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Calcified deposits (CDs) in skin and muscles are common in juvenile dermatomyositis (DM), and less frequent in adult DM. Limited information exists about the microstructure and composition of these deposits, and no information is available on their elemental composition and contents, mineral density (MD) and stiffness. We determined the microstructure, chemical composition, MD and stiffness of CDs obtained from DM patients.
Gain-of-function mutations in LRP5 have been shown to cause high BMD disorders showing variable expression of some clinical symptoms, including torus palatinus and neurological complications. In an extended family, we were able to add craniosynostosis and developmental delay to the clinical spectrum associated with LRP5 mutations. We report on an extended four-generation family with 13 affected individuals (7 men and 6 women) in which an autosomal dominant type of osteosclerosis segregates. Osteosclerosis was most pronounced in the cranial base and calvarium, starting in early childhood with variable expression and a progressive character. Craniosynostosis at an early age was reported in four affected family members (two males and two females). The patients also presented with dysmorphic features (macrocephaly, brachycephaly, wide and high forehead, hypertelorism, prominent cheekbones, prominent jaw). They have normal height and proportions. Neurological complications like entrapment of cranial nerves resulting in optical nerve atrophy, hearing loss, and facial palsy were reported in two individuals. A mild developmental delay was reported in three affected individuals. None of the patients have torus palatinus, increased rate of fractures, osteomyelitis, hepatosplenomegaly, or pancytopenia. A missense mutation 640G-->A (A214T) in the low-density lipoprotein receptor-related protein 5 (LRP5) gene was found in all affected individuals analyzed, including cases in whom craniosynostosis, a mild developmental delay, and/or macrocephaly is observed. To our knowledge, this is the first report in the literature of patients presenting with autosomal dominant osteosclerosis in whom a variable expression of craniosynostosis, macrocephaly, and mild developmental delay is observed, which is most likely associated with a mutation in the LRP5 gene. These phenotypes can therefore be added to the clinical spectrum of LRP5-associated bone disorders.
Interleukin-4 (IL-4) potently inhibits bone resorption by preventing the differentiation of osteoclast precursors to osteoclasts. To elucidate the role of IL-4 in bone formation, we studied the effects of human IL-4 on human osteoblast-like cells obtained from trabecular bone, which showed increased osteocalcin production in response to 1,25-(OH)2D3 in more than 10 passages. IL-4 stimulated the proliferation of osteoblast-like cells in a concentration-dependent manner, showing the minimal and maximal stimulatory effects at 10 pg/ml and 100-1000 pg/ml, respectively. IL-4 also stimulated the expression of alkaline phosphatase mRNA (1.7-fold) and the enzyme activity to the same extent at 10-100 pg/ml. Furthermore, IL-4 stimulated collagen type I mRNA expression in human osteoblast-like cells. The cytokine did not affect osteocalcin production in a short culture period (3 days). These in vitro findings suggest that IL-4, a bone-resorption-inhibitory cytokine produced by activated T cells in bone marrow, may exert an anabolic effect on osteoblast-like cells in trabecular bone through a paracrine mechanism.
In this study, we investigated the relation between calcium kinetic indices of bone remodeling (resorption rate, r; and formation rate, m, respectively) and two serum markers of type I collagen turnover: the pyridinoline cross-linked carboxyterminal telopeptide domains of type I collagen (S-ICTP a marker of bone matrix degradation) and the carboxyterminal propeptide of human type I procollagen (S-PICP, a marker of bone matrix formation). We studied three groups: (i) healthy controls (n = 19), (ii) a mixed group of high and low-turnover bone diseases without mineralization defects (myxedema, thyrotoxicosis and primary hyperparathyroidism n = 38), and (iii) osteoporosis (n = 52). In healthy controls, a significant regression of S-PICP on m was obtained (R = 0.53, SEE/Y = 0.44, P < 0.02). Significant regressions were also demonstrable in high- and low-turnover bone disease (R = 0.50, P < 0.001), SEE/Y = 61%) and osteoporosis (R = 0.49, P < 0.001, SEE/Y = 50%). In controls the regression coefficient for the regression of S-ICTP on r was 0.19 (NS), in high and low turnover bone disease 0.66, (SEE/Y = 59%, P < 0.001) and in the osteoporotic group 0.40 (SEE/Y = 61%, P < 0.01). We conclude that S-PICP and S-ICTP reflect whole skeletal bone formation and resorption rates in a variety of metabolic bone diseases including osteoporosis.
Bone loss during androgen deficiency has been associated with accelerated bone turnover and imbalance between bone formation and resorption but the relative increase of both phenomena is not well described. Serum osteocalcin as marker of bone formation and urinary excretion of pyridinoline (PYD) and deoxypyridinoline (DPD) as markers of bone resorption were measured in both orchidectomized (ORCH, n = 8) and sham-operated (SHAM, n = 8) aged (12-month-old) male rats from 2 days before until 66 days after surgery. PYD and DPD were significantly higher in the ORCH group compared to the SHAM group starting from 21 days after surgery until the end of the experiment. Serum osteocalcin was only significantly increased in the ORCH group at 30 and 40 days. The maximal increase of serum osteocalcin was also smaller than the increase in PYD and DPD (30% versus 74% and 112%, respectively). The two markers of bone resorption were correlated with osteocalcin (r = 0.63 for PYD and r = 0.71 for DPD). Based on these results, we conclude that (1) bone resorption, as measured by PYD and DPD, increased during androgen deficiency; (2) moreover, the increase of bone resorption, as measured by DPD and PYD, was followed by a more moderate increase in bone formation as measured by serum osteocalcin, supporting the hypothesis that androgen deficiency causes accelerated bone turnover and imbalance between bone resorption and bone formation.
Bone mineral density (BMD) is the major factor in bone strength and in the risk of suffering osteoporotic fractures. The aim of this study was to examine the normal/abnormal difference for antero-posterior (AP) spine, lateral spine, proximal femur and total body BMD to assess if age influences discrimination at three different decades between 50 and 80 years of age. The BMD was determined in 61 control women and 60 osteoporotic women (at least one vertebral wedge fracture readily visible in the lateral X-rays of the thoracic or lumbar spine). Measurements were made by DEXA with a total body scanner. The BMD of the whole group of osteoporotic women was markedly lower than that of age-matched controls at all skeletal areas (P < 0.001) except at the arms where the difference was smaller (P < 0.02). The Z-score (the difference between osteoporotic patients and age-matched control divided by the intrapopulation S.D.) was similar (approximately -1.7) over the AP spine, femoral neck, Ward's triangle, total body and legs. It was significantly lower at the arms (-0.8, P < 0.001), lateral spine (-1.4, P < 0.01) and trochanter (-1.3, P < 0.001) compared with the Z-score of the AP spine. The analysis of the results by decades of age disclosed that the higher Z-score on the 6th and 7th decades corresponded to the AP lumbar spine (approximately -2.0). A high descrimination was also observed for the femoral neck, Ward's triangle and legs while the Z-score of the lateral lumbar spine, total body, trochanter and arms were significantly lower than that of the AP lumbar spine. However on the 8th decade the Z-score of the AP lumbar spine diminished to -1.2 and was only significantly higher than the Z-score of the arms (P < 0.01). The study showed that, in women 50-60 years of age--the period where the majority of studies are made for prevention of osteoporosis, none of the other skeletal areas were superior to the AP spine in discrimination for spinal osteoporosis. Proximal femur and legs densitometry gave lower but not significantly different Z-score than the AP spine, while the remaining areas were significantly inferior to AP spine in separating osteoporotic and normal women.
Ascites sarcoma 180 (S180A) is a transplantable tumor which causes hypercalcemia in tumor-bearing mice, and stimulates bone resorption without parathyroid hormone-like activity. In the present study, parathyroid hormone-related protein (PTHrP) mRNA could not be detected in total RNA from S180A cells. Bone-resorbing activity (BRA) derived from serum-free conditioned medium of S180A cells (S180A-CM) was coeluted with either transforming growth factor alpha (TGF alpha) activity (peak A, approximate M(r), 29 kDa) or lymphocyte-activating factor (LAF) activity (peak B, M(r), 20.1-24 kDa) in Bio-Gel P-100 column chromatography. Fractions in peak A and B contained IL-6 but not tumor necrosis factor alpha (TNF alpha). Subsequent separation of peak A by reverse-phase high performance liquid chromatography produced a single fraction which contained both BRA and TGF alpha activity. Recombinant human TGF alpha-induced bone resorption was completely inhibited by indomethacin. The BRA in peak A was partially inhibited by indomethacin and almost completely inhibited by simultaneous treatment of indomethacin and anti-IL-6 antibody. The BRA in peak B was partially inhibited by neutralization with anti-IL-1 alpha antibody and was completely inhibited by simultaneous treatment with anti-IL-1 alpha and anti-IL-6 antibody in the absence of indomethacin. Bone resorption induced by S180A-CM was associated with an increased production of prostaglandin E(2) (PGE(2)) by calvaria. The BRA in S180A-CM, however, was not completely abolished by the simultaneous addition of indomethacin and anti-IL-1 alpha, anti-IL-1 beta and anti-IL-6 antibodies. Our findings indicate that (1) BRA derived from S180A cells includes TGF alpha, IL-1 alpha, IL-6 and some other unknown factor(s), distinct from PTHrP, IL-1 beta and TNF alpha, and (2) these unknown factors resorb bone in part via a PGE(2)-independent pathway.
The object of this study was to determine the tissue level mechanism in which mechanical loading had an influence on ovariectomy-induced cancellous bone loss. Two experiments were performed: (1) 230 g female rats were divided into basal, age-related controls and right hindlimb immobilized (RHLI) group; (2) 250 g female rats were divided into basal, age-related, ovariectomized (OVX) controls and OVX combined with RHLI. The RHLI model immobilized (IM) or underloaded (UL) the right hindlimb and loaded (L) the left hindlimb compared to the right hindlimb. Both experiments lasted 60 days. Histomorphometric data was gathered from the secondary spongiosa of double fluorescent labeled proximal tibial metaphysis (PTM). The study confirmed that IM or UL induces cancellous bone loss, by decreasing bone formation, and increasing bone resorption and OVX results in cancellous bone loss, a higher bone turnover with bone resorption exceeding bone formation in the PTM. The OVX'd and immobilized (OVX + UL) PTM showed further decreased cancellous bone mass (−48%), decreased number (−43%), and decreased tissue-level bone formation rate (−54%) from that of OVX rats. However, the OVX’d and loaded (OVX + L) PTM partially prevented the OVX-induced cancellous bone loss. The cancellous bone area (+46%), number (+29%), and ratio of node to free end (+70%) were increased and percent eroded perimeter (−44%) and bone resorption rates (−30%) were decreased from OVX rats, but cancellous bone area and number were still significantly lower than those in age-related controls. We found that IM or UL accentuated cancellous bone loss in OVX rats by inhibiting bone formation and that loading partially prevented cancellous bone loss in OVX and RHLI rats by inhibiting bone resorption.
The effects of 3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinoline-6-one (KCA-098), a derivative of coumestrol, on bone resorption was studied in organ cultures of 20-day fetal rat femora. KCA-098 increased the length, dry weight, and calcium and phosphorus contents of parathyroid hormone (PTH)-treated fetal rat femur. As PTH significantly reduced the calcium and phosphorus contents of the femora, probably by stimulating bone resorption, KCA-098 seems to inhibit bone resorption. In fact, KCA-098 inhibited the PTH-induced release of 45Ca from pre-labeled fetal rat femora into the medium in organ culture. Coumestrol also inhibited the release of 45Ca from bone into the medium. However, KCA-098 did not increase the uterine weight of ovariectomized rats, whereas coumestrol did so. Thus KCA-098 is a unique, new inhibitor of bone resorption that has no estrogenic activity.
We performed the dosing experiment to establish whether estrogen administration has any beneficial effects on the mass and the turnover of bone in ovariectomized rats taking a mild dose of thyroxin. Thirty-five Wistar rats, 28 weeks of age, received ovariectomies (OVX) or sham operations and were divided into five groups. Group I was the sham group, Groups 2–5 were ovariectomized. Group 2 was the OVX-control, Group 3 treated with thyroxin 30 μg/kg/day (T4), Group 4, 17β-estradiol 0.3 mg/kg/week (E2), and Group 5, the combination of T4 and E2. The duration of the experiment was 12 weeks. At the end of the experiment, serum chemistries were measured. Bone minerals in the femur were determined with single photon absorptiometry and bone turnover was assessed histomorphometrically. Alkaline-phosphatase increased in Group 3 (OVX-T4), but it reduced in Groups 4 (OVX-E2) and 5 (OVX-T4 + E2). Bone minerals decreased in Groups 2 (OVX) and 3. In Group 4, it was preserved at the same level as in Group 1. Group 5 showed a significant increase of bone mass compared with Group 1. Eroded surface and osteoid surface increased in Groups 2 and 3 and they were reduced in Groups 4 and 5. Bone volume and mineral apposition rate were at a maximum in Group 5. This study demonstrated that 17β-estradiol was capable of preventing the bone mass decrease by regulating the turnover in ovariectomized rats taking a mild dose of thyroxin. Osteoblast function appeared to be stimulated in combination with 17β-estradiol and thyroxin.
Maternal and fetal 1,25-dihydroxyvitamin D3 (l,25(OH)2D3) and osteocalcin were measured in guinea pigs, to examine their potential use as animal models for fetal bone development and calcium homeostasis. Measurements were performed on days 42, 57 and 63 of gestation. Maternal serum total l,25(OH)2D3 concentrations were increased only at the end of gestation (day 63). However, because the vitamin D binding protein (DBP) and albumin levels were decreased by 35–50% from day 42 onwards, the unbound l,25(OH)2D3, calculated as the l,25(OH)2D3/DBP molar ratio, was increased before day 63. Osteocalcin concentrations during gestation were 50–54% of levels found in nongravid animals. Fetal serum total l,25(OH)2D3 concentrations were 20% of those in maternal guinea pigs. Since DBP levels were only 9–15% of maternal levels, the unbound l,25(OH)2D3 was consistently higher in fetuses, from day 42 onwards. There was a rise in total and unbound l,25(OH)2D3 between days 57 and 63 of fetal life. Osteocalcin concentrations were higher in fetal than in adult guinea pigs, and reached peak values on day 57 (1023 μg/l, i.e. 4.2 times higher than in adult female guinea pigs). Fetuses of guinea pigs that had received a restricted food supply for 14 days (days 49–63) had normal l,25(OH)2D3 concentrations, but decreased osteocalcin concentrations compared with normal fetuses. The data obtained in fetal guinea pigs are comparable with those found in human fetuses, and suggest that the guinea pig may be a suitable model for studies on fetal bone and mineral development.
Cyclosporin-A (CsA) inhibits in vitro proliferation of non-human tumour-cloned osteoblasts. Our aims were to study the direct effect of CsA on proliferation of normal human osteoblast (NHOb) cultures and to ascertain whether CsA-treated patients' sera (CsATPS) may exert effects on the osteoblast which differ from the direct effects of CsA. We studied tritiated thymidine ([3H]thymidine) incorporation in NHOb cultures incubated with (a) increasing CsA concentrations (1.2 to 4800 ng/ml), (b) the same concentrations as in the previous experiment but with the addition of 20% fetal calf serum (FCS) or 20% normal human serum (NHS), (c) 40% NHS or 40% CsATPS. Results at 96 h in (a) CsA inhibited [3H]thymidine uptake from 300 ng/ml, in (b) CsA inhibited [3H]thymidine uptake from 2400 ng/ml for cultures with FCS and 4800 ng/ml for cultures with NHS, in (c) CsATPS produced [3H]thymidine uptake inhibition compared with NHS. Conclusion: CsA alone inhibited [3H]thymidine incorporation in NHOb from concentrations similar to therapeutic concentrations. With FCS or NHS, inhibition was produced at higher concentrations. CsATPS inhibited at CsA concentrations lower than those of the two previous experiments.
The changes of bone mineral density (BMD) and skeletal muscles were evaluated in the rat model at either sham or ovariectomized stages to attempt to make clear the effect of voluntary running exercise on bone metabolism. In comparison with the control groups within the ovariectomized (OVX) and the sham groups, in the running groups, (1) the urinary phosphorus (P) and creatinine (Cr) excretions showed an increase concurrently with the increase in the running distance; (2) the weight of the quadriceps femoris was significantly higher; and (3) the BMD of appendicular and axial bones was significantly greater. These results suggest that voluntary running exercise could effect the BMD, the weight of the skeletal muscles, and the acceleration of energy metabolism.
To evaluate the effect of 17β-estradiol replacement (10 μg, twice a week) (E2) and treadmill exercise (18 m/min, 45 min/day) (EX) on long bone and vertebral bone mass and density, 10-month-old rats were ovariectomized (OV) and divided into four groups: OV, OV + E2, OV + EX, OV + EX + E2 2 months after surgery. After 7 weeks intervention, the calcium content and the density of lumbar-5 were higher in both OV + E2 and OV + EX + E2 groups than in the OV group, but, only the OV + EX + E2 group had a significantly higher femoral bone weight and density than the OV group. After 16 weeks intervention, the bone-conserving effects of E2 and EX were significant on lumbar-5 and femoral dry weight and density. The effect of E2 on both two sides of bones was due to the suppression of the bone turnover rate, while EX suppressed bone turnover rate primarily on the femur. We conclude that the effect of the two interventions on lumbar-5 and femoral bone mass were additive and independent.
It has been shown that both calcitonin gene-related peptide (CGRP) and amylin bind weakly to calcitonin (CT) receptors in osteoclast-like cells formed in vitro and inhibit bone resorption by a cAMP-dependent mechanism. Osteoclasts are thought to be derived from cells of the monocyte macrophage lineage, in which CGRP, but not CT, induces cAMP production. In this study, we determined the presence of functional receptors for CGRP in mouse alveolar macrophages and the effects of this peptide on proliferation and osteoclastic differentiation in mouse alveolar and bone marrow-derived macrophages. Human CT did not stimulate cAMP production in macrophages. Human CGRP stimulated cAMP production in mouse alveolar macrophages and bone marrow-derived macrophages dose-dependently. Human amylin, which has 43% homology with human CGRP, also stimulated these macrophages to produce cAMP, but only at a 100-fold higher concentration. The increment in cAMP production induced by human CGRP and amylin was abolished by the addition of human CGRP(8-37), a selective antagonist for CGRP receptors. Specific binding of [125I]human CGRP to alveolar macrophages was detected (dissociation constant, 2.5 x 10(-8) M; binding sites, 1.4 x 10(4)/cell). Amylin, but not CT, displaced the bound [125I]human CGRP from alveolar macrophages, but at a 100-fold higher concentration. No specific binding of [125I]human CT and [125I]human amylin to alveolar macrophages could be detected. Pretreatment with human CGRP for 24 h dose-dependently suppressed DNA synthesis in alveolar macrophages induced by granulocyte-macrophage colony-stimulating factor (GM-CSF). CGRP also suppressed the number of macrophage colonies formed from bone marrow cells induced by macrophage colony-stimulating factor (M-CSF).(ABSTRACT TRUNCATED AT 250 WORDS)
The expression of some candidate osteoclast markers, tartrate-resistant acid phosphatase (TRAP), macrophage associated antigens (M phi Ag), and vitronectin receptor (VNR) on foreign body giant cells (FBGCs) was investigated in peri-implant tissues of loosened total joint arthroplasties. Osteoclasts showed distinct staining characteristics. They were strongly TRAP-positive at tartrate concentrations of 50-200 mM and expressed VNR and a restricted range of M phi Ag. In contrast, FBGCs were shown to be significantly heterogeneous. Significant numbers of FBGCs were TRAP-positive at a 100 mM tartrate concentration and some were more intense than osteoclasts. A population of FBGCs did not express M phi Ag such as CD11b, but expressed VNR. It was demonstrated that these candidate osteoclast markers were also positive on FBGCs. These results have highlighted the difficulty in distinguishing these two cell lineages and suggested that there might be some uncertainty in defining osteoclast-like cells in culture studies.
To determine the potential adverse effects, if any, of long-term fluoride ingestion in humans, samples were collected from 25 adult females taking daily doses of fluoride (mean, 23 mg elemental F) for the treatment of osteoporosis and from 38 osteoporotic female controls. Patients in the fluoride group had been receiving therapy for approximately 18 months with a mean duration of 4.2 years and had serum fluoride values of at least 10 μmol/l. Laboratory analyses for fluoride were conducted on plasma, urine and drinking water samples collected from each panelist. Blood was also collected for blood chemistry analyses and plasma lymphocytes were examined for the frequency of sister chromatid exchange (SCE). Plasma and urine fluoride levels were significantly different between the two groups, while water fluoride was not. The SCE frequency, a measurement of potential genotoxicity, did not differ between the two groups. Of the blood chemistry parameters measured, albumin, alkaline phosphatase, sodium, chloride, the albumin/globulin (A/G) ratio, indirect bilirubin, lactate dehydrogenase (LDH), and γ-glutamyl transferase (GGT) were found to be significantly different between the two groups (P ≤ 0.05). However, none of the mean group values were outside stated normal ranges for any of these parameters. We conclude that the risk of developing adverse systemic effects from the ingestion of fluoride, at dosages and for a duration comparable with that of our panel, is minimal.
