Ryuichi Kasai

Synovial Fluids from Patients with Osteoarthritis and Rheumatoid Arthritis Contain High Levels of Parathyroid Hormone–Related Peptide†

High levels of immunoreactive and biologically active parathyroid hormone–related peptide (PTHrP) were detected in synovial fluids from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). The levels of PTHrP immunoreactivity in synovial fluids, measured by a two‐site immunoradiometric assay (IRMA) which detects hPTHrP(1–72) or longer peptides and a radioimmunoassay (RIA) specific to the carboxy‐terminal portion of hPTHrP, were 3.2 ± 0.3 pmol of hPTHrP(1–86)/l and 61 ± 7.0 pmol of hPTHrP(109–141)/l in OA patients (mean ± SE, n = 23), and 4.8 ± 0.8 pmol of hPTHrP(1–86)/l and 164 ± 30 pmol of hPTHrP(109–141)/l in RA patients (n = 26). Synovial fluid PTHrP levels distributed above the normal plasma reference ranges in each assay (0.7–2.6 pmol of hPTHrP(1–86)/l; 16–60.6 pmol of hPTHrP(109–141)/l). After concentration using sequential cation‐exchange and reverse‐phase chromatography, synovial fluid exhibited the activity that stimulated cyclic adenosine monophosphate (cAMP) accumulation in osteoblastic ROS 17/2.8 cells expressing PTH/PTHrP receptors. The cAMP accumulation activity in synovial fluid was sensitive to coincubation with excess hPTHrP(3–40), a PTH/PTHrP receptor antagonist, and was completely neutralized by preincubation with a monoclonal antibody specific to hPTHrP but not PTH. Immunohistochemical analysis of RA synovium revealed that PTHrP was localized in fibroblast‐like cells in the synovial pannus invading articular cartilage. Our data show that PTHrP is produced locally by the diseased synovial tissue and released into synovial fluid at high concentrations, allowing us to hypothesize that PTHrP plays a novel role as a paracrine/autocrine factor in the pathology of OA and RA.

Bone morphogenetic proteins (BMP‐2 and BMP‐3) induce the late phase expression of the proto‐oncogene c‐fos in murine osteoblastic MC3T3‐E1 cells

Here we report that bone morphogenetic proteins 2 and 3 (BMP‐2 and BMP‐3) induced marked expression of c‐fos mRNA in a biphasic manner, i.e. the late phase (48 to 60 h) as well as the immediate‐early phase (0.5 h), in murine osteoblastic MC3T3‐El cells in vitro. The BMP‐induced late phase c‐fos gene expression was temporally associated with the onset of marked expression of the genes for osteocalcin and alkaline phosphatase, differentiation markers of mature osteoblasts. In contrast, none of TGF‐β1, 10% FBS, IGF‐I and IGF‐II, which induced only the immediate‐early c‐fos mRNA expression, stimulated the expression of osteocalcin and alkaline phosphatase genes. These data suggest that in osteoblasts BMP‐2 and BMP‐3 induce the late phase expression of c‐fos, which may play a role in transcriptional activation of the genes involved in differentiation of osteoblasts.

Fracture healing induces expression of the proto-oncogene c-fos in vivo. Possible involvement of the Fos protein in osteoblastic differentiation.

Here we report marked in vivo expression of the c‐fos gene in the external soft callus (ESC) and periosteal hard callus (PHC) at the fracture site of adult rat tibia. Northern‐blot analysis showed that the ESC expressed a high level of c‐fos mRNA from post‐fracture day 10 to day 28, the time when endochondral ossification progressed, and that the ossifying PHC also expressed c‐fos mRNA. This c‐fos expression was followed by sequential expression of the genes for alkaline phosphatase, osteopontin and osteocalcin, which are osteoblastic markers. Immunohistochemical analysis showed that the c‐Fos protein was predominantly located in osteoblasts in the ossifying calluses.

Production and characterization of an antibody against the human bone GLA protein (BGP/osteocalcin) propeptide and its use in immunocytochemistry of bone cells.

We have generated and characterized an antibody that recognizes the C-terminal sequence of the propeptide of human bone GLA protein (BGP/osteocalcin)(amino acid -26 to -1, with +1 being the amino terminus of the mature protein). The range of sensitivity of the antibody, as determined by enzyme-linked immunosorbent assay (ELISA), was 0.5-250 ng/ml. The antibody effectively recognized pro-BGP in cell layer extracts of transformed cells (KT-005), but did not recognize mature, propeptide-less BGP in the medium from the same cultures. Strong labelling was obtained using this antibody in immunoperoxidase staining or immunofluorescence of both transformed and normal human bone cells in vitro. Monensin significantly altered the intracellular pattern of labelling in immunofluorescence studies, indicating that the recognized antigen was associated with the cellular secretory pathway. We also obtained a specific and strong staining of cells in tissue sections of human fetal bone. Antibodies against the mature protein strongly stained the mineralization front, but did not stain cells to any appreciable level. Newly embedded osteocytes were the predominant cell type stained in such material, suggesting that they may represent the major of BGP in the intact tissue. These observations indicate that BGP synthesis is a late event in osteoblastic development and that antibodies generated against the propeptide sequence are a potentially powerful tool in the analysis of bone tumors and evaluation of osteoblastic differentiation.

Socket location in total hip replacement Preoperative computed tomography and computer simulation

For choosing the size and location of the acetabular component, we have developed a three-dimensional simulation system based on computerized tomography and a microcomputer. In 34 cases of coxarthrosis secondary to congenital dysplasia or dislocation, the system provided valuable preoperative information.

The effect of active vitamin D3 analogs and dexamethasone on the expression of osteocalcin gene in rat tibiae in vivo

We tested the effects of 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3), 2 beta-(3-hydroxypropoxy)-1 alpha,25-dihydroxyvitamin D3 (ED-71) and dexamethasone on osteocalcin mRNA levels in rat tibiae in vivo. Northern blot analysis showed that both 1,25-(OH)2D3 and ED-71 caused an increase in osteocalcin mRNA levels in bone: 1,25-(OH)2D3 induced a transient increase in the mRNA levels followed by a decrease in the control level by 12 h post administration. In contrast, ED-71 caused a persistent increase in osteocalcin mRNA level for seven days post administration. Serum osteocalcin levels paralleled the osteocalcin mRNA level in bone in both groups. Dexamethasone caused a marked reduction in both osteocalcin mRNA and serum osteocalcin levels. Suppressive effect of dexamethasone on osteocalcin expression was persistent for seven days at higher dose. Our results represent the first demonstration of the effect of active vitamin D and corticosteroid on the expression of osteocalcin mRNA in bone in vivo.

Modification of strain-specific femoral bone density by bone marrow-derived factors administered neonatally: a study on the spontaneously osteoporotic mouse, SAMP6.

SAMP6 is a recently developed strain of osteoporotic mice, and SAMP2 is a control for SAMP6 and has a higher peak bone mass. The bone mass of SAMP6 was increased until 2 months of age when a lysate of cells derived from the bone marrow of SAMP2 was injected at 1 or 4 days of age, but it was not increased when the lysate was injected at 21 days of age. No effect on bone mass was observed when lysates of other cells, bovine serum albumin or heat-inactivated lysate of bone marrow-derived cells of SAMP2, were injected. The ability to increase bone mass was not in the supernatant but in the pellet obtained by ultracentrifugation (105,000 g) of the lysate of bone marrow-derived cells of SAMP2. The lysate did not change the osteoclast surface but changed the appositional bone formation. In conclusion, the lysate of cells derived from the bone marrow of SAMP2 contains factors which can increase the bone mass of SAMP6, and these factors are present in the pellet obtained by ultracentrifugation.

Severe metallosis due to abnormal abrasion of the femoral head in a dual-bearing hip prosthesis : a case report

The authors report on a patient with a case of severe metallosis due to an abnormal abrasion of the femoral head. A primary arthroplasty was performed using a dual bearing hip prosthesis with acetabular bone grafting by ceramic screws. At the time of the revision surgery the synovia was black, and an analysis using a scanning electron microscope and scanning electron microscope-electron probe micro-analyzer revealed numerous small particles of small alumina ceramic on the inner surface of the bearing insert of high-density polyethylene. These particles, which came from the broken ceramic screws due to proximal migration of the prosthesis, scraped the femoral head away. A line and area analysis of the black synovia revealed that the synovia contained metal particles of a cobalt-chromium alloy as well as a cobalt ion. The patients serum showed elevated concentrations of cobalt, chromium, and molybdenum that dramatically reduced 2 months after the revision surgery.

Expression of the c-fos gene induced by parathyroid hormone in the bones of SAMP6 mice, a murine model for senile osteoporosis.

SAMP6 mice are a murine model for senile osteoporosis, characterized by low peak bone mass seen at 4 or 5 months of age. Parathyroid hormone (PTH)-induced c-fos expression was examined in the bones, bone-marrow cells and kidney tissues of 2-month-old male SAMP6 mice. SAMP2 mice, which have a higher peak bone mass, were used as controls. The expression of c-fos in the bone peaked at 30 min after 60 microg/kg of human PTH(1-34) administration. After peaking, the expression fell quickly in SAMP2 mice. This decrease in expression was delayed in SAMP6 mice and the expression was higher at 1 h than in SAMP2 mice. The phenomenon observed in the bone appears to be tissue specific as it was not seen in the bone-marrow cells or kidney tissue. Immunohistochemical studies showed that c-Fos protein was localized to the nuclei of some of the osteocytes and a few of the osteoblasts in the cortical bone, and that osteocytes expressing c-Fos protein increased after PTH treatment. These results suggest that osteocytes might contribute to the maintenance of higher levels of c-fos expression in the bones of SAMP6 mice and may be related to cortical osteopenia in these mice by modulating bone remodeling and/or modeling.

Effect of dietary cadmium and/or copper on the bone lysyl oxidase in copper-deficient rats relative to the metabolism of copper in the bone.

Effects of cadmium (Cd) on lysyl oxidase activity and copper (Cu) metabolism in bone were studied using Cu-deficient rats supplemented with Cu and/or Cd in a diet. When fed for 8 weeks on a diet containing 0.3 ppm or less Cu (-Cu diet), weanling rats revealed anemia, and markedly decreased plasma ceruloplasmin activity and serum Cu to less than 15% of normal level, showing features of Cu-deficiency. These rats were divided into four groups and refed for another 2 weeks on the following diets: Group I, -Cu diet; Group II, -Cu diet with 50 ppm Cd (+Cd diet); Group III, -Cu diet supplemented with 15 ppm Cu (+Cu diet); group IV, -Cu diet with both Cu and Cd (+Cu/+Cd diet). After 2 weeks, serum Cu levels of Groups I, II, III and IV were 1.8, 0.8, 78 and 74% of the normal control level (1.438 +/- 0.060 micrograms/ml), respectively. Concentrations of Cu in epi- and metaphyses of the control group, Groups I, II, III and IV were 1.45 +/- 0.20, 0.67 +/- 0.08, 0.76 +/- 0.12, 1.40 +/- 0.31 and 1.22 +/- 0.05 micrograms/g wet tissue, in that order. Concentrations of Cd in epi- and metaphysis increased in only Groups II and IV and were 0.15 +/- 0.03 and 0.18 +/- 0.01 micrograms/g wet tissue, respectively. Thus, having both Cd and Cu supplements in a diet did not inhibit each others uptake into the tissue.(ABSTRACT TRUNCATED AT 250 WORDS)