Differences in vertebral, tibial, and iliac cancellous bone metabolism in ovariectomized rats.

Bone histomorphometry is usually performed on the iliac bone in humans and the tibia or vertebrae in rats. Bone metabolism differences among skeletal sites may be problematic when translating experimental results from rats to humans, but data on such differences in rats are lacking. Therefore, we examined the differences in bone structure and metabolism among skeletal sites using the lumbar vertebra (LV), tibia, and iliac bone obtained from ovariectomized or sham-operated rats preoperatively and at various times from 3 days to 26 weeks postoperatively. The trabeculae were thicker in the LV, where bone metabolism was less active than at other sites, and numerous fine trabeculae were observed in the tibia, where bone metabolism was more active. The iliac bone structure and metabolism were intermediate between those of the tibia and LV. Ovariectomy induced lower bone volume and higher bone metabolism in all skeletal sites, but the changes were greatest and occurred earliest in the tibia, followed by the iliac bone and then LV. Ovariectomy caused changes in bone metabolic markers, which occurred earlier than those in bone tissue. Activation frequency (Ac.f) increased after ovariectomy. At week 26 in ovariectomized rats, Ac.f was highest in the tibia (3.13 N/year) but similar between iliac bone (0.87 N/year) and LV (1.39 N/year). Ac.f is reportedly 0.3-0.4 N/year in the iliac bone of postmenopausal women, suggesting that bone turnover in rats is several times higher than in humans. The reference values reported here are useful for translating experimental results from rats to humans.

The slow resorption with replacement by bone of a hydrothermally synthesized pure calcium-deficient hydroxyapatite.

A newly developed calcium-deficient hydroxyapatite composed of rod-shaped particles synthesized by the hydrothermal method (HHA) and stoichiometric hydroxyapatite (SHA) synthesized by the sintering method was used for in vivo implantation and in vitro culture systems to compare these biological responses. In the rabbit femur, implanted HHA was slowly resorbed and about 80% of the implant remained 24 weeks after implantation; however, up to 72 weeks after implantation, most of the implanted HHA was resorbed. The implanted SHA was unresorbed throughout the experimental period, but degradation by the invasion of newly formed bone was seen at 72 weeks after implantation. Bone histomorphometry showed that the volume of newly formed bone and the number of osteoclasts in the implanted region were significantly higher in HHA than in SHA 24 weeks after implantation. In vitro culture of C2C12 cells with the induction of osteoblastic phenotypes using recombinant bone morphogenetic protein-2 showed similar cell density and the induction of alkaline phosphatase activity between the cells on HHA and SHA discs. In vitro osteoclastogenesis of HHA and SHA discs using bone marrow macrophages and recombinant receptor activator of nuclear factor-kappaB ligand showed higher TRAP activity of osteoclasts cultured on HHA discs. These results showed that slow biodegradability did not always correlate to final replaceability in bone tissue, and suggested that the activity of osteoclasts correlated to the bone-forming activity of osteoblasts.

The effect of the microstructure of beta-tricalcium phosphate on the metabolism of subsequently formed bone tissue.

The response of bone cells to a newly developed porous beta-tricalcium phosphate composed of rod-shaped particles (RSbeta-TCP), beta-TCP composed of conventional non-rod-shaped particles (Cbeta-TCP), and hydroxyapatite (HA) was analyzed using in vivo implantation and in vitro osteoclastogenesis systems. Implantation of the materials into the rabbit femur showed that RSbeta-TCP and Cbeta-TCP were bioresorbable, but HA was not. Up to 12 weeks after the implantation, bioresorption of RSbeta-TCP and Cbeta-TCP accompanied by the formation of new bone occurred satisfactorily. At 24 weeks post-implantation, most of the RSbeta-TCP had been absorbed, and active osteogenesis was preserved in the region. However, in the specimens implanted with Cbeta-TCP, the amount of not only the implanted Cbeta-TCP but also the newly formed bone tissue decreased, and bone marrow dominated the region. The implanted HA was unbioresorbable throughout the experimental period. When osteoclasts were generated on RSbeta-TCP, Cbeta-TCP, or HA disks, apparent resorption lacunae were formed on the RSbeta-TCP and Cbeta-TCP, but not HA disks. Quantitation of the calcium concentration in the culture media showed an earlier and more constant release of calcium from RSbeta-TCP than Cbeta-TCP. These results showed that the microstructure of beta-TCP affects the activity of bone cells and subsequent bone replacement.

Menatetrenone rescues bone loss by improving osteoblast dysfunction in rats immobilized by sciatic neurectomy.

Menatetrenone (MK-4) is a vitamin K2 homologue that has been used as a therapeutic agent for osteoporosis in Japan. However, there is no far any reported evidence that MK-4 ameliorates a pre-existing condition of reduced bone mineral density (BMD) in vivo. In this study, we evaluated the effect of MK-4 in a rat model of established bone loss through immobilization caused by sciatic neurectomy. Unilateral sciatic neurectomy (SNx) was performed in rats, and 10 or 30 mg/kg of MK-4 or vehicle was administered to the rats three weeks after operation. Seven weeks after operation, the rats were sacrificed and BMD and bone histomorphometric parameters were measured to assess the effects of MK-4. While BMD of the distal femoral metaphysis was significantly decreased after SNx, MK-4 administration increased BMD in the neurectomized rats. Bone formation was decreased continuously and bone resorption was initially increased in SNx rats. Four weeks treatment of MK-4 increased bone formation and suppressed bone resorption. In addition, increased carboxylated osteocalcin and decreased undercarboxylated osteocalcin in serum were observed in MK-4-administered rats. These results indicated that MK-4 rescued bone volume by improving osteoblast dysfunction and accelerating gamma carboxylation of osteocalcin. MK-4 may be useful for treating disuse osteopenia.

Microfocus computed tomography analysis of early changes in bone microstructure in rats with chronic renal failure.

BACKGROUND/AIMS: Little is known about the microstructural changes in bone tissue in the early phase of chronic renal failure (CRF). In this study, we analyzed the bone tissue in a rat model of early CRF using a high-resolution X-ray tomographic system, i.e. microfocus computed tomography (MXCT), which is capable of detecting microscopic structural changes in bone tissue. METHODS: CRF rats were created by 7/8 nephrectomy by ligation of the renal artery branches, and were sacrificed 24 weeks after surgery. MXCT and bone mineral densitometry by single-energy X-ray absorptiometry (SXA) were performed using extracted bone samples. Images obtained by MXCT were further analyzed by node-strut morphometry. RESULTS: Biochemical analysis confirmed that CRF was induced in 7/8-renal artery-ligated rats. MXCT demonstrated a significant decrease in trabecular bone in uremic rats, whereas SXA was unable to detect the difference. The node-strut method revealed decreased trabecular connectivity, with little change in the thickness of the trabecular bone in uremic rats. CONCLUSION: MXCT was able to detect significant changes in trabecular bone at an early stage of renal failure. The observed structural changes differed from those found in long-term dialysis patients. MXCT is a practical and promising device used for the non-invasive evaluation of early bone changes in uremic patients.

Menatetrenone ameliorates reduction in bone mineral density and bone strength in sciatic neurectomized rats.

Vitamin K2 (menaquinone) acts on the bone metabolism. Menatetrenon (MK-4) is a vitamin K2 homologue that has been used as a therapeutic agent for osteoporosis in Japan. Rat models of immobilization induced by sciatic neurectomy are characterized by transiently increased bone resorption and sustained reduction in bone formation. Using such a rat model, we investigated the efficacy of MK-4 on bone loss. Male Sprague-Dawley rats were subjected to unilateral sciatic neurectomy and administered MK-4 for 28 d beginning day 21 after operation. The effect of MK-4 on the immobilized bone was assessed by measuring the bone mineral density of the femur, breaking force of the femoral diaphysis, and bone histomorphometry in tibial diaphysis. The BMD on both the femoral distal metaphysis and diaphysis was reduced by sciatic neurectomy. The administration of MK-4 ameliorated this reduction in a dose-dependent manner. The administration of 30 mg/kg MK-4 ameliorated the reduction in bone strength. An improvement in bone formation was observed following the administration of MK-4. These results suggest that MK-4 has a therapeutic potential for immobilization-induced osteopenia.

A case report of a bone histomorphometrical analysis after a total parathyroidectomy.

A patient with secondary hyperparathyroidism (2 degrees HPT) underwent a total parathyroidectomy (PTx) without autotransplantation and showed interesting changes in the morphology of his iliac bone before and after the surgery. A 70-year-old man had been on hemodialysis for chronic glomerulonephritis since 1987. He had been administered calcium carbonate 6.0 g daily to prevent reabsorption of phosphorus and alfacalcidol 1.0 microg three times weekly at the end of hemodialysis. In September 2000, his intact parathyroid hormone (iPTH; 1-84 PTH) was 610 pg/mL; therefore, from 2.5 microg to 10 microg 22-oxacalcitriol (maxacalcitol, a derivative of active vitamin D) was administered intravenously three times weekly at the end of hemodialysis. This compound is used in Japan for 2 degrees HPT. However, the iPTH progressed, and hypercalcemia and hyperphosphatemia were observed. Ultrasonography of the neck illustrated three enlarged parathyroid glands that were each over 1.0 cm in diameter. On 14 July 2004, a PTx without autotransplantation (PTx alone) and an iliac crest bone biopsy were performed. Bone specimens showed mild lesions of hyperparathyroidism, but did not meet the criteria for osteitis fibrosa. One year after the procedure, a second biopsy was obtained to investigate the bone turnover in response to a lack of parathyroid hormone. The bone specimen showed tetracycline labeling at the time of PTx alone, and new bone apposition on the surface without tetracycline labeling. This adynamic bone disease (ABD) suggested that new bone apposition in the absence of tetracycline labeling had occurred after the PTx alone, and it had likely occurred over the course of one year.

Stimulatory effect of hydrothermally synthesized biodegradable hydroxyapatite granules on osteogenesis and direct association with osteoclasts.

Calcium-deficient hydroxyapatite (HA) granules with a unique spherical shape were prepared using an applied hydrothermal method. Spherical stoichiometric HA granules were also prepared by normal sintering and both granules were used for implantation into rat tibiae to compare the biological responses to each implant. Twelve and 24 weeks after implantation, the volume of calcium-deficient HA granules was significantly less than that of stoichiometric HA granules, and the biodegradability of calcium-deficient HA granules was confirmed. The larger number of osteoclasts, larger osteoblast surface and larger bone volume in the implanted area of calcium-deficient HA than those of stoichiometric HA suggested that osteoclastic resorption of calcium-deficient HA affected osteogenesis in that area. To analyze the direct contribution of osteoclasts to osteogenesis, C2C12 multipotent myoblastic cells, which have the potential to differentiate into osteoblasts in the presence of bone morphogenetic protein 2, were cultured with supernatants of osteoclasts cultured on calcium-deficient HA, stoichiometric HA, beta-tricalcium phosphate disks or plastic dishes, or bone marrow macrophages cultured on plastic dishes. Supernatants of osteoclasts but not bone marrow macrophages stimulated the expression of Runx2 and osteocalcin in C2C12 cells in concert with bone morphogenetic protein 2. The expression of alkaline phosphatase was stimulated with supernatants of osteoclasts cultured on ceramic disks. These results suggested that osteoclasts produced certain soluble factors which stimulated osteoblastic differentiation and they were thought to be associated with the induction of a larger osteoblast surface and bone volume in the animals implanted with calcium-deficient HA granules.

In vivo administration of 1,25-dihydroxyvitamin D3 suppresses the expression of RANKL mRNA in bone of thyroparathyroidectomized rats constantly infused with PTH.

It is known that pharmacological or toxic doses of vitamin D induce bone resorption both in vivo and in vitro, whereas physiological doses of the vitamin have a protective effect on bone in vivo. To investigate the discrepancies of the dose-dependent effect of vitamin D on bone resorption, we examined the in vivo effect of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] on the expression of the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) and osteoprotegerin (OPG) mRNAs in bone of thyroparathyroidectomized (TPTX) rats infused with or without parathyroid hormone (PTH). Continuous infusion of 50 ng/h of PTH greatly increased the expression of RANKL mRNA in bone of TPTX rats. Expression of OPG mRNA was not altered by PTH infusion. When graded doses of 1,25(OH)(2)D(3) was daily administered orally for 14 days to normocalcemic TPTX rats constantly infused with PTH, 0.01 and 0.1 microg/kg of 1,25(OH)(2)D(3) inhibited the PTH-induced RANKL mRNA expression, but 0.5 microg/kg of the vitamin did not inhibit it. Regulator of G protein signaling-2 (RGS-2) gene expression was suppressed by 1,25(OH)(2)D(3) dose-dependently, but PTH/PTHrP receptor mRNA expression was not altered. Bone morphometric analyses revealed that 1,25(OH)(2)D(3) suppressed PTH-induced osteoclast number in vivo. These results suggest that pharmacological or toxic doses of 1,25(OH)(2)D(3) stimulate bone resorption by inducing RANKL, but a certain range of physiological doses of the vitamin inhibit PTH-induced bone resorption, the latter mechanism appeared to be mediated, at least in part, by the suppression of the PTH/PTHrP receptor-mediated signaling.

Menatetrenone prevents osteoblast dysfunction in unilateral sciatic neurectomized rats.

Menatetrenone (MK-4) inhibits bone resorption and enhances osteoblast-induced mineralization. In this study, we examined whether MK-4 administration had beneficial effects on osteoblast dysfunction and trabecular microstructure as well as on bone volume loss in a rat model of osteopenia. Male Sprague-Dawley rats were neurectomized and administered MK-4 as a daily supplement. On Day 21 after neurectomy, significant bone loss was observed in the positive control rats. MK-4 prevented the decrease in bone mineral density of the distal metaphysis of the femur. The osteoclast surface per bone surface (Oc.S/BS) and the number of osteoclasts per bone perimeter (N.Oc/B.Pm) were reduced and the mineral apposition rate (MAR) decreased in the immobilized rats on Day 42, suggesting suppression of bone turnover. In contrast, administration with a low dose of menatetrenone led to an increase of MAR and bone formation rate (BFR), while Oc.S/BS and N.Oc/B.Pm remained at normal levels. These data suggested that MK-4 reduced the loss of trabecular bone, prevented osteoblast dysfunction to a certain extent, and contributed to preservation of the trabecular microstructure in this rat model of osteopenia induced by sciatic neurectomy.

Combination use of vitamin K(2) further increases bone volume and ameliorates extremely low turnover bone induced by bisphosphonate therapy in tail-suspension rats.

Bisphosphonate is a potent inhibitor of bone resorption, which results in the increase of bone volume. However, bisphosphonate treatment may lead to extremely low bone turnover and abnormal bone microstructure. In this study, we examined whether the combination of bisphosphonate with vitamin K(2) treatment may have beneficial effects on bone turnover and trabecular microstructure as well as on bone volume loss by using tail-suspension model rats. In these model rats, bone mineral density (BMD) decreased with histological evidence of enhanced bone resorption and suppressed bone formation. By bisphosphonate treatment, BMD was increased compared with that of tail-suspended rats. Osteoclast surface per bone surface (Oc.S/BS) and number of osteoclasts per bone perimeter (N.Oc/B.Pm) were reduced and mineral apposition rate (MAR) decreased, suggesting extreme suppression of bone turnover. However, trabecular structure examined by microfocus CT was apparently abnormal. By contrast, combination of bisphosphonate with vitamin K(2) leads to further increase of bone volume. MAR and BFR as well as Oc.S/BS and N.Oc/B.Pm were increased compared with those of the bisphosphonate-treated group. However, abnormal structure of trabeculae in secondary spongiosa was not completely ameliorated. These data suggested that concomitant use of vitamin K(2) with bisphosphonate excessively ameliorates too much suppression of bone turnover while more efficiently preventing bone volume loss.

Maintenance of trabecular structure and bone volume by vitamin K(2) in mature rats with long-term tail suspension.

Bone volume loss is one of the major health problems during long-term spaceflight. We examined the effects of vitamin K(2) on bone abnormalities in tail-suspended mature male Sprague-Dawley rats (13 weeks old). In this model, increased bone resorption and sustained suppression of bone formation resulted in progressive bone loss in 4 weeks, which simulates bone changes in humans during spaceflight. A significant decrease in bone mineral density (BMD), as well as a decreased mineral apposition rate (MAR), increased number of osteoclasts per bone perimeter (N.Oc/B.Pm), and increased osteoclast surface per bone surface (Oc.S/BS) in the suspended group was effectively prevented by vitamin K(2), given orally (menatetrenone, 22 mg/kg body weight). Microfocus computed tomography (CT) and node-strut analyses revealed that the volume and structure of trabecular bone were maintained near normal by the vitamin K(2) treatment. A recent report has suggested the abnormal metabolism or action of vitamin K in a microgravity environment, and our data therefore suggest that vitamin K(2) may be useful for the prevention of bone loss and for the maintenance of normal trabecular structure during spaceflight.