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P15. Acute phase reactants in myositis ossificans progressiva and in acquired ectopic ossification
Abstracts from the Bone and Tooth Society Meeting osteoarthritic cartilage is abnormal. The enzyme alkaline phosphatase is thought to enhance calcification. Therefore, we have studied the distribution of alkaline phosphatase activity using an electron microscope histochemical technique in three different types of cartilage from human femoral heads. Disease free articular cartilage showed relatively little
reaction in any region either around chondrocytes or matrix vesicles. In osteourthritic cartilage, chondrocytes in the tide mark region (osteochondral junction) were highly reactive for alkaline phosphatase activity In addition, many matrix vesicles within the extracellular matrix reacted positively for alkaline phosphatase. These vesicles often showed initial signs of calcification (microcrystals or hydroxyapatite). In osteophytic cartilage there is an increased alkaline phosphatase activity around chondrocytes and matrix vesicles in both the deep and mid-zone cartilage. Again, there was correlation between signs of initial calcification and alkaline phosphatase activity. By using this technique we are now in a better position to localise alkaline phosphatase activity with the chondrocytes and matrix vesicles in different saggital depths of human articular cartilage and correlate it with the disease process.
Pl5. Acute phase reactants in myositis ossificans
progressiva and in acquired ectopic ossification SJ Forster,* JTTriffitt,* C Fergussont and R Smitht *MRC Bone Research Luborutoy, tNuffield Department of Orthopaedic Surgery, Nuffield Orthopuedic Centre, Headington, Oxford OX3 7LD
In the very rare connective tissue disorder myositis (fibrodysplasia) ossificans progressiva (MOP), ectopic ossification is preceded by pain and swelling in affected muscles. Similar signs of inflammation precede ectopic ossification which occurs after neurological injury or trauma. We have measured the plasma levels of a number of acute phase reactants (C-reactive protein, al antitrypsin, haptoglobin, orosomucoid and caeruloplasmin) in 6 patients with MOP, in 3 with trauma-induced tutopic ossification and in 3 normal controls. In patients with MOP the levels of all these components were normal in between episodes of ‘myositis: during which ai antitrypsin, haptoglobin and orosomucoid were markedly increased. The same three proteins were also elevated in the 3 patients where ectopic ossification was a delayed result of injury In all cases levels of C-reactive protein and caeruloplasmin were normal. A more detailed study of plasma orosomucoid in 12 MOP patients, 6 with trauma-induced ectopic ossification and 11 normal subjects showed a significant (p
265
and haptoglobin without any obvious clinical reason for this. We consider that the increase in some acute-phase reactants associated with ectopic bone formation is part of the usual response to inflammation. The apparent increase in orosomucoid concentration in unaffected relatives of MOP patients requires further study Fl6. Generation of osteoblast-like cells from longterm human marrow cultures in vitro DB Evans, M Thavarajah and JA Kanis Department of Human Metabolism &Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield SlO 2RX
The precursors of osteogenic and osteoclastic cells are found within the bone marrow microenvironment. We have previously described the generation of multinucleated osteoclast-like cells derived from a long term human bone marrow culture system (Thavarajah et al J Bone Miner Res 2, Suppl 1; 1987) to study osteoclast formation. After 3 weeks the majority of mononuclear cells possess characteristics of osteoclast precursors. We have found that continued culture results in the formation of non-osteoclastic mononuclear cells with features of an osteoblast phenotype. Human bone marrow cells were cultured in alpha MEM + 20% horse serum in the presence of 1,25dihydroxyvitamin D3 (1,25(OHhD3) and retinoic acid (RA). After 3 weeks, osteoclast-like mononuclear cells predominate as judged by the expression df tartrate resistant acid phosphatase and reactivity to an osteoclast specific antibody (13C2). On prolonged culture (48 weeks) the mononuclear cells generated possessed a polygonal/cuboidal morphology characteristic of osteoblast-like cells. These cells were passaged by trypsin/EDTA treatment and subcultured in Eagles MEM + 10% fetal calf serum for 7-14 days. Contrary to expectation, the cells expressed alkaline phosphatase activity assessed by histochemical and bioc emical methods, enzyme levels being increased by 1,5 (OH)zDj. Treatment with 1,25(OHbD3 and RA individually stimulated the production of osteocalcin, an osteoblast specific bone matrix protein. Retention of the osteoblast phenotype was maintained on subsequent cell passage. The stimulation of osteocalcin production by 1,25(OH)zDs was antagonised by interleukin 1 (IL-l) in a similar manner as described for human trabecular bone osteoblast-like cells. These observations indicate that the long-term marrow culture method employed may provide a useful system for the identification of factors which regulate the formation of, and subsequent differentiation of, cells of both the osteoclast and osteoblast lineage fro precursor stem populations within human marrow. ;f;, e system may also provide an in vitro model to study the cellular sequence of events associated with the skeletal remodelling process in viva.
reaction in any region either around chondrocytes or matrix vesicles. In osteourthritic cartilage, chondrocytes in the tide mark region (osteochondral junction) were highly reactive for alkaline phosphatase activity In addition, many matrix vesicles within the extracellular matrix reacted positively for alkaline phosphatase. These vesicles often showed initial signs of calcification (microcrystals or hydroxyapatite). In osteophytic cartilage there is an increased alkaline phosphatase activity around chondrocytes and matrix vesicles in both the deep and mid-zone cartilage. Again, there was correlation between signs of initial calcification and alkaline phosphatase activity. By using this technique we are now in a better position to localise alkaline phosphatase activity with the chondrocytes and matrix vesicles in different saggital depths of human articular cartilage and correlate it with the disease process.
Pl5. Acute phase reactants in myositis ossificans
progressiva and in acquired ectopic ossification SJ Forster,* JTTriffitt,* C Fergussont and R Smitht *MRC Bone Research Luborutoy, tNuffield Department of Orthopaedic Surgery, Nuffield Orthopuedic Centre, Headington, Oxford OX3 7LD
In the very rare connective tissue disorder myositis (fibrodysplasia) ossificans progressiva (MOP), ectopic ossification is preceded by pain and swelling in affected muscles. Similar signs of inflammation precede ectopic ossification which occurs after neurological injury or trauma. We have measured the plasma levels of a number of acute phase reactants (C-reactive protein, al antitrypsin, haptoglobin, orosomucoid and caeruloplasmin) in 6 patients with MOP, in 3 with trauma-induced tutopic ossification and in 3 normal controls. In patients with MOP the levels of all these components were normal in between episodes of ‘myositis: during which ai antitrypsin, haptoglobin and orosomucoid were markedly increased. The same three proteins were also elevated in the 3 patients where ectopic ossification was a delayed result of injury In all cases levels of C-reactive protein and caeruloplasmin were normal. A more detailed study of plasma orosomucoid in 12 MOP patients, 6 with trauma-induced ectopic ossification and 11 normal subjects showed a significant (p
265
and haptoglobin without any obvious clinical reason for this. We consider that the increase in some acute-phase reactants associated with ectopic bone formation is part of the usual response to inflammation. The apparent increase in orosomucoid concentration in unaffected relatives of MOP patients requires further study Fl6. Generation of osteoblast-like cells from longterm human marrow cultures in vitro DB Evans, M Thavarajah and JA Kanis Department of Human Metabolism &Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield SlO 2RX
The precursors of osteogenic and osteoclastic cells are found within the bone marrow microenvironment. We have previously described the generation of multinucleated osteoclast-like cells derived from a long term human bone marrow culture system (Thavarajah et al J Bone Miner Res 2, Suppl 1; 1987) to study osteoclast formation. After 3 weeks the majority of mononuclear cells possess characteristics of osteoclast precursors. We have found that continued culture results in the formation of non-osteoclastic mononuclear cells with features of an osteoblast phenotype. Human bone marrow cells were cultured in alpha MEM + 20% horse serum in the presence of 1,25dihydroxyvitamin D3 (1,25(OHhD3) and retinoic acid (RA). After 3 weeks, osteoclast-like mononuclear cells predominate as judged by the expression df tartrate resistant acid phosphatase and reactivity to an osteoclast specific antibody (13C2). On prolonged culture (48 weeks) the mononuclear cells generated possessed a polygonal/cuboidal morphology characteristic of osteoblast-like cells. These cells were passaged by trypsin/EDTA treatment and subcultured in Eagles MEM + 10% fetal calf serum for 7-14 days. Contrary to expectation, the cells expressed alkaline phosphatase activity assessed by histochemical and bioc emical methods, enzyme levels being increased by 1,5 (OH)zDj. Treatment with 1,25(OHbD3 and RA individually stimulated the production of osteocalcin, an osteoblast specific bone matrix protein. Retention of the osteoblast phenotype was maintained on subsequent cell passage. The stimulation of osteocalcin production by 1,25(OH)zDs was antagonised by interleukin 1 (IL-l) in a similar manner as described for human trabecular bone osteoblast-like cells. These observations indicate that the long-term marrow culture method employed may provide a useful system for the identification of factors which regulate the formation of, and subsequent differentiation of, cells of both the osteoclast and osteoblast lineage fro precursor stem populations within human marrow. ;f;, e system may also provide an in vitro model to study the cellular sequence of events associated with the skeletal remodelling process in viva.