Showing papers in "Journal of Bone and Mineral Metabolism in 2000"

Glucocorticoid-induced osteoporosis : pathogenesis and management

Abstract: Glucocorticoid- (GC-) induced osteoporosis and an increased risk of fractures are one of the most serious problems for patient using long-term GC therapy, such as those with rheumatoid arthritis, autoimmune diseases, inflammatory bowel diseases, bronchial asthma, and chronic lung diseases. GCs are known to affect both bone formation and resorption. In rheumatoid arthritis, the etiology of bone loss is multifactorial, including local inflammation around joints, release of bone-absorbing cytokines, physical inactivity, and malnutrition, in addition to the use of GC. Two guidelines have been published, by the American College of Rheumatology Task Force in 1966 and by the UK Consensus Group in 1998. Both guidelines recommend that patients 'receiving GC therapy at doses of 7.5 mg/day of prednisolone or more for 6 months or longer should have their bone mineral density measured and begin preventive therapies. Calcium and vitamin D supplements, sex hormone replacement, and weight-bearing exercise are the first-line therapies. For patients who are unable to take sex hormone replacement therapy (HRT), bisphosphonates are recommended by both guidelines. In this article, we briefly summarize the pathogenesis of GC-induced osteoporosis and its prevention and treatment.

The Utah paradigm of skeletal physiology: an overview of its insights for bone, cartilage and collagenous tissue organs.

Abstract: In a 1960 paradigm of skeletal physiology, effector cells (chondroblasts, fibroblasts, osteoblasts, osteoclasts, etc.) regulated by nonmechanical agents wholly determined the architecture, strength, and health of bones, joints, fascia, ligaments, and tendons. Biomechanical and tissue-level phenomena had no roles in that paradigm. Subsequent studies and evidence slowly revealed skeletal tissue-level mechanisms and their functions, including biomechanical ones, as well as "game rules" that seem to govern them. That slow discovery process found that effector cells are only parts of tissue-level mechanisms, as kidney cells are only parts of nephrons and wheels are only parts of cars. Normally all those things help to determine skeletal architecture, strength, and health, and adding them to the 1960 paradigm led to the still-evolving Utah paradigm of skeletal physiology that concerns, in part, how load-bearing skeletal organs adapt to the voluntary mechanical loads on them. That caused controversies this article does not try to resolve; instead, it describes some issues they concern. In that regard, controversy can depend on how one assesses the relevance of facts to a problem more than on their accuracy. If a paradigm added new facts to a former one and the new one's advocates viewed all those facts as relevant, but the former's advocates questioned the relevance of some of the new facts, their views about a problem could differ even though each view depended on accurate facts. Readers would make their own judgments about the bearing of those ideas on this article's content.

Patterns of gene expression associated with BMP-2-induced osteoblast and adipocyte differentiation of mesenchymal progenitor cell 3T3-F442A.

Abstract: The pluripotent mesenchymal stem cells give rise to osteoblasts, adipocytes, chondrocytes, and myoblasts. The differentiation of these stem cells into each of the mature functional cells may be controlled by a distinctive master gene(s) and is associated with temporal and spatial expression of diverse genes. Identification of genes that are expressed during the differentiation of the mesenchymal cells to osteoblasts is, therefore, important to obtain insights into the molecular mechanisms of osteogenesis. The murine undifferentiated mesenchymal cell 3T3-F442A, when treated with the bone morphogenetic protein 2 (BMP-2), a well-characterized inducer of mesenchymal cell differentiation, exhibited both osteoblastic and adipocytic differentiation. Using the SAGE (serial analysis of gene expression) technique, which has been shown to enable quantitative analysis of large numbers of genes in a simple and quick manner, we obtained 1600 sequence tags representing 2107 individual nucleotide sequences from control and BMP-2-treated 3T3-F442A cells, respectively. By comparing the frequency of tag occurrence, we found profiles of up- or downregulated genes associated with osteoblast or adipocyte phenotype such as type I collagen, osteonectin and OSF-2, or C/EBPβ, aP2, fatty acid synthase, and lipoprotein lipase, respectively, in BMP-2-treated 3T3-F442A cells. Our data show that BMP-2 induces not only osteoblastic but also adipocytic differentiation in the 3T3-F442A cells. They also show that the 3T3-F442A cells have bipotentials of differentiating toward osteoblasts and adipocytes. The results, therefore, might explain the inverse correlation between trabecular bone volume and fat volume in the bone marrow cavity. The results also suggest that the SAGE may be a useful technique that allows us a fast and efficient way to generate global and local views of gene expression associated with cellular differentiation of the mesenchymal stem cells.

Roles of macrophage-colony stimulating factor and osteoclast differentiation factor in osteoclastogenesis

Abstract: We have isolated osteoclast precursors (OCPs) from cocultures of mouse calvarial cells and bone marrow cells without adding any osteotropic factors. OCPs expressed Mac-1, Mac-2, and Gr-1 antigens but not osteoclast markers such as tartrate-resistant acid phosphatase (TRAP) and calcitonin receptors, and they differentiated into TRAP-positive cells within 48 h on a fixed calvarial cell layer pretreated with osteotropic factors such as 1α,25-dihydroxyvitamin D3. In the present study, we investigated the regulatory mechanisms of OCP formation from hemopoietic cells and TRAP-positive cell formation from OCPs. Calvarial osteoblasts obtained from macrophage-colony stimulating factor (M-CSF) -deficient op/op mice failed to support OCP formation or the differentiation of OCPs into TRAP-positive cells. Both OCP formation and TRAP-positive cell formation supported by osteoblasts were completely inhibited by osteoclastogenesis inhibitory factor (OCIF, also called OPG), which is a decoy receptor of osteoclast differentiation factor (ODF; also called TRANCE, RANKL, and OPGL). When bone marrow cells were cultured for 4 days with soluble ODF (sODF/sRANKL) together with M-CSF, OCPs were formed even in the absence of osteoblasts. When OCPs were treated with sODF/sRANKL and M-CSF in the absence of osteoblasts, they differentiated into TRAP-positive cells within 48 h even in the presence of hydroxyurea. Northern blotting analysis revealed that osteoblasts constitutively expressed a certain level of ODF/RANKL mRNA. These results indicated that M-CSF and sODF/sRANKL produced by osteoblasts are two essential factors for both OCP formation and TRAP-positive osteoclast formation.

Calcium paradox disease: calcium deficiency prompting secondary hyperparathyroidism and cellular calcium overload.

Abstract: The consequences of such Ca overload in response to Ca deficiency, which can be called Ca deficiency disease or Ca paradox disease, to be more explicit, include osteoporosis, hypertension, arteriosclerosis, degenerating diseases of the central nervous system such as Alzheimer’s disease, diabetes mellitus, muscular dystrophy, and malignancy, especially colorectal carcinoma. In this review, evidence supporting the concept of calcium paradox disease is presented from the viewpoints of epidemiology, clinical evidence, experimental models, and cell biology.

Osteoclasts, integrins, and osteoporosis.

Abstract: It is now known that the osteoclast is of hematopoietic origin [1] and a member of the monocyte macrophage family [2]. This conclusion is derived from experiments initially performed by Donald Walker, who demonstrated that parabiosis of normal mice to osteopetrotic litter mates, or administration of normal spleen cells to osteopetrotic rodents, cures their sclerotic bone disease [3–5]. With this information in mind, we undertook a similar exercise in a 3.5-month-old female infant who received a bone marrow transplant from her HLA/ MLC identical brother [1]. This undertaking, performed two decades ago, resulted in the first established cure of infantile malignant osteopetrosis, and, by following the Y chromosome, we validated that osteoclasts, but not osteoblasts, are of donor (i.e., hematopoietic) origin (Fig. 1). These experiments led to development of in vitro systems that enabled a number of laboratories to delineate the mechanisms by which osteoclasts are recruited to and resorb bone. The precursor of this polykaryon arises in hematopoietic tissues such as marrow, circulates, and targets to bone to which it attaches and undergoes multinucleation by fusion. The multinucleated cell, specifically recognizing bone matrix, assumes the osteoclast phenotype which distinguishes it from other members of the monocyte/macrophage family. These phenotypic features include expression of tartrate-resistant acid phosphatase (TRAP) and the calcitonin receptor, as well as the capacity to form the characteristic osteoclast ruffled membrane. This complex structure, appearing in the plasma membrane juxtaposed to bone, is the resorptive organelle of the osteoclast [6].

Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life: a hypothesis.

Abstract: Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life. A hypothesis.

Intake of fermented soybean (natto) increases circulating vitamin K2 (menaquinone-7) and gamma-carboxylated osteocalcin concentration in normal individuals.

Abstract: Changes in circulating vitamin K2 (menaquinone-7, MK-7) and gamma-carboxylated osteocalcin concentrations in normal individuals with the intake of fermented soybeans (natto) were investigated. Eight male volunteers were given sequentially fermented soybeans (natto) containing three different contents of MK-7 at an interval of 7 days as follows: regular natto including 775 micrograms/100 g (MK-7 x 1) or reinforced natto containing 1298 micrograms/100 g (MK-7 x 1.5) or 1765 micrograms/100 g (MK-7 x 2). Subsequently, it was found that serum MK-7 and gamma-carboxylated osteocalcin concentrations were significantly elevated following the start of dietary intake of MK-7 (1298 or 1765 micrograms/100 g). Serum undercarboxylated osteocalcin concentrations were significantly decreased by dietary MK-7 (1765 micrograms/100 g) supplementation. Moreover, the changes in serum MK-7 level with the frequency of dietary natto intake were examined in 134 healthy adults (85 men and 39 women) without and with occasional (a few times per month), and frequent (a few times per week) dietary intake of regular natto including MK-7 (775 micrograms/100 g). Serum MK-7 and gamma-carboxylated osteocalcin concentrations in men with the occasional or frequent dietary intake of natto were significantly higher than those without any intake. The present study suggests that intake of fermented soybean (natto) increases serum levels of MK-7 and gamma-carboxylated osteocalcin in normal individuals.

Trends in the incidence of hip fracture in Japan, 1987-1997: the third nationwide survey.

Abstract: The third nationwide survey for hip fracture incidence was conducted in 1997 following the first such survey in 1987 and the second in 1992. The purpose of this study was to investigate the trends in the incidence and regional distribution of this disease during 10 years. Of 10 271 orthopedic institutions in Japan, 4503 were selected as subjects for the study using the optimum allocation method. Questionnaires concerning new patients with hip fracture were mailed. The replies were obtained from 2930 institutions by the end of December 1998; the response rate was 65.1%. The number of new patients was estimated to be 89 900–94 900 [mean, 92 400; 20 100–21 400 (20 800) men and 69 600–73 600 (71 600) women]. The number of cases in 1997 was about 1.7 times higher than that in the first survey and 1.2 times higher than that in the second survey. The age-specific incidence (per 10 000 per year) in men and women in 1997 was 0.30 and 0.13, respectively, for age under 40 years; 0.91 and 0.60, 40–49 years; 2.00 and 2.39, 50–59 years; 5.12 and 9.07, 60–69 years; 17.3 and 40.8, 70–79 years; 57.4 and 147.8, 80–89 years; and 128.9 and 281.0, for age over 90 years. The incidence was increased compared with that of the first survey, and similar to the second survey, excepting that of women aged 80 years or older. Concerning regional differences, hip fracture incidence was relatively low in the eastern area compared to the western area in Japan, which was a trend identical to that in the previous nationwide surveys.

Prolonged intake of fermented soybean (natto) diets containing vitamin K2 (menaquinone-7) prevents bone loss in ovariectomized rats

Abstract: The effect of the prolonged intake of dietary vitamin K2 (menaquinone-7, MK-7) on bone loss in ovariectomized (OVX) rats was investigated. OVX rats were freely given experimental diets containing the fermented soybean (natto; including 9.4 micrograms MK-7/100 g diet) without or with supplemental MK-7 (containing 14.1 or 18.8 micrograms of MK-7 as total per 100 g diet) for 150 days. Feeding produced a significant elevation of MK-7 concentration in the serum of OVX rats. In this case, the femoral MK-4 content was significantly increased, but MK-7 was not detected in the femoral tissues, indicating degradation of MK-7. Serum gamma-carboxylated osteocalcin concentration was significantly decreased by OVX. This decrease was significantly prevented by the feeding of the natto diets with supplemental MK-7 (18.8 micrograms/100 g diets). OVX caused a significant decrease in femoral dry weight, femoral calcium content, and mineral density. These decreases were significantly prevented by feeding with diets containing natto with MK-7 (total, 18.8 micrograms/100 g diets). This study demonstrates that the prolonged intake of natto dietary including MK-7 has a preventive effect on bone loss induced by OVX. Dietary MK-7 may be useful in the prevention of osteoporosis.

Expression of bone morphogenetic proteins and rat distal-less homolog genes following rat femoral fracture.

Abstract: Expression of the genes encoding bone morphogenetic proteins (BMPs), BMP type IA receptor (BMPR-1A), and rat distal-less homolog (rDlx) was studied in bone, callus, and the surrounding soft tissue following rat femoral closed fracture, using RT-PCR-based techniques. Before fracture, the genes encoding BMP-5, BMP-6, and BMPR-1A were found to be expressed in both bone and the surrounding soft tissue, whereas the BMP-2 gene was expressed only in bone and BMP-7 was not expressed in either tissue. Expression of these genes was unaffected by fracture. The gene encoding BMP-4 was also expressed in both bone and the surrounding soft tissue before fracture. Moreover, although unchanged in bone, 6 h after fracture BMP-4 expression was increased tenfold in the surrounding soft tissue. The increased BMP-4 expression was transient and returned to prefracture levels within 72 h. Expression of rDlx was also increased in bone after fracture, but at later times than were observed with BMP-4: elevated rDlx expression was detected after 48 h and persisted for 30 days or more. No expression of rDlx was observed in the surrounding soft tissue before or after fracture. These findings indicate that BMP-4 and rDlx are selectively expressed following femoral fracture in the rat, and also suggest that they are involved in the formation of the callus at an early point during the postfracture healing of bone.

Aggrecan from start to finish.

Abstract: terminal globular (G)1 domain and a C-terminal G3 domain that are joined by intervening nonglobular sequences of lengths characteristic for each core protein. In addition, the aggrecan core protein has a G2 domain. The G1, G2, and G3 domains each contain distinctive motifs, with each motif having its own folded structure (Fig. 1). The G1 domain contains two proteoglycan tandem repeats (PTR) and one Ig motif [2]. The folded PTRs in G1 define a binding site for HA [3]; this site engages HA after mature proteoglycans are secreted into the ECM. G3 motifs bear homologies to epidermal growth factor (EGF), C-type lectin, and sushi or complement reactive protein (CRP); each has a unique conformation [4,5]. The lectin and sushi motifs are always present whereas the presence of the EGF motif is variable.

Epidemiology of cervical and trochanteric fractures of the proximal femur in 1994 in Tangshan, China.

Abstract: The purpose of this study was to determine the incidence of cervical and trochanteric fractures of the proximal femur in 1994 in Tangshan City, China. There are many reports on hip fracture incidence in many countries, suggesting that there are many factors affecting hip fractures. We visited 15 hospitals with an orthopaedic department within Tangshan City, and reviewed the medical records and radiographs of all patients with hip fractures occurring between January 1 and December 31, 1994. The population of Tangshan in 1994 was determined to be 1 454 543 (746 015 males and 708 528 females). The population of those over 65 years of age was 88 490 (41 519 males and 46 971 females), representing 6.08% of the total population. This study detected 184 cervical and trochanteric fractures of the proximal femur in 1994 in Tangshan (127 men and 57 women). The overall incidence or rate of the combined number of cervical and trochanteric fractures was 25 fractures per 100 000 population per year for men and 12 for women. There were a total of 147 cervical fractures (80%) and 37 trochanteric fractures (20%). The incidence of the combined number of cervical and trochanteric fractures in patients over 70 years of age increased to 108 for men and 156 for women. The incidence of hip fractures increased with age in both sex groups, especially in women over 65. Severe trauma fractures happened more often in younger groups, and mainly occurred in men, which may be a result of the particular composition of the population in Tangshan, which is young and male dominated. In addition, because Tangshan is an industrial city, many of its citizens are involved in occupations requiring a high level of physical activity.

Calcium paradox: Consequences of calcium deficiency manifested by a wide variety of diseases

Abstract: Calcium deficiency is a global problem, especially in the aging population. Among various nutrients, calcium is one of the few that is still deficient in industrialized countries such as Japan and many Western countries. Calcium deficiency is readily connected with osteoporosis, which is a decrease of bone calcium content. Less well known is the calcium outflow from bone that occurs to prevent decrease of blood calcium in calcium deficiency caused by the parathyroid hormone, with consequent calcium overflow into soft tissues and the intracellular compartment. Such intracellular paradoxical Ca overload as a consequence of nutritional calcium deficiency may give rise to a number of diseases common in old age: hypertension, arteriosclerosis, diabetes mellitus, neurodegenerative diseases, malignancy, and degenerative joint disease.

Alternation in bone components with increasing age of newborn rats: role of zinc in bone growth

Abstract: The effect of zinc on bone growth in newborn rats supplied with lactation by maternal rats was investigated. Newborn rats were killed between 1 and 35 days after birth. Increasing age caused a significant increase in zinc content, calcium content, and alkaline phosphatase activity in the femoral-diaphyseal and metaphyseal tissues, while the bone deoxyribonucleic acid (DNA) content was significantly decreased because of elevation of mineral content. Oral administration of zinc sulfate (2.0 mg/100 g body weight; four times at 24-h intervals) to maternal rats from 1 day after birth induced a significant increase in zinc, alkaline phosphatase activity, DNA, and calcium content in the femoral-diaphyseal and metaphyseal tissues of newborn rats compared with those 7 or 14 days old. The results indicate that the increase in bone components results from lactation with zinc-containing milk of maternal rats. The femoral-metaphyseal tissues of newborn rats obtained at 7 days after birth were cultured for 24 h in a medium containing either vehicle or zinc sulfate (10−6 to 10−4 M) in vitro. Bone alkaline phosphatase activity and calcium and DNA content were significantly increased by zinc addition. These increases were completely prevented by the presence of dipicolinate (10−3 M), a chelator of zinc ion, or cycloheximide (10−6 M), an inhibitor of protein synthesis. The present study suggests that zinc plays a role in the development of bone growth in newborn rats.

Synergistic effect of genistein and zinc on bone components in the femoral-metaphyseal tissues of female rats.

Abstract: The effect of genistein and zinc on bone components in rats was investigated. Femoral-metaphyseal tissues obtained from female rats (4 weeks old) were cultured for 24 h in a medium containing either vehicle or genistein (10−7–10−5 M) in the absence or presence of zinc sulfate (10−6–10−4 M) in vitro. The presence of genistein (10−6 and 10−5 M) or zinc (10−6 and 10−5 M) caused a significant increase in alkaline phosphatase activity, deoxyribonucleic acid (DNA), and calcium content in the metaphyseal tissues. These increases were significantly enhanced by the combination of each compound. The synergistic effect on bone components was seen in the combination with genistein (10−6 and 10−5 M) plus zinc (10−5 M). Such an effect was completely blocked by the presence of cycloheximide (10−6 M), an inhibitor of protein synthesis. Moreover, the oral administration of genistein (100 and 300 μg/kg body weight) or zinc sulfate (1 and 5 mg Zn/kg) to rats for 3 days caused a significant elevation of alkaline phosphatase activity, DNA, and calcium content in the femoral-metaphyseal tissues. These increases were significantly enhanced by the combination of each compound. The synergistic effect was seen in the case of genistein (100 and 300 μg/kg) plus zinc (5 mg/kg). These results demonstrate that the anabolic effect of genistein on bone components is synergistically enhanced by zinc in vitro and in vivo. This study further supports the view that the combination of nutritional factors has a potent anabolic effect on bone metabolism.

Association of bone mineral density with polymorphism of the human matrix Gla protein locus in elderly women.

Abstract: The contribution of genetic factors has been implicated in the determination of bone mass in twin and family studies. Some of the genes involved would regulate bone metabolism, bone formation, and resorption, all processes that determine bone mass. One candidate gene, matrix Gla protein gene (MGP), has been implicated in the pathogenesis of bone loss through a repression of bone formation. To analyze the genetic background for osteoporosis in elderly women, we have investigated a possible association between the CA repeat polymorphism at the human MGP gene locus and bone mineral density (BMD) of radial bone in 460 elderly Japanese women. Genotypes were classified into six groups according to the number of CA repeats present, from 13 to 18 (alleles A1 through A6). BMD was expressed as the adjusted BMD (ADJBMD), which was the body mass index (BMI)- and age-adjusted average BMD. The 214 women who lacked an A2 allele (212 bp, containing 17 repeats of CA) had significantly lower adjusted BMD than the participants (n = 246) who possessed an allele of that size (mean ± SD; 0.303 ± 0.062 vs 0.315 ± 0.062 g/cm2; P = 0.0382). This result suggests that genetic variation at the MGP locus is associated with some determinants for BMD in elderly women. Therefore, this locus should serve as one of the genetic markers for osteoporosis.

Quantitative analysis using the star volume method applied to skeleton patterns extracted with a morphological filter.

Abstract: In this study, a morphological filter was combined with star volume analysis and applied to digital images to determine its potential usefulness in assessing trabecular structure Three digital "geometric" test patterns (square, rectangle, circle) were created on a CRT (cathode ray tube) Each shape was arranged into five groups by size to yield 15 final "skeletal" patterns that were subsequently assessed with star volume analysis Also, three digital X-ray images (background, soft tissue, bone block) were processed with a morphological filter to create three sets of 11 skeletal patterns each These patterns were also assessed with star volume analysis and the ratio of extracted skeletal elements (in pixel numbers) to total pixel numbers was expressed as the pixel percentage Star volume analysis was then applied to these digital skeletal images to yield the volume of extracted "skeletal" trabecular elements (Vsk) and the volume of nonskeletal (marrow space) elements (Vsp) The Vsk and Vsp were compared for all the different skeletal patterns The pixel percentages were then compared to the star volume results for the X-ray test patterns The Vsk decreased and Vsp increased as the number of operations (n) increased for both digital X-ray images and the geometric test patterns when the X-ray images were depicted by pixel percentages Also, all true bone test patterns were clearly different both visually and quantitatively when compared to the noise skeletons extracted from background and soft tissue Therefore, as Vsk was increased, so was connectivity It can be concluded that the application of morphological filters and star volume analysis may be a useful tool in quantitatively determining the characteristics and continuity of trabecular skeletal structures Further studies involving a larger number of bone samples and using models to compare measurements of calculated versus actual volume should reveal the true potential of this method for evaluating bone structure and its relationship to bone strength and also increase the tools available for evaluating bone diseases such as osteoporosis

Epidemiology of cervical and trochanteric fractures of the proximal femur in 1996 in Kaohsiung City, Taiwan.

Abstract: The goal of this study was to determine the incidence of cervical and trochanteric fractures of the proximal femur in 1996 in Kaohsiung City, Taiwan. Kaohsiung City is the industrial and commercial center of southern Taiwan, with a population of 1,433,621 in 1996. The number of individuals over 65 years of age accounted for 6.2% of the total population. Data from the archives of reimbursement of the National Health Insurance program were used to investigate the incidence of fractures of the proximal femur. This study detected 580 cervical and trochanteric fractures (40.5 fractures per 100,000 population per year) in 261 males (35.8 fractures per 100,000 men per year) and 319 females (45.3 fractures per 100,000 women per year), with 420 (72%) of these fractures occurring in individuals over 65 years of age. The age-specific incidences of cervical and trochanteric fractures increased exponentially with age in both genders. The overall ratio of cervical to trochanteric fractures was 1:1.04. The mean ages of women with cervical or trochanteric fractures (71.6 and 74.0 years, respectively) were significantly higher than those of males (59.9 and 64.8 years, respectively; P < 0.01). The age-adjusted incidences of fractures of the proximal femur in Kaohsiung City were higher than in other Asian countries, but were lower than in Western countries such as the United States and Norway. The urban lifestyle and low daily calcium intake may be responsible for this increased incidence of proximal femoral fractures in Kaohsiung City.

A fourier transform infrared spectroscopic and solid-state NMR study of bone mineral in osteogenesis imperfecta.

Abstract: Fourier transform infrared spectroscopy and 31P solid-state nuclear magnetic resonance spectroscopy were used to determine if any structural or compositional differences in osteogenesis imperfecta (OI) bone mineral could be detected that might help to explain the bone fragility observed in this disease. A previous study by Cassella et al. used an electron probe X-ray microanalytical technique to compare the calcium to phosphorus (Ca/P) molar ratios in normal bone and bone from patients with OI. It was demonstrated that bone from OI patients had a lower Ca/P molar ratio. This study demonstrated that OI bone mineral had a general hydroxyapatite structure and that isomorphous substitutions in the carbanoapatite lattice could account for the low Ca/P molar ratio.

Analgesic mechanism of calcitonin.

Abstract: Calcitonin is employed in clinical treatment to improve bone mass in osteoporosis and to relieve its accompanying pain, but its analgesic mechanism is still unclear. Using ovariectomized (OVX) rats that are well known as an animal model of osteoporosis, the antinociceptive effect of elcatonin ([Asu] eel calcitonin, ECT), a synthetic derivative of eel calcitonin, was examined with the tail-withdrawal nociceptive test. Prolonged hyperalgesia was induced by OVX, and the antinociceptive effect of ECT was proved because peripherally and repeatedly administered ECT improved hyperalgesia. This effect of ECT was completely abolished by injections of p-chlorophenylalanine (PCPA), an inhibitor of serotonin biosynthesis, suggesting that the serotonergic system may be involved in antinociception. Spinal cord slices that retained an attached dorsal root were prepared, and blind whole-cell recordings were made from substantia gelatinosa (SG, lamina II) for electrophysiological analyses of a relationship between the effect of ECT and the serotonergic system. The descending serotonergic fibers, one of the inhibitory systems for nociceptive transmission, originate from the nucleus raphe magnus and terminate preferentially on SG of the spinal dorsal horn. Excitatory postsynaptic currents (EPSCs) evoked by dorsal root stimulation were then recorded from SG neurons, and the effects of serotonin on the EPSCs were compared in sham-operated and OVX rats. In addition, influence of ECT administration to OVX rats on the effects of serotonin was also examined. Glutamatergic short- and long-latency EPSCs, mediated by A delta and C afferent fibers, respectively, were observed after stimulation of the dorsal root, and both were depressed in amplitude by serotonin in sham rats, whereas only A delta-mediated EPSCs but not C-mediated were inhibited in OVX rats. Interestingly, C-mediated EPSCs were inhibited by serotonin in ECT-treated OVX as well as sham rats. A relationship between the fact that OVX induced hyperalgesia and that ECT alleviated this hyperalgesia was well correlated with changes in serotonergic inhibition of C-mediated EPSCs. These results suggest an alteration in the spinal serotonergic receptors that control the nociceptive transmission. Receptor-binding assay using spinal membranes and [3H]-8-OH-DPAT as a radioactive ligand was used to assess the change in the receptors. Although the level of [3H]-8-OH-DPAT-binding sites was decreased in OVX rats compared with sham rats, this reversed to the normal level in ECT-treated OVX rats. All the results strongly suggest that the behavioral and electrophysiological changes may be caused by an alteration in the level of spinal serotonergic receptor expression.

Does bone design intend to minimize fatigue failures? A case for the affirmative.

Abstract: Threshold strain ranges help to control the ability of modeling to increase bone strength and "mass" and the ability of remodeling to conserve or decrease them. Whether expressed as strains or stresses, the probable remodeling threshold of bone (MESr) lies below its modeling threshold (MESm), which lies below its operational microdamage threshold (MESp), which lies well below its ultimate strength (Fx). Given normal modeling and remodeling potentials, that arrangement should tend to cause whole-bone strength and stiffness to keep typical peak bone strains ("E") from voluntary activities from exceeding bone's modeling threshold and therefore from reaching its microdamage threshold. Satisfying that laddered "MESr < "E" < MESm << MESp <<< Fx" arrangement should minimize fatigue failures of bones, which might be at least one purpose of normal bone design. That arrangement would have practical implications that include, in part, the following. (A) It could make healthy young adult bones about six times stronger than needed for the largest voluntary loads they usually carry. (B) It suggests a biomechanical pathogenesis for different kinds of osteoporosis. (C) It suggests design criteria that load-bearing bone implants and endoprostheses should satisfy to endure their voluntary mechanical usage. (D) It also suggests features that future models of mechanical loading effects on bone strength, architecture, and "mass" might incorporate.

Effect of ethanol on bone mineral density of rats evaluated by dual-photon X-ray absorptiometry.

Abstract: Abuse of alcohol may derange bone metabolism and cause osteoporosis. Due to confounding factors associated with alcohol abuse, e.g., dietary deficiencies and liver damage, a study using an animal model is preferable to examine whether alcohol itself actually reduces bone density. We evaluated the effect of alcohol intake on bone in rats by dual-energy X-ray absorptiometry. Six-week-old male (n = 16) and female (n = 16) Wister rats were divided into two groups. Sixteen alcohol-exposed rats (8 male and 8 female) were fed Lieber's liquid diet and 16 control rats (8 male and 8 female) were fed a control liquid diet. The bone mineral density (BMD) and bone mineral content (BMC) of the right femur were measured before and after experimental feeding under anesthesia. The BMD of lumbar spine (L2-L4) of sacrificed rats was measured. For male rats, BMD and BMC decreased significantly in the alcohol group (P = 0.0132 and 0.0133, respectively) but did not decrease in control group. For female rats, BMD and BMC decreased significantly in the alcohol group (P = 0.0012 and <0.0001, respectively) but did not decrease in the control group. For male rats, the mean ratio of BMD after experimental feeding divided by BMD before experimental feeding was significantly lower in the alcohol group than in the control group (P = 0.0031). For female rats, the mean ratio of BMD after experimental feeding divided by BMD before experimental feeding was also lower in the alcohol group than in the control group (P = 0.0002). For male rats, the mean BMD of L2-L4 after experimental feeding was significantly lower in the alcohol group than in the control group (P = 0.0210). For female rats, the mean BMD of L2-L4 after experimental feeding was also significantly lower in the alcohol group than in the control group (P = 0.0006). These results indicate that alcohol intake decreased the BMD of rats in both spongy and cortical bone, and that the reduction of BMD was greater in female rats than in male rats.

Spatiotemporal change of rat collagenase (MMP-13) mRNA expression in the development of the rat femoral neck.

Abstract: The interepiphyseal region between the greater trochanter and the capital femoral epiphysis and the medioproximal portion of the femoral neck exhibit extensive morphological changes during the first 4 weeks after birth in rats. Previous reports show that matrix metalloproteinase-13 (MMP-13, rat collagenase) mRNA is expressed in bone and cartilage during embryonal development and fracture healing. We examined MMP-13 mRNA expression and compared it with the distribution of osteopontin and osteocalcine mRNA in the femoral neck. Moreover, we examined histomorphometric analysis in the femoral neck where the morphology changes rapidly. Histomorphometric analysis of the 4-week-old rat femoral neck showed a high rate of bone formation and resorption in the region where shape changed rapidly. Osteopontin mRNA was expressed diffusely along the endosteum. In contrast, MMP-13 mRNA expression was restricted to the medial endosteal portion near the cartilage-bone interface of the femoral neck in 15- and 28-day-old rats and in the deepest endosteal interepiphyseal region of 15-day-old rats. MMP-13 mRNA-expressing osteoblastic cells were also expressing osteopontin but not osteocalcin mRNA. MMP-13 mRNA-expressing cells differ from tartrate-resistant acid phosphatase (TRAP)-positive cells, and MMP-13 mRNA-positive cells are located adjacent to TRAP-positive cells. The results of the site- and cell-specific expression of MMP-13, taken together with its enzymatic property, suggest that MMP-13 plays an important role in morphological changes in the rat femur, at least during the third and fourth week after birth, and that MMP-13 itself is involved in the interaction between osteoblastic and TRAP-positive cells.

Rheumatoid arthritis-related antigen 47kDa (RA-A47) is a product of colligin-2 and acts as a human HSP47.

Abstract: We previously isolated RA-A47, which is recognized as an antigen of rheumatoid arthritis (RA), from a human chondrosarcoma-derived cell line (HCS-2/8). The N-terminal 21-amino-acid sequence of RA-A47 had 81% homology to the deduced amino acid sequence of the human heat-shock protein (HSP) 47 gene, the colligin gene, and 100% homology to that of the colligin-2 gene. Moreover, as is HSP47, RA-A47 was a heat-inducible collagen-binding protein. To further characterize RA-A47, we isolated ra-a47 cDNA from HCS-2/8 cells and human periodontal ligament fibroblast (HPLF) cells. The isolated ra-a47 cDNAs from both cells were almost the same as that of colligin-2. C504 and G505 in the cDNA sequences of both cells and C598 in the cDNA of HCS-2/8 were different from the corresponding bases of colligin-2 cDNA. These differences were also observed in genomic DNA. colligin cDNA was not isolated. To show that the isolated cDNA encodes RA-A47 protein, it was expressed in Cos-7 cells. The produced protein was 47kDa and was recognized both with RA sera and antirat HSP47 antibody, indicating that it is RA-A47 and has structural similarity to HSP47. These results taken together with our previous findings show that RA-A47 is the putative colligin-2 gene product and behaves as a human HSP47. Although colligin has been considered the human HSP47 gene, failure to detect the colligin gene and its mRNA suggests that colligin does not exist in human cells and that the HSP47 gene is identical with colligin-2, which encodes RA-A47.

Effect of intermittent administration of human parathyroid hormone (1-34) on the mandibular condyle of ovariectomized rats

Abstract: Intermittent administration of human parathyroid hormone (1-34) (PTH) increases bone mass in lumbar vertebrae and long bones of osteoporotic experimental animals. However, whether PTH has the same effect on jaw bones remains unclear. This study determined the effect of intermittent administration of PTH on rat mandibular condyle affected by estrogen deficiency. Fifty 6-month-old rats were either sham operated or ovariectomized, then divided into five groups depending on surgical procedure and hormone administration: sham + vehicle (SV), OVX + vehicle (OV), OVX + PTH 6 μg/kg once per week (OP6-1), OVX + PTH 60 μg/kg once per week (OP60-1), and OVX + PTH 20 μg/kg three times per week (OP20-3). PTH or vehicle was injected intermittently for 6 months in 5 rats of each group either immediately after surgery in a preventive administration experiment, or injected starting 6 months after surgery in a therapeutic administration experiment. The mandibles were excised, and bone morphometry was performed using confocal laser scanning microscopy and soft X-ray images. In both experiments, the bone volume of the OV groups was significantly lower than that of the SV group (P < 0.01); also, depending on dose and frequency, the bone volume of the OP group was higher than that of the OV group, particularly in the OP20-3 group. The value of mineralized surface of the OP groups was significantly higher than that of the OV group (P < 0.01), whereas the value of eroded surface of the OP groups was not significantly higher than that of the OV group. This study indicates that preventive and therapeutic intermittent administration of PTH in ovariectomized rats increase the bone formation in rat mandibular condyle without accelerating bone resorptive activity. This anabolic effect was best induced by the injection mode of 20 μg/kg three times per week.

Identification of a novel polymorphism of estrogen receptor-α gene that is associated with calcium excretion in urine

Abstract: A novel variation of the estrogen receptor-α (ERα) gene was identified by polymerase chain reaction–single-strand conformational polymorphism (PCR-SSCP). It is one base substitution in codon 325 (CCC [allele M] to CCG [allele m]) in exon 4 of the human ERα gene. This substitution did not cause an amino acid change. We categorized 306 unrelated Japanese postmenopausal women into three genotypes: MM, Mm, and mm; the frequency of each genotype was 26.5%, 43.1%, and 30.4%, respectively. Then, the association of this polymorphism with bone mineral density (BMD) of lumbar spine and bone–calcium metabolic markers was studied. There was no significant difference in BMD of the lumbar spine or most of the bone metabolic markers. However, the urinary calcium (Ca) excretion ratio (u-Ca/Cre) corrected by creatinine was significantly lower in the genotype mm group compared with the genotype MM group (MM vs mm, 0.247 ± 0.158 vs 0.200 ± 0.105; P < 0.05). We examined the relationship of restriction fragment length polymorphisms (RFLPs) (PvuII, XbaI) in intron 1 and the polymorphism in exon 4. The frequency of genotype MM was higher in the genotype PPxx, which was reported to be associated with lower BMD in the same population of Japanese postmenopausal women. The ER polymorphism identified in this study might be related to some biological mechanisms that regulate calcium metabolism.

Peripheral computed tomography (pQCT) detected short-term effect of AAACa (heated oyster shell with heated algal ingredient HAI): a double-blind comparison with CaCO3 and placebo.

Abstract: Trabecular bone density at the distal radius and cortical bone density at the midradius were measured in four randomized groups of women before and after 4 months administration of AAACa, oyster shell heated under reduced pressure with addition of heated algal ingredient (HAI) (group A); AACa, the same preparation without HAI (group B); CaCO3 (group C); and placebo (group D) in a double-blind system using peripheral quantitative computed tomography (pQCT) with lumbar spine density measurement by dual-energy X-ray absorptiometry (DXA). Groups A, B, and C received 900 mg/day elemental calcium and D received none. In subjects of group A, but not B, C, and D, radial trabecular bone density increased significantly, to 106.2% +/- 2.1% of the initial value (mean +/- SEM). The increase of trabecular bone density was significantly different from the placebo group (D) only in AAACa (group A) and not in AACa (group B) and the calcium carbonate (group C). Cortical bone density increase was also greater in group A (but not in B and C) than in D. Lumbar spine density did not change significantly. AAACa was apparently more effective, increasing trabecular bone density more than AACa and CaCO3 containing the same amount of elemental calcium.

Effect of salmon calcitonin on experimental osteoporosis induced by ovariectomy and low-calcium diet in the rat.

Abstract: To investigate the action and effect of calcitonin on osteoporosis, this study was performed in osteoporotic rats that had been ovariectomized and maintained on a low-calcium diet. Significant bone loss was noted in ovariectomized rats compared with those that underwent sham surgeries, and histological findings proved bone turnover to be increased. In comparison with this osteoporotic rat group, those given calcitonin showed less bone loss; the reduction in bone loss was obvious in the vertebral body, and rats given a high dose of calcitonin over a long duration showed even less bone loss, but this was hardly seen in the proximal tibial metaphysis. Histological findings proved that calcitonin inhibited the bone resorption that was stimulated by the ovariectomy. Values for osteoblast surfaces were relatively higher while those of reversal surfaces were lower. The results confirm that salmon calcitonin has an inhibitory action on bone resorption as well as being effective in maintaining and stimulating bone formation in vivo. These effects of calcitonin prevented progress of the osteoporosis that had been induced by ovariectomy, and the effects appeared differently in each region.

Morphologically extracted trabecular skeleton superimposed upon digital radiograph structure.

Abstract: The purpose of this study was to employ a morphological filter to digital X-ray images to extract the morphology of trabecular structures in a clearly understandable visual format. This study compares the trabecular skeleton extracted by a morphological filter to the original digital radiographic image by superimposing the images. A morphological filter (a combination of a single structuring element and a skeleton operation) based on a mathematical morphology theory was used to extract the skeletal pattern of trabecular bone from a digital X-ray image of a human femoral neck. Subset images with different operation numbers (n = 1, n = 2) were obtained, and then each image was superimposed on the original digital radiographic image using the superimpose function of a workstation. The extracted trabecular skeleton pattern was fairly consistent with the trabecular structure seen on the digital image according to the opinion of seven dentomaxillofacial radiologists. In their opinion, the majority of the structural elements were reproduced on the extracted skeleton. However, accurate skeleton elements were not extracted in the region of dense trabecular structure. The morphological filter was able to extract a large portion of the bone trabecular structure as a binary skeletal pattern image from trabecular bone on digital X-ray image, but more work is needed to improve the assessment of dense trabeculae.