ml GM−CSF. Cells were then cultured for 21 days in the presence of 25 ng/ml M−CSF and 30 ng/ml RANKL. This procedure was applied to PBMCs, CD14+ and CD34+ cells cultured in regular DMEM or in Invitrogen StemPro34 medium, specifically formulated for the survival and expansion of the HSC population. Best osteoclast yield and performance was obtained culturing PBMCs or CD34+ cells in StemPro34 medium, which resulted in a higher number of osteoclasts, which were also larger and resorbed bone more efficiently than osteoclasts obtained in regular DMEM. These cells could be cryopreserved at − 80 °C in a medium of 90% FBS and 10% DMSO retaining the ability to differentiate into osteoclasts with an yield indistinguishable from that of freshly isolated cells. Similar positive results were obtained cryopreserving with identical procedure human and mouse committed osteoclast precursors prepared as described above. Human osteoclasts were also fluorescein-labelled, and GFP-actin expressing osteoclasts were obtained from mouse bone marrow precursors. These cells were injected in vivo in animal models after 1-day of treatment with M-CSF and RANKL, and showed the ability to form multinucleated osteoclasts and to improve to some extent the osteopetrotic phenotype of oc/oc mice. These animals presented with 30% reduction of bone volume, improved haematopoiesis and reduced fibrosis, eruption of tooth rudiments and slightly longer survival compared to sham-treated mice. These results provide first hand information on the feasibility of an osteoclast therapy in osteopetrosis.
doi:10.1016/j.bone.2007.04.021 doi:10.1016/j.bone.2007.04.022
Inoculation of osteoclast precursors improves the phenotype of osteopetrotic oc/oc mice A. Cappariello 1, A. Del Fattore 2, A.C. Berardi 3, B. Peruzzi 2, N. Rucci 2, M. Longo 4, A. Ugazio 3, G.F. Bottazzo 3, A. Teti 2 1 Università dell’Aquila and Ostedale Pediatrico Bambino Gesù, L’Aquila/Rome, Italy 2 Università dell’Aquila, L’Aquila, Italy 3 Ostedale Pediatrico Bambino Gesù, Rome, Italy 4 Università dell’Aquila, Rome, Italy Osteopetrosis is a genetic disease characterised by defective osteoclasts. The autosomal recessive (ARO) malignant form is fatal, and patients who survive with intermediate forms have poor life quality. Haematopoietic Stem Cell Transplantation (HSCT) cures b 50% of cases but often leaves severe neuro-
Bone mass and architecture in children with and without fractures W. Carlino 1, T.N. Hangartner 2, H.Z. Blades 1, Y. Vickers 3, N.J. Bishop 1 1 Academic Child Health, University of Sheffield, Sheffield, United Kingdom 2 Biomedical Imaging Laboratory, Wright State University, Dayton, OH 3 Radiology, Sheffield Children’s Hospital, Sheffield, United Kingdom We have shown previously that children who fracture have reduced bone area and bone mass for body size. We hypothesised altered bone architecture should be demonstrable in children with a fracture compared to those without.