Osteoid Volume Research Articles

Effect of 22-oxacalcitriol on bone histology of hemodialyzed patients with severe secondary hyperparathyroidism

To examine the effectiveness of 22-oxacalcitriol (OCT) injection on the improvement of severe osteitis fibrosa, we studied 10 hemodialyzed patients (age, 59 ± 12 years). The initial OCT dose was 5 μg and was administered three times weekly at the end of each hemodialysis session. OCT doses (1, 3, 5, 10, 15, and 20 μg) were changed in subsequent weeks to maintain serum calcium levels at less than 11.5 mg/dL. Administration of OCT significantly suppressed serum intact parathyroid hormone (PTH) from an initial level of 1,193 ± 584 to 775 ± 552 pg/mL in the 24th week (n = 10). OCT increased PTH levels again to 857 ± 635 pg/mL in the 48th week (n = 7). Among the 10 patients, 5 patients (high responders) showed more than a 50% suppression of serum intact PTH levels at the end of the study. The rest of the patients had hypercalcemia and did not receive increased OCT doses (low responders). At the start of the treatment, the only difference between high and low responders was serum calcium level. Serum calcium levels (adjusted for serum albumin level) increased from 9.7 ± 0.7 mg/dL (n = 10) at the beginning to 10.5 ± 0.6 mg/dL (n = 10) in the 24th week and to 11.1 ± 0.7 mg/dL (n = 7) in the 48th week. Six patients (1 to 6) agreed to undergo a second bone biopsy in the 24th week of OCT administration. In bone histomorphometric measurements, OCT significantly changed bone marrow fibrosis, mineralization (labeled mineralizing surface and bone formation rate), and osteoid formation (osteoid volume and thickness). In conclusion, intravenous OCT effectively suppressed PTH secretion and improved the bone histological characteristics of severe osteitis fibrosa, especially in patients with initial serum calcium levels less than 10 mg/dL. With concerns about OCT causing adynamic bone, additional bone histological data are needed to ensure the long-term safety of OCT.

Daily intermittent decreases in serum levels of parathyroid hormone have an anabolic-like action on the bones of uremic rats with low-turnover bone and osteomalacia

The calcium receptor agonist (calcimimetic) compound NPS R-568 causes rapid decreases in circulating levels of parathyroid hormone (PTH) in rats and humans. We hypothesized that daily intermittent decreases in serum PTH levels may have different effects on bone than do chronically sustained decreases. To test this hypothesis, we compared two NPS R-568 dosing regimens in rats with chronic renal insufficiency induced by two intravenous injections of adriamycin. Fourteen weeks after the second adriamycin injection, creatinine clearance was reduced by 52%, PTH levels were elevated ∼2.5-fold, and serum 25(OH)D 3 and 1,25(OH) 2D 3 levels were reduced substantially. Treatment by daily per os gavage, which decreased PTH levels intermittently, or continuous subcutaneous infusion, which resulted in a sustained suppression of serum PTH levels, then began for 8 weeks. Despite the hyperparathyroidism, the adriamycin-injected rats developed a low-turnover bone lesion with osteomalacia (fourfold increase in osteoid volume in the proximal tibial metaphysis) and osteopenia (67% decrease in cancellous bone volume and an 18% reduction in bone mineral density at the distal femur). Daily administered (but not infused) NPS R-568 significantly increased cancellous bone volume solely by normalizing trabecular thickness, and increased femoral bone mineral density by 14%. These results indicate that daily intermittent, but not sustained, decreases in PTH levels have an “anabolic-like” effect on bones with a low-turnover lesion in this animal model of chronic renal insufficiency.

Induction of Endochondral Bone Formation by Recombinant Human Transforming Growth Factor-β2 in the Baboon (Papio ursinus)

Members of the transforming growth factor-β(TGF-β) superfamily, the bone morphogenetic and osteogenic proteins (BMPs/OPs) but not the TGF-β proteins themselves, induce endochondral bone formation in vivo, when implanted in extraskeletal heterotopic sites of rodents. Here we show that recombinant human TGF-β2 (hTGF-β2) induces endochondral bone formation 30 days after implantation in heterotopic intramuscular sites of the baboon (Papio ursinus) at doses of 1,5 and 25 μg per 100 mg of guanidinium-inactivated collagenous bone matrix as carrier. On day 90 there was generation of large radiopaque and corticalized intramuscular ossicles. Five and 25 ug hTGF-β2 induced large ossicles in the rectus abdominis of the primate as evaluated by key parameters of bone formation, including generated tissue area, mineralized bone and osteoid volumes, and tissue alkaline phosphatase activity. On day 30 and 90 after healing, hTGF-β2 also induced bone formation when implanted in the rectus abdominis in conjunction with a sintered porous hydroxyapatite as carrier. mRNA expression in tissues from heterotopic specimens showed OP-1 (BMP-7) and BMP-3 transcripts in low abundance and with a linear dose-dependent increase both in collagenous matrix and hydroxyapatite samples. Type IV collagen mRNA expression, a marker of angiogenesis, was stronger in collagenous than hydroxyapatite samples. Growth and differentiation factor-10 (GDF-10) mRNA transcripts were expressed in ossicles with a distinctly chondrogenic phase, but its expression was greater in ossicles generated in porous hydroxyapatites, in which bone formation is not via a chondrogenic phase, but is rather intramembranous, without expression of type II collagen mRNA. In the same animals, however, 10 and 100 μg of the recombinant morphogen delivered by identical carriers (collagenous matrix and sintered hydroxyapatite) failed to heal calvarial defects. Thus in the primate, TGF-βs themselves are inducers of endochondral bone formation, although the present data strongly indicate that the bone inductive activity of hTGF-β2 is site and tissue specific, since a single application of hTGF-β, or hTGF-β1 in previously published experiments, did not induce bone in calvarial defects, but did induce endochondral bone differentiation in heterotopic sites.

Relationship between Renal Dysfunction and Bone Metabolism Disorder in Male Rats after Long-Term Oral Quantitative Cadmium Administration.

To investigate the importance of the cadmium (Cd) exposure condition in the evaluation of toxic effect on renal function and bone metabolism, six groups of Male Wistar rats were given Cd at respective daily doses of 2, 5, 10, 20, 30 and 60 mgCd/kg (as CdCl2) via a gastric tube for 6 consecutive days a week for 60 weeks. In the groups given a low Cd dose (2, 5 and 10 mgCd/kg), relatively more Cd accumulated in the kidney without liver damage than in the liver. In the high Cd dose groups (20, 30 and 60 mgCd/kg), on the other hand, more Cd accumulated in the liver than in the kidney. The daily intake of Cd dose from the intestinal tract in each experimental group was deduced to be about 0.36%-0.54% of the cumulative dose of oral Cd administration. The daily intake of Cd into the body was estimated as 7, 22, 40, 100, 120, 260 microgCd/kg/day in the experimental groups of 2, 5, 10, 20, 30 and 60 mgCd/kg/day, respectively. Increase of plasma enzyme activity (GOT, GPT) and of urinary enzyme excretion (NAG, AAP, GST), reflecting hepatic damage and renal dysfunction, was found in the high Cd dose groups (30 and 60 mgCd/kg) from the 5th week. Non-CdMT concentration in the kidney was also significantly high in the high Cd dose groups. In the low Cd dose groups (2 and 5 mgCd/kg), although the renal Cd concentration was higher than that of the high Cd dose groups, prominent renal dysfunction and hepatic damage were not observed. Regeneration, vacuolization, and eosinophilic bodies in proximal tubular tissue were mainly observed in the groups subjected to 20, 30 and 60 mgCd/kg administration. Very slight regeneration was also observed in the renal proximal tubular tissue at the 30th week for the 5 mgCd/kg and 10 mgCd/kg groups, and at the 60th week for the 2 mgCd/kg group. Remarkable decrease of bone mineral density at the midpoint of the femur was found in the high Cd dose groups. Also, the decrease in bone mineral density was observed before or after the manifestation of the renal dysfunction, depending on the dose and the duration of Cd administration. Urinary excretion of Pyr, DPyr, and Ca increased and plasma BGP decreased in the higher Cd dose groups. Osteoid volume in the femur tissue was not increased significantly by Cd exposure. Based on these results, it was suggested that Cd exposure caused osteoporotic change. The results of the present study suggested that the toxic effect of Cd on renal function and that on bone metabolism were caused at different times and that renal Cd concentration after long-term oral Cd administration depended on the dose and the duration of Cd exposure.

Bone Anabolic Effects of PTH(1-34) and Salmon Calcitonin in Ovariectomy- and Ovariectomy-Steroid-Induced Osteopenic Rats: A Histomorphometric and Biomechanical Study

Using an experimental model of type 1 osteoporosis under the chronic therapy with an anti-inflammatory steroid, the bone anabolic effect of PTH(1 – 34) was evaluated by histomorphometrical and biomechanical analysis. Wistar female rats (12-week-old) were ovariectomized and allowed to develop an osteopenic model in the presence or absence of methylprednisolone acetate (MPA: 0.1 mg/kg, s.c., 3-days-a-week basis from the 5th week after ovariectomy (OVX)). The osteopenia that developed for the first 12 weeks after OVX was almost completely normalized by subsequent PTH pulsing (2(20 μg/kg, s.c., 5-days-a-week) for 8 weeks starting at the 13th week; the following characteristics were observed: 1) proximal tibial metaphysis: recovered bone volume, rather increased trabecular thickness and osteoid volume, and normalized eroded surface; 2) 5th lumbar vertebra (L-5): partially recovered trabecular connectivity; 3) femur and 4th lumbar vertebra (L-4): recovered mechanical strength in maximum elastic load and maximum elastic energy. The anabolic effect of PTH(1-34) was not substantially modified by MPA. Salmon calcitonin (SCT: 10 U/kg per day, s.c., 5-days-a-week, for 8 weeks) was anabolic in limited parameters: decreased number of osteoclasts, recovered maximum elastic load in femur, and partially recovered maximum elastic load in L-4. The results suggest that PTH(1-34) pulsing is able to recover OVX-induced osteopenia in the structure and mechanical strength not only of the cancellous bone but also of the cortical bone, and the anabolic effect can be clinically expected even under steroid medication.

Reduction in bone formation and elevated bone resorption in ovariectomized rats with special reference to acute inflammation

Changes in bone modeling and remodeling in the tibia of growing rats within 30 days of ovariectomy (ovx) were evaluated by histomorphometric, mechanical, and biochemical means. Three days after ovx, suppressed bone formation was seen. This was shown by reduced osteoid volume, osteoblast surface, and bone formation rate in the secondary spongiosa, and a reduced longitudinal growth rate in the growth plate. In addition, the alkaline phosphatase and tartrate-resistant acid phosphatase activity in bone marrow supernatants was suppressed in conjunction with elevated serum sialic acid levels, indicating inflammation. Although estrogen deprivation itself may provoke the inflammatory process, the serum sialic acid level in the ovx group returned to the baseline level within 5 days after surgery, while that of estradiol in the ovx group remained consistently lower. This suggests that surgical stress, not estrogen deprivation, is the primary cause of the inflammatory response shortly after ovx. A significant difference ( p < 0.01) between the ovx and sham rats was seen in the osteoclast surface, which peaked on day 7 in the ovx rats. On day 14 postovariectomy, the bone formation rate peaked and remained constant until day 30. In the ovx rats, there was a sustained reduction in the serum albumin level until day 30. Estrogen deprivation may be the primary cause of these changes, because both surgical ovx and medical oophorectomy with gonadotropin-releasing hormone agonist (GnRHa) reduce the serum albumin level. In numerous studies dealing with changes after ovx in rats, we have observed: 1) a transient reduction in bone formation in relation to inflammatory changes evoked by ovx surgery, and 2) a sustained reduction in the serum albumin level for at least 30 days after ovx that is possibly due to estrogen deprivation.

Bone Anabolic Effects of Basic Fibroblast Growth Factor in Ovariectomized Rats

The purpose of this study was to characterize the bone anabolic effects of basic fibroblast growth factor (bFGF) in ovariectomized (OVX) rats. Female Sprague Dawley rats were subjected to ovariectomy or sham surgery at 3 months of age and maintained untreated for 2 months post surgery. Groups of OVX rats were then treated iv with bFGF at doses of 100 or 200 μg/kg·day for 7 or 14 days. Another group of OVX rats and a group of sham-operated control rats were treated iv with vehicle alone for 14 days. Certain groups of bFGF-treated OVX rats were killed at 7 or 14 days after withdrawal of treatment. The right tibiae were processed undecalcified for quantitative bone histomorphometry. Vehicle-treated OVX rats were characterized by decreased cancellous bone volume associated with increased bone turnover. Treatment of OVX rats with bFGF strongly stimulated bone formation, as indicated by marked increases of at least a factor of 10 in osteoblast surface, osteoid surface, and osteoid volume. Furthermore, new osteoid spicules were observed within the marrow cavity of these animals. Osteoclast surface was markedly decreased in bFGF-treated OVX rats, but this finding may be secondary to the extensive osteoid surface. The strongest bone anabolic effects occurred in OVX rats treated with the higher dose of bFGF for 14 days, but these animals exhibited a bone mineralization defect, as evidenced by abundant osteoid and a lack of double fluorochrome labeling, despite markedly increased osteoblast surface. However, the newly-formed osteoid rapidly calcified after withdrawal of bFGF treatment. The data indicate that bFGF not only stimulates bone formation on pre-existing bone surfaces but also induces de novo formation of bone spicules within the marrow cavity, which results in partial restoration of lost cancellous bone mass in osteopenic OVX rats after only 14 days of treatment with the growth factor. These findings suggest that bFGF merits consideration for development as a potential treatment for patients with severe osteopenia who are unresponsive to conventional osteoporosis therapies.

Bone anabolic effects of basic fibroblast growth factor in ovariectomized rats.

The purpose of this study was to characterize the bone anabolic effects of basic fibroblast growth factor (bFGF) in ovariectomized (OVX) rats. Female Sprague Dawley rats were subjected to ovariectomy or sham surgery at 3 months of age and maintained untreated for 2 months post surgery. Groups of OVX rats were then treated iv with bFGF at doses of 100 or 200 microg/kg day for 7 or 14 days. Another group of OVX rats and a group of sham-operated control rats were treated iv with vehicle alone for 14 days. Certain groups of bFGF-treated OVX rats were killed at 7 or 14 days after withdrawal of treatment. The right tibiae were processed undecalcified for quantitative bone histomorphometry. Vehicle-treated OVX rats were characterized by decreased cancellous bone volume associated with increased bone turnover. Treatment of OVX rats with bFGF strongly stimulated bone formation, as indicated by marked increases of at least a factor of 10 in osteoblast surface, osteoid surface, and osteoid volume. Furthermore, new osteoid spicules were observed within the marrow cavity of these animals. Osteoclast surface was markedly decreased in bFGF-treated OVX rats, but this finding may be secondary to the extensive osteoid surface. The strongest bone anabolic effects occurred in OVX rats treated with the higher dose of bFGF for 14 days, but these animals exhibited a bone mineralization defect, as evidenced by abundant osteoid and a lack of double fluorochrome labeling, despite markedly increased osteoblast surface. However, the newly-formed osteoid rapidly calcified after withdrawal of bFGF treatment. The data indicate that bFGF not only stimulates bone formation on pre-existing bone surfaces but also induces de novo formation of bone spicules within the marrow cavity, which results in partial restoration of lost cancellous bone mass in osteopenic OVX rats after only 14 days of treatment with the growth factor. These findings suggest that bFGF merits consideration for development as a potential treatment for patients with severe osteopenia who are unresponsive to conventional osteoporosis therapies.

Function of Fos proteins in bone cell differentiation

c-Fos, a component of the dimeric transcription factor AP-1, is a key regulator of bone cell growth and differentiation, affecting both osteoblast and osteoclast lineages during normal development and in bone diseases. Overexpression of c-Fos in transgenic mice results in osteoblastic or chondroblastic tumors. Osteosarcoma formation in c-Fos transgenic mice is enhanced in the p53-/-background, where higher numbers of tumors per animal develop with an earlier onset, thus providing a valuable tumor model (Jochum, W. and Wagner, E. F., unpublished observations). Unlike c-Fos, overexpression of Fra-1, a c-Fos-related protein, in transgenic mice did not induce bone tumors. Lack of tumorigenicity in Fra-1 transgenic mice is consistent with the notion that Fra-1 lacks transcriptional activation domains required for cellular transformation. Unexpectedly, however, Fra-1 transgenic mice showed a progressive increase in bone mass. This osteosclerotic phenotype is characterized by an increase in osteoblast surface and osteoid volume. We are currently analyzing the direct involvement of Fra-1 in osteoblast proliferation and differentiation. In contrast, mice lacking c-Fos develop osteopetrosis due to a complete block in osteoclast differentiation. To have an insight into the mechanism by which c-Fos is required for osteoclastogenesis, an in vitro c-fos-/-rescue system was established whereby c-Fos-deficient splenocytes were infected with retroviral vectors expressing various AP-1 components. After culturing the infected cells in osteoclastogenic conditions, rescued osteoclast formation was quantitated by counting cell numbers expressing osteoclast markers such as tartrate-resistant acid phosphatase (TRAP) and calcitonin receptor. In addition, bone resorption was measured on bovine bone slices. Surprisingly, Fra-1, which lacks transactivation domains detectable in c-Fos, rescued osteoclast differentiation even more efficiently than c-Fos in vitro. Fra-2 and FosB rescued to a lesser extent than c-Fos. Unlike Fos proteins, however, none of the Jun proteins (c-Jun, JunB, and JunD) rescued the differentiation block. Furthermore, the above-mentioned fra-1 transgene, when crossed into mice lacking c-Fos, allowed osteoclast differentiation to proceed and rescued the osteopetrotic phenotype. Regarding the specific role of Fos proteins in osteoclast formation, a number of conclusions can be drawn from our present analyses. First, Fra-1 is the most potent osteoclastogenic Fos protein; introduction of Fra-1 into osteoclastogenic cell lines further increased their differentiation potentials. Second, the fra-1 gene carries AP-1 sites and is transcriptionally activated by c-Fos in the macrophage/osteoclast lineage. Although the fra-1 gene can be activated in a c-Fos-independent manner in many other cell types, transcriptional activation of fra-1 in osteoclast precursors seems to require c-Fos. This implies that c-Fos knock-out mice may be equivalent to c-Fos and Fra-1 double knock-out in the macrophage/osteoclast lineage, leading to an osteopetrotic phenotype. Note that Fra-1 knock-out mice are embryonic lethal due to a placental defect. (Schreiber, M. and Wagner, E. F., unpublished observations). Osteoclastogenic activity of c-Fos in wild-type mice may be either partially or fully mediated by Fra-1. Third, c-Fos is known to carry a major transactivation domain in its COOH-terminal part, which responds to Ras signaling and is critical for transformation. A detailed structure-function analysis showed that COOH-terminal parts of Fos proteins are dispensable for osteoclast differentiation. Further analyses are necessary to identify the mechanisms by which Fra-1, or c-Fos lacking transactivation domains, efficiently activate target genes required for osteoclast differentiation in the absence of c-Fos. One mechanism by which Fos proteins regulate osteoclastspecific gene expression is revealed by the analysis of the TRAP promoter region. Transient transfection and DNA binding experiments revealed juxtaposed functional binding sites for transcription factors NF-AT (nuclear factor of activated T cells) and AP-1. Overexpression of full-length NF-AT3 and Fra-1 activated TRAP promoter-reporter constructs in nonhematopoietic cell lines treated with calcium ionophore and phorbol 12-myristate 13-acetate (PMA). Moreover, ectopic expression of NF-AT3 in peritoneal macrophages and in a macrophage cell line induced expression of the endogenous TRAP gene, suggesting that NF-AT is indeed a key player in osteoclast-specific TRAP gene regulation. Identification of additional AP-1 target genes during osteoclastogenesis as well as further experiments aiming to define a causal role of the transcription factor NF-AT in osteoclast-specific gene expression are important avenues for future research.

Musculoskeletal manifestations of osteomalacia:report of 26 cases and literature review

Objective: This study was undertaken to describe the musculoskeletal manifestationsin a selected population of 26 patients with biopsy-proven osteomalacia (OM) and provide a literature update. Methods: The 26 patients with biopsy-proven OM were selected from a totalnumber of 79 patients who underwent anterior iliac crest biopsy. The diagnosis of OM was confirmed by the presence of an osteoid volume greater than 10%, osteoid width greater than 15 μm, and delayed mineralization assessed by double-tetracycline labeling. Results: OM was caused by intestinal malabsorption in 13 patients, whereas six other patients presented with hypophosphatemia of different causes. Five elderly patients presented with hypovitaminosis D, and in two patients the OM was part of renal osteodystrophy. Twenty-three patients presented with bone pain and diffuse demineralization, whereas three other patients had normal or increased bone density. Characteristic pseudofractures were seen in only seven patients. Six of the 23 patients with diffuse demineralization had an “osteoporotic-like pattern” without pseudofractures. Prominent articular manifestations were seen in seven patients, including a rheumatoid arthritis-like picture in three, osteogenic synovitis in three, and ankylosing spondylitis-like in one. Two other patients were referred to us with the diagnosis of possible metastatic bone disease attributable to polyostotic areas of increased radio nuclide uptake caused by pseudofractures. Six patients also had proximal myopathy, two elderly patients were diagnosed as having polymalgia rheumatica, and two young patients were diagnosed as having fibromyalgia. One of the patients who presented with increased bone density was misdiagnosed as possible fluorosis. Conclusion: OM is usually neglected when compared with other metabolic bonediseases and may present with a variety of clinical and radiographic manifestations mimicking other musculoskeletal disorders.

Histomorphometric study of iliac bones in cervical myelopathy with ossification of the posterior longitudinal ligament

To investigate whether there was any abnormal systemic bone metabolism in patients with ossification of the posterior longitudinal ligament (OPLL), we measured various histomorphometric indices of the iliac trabecular bones in 19 patients with OPLL (14 men, 5 women). For each index, the Z-score for each patient was calculated, using the SD and mean value for non-OPLL control group (n = 159) who underwent orthopaedic surgeries and had no systemic disease, age-matched for each decade. A Z-score of 1.0 meant that the observed data deviated 1 SD from the normal average, and a distribution from –2 to +2 was considered normal. All the averaged Z-scores were within ±1.0 and there were no significant differences between the OPLL and the control groups. There was also no difference in Z-score among three types of OPLL (segmental, continuous, and mixed). From these results, we concluded that there was no common abnormal bone metabolism affecting the bone histomorphometry of the iliac bone in OPLL patients. However, in two patients, the Z-score for bone volume (BV/TV) was more than 2.0, with increased osteoid volume (OV/TV) and increased trabecular thickness, suggesting that there was some abnormal bone metabolism in these two patients with OPLL.

Hypokinetic azotemic osteodystrophy

CASE PRESENTATION A 61-year-old male dialysis patient was referred from another center to the Bone and Mineral Research Unit of the Hospital Central de Asturias for evaluation. At age 24, renal tuberculosis had necessitated a left nephrectomy; at age 42 nephrolithiasis in the right kidney had prompted surgical intervention. Four years later, moderate renal failure presumably due to chronic interstitial nephritis was diagnosed; 7 years later, at age 53, he entered the dialysis program. He received treatment in a dialysis unit equipped with a reverse osmosis (RO) system for water treatment. Renal osteodystrophy had been present since the beginning of dialysis, and the thoracic and lumbar spine showed demineralization. During the first 5 years of hemodialysis, symptoms and radiologic signs of hyperparathyroidism, including multiple vascular calcifications, worsened. The serum calcium (Ca+) level progressively increased, reaching 10.5 mg/dl. The serum phosphorus (P) level was high, although the patient had received long-term treatment with aluminum hydroxide. During that period, parathyroid hormone (PTH) levels (carboxy terminal) had remained 40 to 50 times higher than the upper limit of normal. Total alkaline phosphatase measurements had stayed in the low-normal range. The patient needed frequent red cell transfusions to avoid symptoms of anemia. Five years after beginning dialysis, he had a non-traumatic radial fracture coincident with worsening of the radiologic signs and his symptoms. A subtotal parathyroidectomy was performed. During the 2 years after the subtotal parathyroidectomy, his symptoms mildly improved, and the carboxy terminal PTH level decreased, stabilizing in the upper limit of normal. Total alkaline phosphatase fell to lower than normal, and the serum P was kept at acceptable values by aluminum hydroxide and calcium carbonate. Oral calcitriol (1.5–3.5 μg/week) maintained serum Ca+ levels in the range of 10.0 mg/dl–10.5 mg/dl. Two years after the parathyroidectomy, bilateral carpal tunnel syndrome was detected and surgically relieved. Amyloid was detected in the surrounding fat tissue, serum beta-2 microglobulin was elevated; dialysis with highly permeable membranes (PAN) was prescribed. Three years after the parathyroidectomy, PTH levels, measured several times using a PTH intact assay, revealed very low values (from undetectable to 13.5 pg/ml) and total alkaline phosphatase had returned to pre-surgery values (in the low-normal range). Basal serum aluminum (Al) was 96 μg/liter, increasing to 234 μg/liter after the infusion of 40 mg/kg of deferoxamine. Because Al-induced bone disease was suspected, a bone biopsy with tetracycline labeling was performed. The main histologic and histomorphometric findings of the bone biopsy were reduction in trabecular bone volume (11%), a slight increase in relative osteoid surface (20%), a decrease in osteoid volume (1.3%), low osteoblast surface (0.12%), low osteoclast surface (0.59%), positive Al surface staining (aluminum 16%; solochrome of azurine, 47%), and negative iron (Fe) staining (Perls). Bone Al and Fe concentration, measured by atomic absorption spectrometry, were 28 μg/g and 405 μg/g, respectively (normal, 3 μg/g and 300 μg/g, respectively). The tetracycline study showed only a single and diffuse labeling. Aluminum-induced adynamic bone disease was diagnosed. After the bone biopsy, therapy with deferoxamine (40 mg/kg/week) was prescribed (18 months of intermittent treatment over 3 years). During deferoxamine therapy, repeated basal serum Al levels were 50–85 μg/liter; these values doubled after the deferoxamine tests performed during that period. At the end of deferoxamine treatment, the serum Al remained at the same level, but intact PTH and alkaline phosphatase levels progressively increased, reaching 150 pg/ml and 219 U/liter, respectively (normal, 10–65 pg/ml and 70–280 U/liter). Before and after the deferoxamine treatment, serum transferrin, serum Fe, and Fe-transferrin saturation were always in the normal range. Two years after the bone biopsy, when the patient was 62 years old, he suffered a fracture of the right femoral neck; one year later, he fractured the left femoral neck. Both were treated with standard surgical techniques. During the following 4 years, the patient was admitted to the hospital several times with diagnoses of diabetes mellitus type II, diverticulitis, ventricular tachycardia, and congestive cardiac failure. Also, severe vascular obstruction of the left leg necessitated amputation of the second toe of the left foot. During the last year (7 years after the bone biopsy), he has maintained a serum calcium ranging between 9.0–9.5 mg/dl; serum phosphorus, 4.7–6.5 mg/dl; serum aluminum, 73.0–90.8 μg/liter; normal serum iron parameters; intact PTH levels of 314–415 pg/ml; and an alkaline phosphatase of 125–139 U/liter.

Prevalence of subclinical vitamin D deficiency in different European countries.

Long-lasting and severe vitamin D deficiency leads to osteomalacia, a metabolic bone disease characterized by typical biochemical abnormalities (low serum concentrations of calcium and phosphate, increased activity of alkaline phosphatase). Bone histology shows decreased mineralization of bone matrix, increased osteoid volume and decreased bone formation. Vitamin D deficiency can be confirmed by measuring the serum concentration of 25-hydroxyvitamin D (25(OH)D), which is the major circulating metabolite bound with high affinity to a binding protein (DBP) and representing the storage form of vitamin D. In patients with osteomalacia, serum 25(OH)D levels are usually below 5 ng/ml and often undetectable. In contrast, patients with primary osteoporosis usually have serum concentrations of calcium and phosphate within the normal range, and alkaline phosphatase is rarely elevated. Therefore, in the opinion of many authors vitamin D deficiency is not an important pathogenetic factor for the development of osteoporosis. However, growing evidence demonstrates that moderately low levels of vitamin D can already have unfavorable effects on calcium homeostasis leading to bone loss, even if osteomalacia is not present. BargerLux and coworkers [1] showed that 25(OH)D contributes to the stimulation of enteral calcium absorption explaining the secondary increase in parathyroid hormone resulting from vitamin D deficiency, although 1,25-dihydroxyvitamin D (1,25-(OH)2D) levels may still be normal. A vitamin D status insufficient to maintain physiologic calcium homeostasis, but not resulting in overt osteomalacia, is called vitamin D insufficiency or subclinical vitamin D deficiency. Vitamin D insufficiency is a common problem especially in Europe (for reasons explained below), and therefore this paper will review the vitamin D status in different European countries. Definition of ‘Subclinical’ Vitamin D Deficiency

Magnesium Deficiency Induces Bone Loss in the Rat

Disorders in which magnesium (Mg) depletion is common have an associated high incidence of osteoporosis. Mg depletion in humans results in hypocalcemia, low serum parathyroid hormone (PTH) and 1,25(OH)₂-vitamin D levels, as well as PTH and vitamin D resistance which may serve as mechanisms for the development of osteoporosis. In order to determine if isolated Mg depletion will result in bone loss, we have induced dietary Mg deficiency in the rat. Adult (290 g) female rats were given either a low-Mg diet (2 mg/100 g chow; n = 6) or a normal control Mg diet (63 mg/100 g chow; n = 6). Dietary calcium (Ca) was normal in both groups (592 mg/100 g chow). At 12 weeks, blood was obtained for serum Mg, Ca, PTH, 1,25(OH)₂-vitamin D, and osteocalcin determinations. The rats were then euthanized and the femurs obtained for mineral analysis and histomorphometry. Serum Mg in the low-Mg group was less than control (0.4 ± 0.2 vs. 1.9 ± 0.2 mg/dl, p < 0.001; mean ± SD) while serum Ca was higher (11.7 ± 0.5 vs. 9.3 ± 0.4 mg/dl, p < 0.001). PTH was suppressed in the Mg-deficient group (36 ± 16 vs. 109 ± 30 pg/ml in controls, p < 0.002). Serum 1,25(OH)₂-vitamin D was also suppressed in the Mg-deficient animals (7.1 ± 4.8 vs. 28.5 ± 8.2 pg/ml in controls, p < 0.002). Serum osteocalcin levels were not different (19.8 ± 2.5 ng/ml in Mg-deficient rats vs. 15.3 ± 3.4 ng/ml in controls). While the ash weight of Ca and phosphorus in the femur did not change, the ash weight of Mg fell (low-Mg group 0.55 ± 0.01%, controls 0.65 ± 0.02%, p < 0.001). Histomorphometry demonstrated reduction in bone mass; the trabecular bone volume in the femur of the low-Mg group was reduced from control (7.7 ± 0.2 vs. 13.7 ± 1.9%, p < 0.002). A surprising new observation was an increase in osteoclast (OC) bone resorption with Mg depletion. The number of OC per millimeter bone surface was 16.9 ± 1.3 in the low-Mg group versus 7.8 ± 1.5 in controls (p < 0.001). The percentage of bone surface occupied by OC was 38.3 ± 3.7 in the low-Mg group versus 17.7 ± 2.4 in controls (p < 0.001). This increased resorption occurred with an inappropriate non-altered bone-forming surface relative to control (% osteoid surface: low-Mg group 2.4 ± 0.7 vs. controls 2.6 ± 0.4; % osteoid volume: low-Mg group 0.25 ± 0.09 vs. controls 0.38 ± 0.06; number of osteoblasts per millimeter bone surface: low-Mg group 0.9 ± 0.3 vs. controls 1.3 ± 0.3). No increase in bone-forming surface or osteoblast number despite an increase in OC-resorbing surface and OC number strongly suggests impaired activation of osteoblasts and an uncoupling of bone formation and bone resorption. Our data demonstrate that Mg depletion in the rat alters bone and mineral metabolism which results in bone loss.

Effect of Etidronate Treatment on Bone Mass of Male Nephrolithiasis Patients with Idiopathic Hypercalciuria and Osteopenia

Osteopenia is frequently found among calcium stone forming (CSF) patients with hypercalciuria. We investigated the effect of a 2-year therapeutic course of etidronate, a bone-sparing agent, in 7 young male CSF patients. The treatment consisted of a cyclic intermittent administration of phosphate followed by sodium etidronate and calcium supplementation every 74 days. Bone mineral density (BMD) measured at 12-month intervals and bone biopsies performed at baseline and after 2 years were the primary efficacy parameters. Mean lumbar spine BMD increased significantly after the 1st year by 2.6 ± 1.0% (mean ± SE, p < 0.05) and nonsignificantly after the 2nd year by 5.6 ± 2.6%. Nonsignificant changes were observed for femoral neck mean BMD after either the 1st or the 2nd year (decrease of 2.0 ± 1.0% and 2.0 ± 3.0%, respectively). Mean histomorphometric parameters showed that bone volume, osteoid volume, and eroded surfaces did not differ from baseline (13.9 ± 2.2 vs. 12.2 ± 1.1%, 1.2 ± 0.7 vs. 2.6 ± 0.7%, and 20.7 ± 6.2 vs. 13.7 ± 1.3%, respectively). Osteoid surface was significantly lower than baseline values (9.5 ± 5.2 vs. 18.8 ± 5.3%, p < 0.05). These data suggest that etidronate given to young male CSF patients presenting with hypercalciuria and osteopenia led to a significant amelioration of BMD, evident only in the lumbar spine after 1 year of treatment. There was no histological evidence of long-term improvement in bone remodeling.

Gross, histological and histomorphometric features of the navicular bone and related structures in the horse

Forelimb navicular bones and associated soft tissues were collected from 3 groups of horses and subjected to pathological examinations. The groups consisted of 38 horses with clinical navicular disease (ND) and 2 control groups, with no history of forelimb lameness, consisting of 25 age-matched mature horses (A-MC) and 9 immature horses (IC). Histological and histomorphometric studies were performed on tissue samples from 10 ND, 10 A-MC and 5 IC horses. Gross changes seen only in ND horses included: full thickness defects in the palmar surface fibrocartilage, palmar cortex erosion, medullary lysis, flexor digitorum profundus tendon (FDPT) surface fibrillation, FDPT core lesions and adhesions between the FDPT and navicular bone. Palmar surface partial thickness fibrocartilage loss and distal border fragmentation were seen with a significantly greater incidence in ND than in A-MC and not observed in IC. Remodelling of the proximal border, FDPT surface colouration, palmar surface fibrocartilage colouration and proximal border entheseous bone were identified in ND and A-MC but not in IC. Mid-ridge synovial fossae and horizontal depressions in the palmar surface were identified in all groups. Histologically palmar fibrocartilage thinning and loss were associated with reduced palmar fibrocartilage cell density and chondrocyte cluster formation. Palmar fibrocartilage fibrillation, palmar cortical bone defects, fibromyxoid stromal change in the medulla, medullary pseudocyst formation and entheseous new bone formation were all seen in ND. The adjacent FDPT showed fibrillation, tag formation and degeneration of the dorsal surface. Necrotic foci were also present within the body of the tendon. Although not always present, medullary bone pseudocysts, separate mineralised foci and most changes on the dorsal surface of the FDPT were specific to ND. Bone histomorphometric parameters were compared among groups. Cross-sectional area reduced from the sagittal ridge to the medial and lateral margins of each navicular bone. IC navicular bones had a smaller subchondral area, subchondral bone volume and a greater osteoid volume than in the AC, indicating that these differences were age-related. In ND the medullary area was decreased but the trabecular bone volume increased. The palmar subchondral area was increased but contained bone with an increased porosity and osteoid volume. Changes occurred from the medial to the lateral margins of the bone in horses with ND indicating remodelling of the bony elements throughout the bone in ND. The histological and histomorphometric changes in the navicular bone and palmar fibrocartilage were considered similar of those found in articular hyaline cartilage and subchondral bone in osteoarthritis.

Bone disease in African children with slipped capital femoral epiphysis: Histomorphometry of Iliac crest biopsies

African teenagers with slipped capital femoral epiphysis (SCFE) not infrequently also have genu valgum (knock-knee). Because we had previously demonstrated metabolic bone disease attributable to dietary calcium deficiency in black teenagers with genu valgum, we examined 29 black teenagers (15 male, 14 female) with SCFE for metabolic bone disease. Each patient had an iliac crest bone biopsy taken (after double tetracycline labeling) for routine histomorphometry, and blood and urine samples for bone biochemistry. Spinal bone mineral density was measured in 13 patients. Compared to reported data, we found our patients to be sexually more immature, older, at least as obese, and to have more severe and more frequently bilateral hip disease. Eighty percent of the children took dairy products only once or twice a week or less frequently, and 37.9% had genu valgum. Compared with race- and age-matched South Africans, bone biopsies in our patients showed lower bone volume ( BV TV , p = 0.0003), wall thickness ( p = 0.0002), and trabecular thickness (Tb.Th, p = 0.0002), and a tendency to greater trabecular spacing (Tb.Sp, p = 0.053). Lower osteoid volume ( OV BV , p = 0.0001 ), osteoid surface ( OS BS , p = 0.0001 ), osteoid thickness (O.Th, p = 0.0002), double labeled surface ( dLS BS , p = 0.029 ), and bone formation rate ( BFR BS , p = 0.037 ) suggested poorer bone forming capacity in our patients. No evidence of hyperparathyroid bone disease or osteomalacia was found. BV TV was below the reference range (14.2%) in 65.5% of cases; these patients had lower values for Tb.Th ( p = 0.037) and Tb.N ( p = 0.0003), greater Tb.Sp ( p = 0.0002), a tendency to lower adjusted apposition rate (Aj.AR, p = 0.057), and had had less frequent intake of dairy products than those with normal BV TV ( p = 0.024). Furthermore, months since menarche correlated with histomorphometric variables BV TV ( r = 0.667, p = 0.009), Tb.Th ( r = 0.745, p = 0.002), Tb.Sp ( r = −0.549, p = 0.042), O.Th ( r = 0.784, p = 0.0009), and Aj.AR ( r = 0.549, p = 0.042). The correlation between Tb.Th and spinal bone mineral content ( r = 0.656, p = 0.015) suggests that the reduced trabecular thickness reflected a generalized bone condition. A greater than normal proportion of patients had spinal bone mineral density values below — 1 standard deviation (SD) of the mean (osteopenia) ( p = 0.0001). Patients tested for parathyroid hormone and 25-hydroxyvitamin D levels were found to have normal values. Parathyroid hormone correlated with Aj.AR ( r = 0.661, p = 0.038) and serum phosphorus ( r = −0.764, p = 0.010). We conclude that sexual immaturity and possibly past dietary calcium deficiency contributed to osteopenia, and that this, together with obesity, led to the development of more severe and more frequently bilateral SCFE in our patients than in reported series of black and white children.

Effect of locally infused IGF-I on femoral gene expression and bone turnover activity in old rats.

Previously, we showed that the age-dependent deficit in bone formation activity can be attributed in part to a decline in local expression of insulin-like growth factor I (IGF-I) and altered mitogenic response of old osteoprogenitor cells to IGF-I. To establish the cellular basis for using IGF-I as a possible therapeutic agent for osteoporosis, we examined the effect of locally infused (50 ng/day for 14 days) on the expression of osteoblast-related genes in femurs of old rats. Northern and dot blot analyses showed that the expression of procollagen (I), osteopontin, alkaline phosphatase, and osteocalcin was increased 0.4- to 1.5-fold in IGF-I-treated femurs as compared with control femurs. Histomorphometric analyses were carried out in parallel experiments to assess the changes in bone remodeling activity. Trabecular bone volume, trabecular number, and trabecular thickness were increased 56%, 29%, and 23%, respectively, whereas trabecular separation was reduced 26% by IGF-I treatment. IGF-I treatment increased significantly the osteoid volume, osteoid surface, osteoblast number, and osteoblast surface. Mineralizing surface and mineral apposition rate, kinetic indices of bone formation, were also stimulated by IGF-I treatment. The bone formation rate was stimulated 81% in IGF-I-treated femurs as compared with control femurs. In contrast, eroded surface and osteoclast surface, parameters associated with bone resorption, were not affected by IGF-I treatment. These findings suggest that local administration of IGF-I into femurs of old rats can stimulate the expression of matrix proteins and improve trabecular bone status by stimulating bone formation without any appreciable effect on bone resorption.

長期経口カドミウム投与ラットにおける骨密度と類骨の変化

長期経口カドミウム投与ラットにおける骨密度と類骨の変化

The inhibitory effects of aluminum and iron on bone formation—in vivo and in vitro study

We studied the effects of Al and Fe on bone metabolism in vivo and in vitro using nitrilotriacetic acid (NTA) as a chelator. We have reported experimental osteopathy in growing rats (6 weeks). In the present study, we used adult Wistar rats (15 weeks). Both Al-NTA (3.0 mg Al/kg) and Fe-NTA (6.0 mg Fe/kg) caused renal insufficiency and osteomalacia. Osteoid volume was 65.79±5.25% in Al-treated rats, 3.97±2.25% in Fe-treated rats and 2.22±0.77% in the control rats. The impairment of bone mineralization was detected in Al- and Fe-treated rats. The osteoidosis induced by Al-NTA in adult rats was much severer than that in growing rats. Osteomalacia was not induced by Fe-NTA in growing rats, but moderate osteomalacia was induced in adult rats. Therefore, an age-related change of the bone metabolism was suggested. The direct effects of these metals were also examined using an osteoblast-like cell line (HuO9) in vitro. The collagen synthesis was kept constant between Al and Fe concentration of 10−6 and 10−4 M, although ALP activity was decreased by Al and Fe. The results of this study demonstrate that Al- and Fe-NTA have negative effects on bone formation in vivo and in vitro, and that they are useful for experimental models for mineral induced osteomalacia.